<%BANNER%>

Molecular Interactions in Surfactant Solutions: From Micelles to Microemulsions

xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20101208_AAAAJH INGEST_TIME 2010-12-08T18:46:41Z PACKAGE UFE0017540_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 19589 DFID F20101208_AABNVX ORIGIN DEPOSITOR PATH james_m_Page_158.QC.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
3f061c118383017b445080eafffbef3e
SHA-1
ae8566190b794596b16dae888bc57576b743bc63
1053954 F20101208_AABMSV james_m_Page_094.tif
b17433e5ce6178cf72bf0b04f6bf92d5
d196c35b746c6cc7a9b02be9ca2a0dfba31263e2
8423998 F20101208_AABMTK james_m_Page_109.tif
adc484b36e6af4417ab037676b9a575e
fa4848760f4eb317b6380a6a373ea577c9287812
3321 F20101208_AABNVY james_m_Page_159thm.jpg
0cb6f609bdaec79e203b9f7be0fb27ee
9cd621be006fc6fb097564af5cd4cfa7ed19094b
F20101208_AABMSW james_m_Page_095.tif
f2ba4e466ba86496e7daa1a244f80e1f
fd140a5caaca6ef1b62bb77c316daba3402409da
F20101208_AABMTL james_m_Page_110.tif
a87ffee0946c4258ce4d140cd949cc73
a9789444c993cafd518ab92913d8680822c5af73
7207 F20101208_AABNVZ james_m_Page_160thm.jpg
a52a26e57d205ae21b855b55bc98050d
452093df01f9f78ec9c77ff8c722837f529fffd3
F20101208_AABMSX james_m_Page_096.tif
883d13310bcf888c8c68bb928a35cd04
b316cbbc1cd910c734e63f9ad2ad9b3ca6fbf177
F20101208_AABMUA james_m_Page_125.tif
2ba2b7528fe490c5bfdd44bade053bc0
f75e0e090a92928cff6590d4c6a94758aed720f4
25271604 F20101208_AABMTM james_m_Page_111.tif
e17bb17e9d101d6fd06fdc3697d463a0
eb4407f5649bc14bf8f71e267f857150f72bf20e
F20101208_AABMSY james_m_Page_097.tif
9b6ee11b8b45af9d80fff44483d22be4
c7608a337b89ad98e41952f08265c0f3af834868
F20101208_AABMUB james_m_Page_126.tif
2c0a81ff9f707d2598e047bb08bd609b
fe4ad33fcfe0b293ad822ab888a032aeb8922603
F20101208_AABMTN james_m_Page_112.tif
dd672a3d9107826b4e3f26937a33140b
ad035e60ff8c10e105cb9625a582e0b0b226a29c
F20101208_AABMSZ james_m_Page_098.tif
1a432d10d864a91d688fbba77d5c583f
36a68106ad0772a6f8b9aa04488718bc9d7cf245
F20101208_AABMUC james_m_Page_127.tif
497fe320df425e98bdf0c3b7b6ab2997
79f0c574a762f63fb50226a8bf7d72e57d579ee5
F20101208_AABMTO james_m_Page_113.tif
3b01bd0f9657a1ee176d649628b7fb4f
cb868715a3af790b8852ef25532c809bd144cf26
F20101208_AABMUD james_m_Page_128.tif
7d07f71f33b5658dc41ad0cfb60cb729
be5a6ce71d08231a52d185ac405cd95824eca5be
F20101208_AABMTP james_m_Page_114.tif
1779366fee341d0c446d1b9cd5082057
a8470810f41659cd41d4ed1c554051150f7185de
F20101208_AABMUE james_m_Page_129.tif
5ecc4ef92846d124ecfad5a779934afc
53c026b94805be694ee3c735def0dfa267f5a45d
F20101208_AABMTQ james_m_Page_115.tif
bc764645355e73e0ae6b921f9f0bef55
fd1cf13411ec6f32442cc725fb24e9f8a72af12a
F20101208_AABMUF james_m_Page_130.tif
7c53b2e9a3cef8558d56bde68cf8921a
ee5e9868afca8994dfdedb997476d3ce0e3b553c
F20101208_AABMTR james_m_Page_116.tif
bf64b3de75492d26428911b7d9940fa9
63eeef420d80d975552e8784601560f9385b6b26
9098 F20101208_AABNAA james_m_Page_112.pro
9b53f8a36953d13e002cbf578624fd00
d1eba931874293d6bcd3a37faac33365aac24377
F20101208_AABMUG james_m_Page_131.tif
823d4abcce9ef414f3e35c6b979ad506
55589581805480287f8b0ee6014699294fa2cb31
F20101208_AABMTS james_m_Page_117.tif
2c59f61911f1a3885b989116a5eb7920
13d627b8b88e5799ceca1201511df68353a9416e
F20101208_AABMUH james_m_Page_132.tif
eeb4cad51a5a106d843e6d40c0f747d0
8fa9fa78833fecc5c53787793d8adcfa3cb898ff
F20101208_AABMTT james_m_Page_118.tif
0a9ef26e4d8026ceba062ab40c30c99b
b57b7e70002771b43dae660ee6969fccb6f59a73
13216 F20101208_AABNAB james_m_Page_113.pro
4cbcd67b09f74c69eab291c50889310d
0ef1c2c8c306765a53ee3dadc68b9def596b2f9e
F20101208_AABMUI james_m_Page_133.tif
e77077a3f9167acabc61ac3b4d36fc15
7f58fee49754c9dd5c14f27472e6d41a2cd5a0c0
F20101208_AABMTU james_m_Page_119.tif
b76e4f1faac72853d872a69b2b6af843
54869cf9db62a8e254456752f88516d396092b67
17385 F20101208_AABNAC james_m_Page_114.pro
5e6b251feb09f497d65898bc164c3a81
964009eb20620f4857e82564ca6ff3aec2ec6e8b
F20101208_AABMUJ james_m_Page_134.tif
7bca5324a4921be5fb1e04286951e769
d1b5213e921950840b85d30b79721ae1f928f776
6799 F20101208_AABNAD james_m_Page_115.pro
d328aa010d8193c131f2d9a017355d31
d95c2d42dca72e5d660f4a8437b52904018f36fb
F20101208_AABMUK james_m_Page_135.tif
bc777376677d73719b194c8b53ea56a5
add2263fa01bb4692ec1660c98b2a2429cdeb88d
F20101208_AABMTV james_m_Page_120.tif
d8a6e6c5c9d9750bd912c9befcd1aa4c
e5a4afb68385fbaae965e51ddb3b7abfdf0a591b
19602 F20101208_AABNAE james_m_Page_116.pro
e41239f22c0b2627cffa877c5b698877
a991785971258ac2b029b97a2b558266bafdef6d
F20101208_AABMUL james_m_Page_136.tif
1248c1ab3c8ed48eaea19104be3b3df4
442b96d719714149c4a447419046e3657920cacc
F20101208_AABMTW james_m_Page_121.tif
229efb867c7c7f23908610d1a9475c1a
cac2061d6bef916b914b7140806a3526c9c23372
50603 F20101208_AABNAF james_m_Page_117.pro
cf09cb36af2a1c8f1096d80052603e48
afc8b7488cbc822b261e3f396ec38f5eeee6db80
F20101208_AABMVA james_m_Page_153.tif
506272ee49f8f0c8c30ebabca32cd110
7ea7a7f8cf150b0a4f48018726dcba6ab63290c9
F20101208_AABMUM james_m_Page_137.tif
f86360f8d3c6f6518b004d05e007ebf2
b00568f9a3b7f2fd71ae44f5a6c053f80bb32127
F20101208_AABMTX james_m_Page_122.tif
1276ffc1c6d45d661368fdb2c05f691a
1b8b26b6a184eeb6f4437939aea001f76c48d1d4
54411 F20101208_AABNAG james_m_Page_118.pro
b5528e1d1ae245e22c123c5d5362828a
3b7a5ea04b8fb478dec73b9ed4fccb013bd77237
F20101208_AABMVB james_m_Page_154.tif
8c4dfc8e5c404b94562a04611732b9b6
c934aaa641c48a9d7c5d7ec7135b9cd1803ff1bd
F20101208_AABMUN james_m_Page_138.tif
876e620bd724d6581e7e4422573c0a63
623ba4b117d03fa51473abcb7f789343724143e2
F20101208_AABMTY james_m_Page_123.tif
5c32f54de00e54174ee90d5ebb22c669
d89c8a12e4388bd4a3216ecec127f6ad46280998
54700 F20101208_AABNAH james_m_Page_119.pro
515a7ab9d04847c0f2f71794bd96eeb3
35f267fecf958cfcba0f9d947409201e94c071a0
F20101208_AABMVC james_m_Page_155.tif
752e7116c44fba99f938d2079a65f81b
06296caa09a685fe6dc270067a9661406299621c
F20101208_AABMUO james_m_Page_139.tif
5936275206199b1464a947ac7b771257
0c9410bddd596750bb3d2d1ff70a85e276c1196f
F20101208_AABMTZ james_m_Page_124.tif
a6b5203f1e2bafe4c073e6eeb9b47e00
1471f2080cee8669359e4e850f6684e945020846
54652 F20101208_AABNAI james_m_Page_120.pro
a04a57c75ba0f86e377514e37f2b20ac
95428dcefae61fdb1800e3bb0baa85a673b12e3c
F20101208_AABMUP james_m_Page_140.tif
ac5dce9f6f4c9a1d98597650da5f2b40
ece08836226dacf8c9bd77fc15fb03c7ce674a46
46743 F20101208_AABNAJ james_m_Page_121.pro
7b9985deafef28364f0dbd260c4e12cb
0a5e5734998514faee671453bc14a6fadc1fbe16
F20101208_AABMVD james_m_Page_156.tif
454189b1490eae289b2079b8937f36c6
bee19943bd6f28723aae8fd628b492e4bd07ba17
F20101208_AABMUQ james_m_Page_141.tif
0629724d2735e83665abf899bc1b5f31
643bcb88b7429936d43dadc1bcbfc4049502bb42
54555 F20101208_AABNAK james_m_Page_122.pro
b3fc06672472bf0b8841d736800706de
642d282d70201edff84f6104ec603b0810c9626c
F20101208_AABMVE james_m_Page_157.tif
0033e4f5efbaa24a326019fd4bddf8d2
20b589db817e54441bbcb625a36b20315b25f284
F20101208_AABMUR james_m_Page_142.tif
964934fd80b1d5a1ac800b9605f4c2c9
cf143f352763f21c72e2385bef432e49d5ebb2ff
7824 F20101208_AABNBA james_m_Page_138.pro
108d1f74cc825754e5d07c421b0301ab
2b73ceeec61708c2d8ff88bebd11523154368937
48310 F20101208_AABNAL james_m_Page_123.pro
72d35100a71ba31681ca310ef95e529e
7cee1856a5e7dc26c6eeba0b17719421c79e1b59
F20101208_AABMVF james_m_Page_158.tif
7e5609ede766b3e5d735c1a8451baeed
1ec4c404ac40f4955f9b28b8a4f495704f656539
F20101208_AABMUS james_m_Page_143.tif
ebe456f2d2e39187d902e7b6d20af0f0
c6812e5d369bfa219f737d5efbd04587b55a613a
8965 F20101208_AABNBB james_m_Page_139.pro
7350466b5a7689c855e52b3d9ec51c66
f869277b1b69c1ad4b063616562d3a896dfbed17
43609 F20101208_AABNAM james_m_Page_124.pro
e3cb3e44156b3bf1e6019ca1e83fc214
5e73921454a663ac953feba5b855440eb6e9a4f9
F20101208_AABMVG james_m_Page_159.tif
83e43e09c50c7c528da93651131ea084
b329dac742e1af87cc4f7a48a9afc479b1d7217f
F20101208_AABMUT james_m_Page_144.tif
28740ad772ce6b8e51f3c5895defbd29
af7e0e5f7189e5d9c0661c23f8c730924b3ec13c
57199 F20101208_AABNAN james_m_Page_125.pro
edf42bb31aa88094f1c9ab7adab25f2c
c35d7651925a2703ea6c048749ddb9e6d64e202e
F20101208_AABMVH james_m_Page_160.tif
f329fd26941c4233c77ca7bee1cd286f
5010c9a71ed578920987fbc433aba181a2060a8c
F20101208_AABMUU james_m_Page_145.tif
8ee2afbc62a527b795ba5d46910f473a
716ba4c4376c44caf85b47454da81a3b9f45969c
13479 F20101208_AABNBC james_m_Page_140.pro
77dbf0680161574868865eec41407e0e
3065509442af186adf6d025234975e040f15271e
55038 F20101208_AABNAO james_m_Page_126.pro
6b8d205ec912c37d9daf445d13bb3668
27e7168775de7250958e3ed92af6921829e6b64c
F20101208_AABMVI james_m_Page_161.tif
f1c2e52bfdc14d0249bca12e1c56380a
e5e324599fac35d6bc825bf6b05cc87e5a6d358f
F20101208_AABMUV james_m_Page_147.tif
a447f009c4c5e18a14c042b83f5352dd
a92ee6029545cfbc7238ec4cfc88f9927dbff65a
9868 F20101208_AABNBD james_m_Page_141.pro
a31e60a246de5da1aa9cfad7886094c3
6599e8972374b00bc5a550a10567344f0cb71d52
55146 F20101208_AABNAP james_m_Page_127.pro
94d25a7b6f2172b1d0a5fd7993eb2d32
2512e8419ef2ea0847c48b981fb41bd915076870
F20101208_AABMVJ james_m_Page_162.tif
607687df18c96d6fa4435ec12a3a7292
d1d9815e612d935aaf3144968c27cf4ffd352597
15883 F20101208_AABNBE james_m_Page_142.pro
85adf4e53c0a91bb18db3ac17321253d
a86a6a95b346cb1be27ab0d3a4f051227676c2c6
59427 F20101208_AABNAQ james_m_Page_128.pro
e890548faa350d57dadfa8563d19f045
38c2eadf7966318cf44d8b7c2845b733df60bf19
F20101208_AABMVK james_m_Page_163.tif
bd58c668784a4118b5eb9cb6a2657d75
5c8d2742583097fef19c1a6840af5d825113413d
F20101208_AABMUW james_m_Page_148.tif
5b021aae77eba99d2e4a4ea554116fd3
8e62d7729e09c5a85b650fa83b70f84e2e8065d3
8658 F20101208_AABNBF james_m_Page_143.pro
2d714117c77e15bd6f337325f34e6525
df67be17a86f472ef792ffe68f74dded849a15c1
54105 F20101208_AABNAR james_m_Page_129.pro
f3a93c9d79c99372ef01b88485e80d68
c2cad2b04e1d04f05210b05f98bb00c1be085e3d
F20101208_AABMVL james_m_Page_164.tif
e4d495e88bafdf84db8d00bb262a5e1e
2b2a925a91849bf7f05dc649ba3a50d3ce89c5f1
F20101208_AABMUX james_m_Page_149.tif
fe13b5e1b574733ac728c41e742b75c0
be76d3a7a37bf9d49758ff0118c1601407c5c55e
24344 F20101208_AABNBG james_m_Page_144.pro
56c0d5b76068f266e67c66fd1ce9e861
706cda6e71702cb7a5f8f8a6c7db0eb43c4ae40f
91148 F20101208_AABMWA james_m_Page_006.pro
3285d9d03cd76af4ec4d24a1aac6d7b3
0ae775ca0080b45e0335d4b030620d9307f0e3b0
55054 F20101208_AABNAS james_m_Page_130.pro
3123d86372014efefe5a776e7aa61e26
7de4d02876bf3874fe57431d15f1c210758ae9c0
F20101208_AABMVM james_m_Page_165.tif
2f44b605760d68c01c3a1abfe54396c3
ca5f85bbca02622faa8882051bcb309aee0e8a79
F20101208_AABMUY james_m_Page_150.tif
05a782833040eef2efd7026e27716977
476c43e95a860e1a6f1c04a86f219d0718ab79e4
25318 F20101208_AABNBH james_m_Page_145.pro
52a1aeec7ff2f3fedcb51a07863d39d3
328ecf05dd97bda42b27c95e1a568d17107d8006
33788 F20101208_AABMWB james_m_Page_007.pro
513d7b288c87ac7daf5e8a605a410f89
3c7d5af13f08b9d165859b8474b6b543c971c4d4
55808 F20101208_AABNAT james_m_Page_131.pro
617dd243ff730214ea8cb7b2781723a8
4d8e549cd6dc395f2d150ba7b6716e928eee216b
F20101208_AABMVN james_m_Page_166.tif
a6eadfd8ba0a511d0498f15ca4eeac26
05c4a2866584dc8068ce9c9e50f3a54ec81a5c5b
F20101208_AABMUZ james_m_Page_151.tif
d64994c283309a6d591ac890dc16e92a
6c85b4267ff5c1e73d45191dc5b254447e62c1d1
50561 F20101208_AABNBI james_m_Page_146.pro
c233d1b7c598b551f7c509b0d21cbcdd
e237d071bd1b613e1d0524989f112b1d5bceda62
49665 F20101208_AABMWC james_m_Page_008.pro
d3e70cb320fe94d692714dd081069c96
5ad36928be833e0e6a3a2e6a03ffa7c43bb56133
74866 F20101208_AABNAU james_m_Page_132.pro
733de51b0b1835f84d2d111785e6b3dd
3db931aa62a746af0f7ffe0c8694f1a11953160e
F20101208_AABMVO james_m_Page_167.tif
4880a4230a4b0a4f979889bc7c0a783d
8bdba34d701fbfdf0aba8e6ab0f04509c911ed68
63650 F20101208_AABNBJ james_m_Page_147.pro
79172bf4eab38decaaab9b6ddf279d30
2a753118875f4d42a09166103de95d5228760579
63838 F20101208_AABMWD james_m_Page_009.pro
e9510a6e20be71162864831e7d8e62a6
b3c0751a9a2b1dee25d3e160aafac9a8c30bcdff
7215 F20101208_AABNAV james_m_Page_133.pro
7a4ca428f51e1050691913d525c77a62
b38e0a78d766febcdc9ab3fe891470d60eae9845
F20101208_AABMVP james_m_Page_168.tif
908668abb1841b9d379a9a9a15dc8013
ff392b1a788fdf383bb495afdf1abe80c93bec2f
78165 F20101208_AABNBK james_m_Page_148.pro
f483b18ac9a8557f458891a9f4d3d9d1
72214287b8552c94550ab328ff23ee13b93734ab
70598 F20101208_AABMWE james_m_Page_010.pro
f2ed2ba3fa9c5232b42ce21203772b3a
00690252a49a9c867c8b445f548a3cb9a592fd9d
13227 F20101208_AABNAW james_m_Page_134.pro
0b3ebb2bb3b4062d0e6efa48164c2d67
7453b5c6d0020a5cc4013464f631d390255c9fe0
F20101208_AABMVQ james_m_Page_169.tif
615d478888c1449ecd9ac57882504beb
5a795afe4214473cd5a0934fd8d65fbca963b170
60412 F20101208_AABNCA james_m_Page_165.pro
11def805e8006e90812f04de5eae559b
0794e267a1176a5d828a065668dfc75a416581c5
53523 F20101208_AABNBL james_m_Page_149.pro
f0fe10fde8d9dced399b101b31bb45f7
cfdf6dfe4eeb6d684da284af90615b5045a79ff6
13582 F20101208_AABMWF james_m_Page_011.pro
2123d5a750971a3bb3b52037104874e7
2342b232c77045a6c79fe44242b423734e1b8776
8939 F20101208_AABNAX james_m_Page_135.pro
c76979d9a2f166ba5ebc3f06eaacba6c
00240e9124a20e7489f65b5a198a2ff8bf2c460c
F20101208_AABMVR james_m_Page_170.tif
2bffc3c577c97c431b4fe22f7cf99c7d
97d22844117ea370c027e25a13a4333d68acc669
54064 F20101208_AABNBM james_m_Page_150.pro
cc9484954a782a1a0a111a9a18e81402
a8f7bd8bd5d59a4c5c9fa3e0deae92ed5af497ef
41070 F20101208_AABMWG james_m_Page_012.pro
e9733ba4ef7cbcee9eb56de62708cb1a
e9b5bb9a1e0deca9c25bf6ea6a4994696c7055c8
6532 F20101208_AABNAY james_m_Page_136.pro
36040734f1082bc1793b7baeeb62b797
359a189811e033d6301422c1cd92f40c4046ad8c
F20101208_AABMVS james_m_Page_171.tif
9927819cc10ddc662bde0b79ca516ca6
3095bfbdcb8f3387dd0c9ec04f28afffe1cba111
62863 F20101208_AABNCB james_m_Page_166.pro
15d7de9882e22b2a91508256eb279b7e
5b4232fe163c26152b000b2179174db6c312d54b
74958 F20101208_AABNBN james_m_Page_151.pro
88766205c1c1f6060045d591d9c115ca
7d17718c5aad9f43ca063d561d2ded34dc4bbd00
27589 F20101208_AABMWH james_m_Page_013.pro
800a06855167eeb16fe7ab2838a3a3aa
56c6a8677dc95a22d18d146a49be87408c614f9e
6547 F20101208_AABNAZ james_m_Page_137.pro
f504dba44187d144d60ae527ee8b7a08
715bdd3a49be4178ae33998c398293a2e0ec771f
F20101208_AABMVT james_m_Page_172.tif
29eff6f0f3cfaf32f7c7c8956f17c224
f6f5d8b7f4b4cb724796935845fd08fa5ea1db41
62767 F20101208_AABNCC james_m_Page_167.pro
9df96d18a48eea942869dd69791612cd
82228033f00e2f1aa38d48b5b89703ed70633211
53129 F20101208_AABNBO james_m_Page_152.pro
42115fd267e95c879616d6e4fa6826cc
f542d7545b7e9f32fbf0612eb534e6ae7214df13
48962 F20101208_AABMWI james_m_Page_014.pro
d56bd246c4f17ebca040adbb242df51b
4c432a608429805c01cc2645b3417762893ca961
F20101208_AABMVU james_m_Page_173.tif
80d524b96e3cb58e4ff755e6d17fee68
881c3429743c3d14c1345f3f5db7af127d2f483d
55639 F20101208_AABNBP james_m_Page_153.pro
65501b0140633de48adaf67d8ec35076
d7a39a002a3e2076135281fbe3e416948d47b4b6
53148 F20101208_AABMWJ james_m_Page_015.pro
1bc5fcb34ce7627dc2d009cc4a4a6656
ce8a89af57699f271e5845b2f366db4bc10e322b
8318 F20101208_AABMVV james_m_Page_001.pro
e24fed195ef970783f15202b8e52f68f
26f3f7b0863abc1ec43d4c92113d6bd0fe4a42ba
69024 F20101208_AABNCD james_m_Page_168.pro
096221cdc547171db07e1954419b9624
fe1cd1c6caee1da6695560eb62c0a53baa622acb
55189 F20101208_AABNBQ james_m_Page_154.pro
e8f99d67ddc8aeaa401d0f4a151c01c7
a5e25dcfff13ce48da571d4762dc78837a700f06
54239 F20101208_AABMWK james_m_Page_016.pro
3e4e9a7581619d8c6922cc1438eb7fa8
54f56899d98010d6b842c46ead4e50cbdd46b1cc
1327 F20101208_AABMVW james_m_Page_002.pro
4f1537885c25e9e165f06f6afc6a3528
e4c059eb0ec9c0c457ee9ce55f5f74cf89f49a4a
66165 F20101208_AABNCE james_m_Page_169.pro
d472896deb91395f96e0387af3774fa4
125400e7116830e04041584f309456478ad7969c
44925 F20101208_AABNBR james_m_Page_156.pro
cb7e4eb82e0cda13fc9c5f2bc5582e54
f9d2deb95b50d4f05c6522d82d16bbdbb849fed8
55617 F20101208_AABMWL james_m_Page_017.pro
a1b509cd224f1cdc76d7f9c71eff144c
bb357f2f6f826423931fcc10cf0a5b7054178805
71018 F20101208_AABNCF james_m_Page_170.pro
a2cca3dff9d20b0c74211d522e6c8c21
a46f498683d4bd3c4c6f30d9c0a1b83c254a49a0
46197 F20101208_AABMXA james_m_Page_032.pro
e5a03abdf4700dc2a341cfd7246b66a1
2f04e060e9d6b76fc543b5af68815d60ddecdf99
30525 F20101208_AABNBS james_m_Page_157.pro
4dcb55da1b41effc40ff4f5e83ada590
eb00606500ec8274025c1e16b11562af13430298
54878 F20101208_AABMWM james_m_Page_018.pro
ba3fa1ccdcdd143343210a72a746ed83
6c248b76d02bdd1132c24442f30cec2a908d7ee2
2066 F20101208_AABMVX james_m_Page_003.pro
b35f5ff0f1cff8da7a136be6393265ec
c260931cc07fae1d9d13b125783b68889cd6095f
59524 F20101208_AABNCG james_m_Page_171.pro
7ab6047a79ddd53668ee635bcffb4fc1
acb28a66b7942d38c4ee5b35857b45fc1e343d9c
50754 F20101208_AABMXB james_m_Page_033.pro
4dbe19d5b3c885b8e5659d96f1759b5c
6ddc123e1f03b933e2f40204a7deb2283c3d5647
28473 F20101208_AABNBT james_m_Page_158.pro
96a7db91eb493861cf8702414b9d4825
fc5f96848e0d1cff115fff6588376f90019374b5
54345 F20101208_AABMWN james_m_Page_019.pro
955918cf4b66c48267c1211ac2add042
956db1a9a031eb1642b666e4b74a72bab07754cd
45005 F20101208_AABMVY james_m_Page_004.pro
e8e99dd74f720356a6687d18dd1150ae
e1de65de4482179ea037eff83465320106af0f4d
35485 F20101208_AABNCH james_m_Page_172.pro
c78309804b6b2689cd87cdddb364d0f1
0f673ebccee18dde0ddb1a3e01098fbf45286037
64361 F20101208_AABMXC james_m_Page_034.pro
bc1d6e04616740caed4977417c827390
7365eeb3949ee3bf611d31b1deb0c812cd262f15
16841 F20101208_AABNBU james_m_Page_159.pro
eac60745b085108e760f8163bcb0430f
8c5f45550fb1a305279531a8cf033d96170a42a4
51902 F20101208_AABMWO james_m_Page_020.pro
b99cd307b36cb1773ce5dd1d31430041
1e6c8c6d75380f3687d81570925ae5839000a22d
74059 F20101208_AABMVZ james_m_Page_005.pro
121230a65cc9a6684b6bc9084b181935
3d56145141c8b82d3d393a7781e657bb62eb2d2c
18135 F20101208_AABNCI james_m_Page_173.pro
872c58247fc158cac2d7c225df260b1c
bc56ca2525b2f2b2983ebfc5e39b8d0b3663f482
49122 F20101208_AABMXD james_m_Page_035.pro
f4a7ecb69384c93b09f42fc09b71c121
5455a58e7cf65e41da7abe1a418710137f493b92
36469 F20101208_AABNBV james_m_Page_160.pro
98a522680736c7259916587c2635d3a0
89b8981efc9af3d87ad17fb00875aff3066c7f30
54558 F20101208_AABMWP james_m_Page_021.pro
124a8a5f81934ff02e08c5d7681b93bf
b22b2a02527929bfea7c91f6201b0abb9dc4cd69
398 F20101208_AABNCJ james_m_Page_001.txt
2522c64e8283e8381e83d3941d9b7e2f
c5f66c708ff2274b90d4603a1c600eff7cd04656
51684 F20101208_AABMXE james_m_Page_036.pro
bbe7e7229b2684463f5477bb181fa4eb
989306327a90171a1e0f47ace732aad580d33a91
23590 F20101208_AABNBW james_m_Page_161.pro
cd3082f9042de3dc82fc41b0e12dc512
c2bce6da924e724fc7752929a3e5cde85897991a
49034 F20101208_AABMWQ james_m_Page_022.pro
2c8efc8a199f43beb0d948e23625eb0f
3f6734a8a059f5d0b02234b7dfc4638192ac3078
122 F20101208_AABNCK james_m_Page_002.txt
7e84272d2e5581c51acb679cb12d57e8
09c458c30769556331114e2096175ecfae6c0f96
41700 F20101208_AABMXF james_m_Page_037.pro
92fd394b74d1cf1788bddfbd485f3c7c
91ea17d3a780f970c5bfa88f6b3ebdc22bb9e456
59690 F20101208_AABNBX james_m_Page_162.pro
64c1274b32e1cd1fd327f8c316b4418f
31d568c7dc1fedc8c88d05f0addaaaa20c8f006b
47794 F20101208_AABMWR james_m_Page_023.pro
fe9828f6b647d7345914ef205fba728a
634e5cc913a2ddd95d4a0d0b1d253ff5cffc97c3
2162 F20101208_AABNDA james_m_Page_018.txt
1f8b6ccf085c7eb4239cbb649785ad1b
e439b19bf1ceaa76c40eb4d6c71a3f6bff164241
148 F20101208_AABNCL james_m_Page_003.txt
bf02ff3b82e5e5f7c1120e4908d5336f
eb1cbea7def03087c6eabff394364e94c2401983
54937 F20101208_AABMXG james_m_Page_038.pro
5f4cf98e5626c5704e23a91cbdbfd584
5513a88d80bba09647a0d171017e9eed0630bd3f
58864 F20101208_AABNBY james_m_Page_163.pro
06e0d9e35356d432f7392c960f48c2ff
b4e68b82c9ec4e87a92b7cadee07a286f25b4e40
50572 F20101208_AABMWS james_m_Page_024.pro
0a57e9d231aa05bcb4cc99ca2e40ec71
aae5f86a939dae77ffb595960b09be74ec72a400
2137 F20101208_AABNDB james_m_Page_019.txt
c465d993024fb1003ecef30d9ea95825
99ee59bf22958108c98852390d2488be9367c779
1825 F20101208_AABNCM james_m_Page_004.txt
c3f5ecb0cc84ff2e7dd160e817e28eec
a1dbf5f48c5a8c4d31815b1e25a3cc127c00c7a0
54187 F20101208_AABMXH james_m_Page_039.pro
0e64933fa2571ee25e12c92ad26ccba8
218edde6a813b37f6acb511b276075ec27b26ad5
65188 F20101208_AABNBZ james_m_Page_164.pro
0ac34aacc5642f49975fb8d50c315e92
81e1a72d5bdd10c71e7a236bfb15452d370f1554
52742 F20101208_AABMWT james_m_Page_025.pro
86050a0fb06343a8853fea247e917764
497df835b1bc1c0e506072ca250369a49ef20a32
2078 F20101208_AABNDC james_m_Page_020.txt
30ccb6d352d666e7f7de75bf355e2e85
74177d47f0cd13cfc08e2d5528c32e27067d77ec
3263 F20101208_AABNCN james_m_Page_005.txt
f78bf06c83bbe52863464c4cd6b223b3
51e5ec8253a884eb7e0a3544d8015ab11ab3f2db
51913 F20101208_AABMXI james_m_Page_040.pro
9b80a89537dd7cf2e39903bd6b06491f
ec9375a8f143abf75096fcced78c4a838593aaad
55257 F20101208_AABMWU james_m_Page_026.pro
2d1d324414b5b5d4cfb78c135682e68d
220377b17a1e9ac2ced1b629de41792b172162d4
2225 F20101208_AABNDD james_m_Page_021.txt
77a816c6f1eb66cee0f0a35dff7b1924
507dafa4e39119d382c0d4500287636f3c2c1018
3923 F20101208_AABNCO james_m_Page_006.txt
2c5a251a0b8b9081e8a7a50223777b41
28f8aeb5015e861afe47d182117cfa3b9597857c
53446 F20101208_AABMXJ james_m_Page_041.pro
093167e0fbb6fd71d34897e6d0ea0589
9cab0cdeadd9089fc99aba4c6d08f9f24c00bdd9
58270 F20101208_AABMWV james_m_Page_027.pro
29fbde752db040f09a64ce475a3a36b1
f5aa85b37d57cd4abdb62594a70fbf80157ce2e1
1394 F20101208_AABNCP james_m_Page_007.txt
e793cbecdffe62a290d4a622bfc72619
42a9fd35c8679e340248273f1be555ba52e5f325
54629 F20101208_AABMXK james_m_Page_042.pro
6801f1f013a9db8d9ddcdb481e777a9b
9a25809086e2834c345b1234aa09397dc9c4c586
45200 F20101208_AABMWW james_m_Page_028.pro
7330a7def60d0caf946c6bb347c7f8f2
1faf3e8bc2634dcd963e37cbaf0231de804f66e0
2097 F20101208_AABNDE james_m_Page_022.txt
a418362e01ffecb4e311f69ca0dd14e4
fac16f815b4c7f56bde821b6f075fde3fbdd89e3
2017 F20101208_AABNCQ james_m_Page_008.txt
479ff4c810e713ef7c10896d88165cbf
fe2a1100e0951bcc2d1c4a50085262927a6c9e99
51234 F20101208_AABMXL james_m_Page_043.pro
136a14a232c8e285dd4d7666572d5add
ff067f5104c815945943c535b791ca8c0a5af80f
49636 F20101208_AABMWX james_m_Page_029.pro
32e1719121e868d05d4d9da3255a958c
077896cd2136fd2f8e729feb94e19620d905e683
1938 F20101208_AABNDF james_m_Page_023.txt
28e706ce4081d8c448db80eb9c8054f7
6f5edd0b77e2790b1dc1ca5d7b4439e58e967760
2562 F20101208_AABNCR james_m_Page_009.txt
b1ababa380e9bdabcc65a777323eedd2
a5eb7d3104df04c996fb04484b81e40a08d6a59d
45746 F20101208_AABMXM james_m_Page_044.pro
852e5b932c0d720079e8647bb6d60cd2
3839824af6cc50bf95d925a14e0d31ff567c6b52
2060 F20101208_AABNDG james_m_Page_024.txt
650fdaf4dd575dcbc32ca46cc073ada7
a4074d17e1e7c85a33f6bce41d6ea42f085e0d4a
30126 F20101208_AABMYA james_m_Page_058.pro
c54398d44f538a93068a6f1600b6e55e
af0b69b39834d300e9c9dc71d67495c4c1b825bd
2915 F20101208_AABNCS james_m_Page_010.txt
5aa87fdbd14af503e481937b6057c299
129a162bdc957ebb0e4f926170a5ddecbc6bf6a7
52396 F20101208_AABMXN james_m_Page_045.pro
a523e93dede644a7a6794b08a4dc7b41
863e74a6096123a02a71c763ad5145e1dabb3087
52717 F20101208_AABMWY james_m_Page_030.pro
887d30b78f552fd8d463373da3e9d9aa
fca570e088cc02c591c4114f7e9911a560b67b2e
2121 F20101208_AABNDH james_m_Page_025.txt
891af1c6587f67c61bb540b512bae1bf
d181704d08636bffbad080e9c662578fdb51a9fa
75487 F20101208_AABMYB james_m_Page_059.pro
fcd06687cddf2f46d171575fece00bc3
2a3c0b39351a8572c0d8420b477f86c8218220e9
573 F20101208_AABNCT james_m_Page_011.txt
a6631aa50e45a9169b64ab31018d43bd
06df5a46d22624fa43e074108c61107c5c23ba9a
51616 F20101208_AABMXO james_m_Page_046.pro
b4d9aaa22401235fcd400c45e1e9ce33
2063237648b4bc5d0c1cb496f2924c8b52718d19
53280 F20101208_AABMWZ james_m_Page_031.pro
3051e02207cf924e58da3c1638e4cd52
12b4f4bb3a32fd07e17e2cad9ba2a92c5ba7c472
2175 F20101208_AABNDI james_m_Page_026.txt
ca8072f600c501262ec8108d855f434b
fd1879edd88fb58fe11e1a69dda92437edf111a0
10054 F20101208_AABMYC james_m_Page_060.pro
57278a747244a075084629ef09b82d27
b934093fa8955ba31a876ee1ce1c9f4f4b8c58ac
1831 F20101208_AABNCU james_m_Page_012.txt
d45e845e214e23babe9cb8e661af7148
68bb1cef9011cdfa816ad00faeee0d75edbd4384
34254 F20101208_AABMXP james_m_Page_047.pro
be032ba15463672065da71a6994f4d72
dab7ea0afe33f48d8c700f5729f9172ec7c4d406
2291 F20101208_AABNDJ james_m_Page_027.txt
658c1846797fa5775733439c1e67b686
844c7e3f371de3d58a50a5c4e36e6ded18ec7a28
51217 F20101208_AABMYD james_m_Page_061.pro
b93e7ad057bdc059b11aacf133ee07af
f48c9404db623d49853f1e6abd6b9fa278e0e6c0
1108 F20101208_AABNCV james_m_Page_013.txt
f46bcc8acc61d94040bd2d7222cb7fff
5ea2353b79f53c9daa44d6c086ebdf43027728c9
13736 F20101208_AABMXQ james_m_Page_048.pro
1943fcb2a59a4dec56cdbdeadf6d4fcb
a39aa44bf55781552edcc3a8e0082b2ff59d587b
1816 F20101208_AABNDK james_m_Page_028.txt
dafe6188296e8b61653de30b2d0a094d
f944b695c00bb597dc3a505b2e71d234d9ca57e7
56364 F20101208_AABMYE james_m_Page_062.pro
753a36737699580b4a5efda333d9615e
389d2d28bb43f4e5ecd78456d806daa114abfdc6
2051 F20101208_AABNCW james_m_Page_014.txt
2aa2c6453349e622ca4addaf3bb200e0
f745c2bb728f42500ee41477b4eb21cffd194df2
10988 F20101208_AABMXR james_m_Page_049.pro
27c1ef5491d600f6ef392c97c0ddfc0e
0a16b91e498d967af90aea7af2cb9de78d656c16
2143 F20101208_AABNEA james_m_Page_045.txt
8e79cd5cfb1ad4df178c333d1eb48e46
33edbc344160637c2d63468bfa40f417ede37465
1964 F20101208_AABNDL james_m_Page_029.txt
4f7582d5a9b553d81866391899ef6b95
3a3324308aca8598ffe8613af01ce0e035cca1f4
47796 F20101208_AABMYF james_m_Page_063.pro
13909eae1e367c94989ecfb465d8e72b
78d401b89ab18995bf1b346b15e7b376f36a640c
2094 F20101208_AABNCX james_m_Page_015.txt
618ea8c0cde243c205a7bb158d8cd6bf
b08821961d00d41c446d3fe7a1c3b7cb83a5971f
20799 F20101208_AABMXS james_m_Page_050.pro
0bff2976e897793b9bb65c85f996ab7c
b4624b5606ecf8221d817791c59070873c01ccd1
2037 F20101208_AABNEB james_m_Page_046.txt
ba5020d118c8ac75bc18c371060dce54
a2807f09ab14a03098cf8b2edb538d674483ed3c
2085 F20101208_AABNDM james_m_Page_030.txt
c452f2bdb273a1c405cfcde89851ec25
4aeee648221d1a48a595fb94d53026240412a832
54231 F20101208_AABMYG james_m_Page_064.pro
51af37a44254255ea55e190d8950b3c4
cd4b66fec9b386e61d84fe5e140360d7e1338c94
2128 F20101208_AABNCY james_m_Page_016.txt
c982e2ace6997065a10e727958e3ef2e
7b908404aa38a33b0d0cde97ec03111b5b37c55f
12856 F20101208_AABMXT james_m_Page_051.pro
a3a8bf3138893bb5811735f8d5ee68b3
bdbb338973c5e6246b73a0b66dd908b4999dde8c
1370 F20101208_AABNEC james_m_Page_047.txt
46892b1a0df9d6c22f4e539b94ccebec
647a00349916ebc9dc7baf1f9eb5f629767b64a8
2092 F20101208_AABNDN james_m_Page_031.txt
ce29600bdea84d39c286d8b6be7dc009
9ed6383a7e74001dabee553df15816ae9a9bcc40
51175 F20101208_AABMYH james_m_Page_065.pro
9a54bd78434f6f96437800af1c199e7e
32ec9dee12f94fa241353c0699bac6b3b56330d2
2189 F20101208_AABNCZ james_m_Page_017.txt
f5b74adc92f918c8241e809912a5060d
3f780da2a3a9ff38f1ab7b48c6ab4a846156c733
8053 F20101208_AABMXU james_m_Page_052.pro
aa8a9252d6aa5ee73db2082c183d61fc
d0774a6f24dda6a63b390506b126aff4a8f5251e
1148 F20101208_AABNED james_m_Page_048.txt
e2964531e0c621783cf21dd3f5e3c711
3e1127a0f7e9c6e386b94ffe74b08cc67c46f488
1840 F20101208_AABNDO james_m_Page_032.txt
89983a9de0e7e8f8cc27ff1745cd71c8
59ebfc67b5d40e012360cad51df06826a9302fb1
54691 F20101208_AABMYI james_m_Page_066.pro
4a1e38619732b80f6a0cfa7e30118342
0b813b5e4c96b63b6cdaabaafe9b07d0c1a1ee08
6565 F20101208_AABMXV james_m_Page_053.pro
7dfdf64ee4b9e2b7fff27efd5f8f52e0
2372ab86ba96fbcd730227956a6026ee82eb71e9
602 F20101208_AABNEE james_m_Page_049.txt
d91379eedbb650c65890b51cb3f5be3d
d956d47739ddd2059fc1affa7997a0e9fc260fe6
2014 F20101208_AABNDP james_m_Page_033.txt
843fbd3a249c3f50f1d002c28029fc98
36ab8e06587f8978c4904b32ab02a7c772bf46f0
57791 F20101208_AABMYJ james_m_Page_067.pro
34f966f094cad63511eb36ad666b07dd
b037482077ea019cb4a3d9c41ef54e8917a85db6
25786 F20101208_AABMXW james_m_Page_054.pro
c4fc6310d532d7014f15f18ba289cc5f
7b512bb1a1f2b9f8e04b54aaa47bd7ff0a462985
2568 F20101208_AABNDQ james_m_Page_034.txt
d9bf5ab615a13ab8af34538bd2ca324e
9a4433fccb4b2d3b4821cee503968897972f3a93
50074 F20101208_AABMYK james_m_Page_068.pro
e06d8a737c390b65ac1ab9e463818fdc
35450579efb2279140369c38223a2f2acdcb9474
39954 F20101208_AABMXX james_m_Page_055.pro
c290e437a60b6c68671da46da2e8d96f
3060dcc6e582297c1a48e281c1c5cca6c6165616
1035 F20101208_AABNEF james_m_Page_050.txt
180c9bcd312061558862ea069759b801
37bdb80ab6cfc8cae6cd351f70557ed277c5c5a5
1985 F20101208_AABNDR james_m_Page_035.txt
e0e3e83b71dd34bcaed16650c56b3ba2
1b9d6cc3d90806b3c9581f34f97a49692e15f90b
56301 F20101208_AABMYL james_m_Page_069.pro
1c3be8872f25c5fcaca37a011738773c
9023192e50b8e27f5f2b4ef0af646759f5fd1f99
48315 F20101208_AABMXY james_m_Page_056.pro
8d3332fe488ef9fb7ea34c31ac3edc7f
374dadcff9c17d3b2090ae9f24d032722d75ea8b
738 F20101208_AABNEG james_m_Page_051.txt
e78889e23d397fcda5666ba8cfa59456
6df30d1bf35c14e33a515828468eb0f49ca64dc3
8107 F20101208_AABMZA james_m_Page_084.pro
2065f0269c70c09097a406c2fa8ee85a
a21d8d1a5b4d086db3d7b5065a243d15d936681b
2077 F20101208_AABNDS james_m_Page_036.txt
4135bea9d02d64f0738a3ba9aabe73fc
868d920b556f256a94a6aec054cf1edef023d941
53236 F20101208_AABMYM james_m_Page_070.pro
5625a77be62b34b6e01253c81e91fb46
e4504aae3484ae2fced30d6ab914cb9ee6d55de7
388 F20101208_AABNEH james_m_Page_052.txt
b154a1d9cee58c171f5a237f46a8ca1b
5629d67b0e01e3b1c03dcf541a29773d42818c90
16579 F20101208_AABMZB james_m_Page_085.pro
dd7764ad7e072c3c76c936d0426c86eb
e845c2706fe3059918127cf9dcb337ac4266be15
1782 F20101208_AABNDT james_m_Page_037.txt
69aad7d57775b35630ebb802d095a3d5
d62fb63873e38c267ab9a3f975835e5863866a4d
55276 F20101208_AABMYN james_m_Page_071.pro
523381aaefa4770c7a4b94eaf4187010
86b25a3d9d498a84fdf82309a2cb003c8cd120c8
47088 F20101208_AABMXZ james_m_Page_057.pro
9663641159120c3a6dbd75a1ff053888
f0bf22329dec0b07738bf2cd70457ac2471c6432
401 F20101208_AABNEI james_m_Page_053.txt
bf3272d1f85c8aca56bb74acf2a87909
42673a4bea17906eac28bb2f69b370f263a50314
7854 F20101208_AABMZC james_m_Page_086.pro
a32f00cff7c0be8a1f50eda6a4c1ace2
d06844e964457e08b6a12818e804d6ab2ce463e2
2191 F20101208_AABNDU james_m_Page_038.txt
dabe0e1d1b4e2214280934ee4692bd7a
193514df409454d48969f61fdb0fd260364a6480
50098 F20101208_AABMYO james_m_Page_072.pro
15c83634fc9d5386b8a3d48334f86117
52968c7e2e581a11d93cac00304553aa5bfb0258
1121 F20101208_AABNEJ james_m_Page_054.txt
8e7b654f9bcf239c07239065b4f05262
7e0b94d572093de0d8f9ac733bf89d872df7ece8
12282 F20101208_AABMZD james_m_Page_087.pro
6c56ff512c40908191a1d06bd80cea92
e78d687425d85cc892da23cf18edb662e90e7a3e
2186 F20101208_AABNDV james_m_Page_039.txt
c0450b592a8892b3dac67f97a4f2e83e
c3289fc7770eb0769e508e9477addb09f33516a9
53247 F20101208_AABMYP james_m_Page_073.pro
eecb84ed15b23d8b5a71a11c377185e6
783a80fbc2707c0c0e89f8a1896f7a0b1f631580
1616 F20101208_AABNEK james_m_Page_055.txt
91a40bb857fa1b1d2346c71eb4c6f911
7c2369a158a1b1f7dfa8b2d7beef508f80ddfefe
12587 F20101208_AABMZE james_m_Page_088.pro
26c9671ec36fa9ca7be8343bc67a99e1
438fc2612105d503ccb281e72e6f76440adcdf02
2110 F20101208_AABNDW james_m_Page_041.txt
9805703dfe4716725e8407f3016a294e
3dbdb099b6803664dfa0f39e2b58c4efff45334e
53012 F20101208_AABMYQ james_m_Page_074.pro
70b2a89e00d798fa2028bbe1cd9008d3
0bfbf58121c821e1d6422ad2028098d924062b0f
2605 F20101208_AABNEL james_m_Page_056.txt
9d01509a5d86b215f701139150a50d6f
867d26f68ae8694a0c26d5c31aecf87d4836e6a2
10087 F20101208_AABMZF james_m_Page_089.pro
95e58e00888d8c03939d64e022a19751
3e7e75a904e0fc9a51401217156ab27ff7655d6f
2154 F20101208_AABNDX james_m_Page_042.txt
894c334d184f1fa31dbcbb33aca53616
5aec4d08ecd947f6f841ce85f71e61ff56cbfd93
55754 F20101208_AABMYR james_m_Page_075.pro
4543a9e52757c91f9dace8f8063e76b9
ebc31b71e30d3739b34a9f23963ba4c6cdb600a1
F20101208_AABNFA james_m_Page_071.txt
ec798fe815711fff3bfbfa2e184f1b12
46dd427eae891539db4411349c0d2e31f2812839
1866 F20101208_AABNEM james_m_Page_057.txt
bdd15562cb967d0617d07f6d88f80516
6acef642991f9e4f317d198fcd41acc194e6288f
16071 F20101208_AABMZG james_m_Page_090.pro
5d54bc8a298da773e5e9185b7d44bfb7
c3320f80c19db9b709892203877bc64210a596f5
2020 F20101208_AABNDY james_m_Page_043.txt
f208fc9541d4377a510425222109bb4b
d3f82891c7f2c5ee57a28e5fd2ffc428f379ecc0
54858 F20101208_AABMYS james_m_Page_076.pro
e2b9ba9cf3441265a1b75000f8153778
254bb2aadadbafcc736ebb02276e7f24defa46dd
1976 F20101208_AABNFB james_m_Page_072.txt
02eb2943e04b258f2222bc299c78c9f5
f117a249a532a56432ad8b266c63f1c16af84a62
1215 F20101208_AABNEN james_m_Page_058.txt
d761e0a4955811ac58b6e07e143d13ae
b11400035f8230897e3a75105c96b7d5b2119a9c
6873 F20101208_AABMZH james_m_Page_092.pro
d651972d85d9e9c144dd8551a1b97b3b
653ba2fe9d9a2ee09da2a296156b9c3cf90cc3d2
1860 F20101208_AABNDZ james_m_Page_044.txt
30f458c26de9f63ad1dbd9cc303c0edf
53ddcb7cc65cc60cd5446360a69d97e6a6f5ea1f
55751 F20101208_AABMYT james_m_Page_077.pro
095b3524bf91d51e287542e2bda2ddc1
0d4221d4b5d6d585cdc59483f8f0e52e8ac27b58
112132 F20101208_AABMCA james_m_Page_031.jp2
518cbe8317851befa1818f7af9de1683
7e341f6ebaeef35bb0363b47cb3bee684709f6ee
2117 F20101208_AABNFC james_m_Page_073.txt
1a602a2fb1862ffb6d59278aca1c6fea
09fc7566d5aee4c943468a67f312abdae0ce43b3
2044 F20101208_AABMBM james_m_Page_040.txt
0cf8036bd24a39ee4ccda26c909b2446
5611a5da9ce8467840435865d43cbdd43848eebe
3026 F20101208_AABNEO james_m_Page_059.txt
83f12661d51040ad3724548f72d165fe
396b8eb54c66f9e63f23b8cd949b8dd64ae6398f
5807 F20101208_AABMZI james_m_Page_093.pro
d93303d84c44a54411708876405aed97
21ea7a22379e99225337eed02e5916ea04082dab
64753 F20101208_AABMYU james_m_Page_078.pro
9f5fbc56342f467d5752bb47613e988a
3e0df2f8ac6f9d247411b26b41737addc2d3ffec
107488 F20101208_AABMCB james_m_Page_042.jpg
30088b36844adb61af7e9b471756f339
da174e5c9be5c811d4e2dbafc4d6b500232a94dc
F20101208_AABNFD james_m_Page_074.txt
4700895fd07d76462954683e4d17060b
0329936117cd208b35bd8127de0e317dfeaec8f6
61991 F20101208_AABMBN james_m_Page_013.jp2
421bd5b9012da52d993fe43ec9ecae4b
f9d5e215a9c8aa2608e081d30a332c5434a307cb
501 F20101208_AABNEP james_m_Page_060.txt
0c062586c45b23a59d07b04b8f20e6e1
70fc4b1af975052d34d168104d5abaf9dd8855b5
54348 F20101208_AABMZJ james_m_Page_094.pro
cc3f05a2c06b712b7d7cfeb76e0eb7a7
06df7fb44e5ec9eb4d99f83bc25016665a2729f8
5799 F20101208_AABMYV james_m_Page_079.pro
d266bc2550949572f1872acac6ae6cf0
5313e3acec62534620387af5c7d51ce924daedd8
108335 F20101208_AABMCC james_m_Page_103.jpg
f95df9ee124572166be6df820a974212
d56c7b4afa03ce4fa9043f77c5f6eb2585147ea7
F20101208_AABNFE james_m_Page_075.txt
8c9d61aea20a1880cc8f1d4762c99ca7
c836437c3c7f2026fdfae896f1e4f4e6592e126e
2127 F20101208_AABNEQ james_m_Page_061.txt
46c3b0428045a0c1035d00a200a6fe20
bbca1c21b7770e86db92f237d551c9bc7f25869a
51387 F20101208_AABMZK james_m_Page_095.pro
5484ce1e88c81ab22f54adb03effa9a9
db017cac45a796799a0041f085ff8646df9d1b96
12661 F20101208_AABMYW james_m_Page_080.pro
c0faa1e9e97ab1c413e07afbeb79fd9e
aaf61964ee2761de530c747fcde305714bc32537
57243 F20101208_AABMCD james_m_Page_155.pro
9ea75833ef34a55c8d57e7c3302a397d
6f3cf49283c522be349f25590b82a6a0662a942a
2153 F20101208_AABNFF james_m_Page_076.txt
e667cc4e8c636fae195b7e2d4909c8b7
0b18a6d484f15963ad4119344dc292bd06d08247
686 F20101208_AABMBO james_m_Page_088.txt
993baaf59f5f64348254782b4492bd67
0b0e5bf5ba5ed7efbc56b411c9e42d89e9824592
2209 F20101208_AABNER james_m_Page_062.txt
c9c058326bd372905baf57b601a78e8d
c38fb94f247f46f3bc91104edd6bfed02859b557
52896 F20101208_AABMZL james_m_Page_096.pro
dbc74f11e9d27a2348a534a6f1c28db5
5c322bb518c5707ea2b632f269cb0159cdaa88bd
19479 F20101208_AABMYX james_m_Page_081.pro
249b75ab9f726d3510320be5791dc5d2
b2c87d6dc6ba884cec6e8ced15accb404a7d88b9
105209 F20101208_AABMBP james_m_Page_015.jpg
c81d502f66b6d91f3366ba9efc49b21c
c68f06665e02fd64320588af89c2fd2f6032e346
1923 F20101208_AABNES james_m_Page_063.txt
d77618e343abd83c78025d2cc3333f6b
526db1c0a7c6e239855f41d600912f159e03ea88
53270 F20101208_AABMZM james_m_Page_097.pro
98c1a8064fbbf3ae02aae44546e3255c
bdb33a38820c1303e29ef9df4a84ccfc27f2afad
39121 F20101208_AABMYY james_m_Page_082.pro
f1b3a98818f9be354daabc66454a2583
895cce954cc24105711d61c3715ec559823472c0
36012 F20101208_AABMCE james_m_Page_009.QC.jpg
d884884d2277396a3f7e9a9a5e99b5cd
95ad976c207ac17420d6a8fb9a4b475b543dab8c
F20101208_AABNFG james_m_Page_077.txt
888e53dee4e3d1cb6502685af3d7416d
6d8765fd12f6d23bbef51c168ec7dcf6f7aff993
8853 F20101208_AABMBQ james_m_Page_110.pro
17c1625e83b0699db7005db1b4ed96a1
f0b75c161411b8418b947c4b63bdb220fffef8e6
F20101208_AABNET james_m_Page_064.txt
a2c81e948a9b122ad1b1481e478c8e11
0e1a8f0db7b541274615f6fac4dd7826f3166f1d
49759 F20101208_AABMZN james_m_Page_098.pro
4c6b1a6d5ebe34630ecbc215084457c2
4355fa32e2e7b1d51929e4616dd85e29378631a9
5342 F20101208_AABMYZ james_m_Page_083.pro
0c485e886cc3f8da64ef197861574fc6
ef09bb94cf7792be27418a7fe5673159a388a703
26499 F20101208_AABMCF james_m_Page_060.jp2
49a2ed54158bb3917f2bf126af394cb8
505a732726990049dde638661fe0126462745fd1
2683 F20101208_AABNFH james_m_Page_078.txt
0dcebf2bc9461a4f79a8da5d8fb13294
a2447086e4978e9da3783aa51b96963753c89e3b
133628 F20101208_AABMBR james_m_Page_078.jp2
61b964b5fdb882101f23db86c75723cd
651b551b65cb9f74a9652d08500e6ea66ba57473
F20101208_AABNEU james_m_Page_065.txt
386e325d2670da624e71ca91199f4980
da6c047cceced1db0aeeeac8b92ef2294f917e7b
54061 F20101208_AABMZO james_m_Page_099.pro
e359e33b6ea609076ac866846592abaf
897db2783cc67443cbd0228a5c70b0e89168e73a
198170 F20101208_AABMCG UFE0017540_00001.mets FULL
2e3191c20e777c4072da25a3e47d8d0d
970e79bff5753c4cd3300fca5d35c5376077a03e
232 F20101208_AABNFI james_m_Page_079.txt
8da7d753a3616a5b612e1afb392691cd
00965871ede82e0cc125b9ea6bc1680bfb68144a
F20101208_AABMBS james_m_Page_152.tif
5b142c9b817bcf6509f2abb5cf30382d
4f3031f8ef1313ae7ae43343c68f1c7fe9c05a6f
2147 F20101208_AABNEV james_m_Page_066.txt
683c32dc9aa3c071eb215f7e62f4ca30
35acd26e43c1efcd942eff6e26ce2af682785c37
52034 F20101208_AABMZP james_m_Page_100.pro
3d4b620e2e2317447524948d66f21e8a
ccbb47134d7ce47c2147b15b098b126606192f67
552 F20101208_AABNFJ james_m_Page_080.txt
71f34ef254791515ef3c74ebcc6663b7
e39f4b60599c98e0189798a3d20434807dfaccfd
27611 F20101208_AABMBT james_m_Page_143.jpg
b3dd043ace35c9f52c9af9ef37e2431c
f668e21d4f0da20dbb17aea126c904e66dcac540
2267 F20101208_AABNEW james_m_Page_067.txt
a547faeee8f3546d8df8b98132317023
d61f907cd4e5b5949dd284d1afae4a61d776d38e
56002 F20101208_AABMZQ james_m_Page_101.pro
3e13572a54ccafab0ae4a1775ada7faa
950070f9f288a9da071d533dc39b8c3a3f6ae667
975 F20101208_AABNFK james_m_Page_081.txt
025a385c36d7d4c7ba1b39b908845ff8
0b0782834ae0bd6ff90d97a7584410e728745557
37081 F20101208_AABMBU james_m_Page_075.QC.jpg
0ebe103e67b8e5aae16c868b8e0dab28
286cebdeb966003e0c3c76ba5916ec8e00a73f74
2000 F20101208_AABNGA james_m_Page_098.txt
a10b2338f7803073a2e17af8fce38e30
d1e16363dceae66f47180eaca039747591fe40ec
2106 F20101208_AABNEX james_m_Page_068.txt
41f5252dfe88b12e710989227abe878f
354966ed951e9ce3a781d318068f6eee2030c7eb
52616 F20101208_AABMZR james_m_Page_102.pro
bcad562400a4a78703c2295a74ab10f0
7b98d2d10c9ff5cffa766f6168ee71a3f3cb80b8
26625 F20101208_AABMCJ james_m_Page_001.jpg
baa3af9e5311015f212cd72ddac0359b
a94ac1b9755ca94037edeae5405cbeca53d235eb
1707 F20101208_AABNFL james_m_Page_082.txt
e91e07ca1bf6f32120c9cf06b05bb153
daa7b6af19dce6845d4261183daa5351ddf1d807
94684 F20101208_AABMBV james_m_Page_004.jpg
ff3c687980124c4f90f582fbe03a4547
5e718c6a5a5b1d8360417b4038ed283f8b4152b3
F20101208_AABNGB james_m_Page_099.txt
e9f5016e0d01a9b5dc17b9d267171499
033e0680f2bafac4d5f2698a5c38ddf8538233ae
F20101208_AABNEY james_m_Page_069.txt
e30dd630be01279214a62b05db09df48
e03c828ee6b3619b69e14940b316f396bf066f54
52565 F20101208_AABMZS james_m_Page_103.pro
609562f96d72131c93bbc8e320acbb7a
ae49f25b767730d5b96510dc0d3631a3c72e3bf9
5169 F20101208_AABMCK james_m_Page_002.jpg
e77475d8680382c6b7806660be50fdc6
86ab46fb0820bd6a5f6dc0f715dc46b5e33dd7fe
347 F20101208_AABNFM james_m_Page_083.txt
776a8452d95ef5a5d579f92331e8a6c1
4be0381a2accc77adcaf9d2c539a60de66a6a1c9
F20101208_AABMBW james_m_Page_076.tif
72d27685c69747f8b587f88420ac9bd2
23f9bd151bde96a7ed3ac64ef8c187166530e32b
2059 F20101208_AABNGC james_m_Page_100.txt
880e7582b9b117fcc2a6fffce71448ca
7ce0b66e85dde70e6d7f2ebd06919952af1a7629
58979 F20101208_AABMZT james_m_Page_104.pro
394f9601501f627f57fe57bb755eb41f
58257427338b6746ac1cc0f0b4dfdb1511518f31
105663 F20101208_AABMDA james_m_Page_020.jpg
37e16b043ff0d03c41b83ece6b4b755f
1e08627d7eb0e8b6d28f416d3858789784ce98d7
6670 F20101208_AABMCL james_m_Page_003.jpg
87cb7f3645226a884f8546dd0cc40676
0530966dc5e8e407af5eddac45f8edf7a3871e41
338 F20101208_AABNFN james_m_Page_084.txt
afa4b46e214ab29df2dd3b60597c1c33
720bc14fbcc2c7f39b04d0791ed214c1d9b3a834
F20101208_AABMBX james_m_Page_146.tif
5e1b7fcd94968159fdeacad47575a77d
746e89b0e8b4132dae025cebfac9abdf2284e906
2096 F20101208_AABNEZ james_m_Page_070.txt
330ad21f551076b9d5d4d43c300ce7ec
7cc514aba363a3c67c4879414b4719ca5b7e5677
2201 F20101208_AABNGD james_m_Page_101.txt
722c079e28d0b663574da9d490fb2ed9
25ec9a541533e46b28378c234568729cfa00b658
57852 F20101208_AABMZU james_m_Page_105.pro
2036eb087084bd4519aacd9fffcfcd95
300d14ba95c4201ec535e29fedf1334a90a3d116
108267 F20101208_AABMDB james_m_Page_021.jpg
49d2fd129b1260cf708be649f23cd647
422b068e4b970bebaa720687885db522e0911dcd
126703 F20101208_AABMCM james_m_Page_005.jpg
98013bf58e72da8170dd53c33502f26b
3e32ffc3df0a9ed07f49000a19816ae10daacba7
856 F20101208_AABNFO james_m_Page_085.txt
24626e4ed8cb9ee4a51d41287becaa7b
771d0d78c0ab6e4b908c5eec761d3896d3388e1c
8757 F20101208_AABMBY james_m_Page_091.pro
11b07d1e6f6edf1de1134087f463ee14
d2929c308ed266e5155fecd4231f9fce8c480eac
2073 F20101208_AABNGE james_m_Page_102.txt
f3e986db8c14f000d96c64a19e2da6b0
f9e0ceb9da869f9c27a690666327c794f9b3bd9f
56455 F20101208_AABMZV james_m_Page_106.pro
895e62527a76e6f676ccbb0f7e0feafe
ddedd105a861d7b8773f5c4da2a7abd1161eae90
96062 F20101208_AABMDC james_m_Page_022.jpg
4c57c4d259d9b7b75c9f9e131ef4aa51
35a40e51479ed35fda00da41bfaa52bcd1895776
161137 F20101208_AABMCN james_m_Page_006.jpg
c33851233899c023371c7ed36a3977bf
53e867b8866c6c1ac2b97d0a14788d774d6f4e4c
458 F20101208_AABNFP james_m_Page_086.txt
e72a175f22f12744375c1f318f186495
e6485f4aae258fd3ef9f3abef452dba955f51154
F20101208_AABMBZ james_m_Page_065.tif
6ced852223f7d03f4c5d10ba53622e15
dc236266c359be823dba0b39b05f7c9ae1d14bff
F20101208_AABNGF james_m_Page_103.txt
be3dbd8be713f80d6ba89c5815fbb0b9
01d44ef8a7711cf7f2ef7220255dc4e30877eb5d
54955 F20101208_AABMZW james_m_Page_107.pro
ca9bd52a143215d69458748131868ed2
777d0e737d84b065815370b5fee560cabbe521f2
97733 F20101208_AABMDD james_m_Page_023.jpg
afb58f9f806e49a31f6494dbdf736a29
342f62feae8559ac412b92e835ef5d0c6cbe4e36
73292 F20101208_AABMCO james_m_Page_007.jpg
61c15e1a068211b49b2e379fabb45e0e
423eca442b8282dd1c6baf9c353faa957936bec5
694 F20101208_AABNFQ james_m_Page_087.txt
6deb19ea08e5ecaa43ded118fc01e1b3
d502d308215cd0b1f3e04af9b234c06c91822b83
2309 F20101208_AABNGG james_m_Page_104.txt
d50ddd2576e315afe54aa67a7ca0ff57
0a1f4d062f3a90c8d89792f5e2c57518424a2fc3
16570 F20101208_AABMZX james_m_Page_108.pro
4cbdf6117871c180c6a5b2e523184750
054ed92f86707331b746fcc104237769f9beecfe
100818 F20101208_AABMDE james_m_Page_024.jpg
b7a7cba1f138791ad0f170df339266f4
cb4844400fbd1f9b2a2cce2293add0291b462bc6
98103 F20101208_AABMCP james_m_Page_008.jpg
1ee83c62535a86f0bee92988218cfc78
b709c7d557004ad85da13574187f84523c2dd848
462 F20101208_AABNFR james_m_Page_089.txt
608cfea4b6b699f5bc6698c3a6df4f5c
e4cf3b949387ee684723856bca39b141f73cc494
809 F20101208_AABNFS james_m_Page_090.txt
db3c6c519b04c8b4e6531e06daaecedc
9080916bb45fdddd66890abbc7c597c2a0f9f820
10352 F20101208_AABMZY james_m_Page_109.pro
6aba4a259e497cc1fd7b151b1c6f3b52
086a6421e2e811c5ab73e4a56a13f300988ea461
129564 F20101208_AABMCQ james_m_Page_009.jpg
89f5cd2d2435766280e3393add2f6455
d9acf37024b052226bfd24491ceaf51433042d94
2265 F20101208_AABNGH james_m_Page_105.txt
79f707f95946da468ea4bb17dd00f113
983434f82441830ad993e1dbdeb4b92e3cbb213e
438 F20101208_AABNFT james_m_Page_091.txt
367807fef78b21fa0c5182491ba7f3ee
c3883cc841bdd86f5f9775c51c2586abf95bb2a2
17876 F20101208_AABMZZ james_m_Page_111.pro
94851b29ec101a0b8ae58e18054f475a
d52ed94520d03efed10e15bd21f42baf6f70ad41
106942 F20101208_AABMDF james_m_Page_025.jpg
4af28c7912d046f9f4c0c8293cbbbbb2
80d3e711b6b64bc2d39a4b8802d9ea76d5a561e1
144263 F20101208_AABMCR james_m_Page_010.jpg
4c75867c57763b4b1f1d722b6c27576c
8f7b3632657c7604a450b26c52a4333ae4050f3d
2264 F20101208_AABNGI james_m_Page_106.txt
b7cd2079a292700e7e2d37f5c7e362e5
17e0d17aafcda23f04f501fcfd675133b3b8fa89
423 F20101208_AABNFU james_m_Page_092.txt
995aa060c5ac70d7752c666af553b43c
4c223683a9814bb4dd604204008f64eff0afa6d6
111436 F20101208_AABMDG james_m_Page_026.jpg
30cdba8aa548bdd780b714b0bd7da1bf
309096b4b51af4063c8b47053cbdd6474e73e177
35143 F20101208_AABMCS james_m_Page_011.jpg
45511275077f5c21df1ee58841f86062
4b5a34012ec25611b69c3ecea74dfa6cf384bd9c
2159 F20101208_AABNGJ james_m_Page_107.txt
0d5c86fd83ef3600e6166920c0f89964
ec9a014483b3fd2f2e0764a8f74da7458a884c26
256 F20101208_AABNFV james_m_Page_093.txt
4ae5a0270943e5e1d7be8aec7b56c033
a36f4bc6ee33381db6c9c5a8afeeacc2ae4e508a
115604 F20101208_AABMDH james_m_Page_027.jpg
4f93889324b56bafb04cb5712d1227cc
12e50782891bd5455674c0a964e864aa6aa8e597
88147 F20101208_AABMCT james_m_Page_012.jpg
182158ee0629debbe8136cd5aeb9024b
33b4196b7bd2d118b567c650466016c5f0ad6e3d
658 F20101208_AABNGK james_m_Page_108.txt
3785963456682551b0458693d982455b
a6cc3ea7cdc1265d2117434adac7048d031cea49
2223 F20101208_AABNFW james_m_Page_094.txt
267db88c2a558dd6204bc0043e7ada27
a7bb6c47d1f0ce5d7ee2ca4ad262736a1b0fd9bc
91207 F20101208_AABMDI james_m_Page_028.jpg
4b9af28dc37b6b09401f04f5a46cfa6d
057cd2794817bcc7dd970985c424041a5a54d40f
58626 F20101208_AABMCU james_m_Page_013.jpg
f33cb622c807407bf09a122c1cbf092a
577586d906b242e6b32600b98a8c808ee4ac699f
1944 F20101208_AABNHA james_m_Page_124.txt
507f071c3c7ddc33aee0f0c9150d1f15
658ed620d5912b5a089cbaa204a89807ba9e0489
723 F20101208_AABNGL james_m_Page_109.txt
cdab75b93b1b021a4dd6dafa1a62b817
36a5d0b09509ddcce57c97c7068e4976de64d3fe
2025 F20101208_AABNFX james_m_Page_095.txt
c6b59ea2af7bad8ff3ff63ca04c4d50e
e39bc1ad188bd6072d4b72d9b2117e814a619e41
99806 F20101208_AABMDJ james_m_Page_029.jpg
dd9e8265797d0148a5f7032849cd4d59
c78df677b5d1120bee8112503b952f917da51b60
98779 F20101208_AABMCV james_m_Page_014.jpg
56d1258eba9fd9ec8c090b4492976c50
ff3550d492ba703a76f944aa576cc4edefe8949f
2251 F20101208_AABNHB james_m_Page_125.txt
e09bd1fd0a8c8b7a37d40c5d260655e8
2764d11e7d7fd9ca214187ec225a6879ad7a7d40
604 F20101208_AABNGM james_m_Page_110.txt
be40c83086104e5998cdf14576d6ca27
f42e062053dc2bc573a0676f00e2c801fdf4c603
2114 F20101208_AABNFY james_m_Page_096.txt
5c2209468f45be1480762a1367d0bebf
2a1e7719013765008c2aebffa2040e4499a2783e
105301 F20101208_AABMDK james_m_Page_030.jpg
9c6f108a131e058b0d0005517bec798e
32895b6ed1177a117e72ab96947501260880468a
108164 F20101208_AABMCW james_m_Page_016.jpg
d6212a67bf9f2c3a21131fa7c5b245ce
e7f16017646c62a8775d642c6f910ab5bb9ee95a
2157 F20101208_AABNHC james_m_Page_126.txt
68e3d63a0f4ac70262334e41ec8b986f
0c911e9796d581bd2f46c7d38e41eec25b91b54d
780 F20101208_AABNGN james_m_Page_111.txt
9e4c57d47f8fbe3e7d8f0d8d1e125e20
e3e9451d915e3351b869d7ea6e86190718ab47a9
2095 F20101208_AABNFZ james_m_Page_097.txt
926f4d55ba0909c900b9138720e7eb00
44f7a6be52b898170ea1732438efee4fa9ffc38e
107217 F20101208_AABMDL james_m_Page_031.jpg
7c64d0efcaa5781b1c8acc88443c7ff7
a0835581ac3b05521443375f13d4162028538a70
111002 F20101208_AABMCX james_m_Page_017.jpg
261bb1ba4dfeaab8b3a48833fde8dbf3
ff10cfbf053bc0bab590f5ddd9d6facb422fdbb5
71828 F20101208_AABMEA james_m_Page_047.jpg
ad50d1f51401bc61ac4baa6ecec511bb
c87622cce04382f57bb2887de892bbc30a69d277
2167 F20101208_AABNHD james_m_Page_127.txt
d03c1897bec5880ea9465e2912c5fdd9
0e0c92fab4d92d28778f1e3758a400498ace9172
473 F20101208_AABNGO james_m_Page_112.txt
5d0924072798e90f02b751fc80bc8b77
e5cc8b83a6e5597b12e898cf1b30b7f20605b4a4
92700 F20101208_AABMDM james_m_Page_032.jpg
ee6e37ddbfd40c68b69b9482ede7834a
de542b26f8098b4d908f3dc76b85d84b2e853026
109781 F20101208_AABMCY james_m_Page_018.jpg
1f45de68ee12f1cd167214f003ec02e4
85f1070544cf1dba70c90be44a184822e5f81279
65902 F20101208_AABMEB james_m_Page_048.jpg
ce8b72830f871af6b10c816f290a9272
920dbda8b23992c56e1eaf2ec316b4edc81ca392
2332 F20101208_AABNHE james_m_Page_128.txt
479640504436d1db46bf6491c2104be9
8a746bdcf48f14ddadb2b469e71886c56fe8d680
617 F20101208_AABNGP james_m_Page_113.txt
c0bd52f633446a41e1fcf6f73d1e07a5
31a2077f0d9641d8c4c3463468ed223bf1af6680
101531 F20101208_AABMDN james_m_Page_033.jpg
baa597e5bfeaf93c1f54698090a03b01
98d43bbe97f4ed6cc31638d1ae8e90216de9ee70
108445 F20101208_AABMCZ james_m_Page_019.jpg
2d19cd0234b50b735972468f2ba5db9b
2a201334a10981ebde735c56439ea09f16c997ee
53998 F20101208_AABMEC james_m_Page_049.jpg
582f06d7d004d14a20bde160d904b59a
009cb9cfa884d420ce64ba1fdf10825dc416a1ea
F20101208_AABNHF james_m_Page_129.txt
681fe5d863c8ddf9d592b7fc94641238
2cc2b0da12fcc10328848ae125804007516ab97b
926 F20101208_AABNGQ james_m_Page_114.txt
cdd984acd0a4a8950ecb3fe5df1a2338
dc1c088d076351c5e2f1fe018a17c3d3d5971280
122572 F20101208_AABMDO james_m_Page_034.jpg
f949c556b71b18ec8fb1c3d8f979ee67
8ed0b41df18a684409d7bcceea9239b68d846e13
62822 F20101208_AABMED james_m_Page_050.jpg
6450ded0704aad184e922a6b9959c013
c35f92e2ea9d85a10c7dbd9de12985efc61979dd
2165 F20101208_AABNHG james_m_Page_130.txt
8dfad09676c81493d5658aae02825d36
edbc5853ed11d491ea207f64d7f88f468ac204c8
303 F20101208_AABNGR james_m_Page_115.txt
54800e14e3cff529efac5fdcff5446a4
32fe480eecaf5af7de979c71e136af9b96b1d0fc
100539 F20101208_AABMDP james_m_Page_035.jpg
c941c4b0ae8492fbdb5b6e7e175e42f9
c0d3d434698ebfefc326bcc550419eec5a965889
79770 F20101208_AABMEE james_m_Page_051.jpg
7c9caf37f33042d92b13109383162aa9
316134445999a56be8d311f11becd61804057e22
2238 F20101208_AABNHH james_m_Page_131.txt
d0031ca4bd5d9d4e39d7224069fc122a
18ff135c10206f918f57d2d3f36d823ca70de438
883 F20101208_AABNGS james_m_Page_116.txt
cff810582fcbead0bdb27acabdacbc75
e0f8dbcc83bee293dedc156bab377e857af31aa2
104388 F20101208_AABMDQ james_m_Page_036.jpg
14395c7e8e56822a83be4e3e2f11d9a5
919406fc0f72199b122edcbde9c9d9365a43a713
46871 F20101208_AABMEF james_m_Page_052.jpg
55ad36d48d8a54f8b5afa10b839003a5
07fcd1d3d3211ca1938b2103dbdede7d9ac3a5f3
2125 F20101208_AABNGT james_m_Page_117.txt
dbcf5c21e7438b24a54c93a9b4859b20
1a461aa567c889fc320b2777b2e16ef29d06c083
81452 F20101208_AABMDR james_m_Page_037.jpg
75eb19b9afda21dbcff8fe8a67dd8d92
6563d2b2dcd167d26fa1516f2cea20c3d5ac71af
3266 F20101208_AABNHI james_m_Page_132.txt
71b8a2917547e59eaef52a5af19be858
1edcc41246336c3aba6bde73d23c484216385b5b
2178 F20101208_AABNGU james_m_Page_118.txt
d75de3d1327e1d6be712da2edc662c46
1e81e069c98c5cf552b23f71e818156dce8bc1c6
108643 F20101208_AABMDS james_m_Page_038.jpg
218ebe7007473f58bd22f4270dc7ee44
1628c3a3951545c2474fc37e57b5157625ad43ba
23939 F20101208_AABMEG james_m_Page_053.jpg
0a3dda5a8e0341521479387dc6e06a82
4d04a2038a59aa7d66bd2a144a6d5543a05c4460
301 F20101208_AABNHJ james_m_Page_133.txt
95891f04522cdebf16c16bd3df6ffd77
09d11b6ac43101d41819b83a5ba91064af379877
2183 F20101208_AABNGV james_m_Page_119.txt
c3e2afe9601db4e057fda739f113c2a4
f1844fe994f424f7919809d411af01f2b8689eb3
106791 F20101208_AABMDT james_m_Page_039.jpg
006f84ebd045f6c1a29c293a792d24e9
0c3e1e8518d33fa9e050bd903664a6ca8848f48f
53613 F20101208_AABMEH james_m_Page_054.jpg
fee1b5fd120632957d7f16f6a393bf77
d8a4f49433406463df6e61dd52df3933e2e11696
660 F20101208_AABNHK james_m_Page_134.txt
a7b8217eaed2e4d02064d0a179fe3ce0
4b137875af8d7625c7fe9363320c2dabf1b8afad
F20101208_AABNGW james_m_Page_120.txt
fd46354d06e23f1f9790daa26742f90d
f6474f4d313be143eb3a31457df3ba422d115367
102483 F20101208_AABMDU james_m_Page_040.jpg
793903499792302bdbd02bec8285302b
76b04ad53b2d503b35ed068faa47c17c137d8b0f
75784 F20101208_AABMEI james_m_Page_055.jpg
6ea4a840c99da15c358f391d5f8ceae6
49988bcd211023813d30290aab047a80466ce871
2123 F20101208_AABNIA james_m_Page_150.txt
817e18b55f0c2592c1c2937db50d4805
7e73f12a3f4197c189cf12eccabe2540457c2af8
455 F20101208_AABNHL james_m_Page_135.txt
2a040e7d8e171de41ce591eb6cef6ab1
b620d61ad5c8bc5e19fbfdccb4e63c611a60e3ab
F20101208_AABNGX james_m_Page_121.txt
37059376d67907579a154976c121302b
668f9d5f8432a495c1e6ca03a255b957c3d26b9f
106958 F20101208_AABMDV james_m_Page_041.jpg
383fe6c68271809ef2950cba0d53d93b
60ed1da339411d6d2875e6eb53958a3decad803b
89703 F20101208_AABMEJ james_m_Page_056.jpg
02eafda648c1e2f3738451ea785685ac
532082af2c971696f4f1df0dcfb9b32e82bb0852
3170 F20101208_AABNIB james_m_Page_151.txt
ecd458cca8186b5d743db579a4ddeaed
7a936992dd17322c307b8f463afcc9a2c6ffff0c
307 F20101208_AABNHM james_m_Page_136.txt
d97f68e1ab2f472dc2b583e032fa644a
34621d63e13fd44f0b59eb4435fe2915ffbc5af0
F20101208_AABNGY james_m_Page_122.txt
f2f71cf916245e21104cf4229b895335
7e4fb42ed4bdf03634855f02b1835a2a63debf04
103658 F20101208_AABMDW james_m_Page_043.jpg
0a9d3848b8774d0d1e593e8aa8a29d47
65bbf30db1900102d50f2fcf74546a5a9357facc
87870 F20101208_AABMEK james_m_Page_057.jpg
1c842cf3a922d93e35fb4b3bccf8c5ce
697688bd7439747a99c5a80f83883c5986010dbd
2160 F20101208_AABNIC james_m_Page_152.txt
a703240ffb461f4b6cfb3392ec36bd93
42a2ce2ede54eb056c83cd40bd539a60df901044
321 F20101208_AABNHN james_m_Page_137.txt
8b042d52af3507c4ffee35657f226964
65df28644988482e7bee071ff813ba55f7aac138
1940 F20101208_AABNGZ james_m_Page_123.txt
c6b809fd33c69422ebcced17919d7537
1e1d653239a07f87fdd622afdf44f62678b686a9
93570 F20101208_AABMDX james_m_Page_044.jpg
39a8330c3280cc99879c680232bafc08
cf597be2f9289527f1c65159ad61059439e5facc
105702 F20101208_AABMFA james_m_Page_073.jpg
c90955db179a7250414482fd9e5cf64a
c0d6e3d831ebcad536bc37250d54188089da7f6c
63629 F20101208_AABMEL james_m_Page_058.jpg
9f1167066e4f87d8bab476bd0bff08bb
60057a7fb0de95d0a0fb2bc9b59c3ffc59668a65
2184 F20101208_AABNID james_m_Page_153.txt
d621c26cda8c5db21367dc192059c732
69f2a870b8a98453022f3858ac3b25e6cfe1f696
340 F20101208_AABNHO james_m_Page_138.txt
c34e20a557aa1ef43f78b64cc32ed1f0
aa8083e0aad7e51b98ef6499155e3ce19d8f54b7
105745 F20101208_AABMDY james_m_Page_045.jpg
8e09aefd22653ea04b50ddbf83ca2707
a29256abaec578d6b317fb2b468d7e15336f8415
107916 F20101208_AABMFB james_m_Page_074.jpg
bdd3bac4d010b9de193e84dfcd4d255c
fa47dc009eb4b892bdad3b97a6d591d506469b87
118119 F20101208_AABMEM james_m_Page_059.jpg
341065fe90b6c395c00a00fc871d8535
7ee536c609a40a27ce2a5dfb7fe1e68a0429ee48
2169 F20101208_AABNIE james_m_Page_154.txt
13db21ac09a2cea0666e473c4b9fdaa7
6f7282f55ad1ce311595f43b91204b899c37411d
461 F20101208_AABNHP james_m_Page_139.txt
5172743e14690b121259c802431a11c4
30cae0f76338fbf209cc81045d2b1d00b9d9ca41
103520 F20101208_AABMDZ james_m_Page_046.jpg
15bd0deb10e9ac5e4832b4c74859d111
2b81851def47509fc952f15179b9e262e170c9e0
113257 F20101208_AABMFC james_m_Page_075.jpg
4280863ac4639cb4397aab935ab2bc8d
cb2cd749ac05e04926b9092372cae26f733a69a5
26122 F20101208_AABMEN james_m_Page_060.jpg
b73127e26672b87d08ba196f111aa70d
c93d1faee1d62067c2dae41d307de2f7feeb4c4f
2315 F20101208_AABNIF james_m_Page_155.txt
f075993282a625c44d5882dc9d92134a
5fb7933033d9ee5fc3902edf215b112b6cb0ea03
895 F20101208_AABNHQ james_m_Page_140.txt
fc4514c22b89a6fd73124d01676feba9
d39c560703dab3d0f8e1b7ddafffa4c47e72d810
111420 F20101208_AABMFD james_m_Page_076.jpg
5ce98130bf31cbe2fef345586eeaec0c
7beeff53c831635ef8d438316b4f4667d136f823
105739 F20101208_AABMEO james_m_Page_061.jpg
af5f7fa8716b949fa3b6a268c91dcc7f
a1c93d5dc5d55c3801bfda7bc632ed17324469e0
1815 F20101208_AABNIG james_m_Page_156.txt
0b77986e103d319580b58e6cd98258c5
9766e95fec3c6af1df31c1282917482ce951399a
468 F20101208_AABNHR james_m_Page_141.txt
533f8084d9aa4487a8cbf200bc41e29d
52ea2b3e4b9f81be98b05e8e2aaf0822d652f80d
111000 F20101208_AABMFE james_m_Page_077.jpg
62ffc63b166c028754c6edbcf94bc3d1
6757d4df8e900521867da5060fab12954e0c7b02
112936 F20101208_AABMEP james_m_Page_062.jpg
aba1a9f38cd06c49bf8296097c3a24cd
9235411c137ea54e550787b38712eac2ff6f7ff0
1425 F20101208_AABNIH james_m_Page_157.txt
9ed7aaa5975a26575172c9e3f530f318
772bfe3323c7b680854c9ba6cdd102329f6ea8d6
1177 F20101208_AABNHS james_m_Page_142.txt
b2d7aa30612a4559ceaccf5957befbbd
14f13e06f40ea553019b834a2e35c154d3a588b2
124417 F20101208_AABMFF james_m_Page_078.jpg
e38b12eb05aaf2f9eeffd760ba754499
16f871130affc8ee02a591d94b63a40fd366f966
99678 F20101208_AABMEQ james_m_Page_063.jpg
d678b2335256107ad5bf8da794bca8aa
4bbd49887e38d35155cc64c27aa00262f66b6ae4
1485 F20101208_AABNII james_m_Page_158.txt
3ea4328decec6889f97102cbe5374325
4a4d1b1f245f2b7bb78626efa0d8556b46fd9084
535 F20101208_AABNHT james_m_Page_143.txt
ac5e3070c1d48876d9dd80ab71aa0384
431e86d4bb03148b931dbdb5bc8962ea2eeb6bca
14844 F20101208_AABMFG james_m_Page_079.jpg
3aa7b273c626c115e919cdebf9748600
c1029418fa83ceed5d26e8574e4140970c38886c
112395 F20101208_AABMER james_m_Page_064.jpg
821acaef3693fa7063f8138ffe8ec0e7
9ff49c275ecbeddfb4e0cf7c46b8e3ef339f5190
1069 F20101208_AABNHU james_m_Page_144.txt
0af750f60f09df7db538e6678e798de2
b1ffc9ba9c161053be74767b3ae932b3b51cf2c8
103460 F20101208_AABMES james_m_Page_065.jpg
175372e4cc4b4415b901c54a3ee94620
e284565a7d1ffe454cab86bd34decaff922ff05b
897 F20101208_AABNIJ james_m_Page_159.txt
0a85273140ce9cceca1d531b7d31cd1c
19ff25916241ff97669986006b6d32b2c379f0fc
1138 F20101208_AABNHV james_m_Page_145.txt
49eb1c5b838e6ea233a1f80afcf1c23d
5fc2d2386778c25aec183f4be90a39c31740e537
37678 F20101208_AABMFH james_m_Page_080.jpg
763bd19ec42bc7d2af64a05abae0177e
9511474522d6bd605a5fa8b379eba85c3a756f35
107657 F20101208_AABMET james_m_Page_066.jpg
0b6400ef659fe72c1df267d588f85b1d
12f8b403b779a443bc91d116fd3286cba990fb50
1582 F20101208_AABNIK james_m_Page_160.txt
e1745b4754bb9c90d1d7643ab245678e
7cf9578b76efd2360d084e69e09f7d192d0fa983
F20101208_AABNHW james_m_Page_146.txt
b7b37b758e2d8d8b030f356232deab3a
953dc587df791c6b86e35733ad8973014bc1c38f
48106 F20101208_AABMFI james_m_Page_081.jpg
0a94919d5457875b6e946ef277044d18
eb4882860be363aae62ec00fcddb00c3a6cb3c98
113868 F20101208_AABMEU james_m_Page_067.jpg
cfb096d2b97869ee62c407d313ab0398
fa468d3f991b7b3aabaac7e4720f83283b3f1ad2
11235 F20101208_AABNJA james_m_Page_134.QC.jpg
641d5ba184449e162f2cf3473a7563a8
0828d506d93d4c4b9b02099f33e72b00c4cb66f0
1110 F20101208_AABNIL james_m_Page_161.txt
d44169d988c60f2c79b7fca03575b9dc
69a9695fd5b94353c336cd5e14c7b650230a6f7c
2701 F20101208_AABNHX james_m_Page_147.txt
f590273818db285ab5e75a9aa36528e7
20add73d83719278fa6f997aded7bfd9e0be8bb7
76162 F20101208_AABMFJ james_m_Page_082.jpg
ca48c44c05672cdd21b6c460fb12e4e9
1f25cd28aa7f2655bfdd9cbf5a08cae0dc1a4d6b
99180 F20101208_AABMEV james_m_Page_068.jpg
f35952e3e159f874d8d71b202a02c428
224d8b55cc37f5ce82ec83d4f016cf3183695ade
10826 F20101208_AABNJB james_m_Page_091.QC.jpg
f073f647c1b5b2d5b09e36b373fae598
cd8360c3251a37a1139a14eb2395e08390c7f7ea
2325 F20101208_AABNIM james_m_Page_162.txt
12d241250102e9c5046684732a396741
30c6d590ace0938224713b00b36466e5ded04982
3139 F20101208_AABNHY james_m_Page_148.txt
991266a3820346c4572888cd75ab0d54
86439a3cd265c2387b849e38ce4b75d47913c4d7
83414 F20101208_AABMFK james_m_Page_083.jpg
d6c22e4b23221e50b675c6cfb6a4de8f
fa6996fa431efd641d4a5cc12d1f5713fcb5f250
111622 F20101208_AABMEW james_m_Page_069.jpg
db0f87a2a9552db66e5fcff03079f69c
755081e8661bd58c38fa9876edb31ee547deefa0
29562 F20101208_AABNJC james_m_Page_028.QC.jpg
59646491ab1c29a3d9521dd3ce3326f5
a52ffa3fabfb370dc4c83259c2bb9741b9c1d2f7
2285 F20101208_AABNIN james_m_Page_163.txt
8ff60d8c3a9b0b2f5950a7dcd264e0ad
cf686f3436c9d0f3f5cbfb70cd43a15b44beed0a
2099 F20101208_AABNHZ james_m_Page_149.txt
889e38694f58f1bd77323213e83a0ff9
a7de628c2a0ebc9cc4e91acf71bdcbf4f2e4061f
110194 F20101208_AABMGA james_m_Page_099.jpg
0748cd382b39c515308cc7895e41693f
49eb94c7814ef9d7eb712aeba2d493ac3434353c
28857 F20101208_AABMFL james_m_Page_084.jpg
f6f88b965cc8f88e5e9ee15351531aff
e4379339ed93804bfce42ef839abd963e6c089b7
110626 F20101208_AABMEX james_m_Page_070.jpg
afb3c6bd78863b15b88c089f5dda3ec7
d8c07842847ab06831fd1dab6a93ca040e9a583e
9357 F20101208_AABNJD james_m_Page_164thm.jpg
a37f4ee15e27bb9e4cefd115e0775e0d
edb6029926023b02a01674b7aa80efc93a47da9e
2530 F20101208_AABNIO james_m_Page_164.txt
68852c81d28ee5bfdd8b1bba86133e5d
1b8881edc0ec34e97d4562c9c688cfcbd60a5aec
105027 F20101208_AABMGB james_m_Page_100.jpg
da3d537a8f6e6c774164084cf8a447db
64918a87c57ecaf4eb97a180d75be9e7f0399f87
45231 F20101208_AABMFM james_m_Page_085.jpg
c81830ae9a3eee6fdf5d1a8df22aba80
4f89b0948b2aaac301f5d13b2ecdf34ff87df150
115057 F20101208_AABMEY james_m_Page_071.jpg
d73e81896e90f6fcdcf7d91130f67d43
e7f9a36fc43a5cab3bf27bbfb9afbaf5d204671c
7646 F20101208_AABNJE james_m_Page_004thm.jpg
45d31f7c509c34ca9981ac6c68d5a18b
d56d11562983d7dea77129d4de4a90011a689634
2355 F20101208_AABNIP james_m_Page_165.txt
54258e6e6038e7307c7016da803c0440
c094426969e3ff10dfd8bc1540eb0398b7bd7164
112606 F20101208_AABMGC james_m_Page_101.jpg
1226fb8e1a5a42e4b29797030007f6b0
f1b1a010d3233144b4299c20f6d690c98aee171f
31015 F20101208_AABMFN james_m_Page_086.jpg
7fa4c76b087caf53986f5c800277f64f
92b635cd0c4c68a2baa781d04c5d47d52e96f79b
102735 F20101208_AABMEZ james_m_Page_072.jpg
b4586796dbde9d43b00c934b579f01d3
37b27bd52f371002f9311d83d966fc8a261b640b
9146 F20101208_AABNJF james_m_Page_067thm.jpg
754e99b36ac0ae2dee1deb582d7225a2
b03493a8d32e587bd553dc14a08698571ff93de5
2440 F20101208_AABNIQ james_m_Page_166.txt
31b4ee48cfe4de31dcf2958dcab63543
4809cb0df735709894169b99fdfaffb86d202d0d
104238 F20101208_AABMGD james_m_Page_102.jpg
f6141f428ecdb25bfa6b47d1e882ad5b
762cd3a256830684abcfd1a7b2a4993fd4e810d0
41766 F20101208_AABMFO james_m_Page_087.jpg
c2f23723996f9f764cc1302490e9608f
13aa27d69eca27e52ff40b7a6f02cc9928dea7a6
30882 F20101208_AABNJG james_m_Page_044.QC.jpg
553452b44d3418f2222cef496332367d
984887698216b8350149d67040a3c32ed0601294
2435 F20101208_AABNIR james_m_Page_167.txt
955ca6eda23a4520321239cd0cee47c2
7da0590a52c3d9b97598a312e499c14f760116bf
118681 F20101208_AABMGE james_m_Page_104.jpg
2df282ebc1acc43527be5492622a3d5f
f43f90378c49548f489daf471247001ea431a406
41718 F20101208_AABMFP james_m_Page_088.jpg
cb91c632ca5a6459364801b3e79add69
8f7b67acafdc5a68180d488883e58105afeedb9d
33525 F20101208_AABNJH james_m_Page_098.QC.jpg
73df680d304e6250ac6acf476914fce1
f178534f23f6823880c11a6585aa413a88b74bec
2667 F20101208_AABNIS james_m_Page_168.txt
82d2fe1afc6b630bbb9b77fd2d5a2c58
19f9fff3f40b73ddcc855273de49ad710003ce3f
118601 F20101208_AABMGF james_m_Page_105.jpg
28cfc3781f7e169a35ce497e7fb6bbdc
4fd0f5b1fdfdda3eea7cada39adcdc7ba6aa07ce
29321 F20101208_AABMFQ james_m_Page_089.jpg
f3554a383b87e75c939fb4b3663e88be
0b97bdc8decaf604fa7cbee9c7cdf9ca19a05108
37511 F20101208_AABNJI james_m_Page_107.QC.jpg
c16e2057b555adf1a3deb8c5370d6be1
851c37cbdda444042c6860fde3880623a40943ea
2567 F20101208_AABNIT james_m_Page_169.txt
4d9d1d4a60a5cd7e7e88bd9763677d9b
fa8cdb36b7e400f5c7c989e636ff25d77b6947a1
114751 F20101208_AABMGG james_m_Page_106.jpg
483152442c33ed3eada102a0bbfabcc1
f93a676d5f65894f88c3a8fb999a10b6f8aa2871
61842 F20101208_AABMFR james_m_Page_090.jpg
dc1aec3672b50fd1c5c051d80385c743
0b2ad44b5a351432389152c1f658a6192f083e4e
32400 F20101208_AABNJJ james_m_Page_014.QC.jpg
c63245c31952ac04018c16e9b8222476
8df3544649d842b99686d3997b7562f5fd7078c3
2748 F20101208_AABNIU james_m_Page_170.txt
fceaee5f608bc67ea67e01f2f3a2fa1a
1da4af394e918fe128d6dde338f66025d312ac63
113193 F20101208_AABMGH james_m_Page_107.jpg
cfd3ef7bb1f8f2c1e3ef4e51c7101fde
b6e94375604ba74f794a8cc63abfe11415f7a4da
31008 F20101208_AABMFS james_m_Page_091.jpg
7314e1369e0a62943c48cc651c873398
b40a8d8987e90b7b498bf832572f6e404eb29252
2317 F20101208_AABNIV james_m_Page_171.txt
f1e067c74788956a5808c05af1ebe005
de4e5a661bff1fd434086bf9d3c2b281261e2609
31502 F20101208_AABMFT james_m_Page_092.jpg
eeb240fd43c1f3027507a6c8804d1388
1ce771472957c420800a45af14a2e94b8cc3be96
38969 F20101208_AABNJK james_m_Page_010.QC.jpg
aa7da4265f93292f045f18e07531696f
487c7390938d7fccf05170620534a756547562ad
1396 F20101208_AABNIW james_m_Page_172.txt
2d287b534eedc1df37b34f8ccbfb1333
10ec1cf0f6be26e1f2cfffcc60542b2b9e8c915a
36619 F20101208_AABMGI james_m_Page_108.jpg
be5c4ea8f2f505fe7a15830cf9660d26
7a59146e4904903782636f9e5f1626cb877fe209
15954 F20101208_AABMFU james_m_Page_093.jpg
ffd7b85e1655baeb480c252201fdbf03
233fcea7916e872eaf3401ef8d85e10e892d364b
7956 F20101208_AABNKA james_m_Page_148thm.jpg
bb545057afef6155f6264718a6c1e6db
14bd8ff43b95e9b49d23f7fce2de42d52e6b5467
36308 F20101208_AABNJL james_m_Page_096.QC.jpg
5bedd0ede696eee39e6c480442bf86d9
1472232a3463215b45998fdf38cc779e9165691f
758 F20101208_AABNIX james_m_Page_173.txt
a5f2fcd14a27b9db9d4a35c2f9137912
808f5150d046ef89d22d5b7d87fdd52a3bd389b5
35700 F20101208_AABMGJ james_m_Page_109.jpg
f0a477a70b4372987479b7bd46c3078a
faeb6cb474efae0fa96b440e41295c1cb5f24bec
111025 F20101208_AABMFV james_m_Page_094.jpg
815bef4272fdcb603c31680b919dedc5
6736f9ba8448a42349b6338942167b304a29a2b1
34020 F20101208_AABNKB james_m_Page_065.QC.jpg
98bfaf84ccd0237bb800e58690763e2d
8f3468442aa714b0a10a68fa2aa1f40d6bd04cd1
8052 F20101208_AABNJM james_m_Page_014thm.jpg
a431081b9b32dd616365bb8fb2950d54
8a9fc3cad86dc5e4882bb958adfa3d3481770f5d
1241559 F20101208_AABNIY james_m.pdf
1524e1a0b69d076b52794c993d32de62
b0ebde8f6d0a71a855a6b8bd239c75fc95681a11
31347 F20101208_AABMGK james_m_Page_110.jpg
7c57498851a75e210764a2040b797cde
ffbd3262b11c161d294b222da5ca33d1ee7f0446
104393 F20101208_AABMFW james_m_Page_095.jpg
b76c85a7bd488baaf036aa83a8d8f679
6c191be78b2af2f6ec9c32ffde5b930080fab2cd
8750 F20101208_AABNKC james_m_Page_132thm.jpg
07efb4bec1ca79dceb5dc9311da275e4
aa11d70a4c7e5a98e634f5108ff5afc9e1f508f9
19441 F20101208_AABNJN james_m_Page_013.QC.jpg
f26f839225b89f17803760d775eed5da
019bbc74901530cb657f11ebfc3ef71096069416
38286 F20101208_AABNIZ james_m_Page_034.QC.jpg
e762ad2f229c36710c3cc5011da33c12
74488e5dfe434694013d53ca0b066393772a3faf
110660 F20101208_AABMFX james_m_Page_096.jpg
a0f83930f295fd45fe275b5934463533
802b4126a26e00d85599babba6aa192943818ac3
110126 F20101208_AABMHA james_m_Page_126.jpg
778e05f21e68552138f3b37ffce000b3
82e58b0642de4e22b85b672a1b6a722a13967746
70880 F20101208_AABMGL james_m_Page_111.jpg
70d053d81d20835762a2b9d8be54a9d5
7faad4427ef226275dea66f5d97ed988e981a489
32481 F20101208_AABNKD james_m_Page_029.QC.jpg
416e83934946b35b8e486d395a1dd351
57158fc940f51dbf898f24c18a9bb6131abd4bed
35574 F20101208_AABNJO james_m_Page_147.QC.jpg
e8861761d058be131652b3b8ef53ef49
4503ea0bb80a9f67923f840fd3895ecc05abbebf
107014 F20101208_AABMFY james_m_Page_097.jpg
69ebb2e00da463c525b660e532dc18c7
8719e57fd28195f653b94151a4e36ad0c463afef
109901 F20101208_AABMHB james_m_Page_127.jpg
ace376a8665bba6c457771f58e648619
c8230c322d7a554b949a5cca230bffd11444258c
42480 F20101208_AABMGM james_m_Page_112.jpg
79d7ec42c1d4c7c32163bf8f827bdce8
d3c0aeab88bf2972e41a5f9e201fb7c70c303ab6
8718 F20101208_AABNKE james_m_Page_155thm.jpg
edcbefaf20aa415712166458427d93b9
5793b3356243ba63c08a311c9741969995c31959
39360 F20101208_AABNJP james_m_Page_151.QC.jpg
23c8bd7f321767cbb965540e1019cf53
34111dfd05cb36d0eba8c0f0287baf74fb94cc9e
101597 F20101208_AABMFZ james_m_Page_098.jpg
f2550a606cf8f08132dce04ebbe2376d
479d99bd19fd257bdce9436b1cf5f3e2d5985578
117883 F20101208_AABMHC james_m_Page_128.jpg
522ef1946144c5d9c4e347b1c2237fac
73897019f91f435156bc277f8349242fca4ee6da
58353 F20101208_AABMGN james_m_Page_113.jpg
852bf3ecba03438df3ece6c950b25dc4
6263554bae6b822d7803c131cdd00d07609ca5af
27289 F20101208_AABNKF james_m_Page_012.QC.jpg
e8b773f7af85ca9ef2cd67278b6790d2
0cf67c5ac3cc3f7e56032f69fa09c245393684fc
27658 F20101208_AABNJQ james_m_Page_037.QC.jpg
269df82af79acf112073b9a6378ff18d
df3276313dcbeb181ee60b74e351f335a2ef2326
108270 F20101208_AABMHD james_m_Page_129.jpg
76dd5242081cfa7cd8ccbeebdf04ea98
6a50092559f15c49e7ca2c5933cce1eac44dc588
51671 F20101208_AABMGO james_m_Page_114.jpg
a9924b380dc38142413872d146360648
b4f90862d7a714549fe78232ebe7260a593e1ac9
9002 F20101208_AABNKG james_m_Page_165thm.jpg
53c601704ca81df2ec41747cc41dcbab
a873bd191975e89e77602e5229b511489718e5d3
33158 F20101208_AABNJR james_m_Page_063.QC.jpg
75eae1ed16c21b2a16710fd38dc6a7e6
31d0695466a07ce5e4473069574abd0365a51f8b
113408 F20101208_AABMHE james_m_Page_130.jpg
a9909d76af5844b0e03b32a3071a5a76
aa84fc2a46e0493068534286b0e70c825e6c0ae3
43757 F20101208_AABMGP james_m_Page_115.jpg
1d67b512d4a95962c4f5cd327cb5b986
155c075e4fbaec12ee8986a7aa97c8639a291d90
8909 F20101208_AABNKH james_m_Page_073thm.jpg
b8fd10c94b140c3a348b76ce210c3f53
697c27e73a10c0db8c765f82d87b25ff9f81db20
35559 F20101208_AABNJS james_m_Page_171.QC.jpg
3e829dc0f01d5c232e5dc0a4c02b0ae0
ef296de57811d5f67850b13f3234e0139dfcff67
111212 F20101208_AABMHF james_m_Page_131.jpg
0878fabd41604fdd96a5ef935db44eb3
3bca416bb0be3891bcf63e67605f6f1198187295
50274 F20101208_AABMGQ james_m_Page_116.jpg
9093360fa22a08a9270544cef3f92d92
b90e09f72e83a70b7397003ef934b3292141709a
8795 F20101208_AABNKI james_m_Page_122thm.jpg
723d6fddb2b0479d21678c101ca8aca1
fce382962cd576db6923328c26d88da7c36214b3
9088 F20101208_AABNJT james_m_Page_151thm.jpg
d36995610f6c0c3af9b4386d589ea68a
e9e789450edfa8a175e2291b6d9f23f002fcc58a
139912 F20101208_AABMHG james_m_Page_132.jpg
ec5ce86a257485c915ff3d8aee693bfa
a8d0ce2a94539032a1bf0a680ed0fbdf37e987c7
105180 F20101208_AABMGR james_m_Page_117.jpg
d2f509a995d90941bd59f111e963eeac
ff1507bbe32876896c2eb8e16ecc49a57574dd36
8870 F20101208_AABNKJ james_m_Page_163thm.jpg
af7b467855c49801bbb71f226efc43d7
f045cb0fc207f78315e760e4bac791c8bd720efc
7708 F20101208_AABNJU james_m_Page_032thm.jpg
97b977b834f498acd3f0f46ba9be4444
6231ff8bc7f12486f062c8a4e25636e21fb37569
16918 F20101208_AABMHH james_m_Page_133.jpg
4ce97723832d419141ccc1c571294c62
2eb4c60cf8a5ca08f06d655fb4b50d4eaf4d9c41
107028 F20101208_AABMGS james_m_Page_118.jpg
036237dc00626637cdeac9fafefb8dd0
5bd04524172c53584060fc3c5abd458d94349962
4943 F20101208_AABNKK james_m_Page_112thm.jpg
088b57d52e70f80b4c28bd8c68755539
ca164e2fc1950d8ccbe45a54942d29622b49828e
9224 F20101208_AABNJV james_m_Page_027thm.jpg
ba339e4e1a9fcca7da2ec07eb893ce9f
eb065c59b4a92cb503d4e8771b08c0788bf583ff
38277 F20101208_AABMHI james_m_Page_134.jpg
72f005bfae24d590784f806523d296e5
c3d73d31bd1d60d06dcc294876de3c891f1295ec
111835 F20101208_AABMGT james_m_Page_119.jpg
5392672f37fab609e4fb5fd0e7a213a1
fe12391a32ea0c25dd6111cf6afe6060e9f87409
36780 F20101208_AABNJW james_m_Page_039.QC.jpg
8bacd93063b87da935042dbc6d5788ef
5aa7ab2d3cc7f89a809fa129a007b575c8b39fad
111917 F20101208_AABMGU james_m_Page_120.jpg
76a7c8b783587698788a69c47061f85c
c50f0448dad343ec2a6549ebe2fecc0e99985131
8792 F20101208_AABNLA james_m_Page_137.QC.jpg
e57616b2aaba891c157ee6cf4c3d79c7
40acf9d4d655057a9af29ad0e869951831667a05
8276 F20101208_AABNKL james_m_Page_029thm.jpg
be89718fcb36fa9087f813efe064bb4f
7d77c8b9cec7ddad0f25f541c9b956e7f5e353da
8344 F20101208_AABNJX james_m_Page_136.QC.jpg
65352a1ef6637c960cf9527ac8020837
04fd7f401180843697ce26ebb4b98a8fe337aa1f
48848 F20101208_AABMHJ james_m_Page_135.jpg
f32664e5dd0276b14d1b253835da92c1
326a596a7b55e7825d8de1bda7076fa5453fc422
94134 F20101208_AABMGV james_m_Page_121.jpg
69a0dfabda5a07e5feb73b2f5ae483a7
82c7a9165f6bd71b2ad91278254d89a95555f353
32758 F20101208_AABNLB james_m_Page_072.QC.jpg
d7060b09e91755a1aafb58320b80a824
09f94835fed85cc1a9cb00d403b753dcb8165d76
34403 F20101208_AABNKM james_m_Page_045.QC.jpg
31802d4a5871e7b84caa72fd0113b156
d6b26b4f52395a74aa7d6463962312c2366c3e3b
4497 F20101208_AABNJY james_m_Page_058thm.jpg
8106f8914803daaacc5c2514fb20ba6f
604d7bde904d658d421b6916568f6337a7ba4f88
22963 F20101208_AABMHK james_m_Page_136.jpg
5386aea3879e30c490ffd52ea50f8649
980e44fc0c9f25fb9a8139ec02b47b05033fc0a8
110095 F20101208_AABMGW james_m_Page_122.jpg
0caa69188ec98cf1cab52526d1774e69
e554481e4d7d12ddb58de72d3fac293798167b38
32365 F20101208_AABNLC james_m_Page_123.QC.jpg
995739883d05cb6b36472bb26b26dfd7
0b00df83c82a5e312ac8979c6d91c600ec492553
4889 F20101208_AABNKN james_m_Page_145thm.jpg
f0c8e4dd6c00c3fd03728d8579b912a8
93471778ba4118b450df30e4aaafed03d34acef9
8677 F20101208_AABNJZ james_m_Page_117thm.jpg
49b937f2e244b3aec9a6e374360c4336
ac6788bd561730aaad7daa171b70f8d752e56a8c
110611 F20101208_AABMIA james_m_Page_153.jpg
989217244a5c9a7439b3f45336833612
398d981108fb1d7216a1ef6cdb0c1024664c1c6b
25044 F20101208_AABMHL james_m_Page_137.jpg
c4c08204378b8cff5196e0f831116ef5
adead4e0d35aa7e667f940e86295f24c6f4e3b67
95810 F20101208_AABMGX james_m_Page_123.jpg
89ff197aeac9ec3bcaebb9d56c539247
75deb67bd725753dca289d5910587620a24b3adb
33047 F20101208_AABNLD james_m_Page_024.QC.jpg
91b19e0ecc1a1a9adb61c63d7d697f9f
8bb9d1f5edaa5d59fd38f5059c961e121b09a7ce
8586 F20101208_AABNKO james_m_Page_041thm.jpg
3dde5765bbb18767c9be9f53aab038a2
a3af14d2e43059354e834c4eeadfc3dc1028b6e6
110487 F20101208_AABMIB james_m_Page_154.jpg
5c7e715a92dc68495ee007b309c6e8c2
cab83cf13007e00e83d0d154dcee53b76ce15d1b
24636 F20101208_AABMHM james_m_Page_138.jpg
6266c015416f5ae924ea40312071b69a
8dcfee69107acd9b1f561438a37e02e1c9e50458
86619 F20101208_AABMGY james_m_Page_124.jpg
be394e93f7e06ca8be0b4c7f8dce0833
daf48ef5eb255708262b11c8892a5c20af97f8bc
35680 F20101208_AABNLE james_m_Page_166.QC.jpg
68c504a36fa96a2643b3df2b85f45185
b86e0adced61fd3a100173395c2b6ca1e9d7e836
4355 F20101208_AABNKP james_m_Page_079.QC.jpg
dc5feb1aed931dd7a6d6eb397350e82e
dbd4bfaa05a9d7d5d77e96e385afcd99432a9244
111551 F20101208_AABMIC james_m_Page_155.jpg
1db136914674713179cd33993ae7832c
b366a873eda931216449517ef246281de7441b42
24577 F20101208_AABMHN james_m_Page_139.jpg
a3d4467036d49246099ca73c9eeb1287
e857e22a1489d8aba43ac7d473cc53f1b81afccf
115082 F20101208_AABMGZ james_m_Page_125.jpg
3a2f5c45846eb9c7be736834eb7eb93e
6e853e2a359077be8d1ddfae85271a79bc21f513
9181 F20101208_AABNLF james_m_Page_106thm.jpg
446cbd9b81509533e9216ec95aba0f73
cc8498d1fc5e82801d590ba347ebb6cecf660da5
9057 F20101208_AABNKQ james_m_Page_166thm.jpg
cba29530b43a94802441b6485fb25637
e0009029dd4ea3299b70d43090df132d4fcb4d07
86085 F20101208_AABMID james_m_Page_156.jpg
3ba3afb3e044890a0b96b47abdaf3520
dcd43e1d88ee77fb862d9c7ac14d5c045dc97078
39687 F20101208_AABMHO james_m_Page_140.jpg
e6b5be37d0b96666e8fa8e199c2b81a2
36ae693622030f2ea4ec5d0a02d52bb8fd84de93
39212 F20101208_AABNLG james_m_Page_104.QC.jpg
bbb8c589bca582e3d8bcd88c42c9c1c0
5a4106fe0650009d36d6718670abcc1918ac06f1
8578 F20101208_AABNKR james_m_Page_097thm.jpg
9a97eb579f32e865d7fd49a5bedb12e7
47cb8213c5c913899f2eca65d639c9c9e837fe6d
67598 F20101208_AABMIE james_m_Page_157.jpg
5562e8804178fc24414cc740b7bed206
a4875f7974acafd93c9bd4c5a003144224118f46
45172 F20101208_AABMHP james_m_Page_141.jpg
ad3824a72992de5a10a810e4c1e5d05f
7001a2a3172d7a7d7ceccfb8c7829c43a5eaa0c7
37869 F20101208_AABNLH james_m_Page_132.QC.jpg
24ab23a133d88a9138e930be381d00f2
3168c902c363c2fef4f09ca25ae4c1660542888e
37714 F20101208_AABNKS james_m_Page_165.QC.jpg
9e034b18fd01fb22cb19eef5946f7ebb
494f5f8e4094c5213def6e75839ccb4614df756c
59173 F20101208_AABMIF james_m_Page_158.jpg
f97518a60acf9c4a2e9d954ab09d3599
ea5cc8618d8607b6de045ededaf6d7ab45395e53
40751 F20101208_AABMHQ james_m_Page_142.jpg
d9eaef9170071503324c504ce061eb4c
2e1678a7fa490b6cbb2cd8fe8ce40f71363f6b49
9090 F20101208_AABNLI james_m_Page_034thm.jpg
7d2e8a9417d382e6219bc10ac790fe93
04c62658b190fdbefb68c1c343f7ce34dd2eb09a
1444 F20101208_AABNKT james_m_Page_133thm.jpg
5d92888c776a94c7adcbcad2cf0ff880
a4d036b86245b750ed699e84b9ce5c50d4539996
37824 F20101208_AABMIG james_m_Page_159.jpg
b961844605d8281bb7388ac95fa899ef
d60db81322fb4339f05075d805e5a878f9f24413
56085 F20101208_AABMHR james_m_Page_144.jpg
a77e74f67134d0495426f3f40fc586b1
00cd670a03cd8e8b703924534a6f032a3217a497
6222 F20101208_AABNLJ james_m_Page_048thm.jpg
d5b8d09eca745379b4127ffa28263d82
66c89777430221361105e7c9e9fa9fe361a901e2
33545 F20101208_AABNKU james_m_Page_068.QC.jpg
2be81ed22557b0e492e2da935c73166b
732391b2f6fe6b54812173083fd7ac359fd51b5c
78211 F20101208_AABMIH james_m_Page_160.jpg
06bf90f074d1595b100a3332c3672cdf
03e866d2931a18d2cb692accc95a766270ecbc0a
59779 F20101208_AABMHS james_m_Page_145.jpg
6603ef6c75ddef1f1a2455a7728b59eb
cf65883c31ab029ef61d578a8a111350fe9428f6
9318 F20101208_AABNLK james_m_Page_167thm.jpg
a68b9e55ee9007714ad802eff388ed65
b50aa3b7011bfc4ae6788e75259b6b79cd460011
19656 F20101208_AABNKV james_m_Page_050.QC.jpg
3fa44694703a89e162fef6eeee48f408
a9e51c29486c9429616c12cce6008dddc5ea349e
53677 F20101208_AABMII james_m_Page_161.jpg
1a6a21c58510a58b495f7423f3558587
2162507b91a99157b4738f0d9ed14c4741ca088e
102796 F20101208_AABMHT james_m_Page_146.jpg
c255044a8e0863f93400a933e0b14251
39f4a9c60d741962dfd939979454635b84043d5d
8595 F20101208_AABNLL james_m_Page_150thm.jpg
1a349b33f0e0176ab8e185bf24882a52
9de366a75269ba13cc517bb9df5d6766c01d5be9
4112 F20101208_AABNKW james_m_Page_082thm.jpg
387cbf633a87667b43dcf82690810af0
a510f1e2f10ee62ee4a5a2ef6520d8783b13d22b
116744 F20101208_AABMIJ james_m_Page_162.jpg
2c48e6d33f2679efcf4c4af708943bcf
31fb62a87cd3504898a24accbb73bcebe593beae
125165 F20101208_AABMHU james_m_Page_147.jpg
0de031c34209a68d6d766f7d3349a3ad
612566cc41057792480de05265beda0d6caddfc3
8794 F20101208_AABNMA james_m_Page_102thm.jpg
c83ec817f4452e34cabf4684df6b9e12
db2c14b0d724ef31b54714ec6a850993b3a60615
36706 F20101208_AABNKX james_m_Page_120.QC.jpg
8bdd4bba26a32e1f2c66645820b2e1fb
51b6a5061b5e9bd160d5f9f5024c99c16e8b3de0
148615 F20101208_AABMHV james_m_Page_148.jpg
62d91c6af2351074985c9c9b15b28414
f7f0b334b2db8a8e1586fd75fea4e4324e7445f9
2932 F20101208_AABNMB james_m_Page_086thm.jpg
bce2a6cd15571521a2f574d849bc9a3f
c97d28d7e1b19cb4cf95832da10557503bcd4478
8896 F20101208_AABNLM james_m_Page_125thm.jpg
2ac227e9eb5658713b91e43c89dca8dd
58d57921292a231627e4ea8d0b314e52d5fafe70
35436 F20101208_AABNKY james_m_Page_099.QC.jpg
ed49546d4dd95cbf05e506521559d0d1
860d09ea6d3bd37524c0deecda9c5e6239bd7174
107506 F20101208_AABMIK james_m_Page_163.jpg
be657e14e5e50ecc445e82f22bc9476e
b4472e4d401027c3a0a98eb06b0bd10478187a0f
106882 F20101208_AABMHW james_m_Page_149.jpg
0db9081b33715edbd433646e3c982f5d
9c308876aa7b1b277c33a60b76c697bd9530612d
34846 F20101208_AABNMC james_m_Page_149.QC.jpg
b558b816a2ac040abacd2ef76df6e772
58c841408a1556d36191b3a24fca755c822698bf
34751 F20101208_AABNLN james_m_Page_074.QC.jpg
338ab9b6f673941404cc117275c26962
b1c92b818f454c20e924f0ac5a84db5eeae45f64
12655 F20101208_AABNKZ james_m_Page_109.QC.jpg
432b28177373fd7c4d4e698a9de66dc6
cabdc0e143719bd368efdca7434eddd5b35503f9
127122 F20101208_AABMIL james_m_Page_164.jpg
10291b96b6271bb91f38a7409545cdb7
2d247e2ffd23d6b09ad63b81e6fca7a27f4edcdc
107827 F20101208_AABMHX james_m_Page_150.jpg
60ca092eb29641b3389056c5b41fffb4
0341e9198adc5be79c24f293d959b9bd89e14f1e
1051934 F20101208_AABMJA james_m_Page_006.jp2
2dd01b9ad8ec455185dbcdeef5b9fc8d
b3ab6738dfac6ef22b1c16688bf12f51c5da2f2f
2878 F20101208_AABNMD james_m_Page_138thm.jpg
0a66095db14e07c1bc6c07249e4feca7
9400c66b692f90a7c25ddfc0e8db9c7102210fcc
4937 F20101208_AABNLO james_m_Page_049thm.jpg
17e5a89b2a3257bc1d2d4c0247a9ceb8
9d3cd9e52f2a9ef770d7065d1a4a7d09dda28af3
124540 F20101208_AABMIM james_m_Page_165.jpg
8d64ede9805a428ac3816397039ebe9b
62a115d89687f12e952dc3608efa9bfe4360e52c
143837 F20101208_AABMHY james_m_Page_151.jpg
0f354969884724ab54182c15ad009f1a
fc709df3b3f069972521be0abb43803fca59c912
1051974 F20101208_AABMJB james_m_Page_007.jp2
62979550dbbf2cb5a92d4feb8656c129
a23baa59f14ae0339e85c318bdbd3614f58f0660
8356 F20101208_AABNME james_m_Page_152thm.jpg
c8d0069f4ab3d7e0bb7586f7b090ae1d
29459a338609d699891aa732db0d2110c11526ba
3727 F20101208_AABNLP james_m_Page_089thm.jpg
781e851be72c1b73ee1b7f512cf1eee5
6d6adef80167961bfb9f799bd50c7553d3e428fa
119661 F20101208_AABMIN james_m_Page_166.jpg
b1a7345027c3198b9636882b651162e0
388e2d7160ca45f7aee4c63e6b4f893d73c4c3d6
105673 F20101208_AABMHZ james_m_Page_152.jpg
7e709581d9d2179177d892dcb75b55e6
c95466208d6aac22df856dca4f4b8325466f095d
1051977 F20101208_AABMJC james_m_Page_008.jp2
a521f45687e1fe8146b16c9ce5644528
2d8c218921faae7f09bdf44d217385ff6470a984
32560 F20101208_AABNMF james_m_Page_023.QC.jpg
4f9df2ceeba885e71beee0738c3ffd4e
7abed3a3f142731ac19501873a164f00d18d08d0
12822 F20101208_AABNLQ james_m_Page_141.QC.jpg
689392f72dcd317908d4b9e2379c9a35
e988a9b17e9af94518fae4969e36128c870b79ef
121697 F20101208_AABMIO james_m_Page_167.jpg
10c65708720c0d79b952ccc4bc84cd5e
045c49f77639a873ee84b2082ef1cb40ea47db23
1051972 F20101208_AABMJD james_m_Page_009.jp2
0fd1c2f5a5eb6418f2a931e923e65577
52d9abad441d310ccbc98d432c5b418640deaea2
37484 F20101208_AABNMG james_m_Page_064.QC.jpg
d3f11998aa4d88eed7ec9eb28d3d471f
1ea2e2c4f353435082964394bacaf0426ab0de22
8634 F20101208_AABNLR james_m_Page_038thm.jpg
477532f42e56d34f4a198db09159820c
c75957a9e0a31c9ce8dc45f7eb0cefcfa85dea6e
136119 F20101208_AABMIP james_m_Page_168.jpg
9f92b036234ea586441792a7482008c2
7120ceacdb65afb9d17151298a30bc850c3d4236
1051986 F20101208_AABMJE james_m_Page_010.jp2
d8798871510afc48109ff4d59fb3ad6f
a593a2b5c1fb8eac42ef2d7f902e41698f925382
28958 F20101208_AABNMH james_m_Page_005.QC.jpg
584abf3af39c537827382d84627ef80d
26a60a8a3008b86770bb6aa2d4cdb0e44e31534c
3852 F20101208_AABNLS james_m_Page_091thm.jpg
08e610d42d8562853591e3f2dd9dec94
6ce93e806c5c1a45e3d0e103e3754e4b3f300894
130567 F20101208_AABMIQ james_m_Page_169.jpg
38537cac1dc53225143d213b950f62b9
3ea07c1c0f8981d30b459f444af1ec80dee02699
578485 F20101208_AABMJF james_m_Page_011.jp2
2ec936277364bde435e75afcde53349a
5dc43d03456a3e389ad5e94e37a097b8ae6c5462
35497 F20101208_AABNMI james_m_Page_129.QC.jpg
fac0c4bc03474b28b674f9e1324c9bea
1ff46d79e413f5bdd34dae1eb6c6a10eac5db1f7
33961 F20101208_AABNLT james_m_Page_102.QC.jpg
1de5fde1b385fb5ff6bc1993428f3ef1
72e0efc6aa518e55a8e054330424198e5df46cca
140033 F20101208_AABMIR james_m_Page_170.jpg
e2c41236945878a722d850ee7902c49b
e2fe948911947daa7c817f78e3474ed281f211d0
91520 F20101208_AABMJG james_m_Page_012.jp2
327984df0a2e0671ab465a89bea1fb8e
98388916a7ee58141eb965a9f2e34bd91a102689
7429 F20101208_AABNMJ james_m_Page_044thm.jpg
c6eed9d5ee71e829e3ad2ca57bad7762
386edd4acbeacddc3470314b31b4424c6b77ec24
21035 F20101208_AABNLU james_m_Page_157.QC.jpg
79c0589ba4563afbbb2d7765d5a37770
425ccdbb5528a72c758388899802a73d7646dade
121745 F20101208_AABMIS james_m_Page_171.jpg
ce0f204e643b747552c3066b3aecb9be
f5e9a74de228105811b1ed8b89347fdab25e543b
105071 F20101208_AABMJH james_m_Page_014.jp2
1ea9e256404d17f15e7b34dc579c48fb
f9cd0bd4010bb90816fa32f0eb17043a3f577c7e
37757 F20101208_AABNMK james_m_Page_026.QC.jpg
8c8ed816662220b47f52679ad72b5b52
08e12c384936f2b4b284707bcc0d1fe5cce3297c
9150 F20101208_AABNLV james_m_Page_128thm.jpg
de069551c3543f595d456a5a8ef9146c
d7117ca230d094ebdfe8cdb7b39b238890071052
68611 F20101208_AABMIT james_m_Page_172.jpg
62e91e541a3236dabe0045f79db7ed42
4d8293abbad1c0ee91baf87b28c0c70dc49eefea
112937 F20101208_AABMJI james_m_Page_015.jp2
f5ecedb3c26262ba7fa46ef9c7315dbf
b2eabec303e73a1206e0419377b73f8c42518c78
F20101208_AABNML james_m_Page_003thm.jpg
dcd6e9a113067b9021fe8954cde2207f
74eaf6d3fd4acea688139e473378a8627541a687
8550 F20101208_AABNLW james_m_Page_045thm.jpg
3854b83444326703b247813866ea1f8d
3372b42e4020d5f604841661ee9340cd0cacc23b
42543 F20101208_AABMIU james_m_Page_173.jpg
20945fb66decc6b0696a0a96bcfbd0b2
79d0ba21b2530e3358bba41776ab0489d43bf2ac
115855 F20101208_AABMJJ james_m_Page_016.jp2
aa89203c616a212f02825a44eec5404f
7990a6b5ee2c1e593afa0c1c3dffc439f720ecc3
38967 F20101208_AABNNA james_m_Page_105.QC.jpg
422e916caeb3ed9179668b52729b5776
b2d9196920c5e6bdd939922760693f4d2b6c2291
16304 F20101208_AABNMM james_m_Page_116.QC.jpg
a87d53e1931c81262df34599f4f5357c
897c7c5fbfb0332d59547b625bb2ff8e0c15d10a
8913 F20101208_AABNLX james_m_Page_060.QC.jpg
019c90d8b3d0ba1b9222ccf6cbfb8ff7
476f10190502b58c4f0ea3c53b8243a96144691b
25425 F20101208_AABMIV james_m_Page_001.jp2
a2127545b129a672c96209be4b9d736f
b75b92bb8b2dfc3b23eec08494dc02509ceaa2bf
118426 F20101208_AABMJK james_m_Page_017.jp2
c9f5b5182d315d188b8017e37d155dd3
f721b7ed52d056ceb125a16cc1d36f7248620a12
3393 F20101208_AABNNB james_m_Page_134thm.jpg
22ebdd86d91fc20532d2f570e80ef008
66a300b7fc6084f28c6dcdbca0b327cb15ddd054
12141 F20101208_AABNLY james_m_Page_159.QC.jpg
4d3641f8c1040d72fcf3b66a4a0889b1
8514e65c58f9f6f565945042b0d760b181a08990
6223 F20101208_AABMIW james_m_Page_002.jp2
4e871e08095ae280758aea73c5850c79
ed796dbbdf3324809ea6e8e8e8644e1b08ad557b
36370 F20101208_AABNNC james_m_Page_041.QC.jpg
635142bc6197623bab7453ba22495126
480971943df755132102d79fde393850e1bce07b
13208 F20101208_AABNMN james_m_Page_087.QC.jpg
9b0e6d8c99b439939f873250a5306603
0ae69be230ad42dc69e4fdf527992cf370a0ad99
34528 F20101208_AABNLZ james_m_Page_152.QC.jpg
6957c60a5614fad9444f67683cdcdcd9
1a28c5b80e95352a076d582e24e9148753057b19
7894 F20101208_AABMIX james_m_Page_003.jp2
59eae2d5d8f57ce254962faa80214345
ca10e98dd03a2d8eb7c5aec62533f137cc444ce3
130309 F20101208_AABMKA james_m_Page_034.jp2
36b8df75f565664d5ef9881898402cd3
d606f890e9cde3fd956d3f6030fc37959cd10133
117176 F20101208_AABMJL james_m_Page_018.jp2
abbca8e599f41d617faa0a412608cab7
f1880b272d4748db310e405dbaf86402d8879205
9258 F20101208_AABNND james_m_Page_139.QC.jpg
16c9ab4fc469be2fb09a448bbd5cfc6e
c54589443c54c96aeb4578a087c98c510abaa988
24224 F20101208_AABNMO james_m_Page_056.QC.jpg
c2c652565225e60bacf6d18238dd8aad
573b567a91119647de88526c11f393ec33487fb1
98548 F20101208_AABMIY james_m_Page_004.jp2
5271600e862296b400f81b48c08925af
605412a1f423e7388de7d20bef18777079816a60
106029 F20101208_AABMKB james_m_Page_035.jp2
7464d4e1c076d044953accaa26f55d60
4fc255794bcc6b13f8b4fc3556f3a52d87f71464
112797 F20101208_AABMJM james_m_Page_019.jp2
1b46594fdc5236ac9d0b423dfe62eba5
0ab58aeb93a17058dc06d2edd3d1b47c6ce6cf91
15102 F20101208_AABNNE james_m_Page_135.QC.jpg
61e9352357d71885bbb3a3ddcc9adbea
7ad565fc4a1fd6070fa31e08b9ed13820f00589b
1771 F20101208_AABNMP james_m_Page_002.QC.jpg
76fbe55603816abe87fdb2326f323553
8661dbe2e87973d7783537d669b6e6660eef9c06
F20101208_AABMIZ james_m_Page_005.jp2
229ef0e26474ba40ac5880e88f0fff46
404583ccab935c257238662ba38c35f618ecdaba
111208 F20101208_AABMKC james_m_Page_036.jp2
5f9084d4ab37710176514536f1755570
be6307e8eed480aa6118c6ecf7fcf95ba6f8d409
112724 F20101208_AABMJN james_m_Page_020.jp2
8101d578ebe8521772df0ba0ca20608e
793a4da379ba6c947fa6c874da2f4282f632c0b7
36926 F20101208_AABNNF james_m_Page_006.QC.jpg
034aa26a0067f728046ab95ded2e0192
7ee88a7914c20e9ee071cc42dfebc572d7c93086
37006 F20101208_AABNMQ james_m_Page_148.QC.jpg
4ded2bfea962b1fae3cc39a45a1f6cb2
583542d0cd2acdc816d58da4960fc04010174930
89696 F20101208_AABMKD james_m_Page_037.jp2
73398e34f8ff51569217c89f30f5541b
8334d504efea68897e3f5b88f3993b24b0d5399c
115633 F20101208_AABMJO james_m_Page_021.jp2
89e85daad2cca035545d182e98440b9e
56c36bf8da0896db5533f2a0892317288ff95d4d
24227 F20101208_AABNNG james_m_Page_057.QC.jpg
abb52b0d978580b87fa51302665db311
d1c4a192e80932e11596830d335541282d9e18a2
6408 F20101208_AABNMR james_m_Page_157thm.jpg
b94d0a8654b652069f753cbb65f578c6
d9cfe564a71d2065576ab7eddc4af9655e724e78
114793 F20101208_AABMKE james_m_Page_038.jp2
7a64a2b2f1639ba8d8d6adb83f23e74c
bf0fc3c874d97d4927460f6fe64b9989c60e74d7
102883 F20101208_AABMJP james_m_Page_022.jp2
9cfbdfb5d671e574fdaf918af44136fd
dcc36228c0da875ea1dc983b7dade6aa48346f41
17298 F20101208_AABNNH james_m_Page_049.QC.jpg
81aa275dc2bd8c4ff55165c71822b6e6
10e7082f9f08b144782c1f56837327854cc2aaa6
6098 F20101208_AABNMS james_m_Page_051thm.jpg
e792e262578e5ff9b7ff8d32923651ac
03e89d242b998c3189c82a78de5df7d2c5cac4da
115686 F20101208_AABMKF james_m_Page_039.jp2
69f54bb6b2d9ef5901c64cf9e3388236
cbda09bdb77984ce20a936ee850b3db34addd470
103572 F20101208_AABMJQ james_m_Page_023.jp2
ae4ac31b044dc576584b22101033ac7e
e0bfeda38329545ef15b9acc1a2ec8428b1ecdf3
9170 F20101208_AABNNI james_m_Page_154thm.jpg
242ac83aa715aedeb83a7220015100c2
9cf6607614a60c6da05e6466a1a7a3b8a5e04795
21318 F20101208_AABNMT james_m_Page_172.QC.jpg
2555ad4e3b606c5b4aaafcd3a06ce4b5
1b6c45d4672cbf1aa56f50c2c9718f9543c0c295
109120 F20101208_AABMKG james_m_Page_040.jp2
cf7c29b93e39df4d23afe2a109ddacf9
eaf7c478466a36086b4a4035cdea9473fc63916c
109277 F20101208_AABMJR james_m_Page_024.jp2
614a158b02dd807201307b6082518015
11696bdf4f13fe606f289cc7a4cafe91a9843ed4
4737 F20101208_AABNNJ james_m_Page_085thm.jpg
4e71ed84a9e3b445bdbd5e5cc8843b64
f422802b5395dc9989b182cc1c2fa198425f6f1e
9046 F20101208_AABNMU james_m_Page_107thm.jpg
5cfc61aa09fa1cc911b1ba9962b5f055
0368b35380fde1c6336cda0801bf6eb2a15e49fc
114483 F20101208_AABMKH james_m_Page_041.jp2
ae87b8db605a46ff4fbe03250c3f84e5
bac6f6f1fbe80a203315ec46fcaead68a9dac274
110934 F20101208_AABMJS james_m_Page_025.jp2
8385022dfca0e12a44e38d2a52985b79
ed1c7c976873a8aeae72852018b0a35e6dad7b3d
4881 F20101208_AABNNK james_m_Page_161thm.jpg
1818771b63c5c27c5a396dbc6c9d009f
9426d1790b4acdc425032712429df495167872aa
18225 F20101208_AABNMV james_m_Page_082.QC.jpg
35e49d5385ae32315bea01325325d464
ca9644962282a07b0c0a34bdfb506debee33748d
116129 F20101208_AABMKI james_m_Page_042.jp2
40b48e197301ab417d161109db0ea867
15266df9d96ee3ee212243923e949fee09680c83
116857 F20101208_AABMJT james_m_Page_026.jp2
31549e0d3578e790385843f979a456b1
7f1e552b8c122e9df0e4a42ec0169974ce94a16c
8754 F20101208_AABNNL james_m_Page_131thm.jpg
65db0b9e115921efe1b6031943eba081
25792fdd169632e0a6d0f3c0958044bfdc6e6bf6
34014 F20101208_AABNMW james_m_Page_033.QC.jpg
5b667fa624f38738ce810a006b1ef570
2e2bb6d3eac3e7fe52b38806b788c74deead4507
110883 F20101208_AABMKJ james_m_Page_043.jp2
97305c5c93744ed3dd2ff5f8d74df768
5bf22353d9354cd791fa6201184eee2dff01e549
120562 F20101208_AABMJU james_m_Page_027.jp2
5696e1bd210afbde98febbe460c21b9e
d60606d47df760be8af4cb9583ba28d3c9d20a74
23439 F20101208_AABNOA james_m_Page_051.QC.jpg
c1e85b468fe315225ac811680aa2f629
f71fe949700a4249de1b0287b96c998edb746ba5
8947 F20101208_AABNNM james_m_Page_076thm.jpg
3e9ed6eb2b51256576cb898d6ea96240
adce8c6874f55b52ad5c97fa502338837c2b7c15
5780 F20101208_AABNMX james_m_Page_093.QC.jpg
80c18d8a3a689fd7534de929bf33c7e5
0fdc37a91c2c16cbe77e10c91dcec1e983ec59eb
100751 F20101208_AABMKK james_m_Page_044.jp2
af1db15a26852906581555d340584e54
f04659c4b6feadf039acdb6ba3f51abede11e4c3
96999 F20101208_AABMJV james_m_Page_028.jp2
7ff12e2a016dda5a389388c7f71a8741
d4e30ba1bcfe630cdb6570a431214350c1b52587
37124 F20101208_AABNOB james_m_Page_018.QC.jpg
1ea9934a32e7e6f5d7d77d7fc6dc7a74
677ee933903146f76544ccddf68e6891f70a6580
40059 F20101208_AABNNN james_m_Page_170.QC.jpg
b1796cb7778dde751073d448dac5293e
96122070a7ad848e493b0bfa0c05a733d818b3ae
7997 F20101208_AABNMY james_m_Page_123thm.jpg
ca91fc1d9f18e049344910cec92b4891
25f80a50680d8c87508b6238b56def0eb63f79bd
111539 F20101208_AABMKL james_m_Page_045.jp2
f8c21ee140257c2c0c10175407df1942
849cafd3458d91a834f32bfa004c761952dd1a93
105708 F20101208_AABMJW james_m_Page_029.jp2
7c399429acdc1cf178d60b095ec95b97
e269e893c15a793de62e49b9652293e36aad23c3
8637 F20101208_AABNOC james_m_Page_064thm.jpg
911bfc52460169cf9359f9a278ca8298
1fa962b6a27bf39c24540950d9f68ee03db7b288
36092 F20101208_AABNMZ james_m_Page_076.QC.jpg
31327ff3d310ebc8e471a90fbba30c35
dea007b8501cddc8c2a8c3911bc31fe8c99e9e97
111156 F20101208_AABMLA james_m_Page_061.jp2
429b41c26d7c908dc66d74cb6364040a
71408ea8c210a52f1481f7e13c634cb2ed45b335
110276 F20101208_AABMJX james_m_Page_030.jp2
c8328010d88e552f451ac09ddd4f5b3c
91e59cfa82f9b14ed4e1de0d3bfa2abe99f2d284
36719 F20101208_AABNOD james_m_Page_042.QC.jpg
73e08bb3a1609f1494b892988d56f2f3
83d67906ef65484ab3e928c85c5a73c737ffe832
3172 F20101208_AABNNO james_m_Page_139thm.jpg
e574da5a8b9be1ec13bc61a3d43b0706
0f91b50c498bdc44339bee43a9bff23d6f0b316a
119646 F20101208_AABMLB james_m_Page_062.jp2
ddaadb6575b37709f3c873d59fc81dab
d30ec1c6efaf2827bc5fbe491e75b51793293a19
111257 F20101208_AABMKM james_m_Page_046.jp2
51ff184ec4577f9044955f5629f23ab2
4309f4032ebd78f2c23f74edf9b73836e6c70cc0
100749 F20101208_AABMJY james_m_Page_032.jp2
06a5f32c13b4f17e8421099efd4b60f5
ea4e7ae9c4aa383a06c511745b4d05543010a8ec
3104 F20101208_AABNOE james_m_Page_137thm.jpg
91b34ed824dbac441c3cd4e95df1bb40
c7d3138a30ceeeceb01f7a38d27aa11fd29d960d
8193 F20101208_AABNNP james_m_Page_035thm.jpg
c68d35f41361ad768ece69140bececb2
76c24f6af933a50fa454d653bad4de4a2fa294a1
107329 F20101208_AABMLC james_m_Page_063.jp2
4c98a94470404a12c2d454a7d33dc3e4
d3c527009ea51a06b26343ed67d1b391e8440a5d
74117 F20101208_AABMKN james_m_Page_047.jp2
1584fe92a56c3dab454db9d64f04db4b
925a05714563e642e0fb75a7050e1c36edb24a04
108867 F20101208_AABMJZ james_m_Page_033.jp2
94976e29d20c8cbc2c7a822b662ffb66
5715bb87da31edd9c3b0b6b9e8f2ab3b47c69e04
8893 F20101208_AABNOF james_m_Page_009thm.jpg
211cee009be61e25cc14681d60ed2ab7
12691661f84a5da4e48f5d8b44175a95b66d4c0d
8189 F20101208_AABNNQ james_m_Page_040thm.jpg
0a7a5a1bdeb90817f7114e6c64ea9da4
9233ae26c91151a0abe2decb49db826111d61dcc
118467 F20101208_AABMLD james_m_Page_064.jp2
7492a1c0fb596c5fe8cb40dac637a137
161a85145806fe3d0dd9a7d2db46b193e815a159
985123 F20101208_AABMKO james_m_Page_048.jp2
f77d88069dceb4383f9bd7ab2ba62762
09b2875926aaeaa321f35fcb3f6a72890345d9e0
35645 F20101208_AABNOG james_m_Page_016.QC.jpg
875c0d0c15b9deb931178afe3d90f3ef
8d5e615b7647659b7b1b1b0eb301a1e05c7b6bf4
35836 F20101208_AABNNR james_m_Page_070.QC.jpg
b19fa8ec73095c26191a40591e53deea
589f8190a2230746e2dcc1d896ae74f4aa04e929
111324 F20101208_AABMLE james_m_Page_065.jp2
8413975130f69e4f35a8b7ec63c86f32
1852a6a2eabd3f97214363aae8d2fcb4ed60efb7
692581 F20101208_AABMKP james_m_Page_049.jp2
fe0e403a06f12c315f202e49a88d4410
9aebe1687b7ddd3cc0c82fcf6a8686f4a2687f1c
38486 F20101208_AABNOH james_m_Page_168.QC.jpg
1526a60cf19104c7c1b9fe2821558f7f
c5515d8d9b4231c23d174fbaff0a33ab476298cd
19402 F20101208_AABNNS james_m_Page_145.QC.jpg
f5d124faa26d0db441e7d217c64b1076
d20481c9305aba2da927815d9193e46c8d9102cd
114647 F20101208_AABMLF james_m_Page_066.jp2
76c85bb7db817615061b353d7531bbeb
db1f680ee53493e3574927d6ec454b3b8730c36c
683328 F20101208_AABMKQ james_m_Page_050.jp2
5e6b09712a3ca26e2d68639dd4b2d556
5502975d716cabe65216151f0a8f3ee8df62c7c3
17234 F20101208_AABNOI james_m_Page_161.QC.jpg
b927c7519d7ef24ae058adc305b27f60
62631bf8156b0d05eec502cdff42d85e5cdac6e8
8317 F20101208_AABNNT james_m_Page_065thm.jpg
0bcf643d3b21f72cec070ba24e255d41
5c894ae80d09565f601b7d0287ea21a9b7c4d56e
121288 F20101208_AABMLG james_m_Page_067.jp2
6de99d4f29d10b954c3f07464018c325
f3c3c066300f5808639a53b2c995cde2eea657eb
1051810 F20101208_AABMKR james_m_Page_051.jp2
ab6b62a3ea3832c6fb556ee3be320e2f
ec8c44df8564f01a5d3bd92d919396a231f3e63a
38577 F20101208_AABNOJ james_m_Page_106.QC.jpg
d0d6c0f949a761a10f36da2a4f10e7ec
3ddb823a09fcab5984580178a0bf46a091d02d49
F20101208_AABNNU james_m_Page_162.QC.jpg
ecda3ebd342297081bf5c3d8c9ad72d2
063568f6aaf7438e2bb57d99d824b03d03453d1a
108025 F20101208_AABMLH james_m_Page_068.jp2
b76bf29a268f81b5bca53175f4dd6e45
a20b44d98c792fae638f65cffcd7cb720e6cd285
584430 F20101208_AABMKS james_m_Page_052.jp2
75949bbc620e0196b74b6863debc6796
9bfbdc74e60e4e71e0b1e055f16f4733411896a8
2757 F20101208_AABNOK james_m_Page_053thm.jpg
c5bf039fcfeb946a2ce112856b7f4296
b57ddd74445fcd61989275047e297d0e7755d845
9758 F20101208_AABNNV james_m_Page_143.QC.jpg
bc8d436697152748185e9fa9871acbc7
4cb5fb3f607b54214dc1b59f57dd7cf662a8f631
119692 F20101208_AABMLI james_m_Page_069.jp2
d7d1d5e0ccaa88fddfc0bc20cfdc385a
c0d597d77534c75becb281a2a040030c32ccaa54
236036 F20101208_AABMKT james_m_Page_053.jp2
14d7b2b3709672df3681dd6fb2f2f284
509c22ecb4fa98b25ed42f61cd434eafa5719294
4042 F20101208_AABNOL james_m_Page_109thm.jpg
d5e5f565f7d0fbe67528a67165309b74
25e16467fc2d2e549cd22f80f12f9ccedd5e6697
9402 F20101208_AABNNW james_m_Page_170thm.jpg
110d57170442fd5ff20fb4b14e91fa92
ab7aa491ff8b9d3a26133e8c1e98d495eda0c9a9
517684 F20101208_AABMKU james_m_Page_054.jp2
158363cabb5683d5fab56b68422d9dc1
92f022019d3f7eabaecd0c9133d710b9832fa61f
117039 F20101208_AABMLJ james_m_Page_070.jp2
afc2bc9184a61631762bae3038a73c33
6a1e6eb715d4b4882f3e5574d1c1ce6c3f2eb653
27614 F20101208_AABNPA james_m_Page_160.QC.jpg
cd4a1d105fce950645de637ee101d480
b590162f447d19c08c1ac16358bab9264b0a8521
8895 F20101208_AABNOM james_m_Page_062thm.jpg
07ff41ef151e3404b0e072c530e74f57
5f832e6d85904f8118f17c96a0d133f6d1103760
35377 F20101208_AABNNX james_m_Page_030.QC.jpg
d0513879274d1a775550e52f5c114ea2
d17a7ae66af292bd849b1e090f44e6df7a503dfc
849652 F20101208_AABMKV james_m_Page_055.jp2
09618977cf284227369a92c6c8ebbc78
20329b0d4d3d73659199789f5943a4d09d88933e
120750 F20101208_AABMLK james_m_Page_071.jp2
9424a3194eb3c8591218097798230719
03f855eeba38cb61c2b7f9b2c6379c41e8ae2306
8521 F20101208_AABNPB james_m_Page_061thm.jpg
b9353fa126aebdb151c8a3647abab298
372273beca967888269a1ba6c35dc62cf07ae812
8845 F20101208_AABNON james_m_Page_031thm.jpg
6248208c8e2f28e1339bf4121f3806ee
bcf1146f24245f7ebc52b1a126245eafd98ec3e4
8924 F20101208_AABNNY james_m_Page_066thm.jpg
7f01c3d9cfcaf36a84ffbe561cb807e4
cd764f712411901187cdfbd26ed272e4f85fe369
98586 F20101208_AABMKW james_m_Page_056.jp2
678bb2f5b4c6b86ae59707b06eec330a
7394280b561dfe94f72b7047d247f00900a88c66
107871 F20101208_AABMLL james_m_Page_072.jp2
698b4af6f2508add589f2d6f64c2cf41
d01ed62bcb4ea24e75b865107d56280c58a2cb8c
35886 F20101208_AABNPC james_m_Page_154.QC.jpg
4da37d474602b910d178b6c414d6cacd
64ab588410c4779fc73fa99c669f941148703235
8056 F20101208_AABNOO james_m_Page_023thm.jpg
48414941b5bb4102ce214934ee94f6ac
3b4d06de6a3b8e3319ec5c156011fd806280215d
6822 F20101208_AABNNZ james_m_Page_012thm.jpg
1d4934c3d7793505ac91923a942aeef4
cc961c524c8b312f39b91c162c7561dd6acedf19
96322 F20101208_AABMKX james_m_Page_057.jp2
5be63db46868e74c7227215253d3c075
d4463f11051ba76ae63f699223095faee8d7eaf7
380221 F20101208_AABMMA james_m_Page_088.jp2
6d93f51d5b956b542fd07b84bcca7b23
ee89a797aa4ae9b7ea9f4765b9f4eee9d8b00ea8
114287 F20101208_AABMLM james_m_Page_073.jp2
f4edbf70f11693a86b044fc0ffb06050
001f14a823133fd8ba2904b04a7f1a45d1e0aaae
8618 F20101208_AABNPD james_m_Page_019thm.jpg
b93c711583b9e0871f9679ba6010be75
d6e65ca2d5854aec57909c1f158d9c8658055608
65855 F20101208_AABMKY james_m_Page_058.jp2
478febc13c24e64a0d45a9ffe7489e82
bffcce68702f75b909375a4cb4370c1094b67ffa
263614 F20101208_AABMMB james_m_Page_089.jp2
aef944c37b13c1361158f53deac62406
f1ddb4e79f217abe7d20a1f6c7d34a4d65861a8e
5401 F20101208_AABNPE james_m_Page_172thm.jpg
2613bdf625755a7288107e1f0f0fd999
506fab46a7e39c3c40336f832fa44fe47ab354cc
4339 F20101208_AABNOP james_m_Page_142thm.jpg
82c6ba0da071fd340c7dfd35c50f7368
a7f74ecb9189952f95bb3abe70b03fd4e602b313
131107 F20101208_AABMKZ james_m_Page_059.jp2
cb87b1104a81ef67b4b531d502741d18
f3b255b6bb462fdc663d20604b408922877e3152
611849 F20101208_AABMMC james_m_Page_090.jp2
0f7961232d823d46a7b8a1e53d0decca
8d16810dd6e1b731564d9af01de6d5f9e65de30c
113827 F20101208_AABMLN james_m_Page_074.jp2
3fee526f35350b0b1e2476d29065b670
6d529946edcac1a9d6d1eb80978c56b78b2fab83
14829 F20101208_AABNPF james_m_Page_085.QC.jpg
c5f0aa8e2ebb41a95f4b285e3041e726
38fd4e82766f992efc6a02a6c71fa53f675f7d8e
9958 F20101208_AABNOQ james_m_Page_011.QC.jpg
733a62ae56297061a803677d8b9713b1
199bf3b152bffcfb11e441ce30bdfcb8675f0daf
263793 F20101208_AABMMD james_m_Page_091.jp2
ad3288cc66a768c0a6179a3ee43198ee
45ed2b8d30d68df36156b46b79f02a7d4b3491cc
119916 F20101208_AABMLO james_m_Page_075.jp2
84b1bdf14aa1811393a25518f80b34cb
4de862c98cfafc56455f3ca06ae8c93fcba803b5
5083 F20101208_AABNPG james_m_Page_113thm.jpg
52801c2587811a7650503bb87ce7e5cc
919849cecd5e882f71315f0e13751efeebc1f7d3
34476 F20101208_AABNOR james_m_Page_146.QC.jpg
dc993806b8eea072fb11b86daaec9214
38a451b6cd68528e268afd992b6cff3481dd5edc
250341 F20101208_AABMME james_m_Page_092.jp2
b9591cbd4970656cb317e4eb0e1df9e0
01e58986fd7bcd79a7789130994b52b1998c6ebb
118759 F20101208_AABMLP james_m_Page_076.jp2
940848c2955910c0f898b87cda02c87f
22d1aac87d095a5df1663d0bf61a7ca14d5e6c6a
8206 F20101208_AABNPH james_m_Page_121thm.jpg
778fc4675d96f3c47b5602c40746529a
9095911018294acb9baf4708cb249d774f75d9ac
8187 F20101208_AABNOS james_m_Page_095thm.jpg
01eec662b4aeefb14ea2968b8b37a763
ea9a3de13b72d5826475627c294969a2555dd534
16993 F20101208_AABMMF james_m_Page_093.jp2
0e4bac749d380df844c4fc66af67fd6b
f4dd9bfa3ec4917a5415dcae9d027001e192cdac
118882 F20101208_AABMLQ james_m_Page_077.jp2
f42caaa37c1a7fbeb41040307d8fa0e6
3b96593217f1c89045dca8088472ea1e04ecce04
27900 F20101208_AABNPI james_m_Page_008.QC.jpg
3665b27fe7edb42201328fdd58fbecca
06f1df4f43b40b57674437cd75380a1552a5f3c9
33248 F20101208_AABNOT james_m_Page_043.QC.jpg
20832dd4c42406cc6cf63799082e5c7a
0fb4a77c482844fa0057f16235951ab1afc88185
117145 F20101208_AABMMG james_m_Page_094.jp2
207edbb955fdbd07d375a21bae0d37ca
12a7c38973132db01af1ea31648df2622c3c213b
15328 F20101208_AABMLR james_m_Page_079.jp2
278a8b30d9a7961086c7ac341ef950c5
7a29e1dce93056ed89327aab8264d4d6eb655ab6
8681 F20101208_AABNPJ james_m_Page_021thm.jpg
10da84c79e4ed111b3417d6c2b96edc7
af960419ad17f1f5197336f4cf05ca0b0031d3d4
8670 F20101208_AABNOU james_m_Page_025thm.jpg
7f1ade72e0bd805d5c30633d12d2925c
3f3cb57d360e1fdb9dc650f6c7bfe1389e0b573e
109524 F20101208_AABMMH james_m_Page_095.jp2
01bb0b70a81794c2c351f8762f083cb9
a24583ddcf1bb3ed9ef99a82741ab860434b8a9a
401008 F20101208_AABMLS james_m_Page_080.jp2
b85e36b0f59cf77453f755ef73198457
dd082ccbd2f35c2fca23eb560eacbfab3e6dccfb
8642 F20101208_AABNPK james_m_Page_030thm.jpg
7bfad71e47fdcf11e3a1f53aafe5faa6
ae9544f756c2dde166faf530e1aa3e4c77dfc830
36340 F20101208_AABNOV james_m_Page_069.QC.jpg
b028f9459b37af8987a8616e3cab8b93
9ae9d5e066783bb0a5237edf6f6326eb0879426e
116013 F20101208_AABMMI james_m_Page_096.jp2
d4073113d437c29ba3b8c77250c63085
954d234e98a4f7737e890ef75264cb6a78e856ad
48290 F20101208_AABMLT james_m_Page_081.jp2
13f1b92bbd0c64a4d444126bdfb7b98c
c7ff015ccdc4733514a170b7c0120463bfb9c3b1
3510 F20101208_AABNPL james_m_Page_173thm.jpg
6146cd748e7c45322b644cec2ccb5c09
1d6512426bdcc8d6a6d85cf7fc100e5fdfc44b65
35998 F20101208_AABNOW james_m_Page_021.QC.jpg
6805da78b030b1e57a3cbc8b62f36da2
7104e80544c909030dacbca2a77f980d04d2db17
114546 F20101208_AABMMJ james_m_Page_097.jp2
b038c1380cfd5b0a8589d99cb5c3d468
8bf998a5e4bb81aa89c918eb1679eb627f5819e0
78509 F20101208_AABMLU james_m_Page_082.jp2
3c963bd58a7db3a742372dd543f2de4b
aa8484a43ccb0926ab85a3fe0b38e6050a7d8aa5
33636 F20101208_AABNQA james_m_Page_015.QC.jpg
d75f479dab2806784dbd5888c23328e9
662c937cdf2423e469954c102f6e57f7ef85f00a
8609 F20101208_AABNPM james_m_Page_006thm.jpg
3d3b96e25ab896392560d7685b077b2c
19a4fb53d72e2a7a0b060a655f7f8f91d877a16d
5724 F20101208_AABNOX james_m_Page_047thm.jpg
d9650cd2f95f62df14dfb17214671d0c
3561e2a54722ca77d2033cf02af45265a53d8c8b
108143 F20101208_AABMMK james_m_Page_098.jp2
4e827ddee0a611a140fc0a307b91b8e6
28c98d65e9dce5899efccc5ce27d49fb6d405726
914391 F20101208_AABMLV james_m_Page_083.jp2
096fbbc94e08cff39cdbf9beb0959d5f
8139848646df6553502ca0f912396a90b2567ff2
7270 F20101208_AABNQB james_m_Page_028thm.jpg
73c16d3104ec2d301e49f13bc2fdc079
7989357c42d6d609ef09ff5c0c4af0c03d6a2aae
12123 F20101208_AABNPN james_m_Page_140.QC.jpg
1c0f6931f4ea56c018e708fd4a82237b
e178ef9bedadc0528d0b807258c9a8103ef59e24
8843 F20101208_AABNOY james_m_Page_126thm.jpg
853db5aeaddbbd5c07400ac870a84bf1
ba24790cdf3a5f9672e1ad0876d08daf80b1e842
114380 F20101208_AABMML james_m_Page_099.jp2
d686ab1f382a880cae0df8c34ce23430
e9601b453bb5816a0925024f5ed9024373cbbaa3
32379 F20101208_AABMLW james_m_Page_084.jp2
0f194be390484744efd5d04fee1a6acc
db28e7502de2c0112b70ac5768293b46c6ffdd8b
8951 F20101208_AABNQC james_m_Page_127thm.jpg
f6ff70b6dde66152397eea3532fe9329
6b11b9ea14d7d2880b60cdf5bc1e4c715b9ee459
33431 F20101208_AABNPO james_m_Page_036.QC.jpg
c0cd17adc91eaab434486395f3abd2ab
5470b0c86982cc58f9331e8ef4a002cbdb0728cb
37144 F20101208_AABNOZ james_m_Page_131.QC.jpg
b8839cf4c0c749a97024d37cbb3eb2ff
f3752daf6cec21561b3def10485039ba5e1a90e1
510729 F20101208_AABMNA james_m_Page_114.jp2
c0f0fee4a2d5151f35786eb67ac9f9d6
7b5825e022441485ecf4eb093327b55aa6399b12
110410 F20101208_AABMMM james_m_Page_100.jp2
7465ff8a0c6d0ab8be92215474f24dab
5d443d81b7f027c65da3a9639839d4cd0bf7d5c2
458811 F20101208_AABMLX james_m_Page_085.jp2
5033d73112dd85d91e0115f0732e133a
0f6f3fdc348fcef1067387aa73b9def879aa9190
8796 F20101208_AABNQD james_m_Page_075thm.jpg
23f2a1d5940226dd6696e2be37a3e3e5
7da1b7248914f1d95521c7f26c48ee8c0e01269d
37376 F20101208_AABNPP james_m_Page_094.QC.jpg
5b0183e434d0729a34be68494ca799bb
0502d3476df749e6066abb5071af2a828a4f3e2d
453889 F20101208_AABMNB james_m_Page_115.jp2
5029e10ae6d8fc5ea3825dfe9174b7eb
c794a6f9abad129fda1ac0f6edea10a88a5b9036
118312 F20101208_AABMMN james_m_Page_101.jp2
09d7f54340d984e22f212a7eb30db216
5bbc1e382a43a70ac91a438ba5f5ac412a15fe95
273092 F20101208_AABMLY james_m_Page_086.jp2
f670b7de8b07d7c6dc30d1052f76fdd9
82e0a58147162cb33bdb1cab05911c4eeecd7ba9
34397 F20101208_AABNQE james_m_Page_019.QC.jpg
a85c366f431fed88e9cd99092bad6312
0d3fd089605a3e54d60b9e2ef2febe1269648f3b
45695 F20101208_AABMNC james_m_Page_116.jp2
017f500796da65d9da90ad295ac4467b
d9b8ae78372bbedadc5430a787bb86ba7421db7e
403454 F20101208_AABMLZ james_m_Page_087.jp2
9e5e2476468d0c75e36714f6bc6acc72
4a1d503a844d45e6d40640d8f9defa9dcbb10cd5
5231 F20101208_AABNQF james_m_Page_054thm.jpg
79d174aee4d1810f67d754e0184be71a
a925dcda761010f069fb8c40be3901a0edb63504
2548 F20101208_AABNPQ james_m_Page_003.QC.jpg
6445947a6d1b4b0893113245ece8da3f
dea8d93f94a34e2952f0cec86991cf641b50f20e
108647 F20101208_AABMND james_m_Page_117.jp2
a2b22b929214960e5396cdda8f2865d7
df4f67fbc3f98b779a17d53985ae1d4eb50269ba
111341 F20101208_AABMMO james_m_Page_102.jp2
77b0dc0e11d280b1b75a15051f467e20
4ef99b5c1da96a491de26bebfa0778471fc1515c
28312 F20101208_AABNQG james_m_Page_124.QC.jpg
58836020baa3c129b7c70456a65cf340
8623d132610f14c6f7b9eaa4d131cf6d95c73f6c
2399 F20101208_AABNPR james_m_Page_060thm.jpg
2a3af43ed2e7f80fe9173a4427c6ec63
a0e896e9abc14b90119df58152e947b6c9b76f38
113590 F20101208_AABMNE james_m_Page_118.jp2
f1697d806b770e5db9a77e821dab5a62
d7bac46001233c99bbefd73747b321f34caa8c2b
111880 F20101208_AABMMP james_m_Page_103.jp2
e4dc837b38519239c4b9a5737487fcc6
e69e46d1541f1132ba973214dbfb65bf7a2955d7
9262 F20101208_AABNQH james_m_Page_010thm.jpg
e27b4075f15d7bd1ddb8c7286f0bd515
c75b234f5dee40ccac88955c0e759f459b2efe13
30590 F20101208_AABNPS james_m_Page_032.QC.jpg
8c86a93601261a2ce609c2901e0f8e65
d05f1c4267f0b8ac3041df06d20efef45b79b26e
118546 F20101208_AABMNF james_m_Page_119.jp2
9c9009fda8c44d004b942110f4290db6
320a32540c579bd5d1fd8c21d8692cce5ddcc1c0
124139 F20101208_AABMMQ james_m_Page_104.jp2
8d3c54833edd0df6485b666db2f5de9c
fe9c0c3ab5db48cbfba8fbd44bd896b4868bdeaf
37746 F20101208_AABNQI james_m_Page_027.QC.jpg
d9b2e1d40e0a54743af346edaeddce27
b69c4084624ecb573595dbfba023a9792a4dba2c
5681 F20101208_AABNPT james_m_Page_158thm.jpg
59280ffc91ec690e332651ad9aea3e88
bacb6c6cc107c59e76cf3437dcba460159b78487
118635 F20101208_AABMNG james_m_Page_120.jp2
5f442a2486f6e904135b82d023f4013d
b0bc0854395437e11fdc191e2334c1d520655bd5
123323 F20101208_AABMMR james_m_Page_105.jp2
ebe9556ba9343e6d68cc22e8fc4e6327
f374f51e52e0faeb28b615368da94fbe87833609
8709 F20101208_AABNQJ james_m_Page_162thm.jpg
4a7e9a49f9d64e492f8a02cf091411f9
4c6813fb435ccf018829286e2425764162078078
34707 F20101208_AABNPU james_m_Page_066.QC.jpg
bb25970db7228d57af377da514e46301
412b3121d7b9b00ce99c06f25b57c5a25b689e7d
101703 F20101208_AABMNH james_m_Page_121.jp2
3557a5a35a550eff6813758a8aadd197
0db9a1a5a5acf70c8d9e32f18daf09fc7dbb2728
118506 F20101208_AABMMS james_m_Page_106.jp2
24d97d3bfd3ddfa731b1f7af4b224f62
e5bdb36e7878324a12e83dd71bd43b30553af52c
6193 F20101208_AABNQK james_m_Page_111thm.jpg
b30c53f916b8586bee6065091ce035d0
38d8e3800e3e06e72271b3d7992002365cbec47f
8925 F20101208_AABNPV james_m_Page_094thm.jpg
e6ad3d1f01ecc6a206db89906aed5b5a
83b013aa644d1ac5520878eb77158cd8cbafccb5
117030 F20101208_AABMNI james_m_Page_122.jp2
e4c52dbd49768901df2f80eb0f8ec290
6049666579a6ca0935676ed00e67c3ad53b9dc09
116987 F20101208_AABMMT james_m_Page_107.jp2
f29c4ac4a5d4641031fda129cbe96320
ad015426cbe053c9f62d054c6bee29ce3191c5f6
5034 F20101208_AABNQL james_m_Page_114thm.jpg
9b325a0c91268b4376d51f7c9e57cd38
c26bdc3a351927111fa689a39c16e74d2418c1b3
7359 F20101208_AABNPW james_m_Page_037thm.jpg
ce29a63c00b6790a6296657287d35e7e
31c30777513097eff37e02c3ed750c7e24af8ea0
103814 F20101208_AABMNJ james_m_Page_123.jp2
e65cfc0cffac766424296366f4346b34
1cbc3b214a3b269fd1d9e764cede4deeafe2b7eb
39260 F20101208_AABMMU james_m_Page_108.jp2
fab659c0f308d8d7958b82501880b0e6
f6b941f34178b1c7bddc5a58c6b8bdf1758a7e83
5369 F20101208_AABNRA james_m_Page_052thm.jpg
20b87bf83cbda7340dba48ae2f1072e1
4572624c249b49bbdadb148661e6b6acccd3551e
36803 F20101208_AABNQM james_m_Page_119.QC.jpg
6e1eb06f0ce797513b3fecc6d34896ee
16ad152a23765df788da346d694223df341d73bc
22169 F20101208_AABNPX james_m_Page_090.QC.jpg
2a6d6f93a7fb34e25adff53066c33c37
789fc1e9c289b9b9cc8ded40a12ffe49a6337fc4
92493 F20101208_AABMNK james_m_Page_124.jp2
10e95d77f1fc71639bf3093ef0f43ada
5518d6e91eec6082c233bedb18851781395d39ad
323556 F20101208_AABMMV james_m_Page_109.jp2
683d5ec3da118bea535447f32cbace59
10cdf7c2fa4a461f723a9705b6e3404f22b40383
4945 F20101208_AABNRB james_m_Page_156thm.jpg
3a0a3025139c74b561412c7f1a7cf406
ec86f24de1376dd7857d3481ec3ef4f27c82df22
8774 F20101208_AABNQN james_m_Page_098thm.jpg
aaebdbc9babb82feb96d797d52e55cb4
9b07189d1979f8af0b21cd7b68e6a0b6140520be
1113 F20101208_AABNPY james_m_Page_079thm.jpg
30bceb0c3a4a8968ba0fb41788ad64f3
06ed61de47b5c4612a9ba2d9681d48c60a48210b
119871 F20101208_AABMNL james_m_Page_125.jp2
32be524535b80749aaf304f09323f8b8
7786bf41c4ef2a13b766c80ed6de1a25780a3c29
265594 F20101208_AABMMW james_m_Page_110.jp2
f62873ca25c1c619e993a6d73d4258b4
f600c5383f93b88b2e703db79e1fe46434200387
9079 F20101208_AABNRC james_m_Page_071thm.jpg
fad533273b9231c0593c836cc49ee546
68be3918dce5328eea07ca497936dc1392474389
8684 F20101208_AABNQO james_m_Page_039thm.jpg
3339f0eedefb2048fcde3339f5be0475
bdb80cb8adc49bd7da326a5c51f3cfe083711e65
33346 F20101208_AABNPZ james_m_Page_163.QC.jpg
b821a7f7e967e5465e38d2ca7389c43b
bda6d0bc7b6b70d400c507243a705b7d5391d796
118468 F20101208_AABMNM james_m_Page_126.jp2
52f906d816568b16d6da7c0d42f16e68
f62954a719c939eaf1f46b1ab5b4c2c919f2ece4
883294 F20101208_AABMMX james_m_Page_111.jp2
bd614df43063098598c3435bd29b8c5f
41d63651a610a80c395f0ff6cd00ee7044e9c486
34079 F20101208_AABMOA james_m_Page_140.jp2
cc77868f6e3ea47b239904e0238fdb0c
584a565c8ae2b25214c39a188cc5b07e4746119b
35346 F20101208_AABNRD james_m_Page_025.QC.jpg
22f9fd6017e355021cd531123ccad48c
cb19308187331003758aba8873db1af46bd87f25
12692 F20101208_AABNQP james_m_Page_108.QC.jpg
47fdaf53f2d7cbdf3d00bf6058913bc8
b7b7d67f75d6ea40e2079764f074faa745a0f986
116473 F20101208_AABMNN james_m_Page_127.jp2
eaf3d6a86c62ff0cb33e602b3c32289c
ef0ece8d36c7f743e34f345091814ac479498f83
449454 F20101208_AABMMY james_m_Page_112.jp2
22714e013ad5ffb2e31549045246dad9
16d0f5bd2a536f7653d77506cd21c14e9ebee689
496303 F20101208_AABMOB james_m_Page_141.jp2
568fda4f4cca57331676ed4afc29295d
d64fb3a9c3a6f8dc06f8ffd674a56bd88eecd881
7356 F20101208_AABNRE james_m_Page_124thm.jpg
71a77e09f137c98665f650b7e6ef4032
3d2b0bfde2257a15be4c81dedad71f3fbec4fb15
36891 F20101208_AABNQQ james_m_Page_130.QC.jpg
b406f988fc5e1f69564da77a084b3cfa
0989c65102200146fb8b74267404986b2a63eecd
124135 F20101208_AABMNO james_m_Page_128.jp2
b48d38f2b21765186866f247a51d017d
aa3350cdf4df2bcf9595141139da10fbdafb84b0
606599 F20101208_AABMMZ james_m_Page_113.jp2
995f4d30b1f0303f4330c955ff293ded
19b3afbf26b2104f2c64f8730c109e06c3d42859
37925 F20101208_AABMOC james_m_Page_142.jp2
21b02ea9f2e0d505ad30b50de33e5ae3
f59560e5d0f6dc52ccf0b04a0679354221162183
35586 F20101208_AABNRF james_m_Page_122.QC.jpg
a1954de6b78b0286639a9a2f79dd4c96
8139a14d9d2a24bbabb35bf3d600b41e4bc5f906
25390 F20101208_AABMOD james_m_Page_143.jp2
e60b5347b23ed5adfbf2925c57d2ec57
6c888412e9b22270661a8f0b95cb2cb28bf3ddaf
36490 F20101208_AABNRG james_m_Page_062.QC.jpg
7736a13bed07c00c685d669dddba0709
78498f61ee5d2c5f26d6c4d6778d78ecd2f61ff2
21409 F20101208_AABNQR james_m_Page_055.QC.jpg
7510d465f9b4af983568cca94fbb2f67
39de2336c4058433068c6fa1e7d56376fd599d90
115719 F20101208_AABMNP james_m_Page_129.jp2
4a8a338633ef45c8e2df725835c1e110
759216ce1c2911637a2a4d6f116e35d2cb68848b
57018 F20101208_AABMOE james_m_Page_144.jp2
89a06c3af78b3eee5209a22e9266f7fb
f96753e45ad8bd125b9427c4db478035435276d1
37378 F20101208_AABNRH james_m_Page_071.QC.jpg
2ca8d8720a666e703d6934b395ad42b7
c1c00458ba28c783d8d9bac58918946753cd1efe
16207 F20101208_AABNQS james_m_Page_114.QC.jpg
9532bdb2a626d44765e1023ce52a5d91
b098a0c48584adb526a628837c09740d4060c7d5
118252 F20101208_AABMNQ james_m_Page_130.jp2
fdc854d310ac29da9d9cbe167d577719
fe65f098a340141e3563c9765e6e0cbb3eeada4a
60190 F20101208_AABMOF james_m_Page_145.jp2
83fa0c48a45d47f67150cbe7e4f19173
5f65f1cb4767fc16038adf719522ebb5d29940a6
8626 F20101208_AABNRI james_m_Page_101thm.jpg
004ff1ae5340b79b0b4c244b34d1208c
078cc324fa63edf50fa67b2a590810e2e9848c5a
37592 F20101208_AABNQT james_m_Page_077.QC.jpg
0ac514fcf708d13a97a5c4d0674fa2e4
f81603e10070f67cb0a895c97a899b92c64f1ceb
118035 F20101208_AABMNR james_m_Page_131.jp2
0e438e74310a318c6b6dfff32edfca26
83459a65656bea64b587a8bc724e06f34a6643b6
109217 F20101208_AABMOG james_m_Page_146.jp2
51074d6895d5fc38b8849453c66ab9b9
bd9c657f2f1526fb2d126593aee4af3de9f0a859
3606 F20101208_AABNRJ james_m_Page_110thm.jpg
254f1c53c2c2b4af354c603324bb0c71
bb60bb8464e96c9dd9de6310525531d990e675a5
11908 F20101208_AABNQU james_m_Page_092.QC.jpg
63d5cdc67c3d6ccf98282947755485a2
07137c4b373fc0515648533959412e595a7a3c3b
149468 F20101208_AABMNS james_m_Page_132.jp2
a2f108c9c75d80fae20933873145c939
4332f5920d2f0c1bbd71e12dd7a2b274200b9187
133036 F20101208_AABMOH james_m_Page_147.jp2
0c6bc425307f187c89ef261673bf94fc
0ca8509188af6924a6bbcda6a1bfaaf5de55e28b
F20101208_AABNRK james_m_Page_001thm.jpg
0ecebc4c3a1dd78b8e631b576efacd83
7c87a1f1077c3b06bdb06afad476dc69b021e786
671 F20101208_AABNQV james_m_Page_002thm.jpg
7d4af13d775b186d80173a7188262f73
d15bfe1fd2d64b7837570a6bf0648718c181cade
18943 F20101208_AABMNT james_m_Page_133.jp2
793fdcafe1ffc78cc726809416b7f3af
561c419c853cd007ec49a05ffa7cc642855530de
157855 F20101208_AABMOI james_m_Page_148.jp2
2ca17f13c84e6f4bd916ad924d518265
85d69a0be64e453b4330b7555f5241dd93c8eebf
9439 F20101208_AABNRL james_m_Page_105thm.jpg
dfdd517fab35aeb2cec8f423cb7087a2
20deae9a47c747f21c02efc06e96d9b657285f1e
10005 F20101208_AABNQW james_m_Page_089.QC.jpg
3b924a82944f0aeaea907266e8589da4
a29eb68c9ab7633e37e900559f53c974f9e1890b
409885 F20101208_AABMNU james_m_Page_134.jp2
cfacb0dfd5d714526a5fda5f3b7c1369
746ca8c9155af11ed5eee2833f2c2dc0349f8b04
112976 F20101208_AABMOJ james_m_Page_149.jp2
6c07721612500641d787961970db642b
5783e6b3d29c90d97f4938f1a72fdf54c912a999
6360 F20101208_AABNSA james_m_Page_056thm.jpg
082ad60092662a2017c29e3068095bd8
c8413a76bef66b4bebb15cd206c28071c705dd1c
36278 F20101208_AABNRM james_m_Page_127.QC.jpg
88d98b82beddcdf70da703deab7f7f2d
b6995180ce9f34d5e33e8bca9fd94ef8555f0271
8593 F20101208_AABNQX james_m_Page_078thm.jpg
2239d2c352defe82d6c61862dc1265ea
669760ed63f6101eec282c4962acee5cc62fa95d
515894 F20101208_AABMNV james_m_Page_135.jp2
640390746927067686c4c73ca322a087
4dd71f812c35265ff68827cf995d038f1b16ca86
116119 F20101208_AABMOK james_m_Page_150.jp2
62b2071157014d976b416cbb2e18a7c5
b5ec2760987926d68607e8dd7becf596160cb0d2
4283 F20101208_AABNSB james_m_Page_092thm.jpg
61574ca89acc256d8c9bd79c8e9013f4
c84746a6bbb701f682150a343fd3c66adca96d7e
8976 F20101208_AABNRN james_m_Page_120thm.jpg
46604c3ed1f28fbf5d433a78cc3a537c
6a0a79d7588f79ce1d20a0cfd47640a0252f7450
10317 F20101208_AABNQY james_m_Page_110.QC.jpg
0ede84ad568e3b40bc5e9201373f3fc7
c2407abebf7b76a2d64b806a3ee59cc351280ed3
198031 F20101208_AABMNW james_m_Page_136.jp2
cf8f886d3f4acda9719ab749b900eba0
64f8e342a6bbcdf45e20373dcd75d0ed167b6039
148885 F20101208_AABMOL james_m_Page_151.jp2
4053a6997564445d716d3c8c3e7bc686
3a875c6952973c2bba17f084af84365b1cdc26d3
2537 F20101208_AABNSC james_m_Page_011thm.jpg
342180522ab55161b3f21cb8554c0f78
42d8b0243b293554b0d2e7f5a636c577845518a8
2958 F20101208_AABNRO james_m_Page_084thm.jpg
ac47cc81c55a18d9ab73f9368224dd61
949f56295df2ff3f826b0a5ede0e6115bc236f39
2842 F20101208_AABNQZ james_m_Page_136thm.jpg
fa6b60d221c15e88a7764ce1f05d8254
3962bdf997f4e909214235c40e9e42fc02df8e73
331716 F20101208_AABMNX james_m_Page_137.jp2
712f6dfee8925c82b68e529c85a4068f
322b30e4ef9ae52c9eebf62ec9b86a71b2330762
129678 F20101208_AABMPA james_m_Page_166.jp2
0ed96a2f27c2604952d5a0d97811121b
c3437f1fefd77916801e14f36588ba19ec2d3941
112195 F20101208_AABMOM james_m_Page_152.jp2
6bea792bc0e14e792e84e7d8e28c4514
7626068b24345dae03b1f3bd9396059c8982e2fd
8900 F20101208_AABNSD james_m_Page_069thm.jpg
58edf28c7e30c47ee295f3523bf0843b
1a24081da8aed3627893c701d705180dea4a5a8c
36550 F20101208_AABNRP james_m_Page_031.QC.jpg
b691b95d41b9b66e8038e2120b35a218
0630394402f5d44c17fc8e7f230e7fc1cfbae9da
23510 F20101208_AABMNY james_m_Page_138.jp2
a47f3f543955e9b8cfe68ab6a38d9eb5
f2d2e25f12b25bf492a63e1b0a0bd2eee487bab5
132051 F20101208_AABMPB james_m_Page_167.jp2
eb54953b4ed22d744eb6cb22bee28de5
19e90d4e43531afcf21537f56a6d12e574778e77
116795 F20101208_AABMON james_m_Page_153.jp2
63f91598dabd9b2d684df4a54f886d54
9026d2b677422f151f368a13e9d6190feff607fb
7887 F20101208_AABNSE james_m_Page_059thm.jpg
de491d3215618ab9734fa07cfb3d01ec
1d26398788f9f769c99b54eebb8f64f5e5d8b779
8423 F20101208_AABNRQ james_m_Page_033thm.jpg
015dffb4604a72ab80369cb5506b9ec4
c5d5d83fbd41eb832c544f27810ffd0182bb7598
24392 F20101208_AABMNZ james_m_Page_139.jp2
bdc8ef4be1d08868e13ab12d0f9736bb
0727d5450ee00892d838bb022225dbd001b2192d
142705 F20101208_AABMPC james_m_Page_168.jp2
639321a4c4a000aa3cf1a37eacd0df27
4aea410dcbf2cbb65939dac2cbc52507de7ee1cd
117573 F20101208_AABMOO james_m_Page_154.jp2
fa6b092c4077351a0c38c6f5b71dbfcf
0974cd3c731ca9855bde11c993b309047c66f322
256809 F20101208_AABNSF UFE0017540_00001.xml
0f1f8e33a2920422f61a825d8af7611a
add72c56d7a5b606a8bf6d1331df125ebd32a5de
17647 F20101208_AABNRR james_m_Page_144.QC.jpg
e39be2c785e0bae7e62988b113151764
ca424fb1c872ca064c30af643de40a2d8bbbf3dc
136472 F20101208_AABMPD james_m_Page_169.jp2
38d96e1eb1c426c7c6162dc5fede4fc4
2638be4c1f94417ed6a9cfa3e294c9957d33b00e
119127 F20101208_AABMOP james_m_Page_155.jp2
dd101d41a5ebd9577febddb8574abbb4
ee9867fc711efd63f206e3e8bff940e1673f7681
8507 F20101208_AABNSG james_m_Page_001.QC.jpg
06852509136bb09a1ee7949df08925ba
fc2ed13bf7ca1f045706ab7b5f22d261232251b3
144111 F20101208_AABMPE james_m_Page_170.jp2
ada9075d15eff1d0e61d5d489f87d9f5
81417a5c1356fdda01c2cf69813ad94a24268f8a
30634 F20101208_AABNSH james_m_Page_004.QC.jpg
3ef577fd9ba4db2d3ccad749832c9b63
819826fc6a3972f2e87f0ba49ebf9b98fad9c8ac
8847 F20101208_AABNRS james_m_Page_017thm.jpg
c68ce640c8ab5f48f8d5064a8d388e3f
e1a07ef32e0aec399c2208f83942ed7e97ebb8c7
125979 F20101208_AABMPF james_m_Page_171.jp2
c2485853b1afda1151534ac213546285
036a77ee128af32434f0c2c1e0f9ff3504bd34a0
92842 F20101208_AABMOQ james_m_Page_156.jp2
cd4df256430d4f3164848dbaedfadc83
8ec1d4c3cab749ec08f9c360b5ed89c378d53e1a
6796 F20101208_AABNSI james_m_Page_005thm.jpg
ece23904d659d74def50eda0cad5b066
56c933e135c3c1b2460b060d9bdee88e7407ca25
31528 F20101208_AABNRT james_m_Page_022.QC.jpg
c39dadc7bc9da0e30ef3e990c6a058a1
f9a259ea478758e4620ef20832ebb6586c3f7fc4
74020 F20101208_AABMPG james_m_Page_172.jp2
1367c0acffdae9fedce66bccbd37667e
b51f73752bf9207c2d763086254923f00b7abb03
70808 F20101208_AABMOR james_m_Page_157.jp2
2bd76cc58a24abaf90f11781391d4887
379fb3e4633e94cae93d0a06a024415b2e25e115
4376 F20101208_AABNSJ james_m_Page_007thm.jpg
41abc47149e903dfcbfffb6975a28dd2
ac86c3542650222b88c5faa9989c87d45dfc0281
34347 F20101208_AABNRU james_m_Page_040.QC.jpg
241ebc9ffcc119454e17f0c83f989569
c945aac853553653e2fafd0360380827297422bb
43641 F20101208_AABMPH james_m_Page_173.jp2
3a78cc07c7b8e0c4fb99857a0540429d
aff6fdfe969c38ea9f4bb562726ba91a80e2940e
63802 F20101208_AABMOS james_m_Page_158.jp2
2fa49536670e7323d6200c1310f799b6
4368919eee3917db3d18d40bde146e4ccfab5df8
18222 F20101208_AABNSK james_m_Page_007.QC.jpg
cd1b60d9b0ce7bd2e32c46cd18f2527d
76fddc013a25d807725fbbc124f1fbd744c11466
34613 F20101208_AABNRV james_m_Page_061.QC.jpg
9eb514b6195da5bcee790ba32736d6e3
fa45fa1790cb567c76a7bd275eb5a32f863f954e
F20101208_AABMPI james_m_Page_001.tif
ebd435ffc7d1eedb211824a549138e79
57047178324b9b61f51c1b7f726ebc859f3ed59b
39666 F20101208_AABMOT james_m_Page_159.jp2
ebefb96e33ba0f82354b5958229981b5
f8b5cd4a14be38bd7a859edcb6266f34f519fc8c
6784 F20101208_AABNSL james_m_Page_008thm.jpg
34a4633a60e1ed1cb6d7bf66b8d31487
4461bafcf76a3bf0804e71a60c0a311c6652cc81
37346 F20101208_AABNRW james_m_Page_164.QC.jpg
43f22db84d63455d5f710771d2552641
22757d392a1cb1e26c7f06feb5d739f4f423de54
F20101208_AABMPJ james_m_Page_002.tif
e746c0a06174d8d653268d26be086037
23391b4f681871105d6a7b28ee80431c6cdbea45
83429 F20101208_AABMOU james_m_Page_160.jp2
340827dc546c243164b0d749a19e0a6e
a891375035247ba5bb376e36fe6689f588440bb5
8230 F20101208_AABNTA james_m_Page_043thm.jpg
f746e3dbc612876de1bd911e44e9fbde
dfd55af5aba88cfa9dab0eecdda311d4f99e4ba0
4599 F20101208_AABNSM james_m_Page_013thm.jpg
c5f9a7302d719a5264f92baab75c6728
811562f6337eb68454d905ea12544e82b69f0272
8494 F20101208_AABNRX james_m_Page_103thm.jpg
65fc4665e7afe9d5a09dcfea8d0e1f04
c58e00732d0caeef7cdd519ab53a029797e63f7c
F20101208_AABMPK james_m_Page_003.tif
61257b8c1fb51d7e052b0656c2a7e8de
55484181781e11d5dd680d21cc4f18462c1cd726
56743 F20101208_AABMOV james_m_Page_161.jp2
217b3795f87fe2cd795800e96bf1b16f
79146bfed1fba2395bd2ee1872afb90ecf076af4
8457 F20101208_AABNTB james_m_Page_046thm.jpg
b660f26b4d096910bb8292579d18b18f
0026d7427887cea275924d8ee2f4f48b33014ea0
8744 F20101208_AABNSN james_m_Page_015thm.jpg
8567994d2500666c9a00a1319f9fbe37
86140e52c0fcb5df33abbecfe3076d1c6d731bc2
31962 F20101208_AABNRY james_m_Page_121.QC.jpg
673186369cc213487a46302a1d223ed2
21148f7de4c035e47b370c6709b8b0a76583b589
F20101208_AABMPL james_m_Page_004.tif
6cbe3e21236ce098274d22ed87373f18
3fdd3295a418f27508301ee00a21472e455a9b44
124273 F20101208_AABMOW james_m_Page_162.jp2
e6356a0786970d31c4ca33b7776c2beb
8367c6127a621d89632cf4dc9b4c4dd6e200891e
33434 F20101208_AABNTC james_m_Page_046.QC.jpg
8c7f7a28ae92494c63d61f0cbd2c53cd
2ca45c6ff340523296baee9bb1e0a470f4294b37
8572 F20101208_AABNSO james_m_Page_016thm.jpg
eb17f41da1338dbc459f652ad2f241cb
6cd067287d9de566546d60865cc6869ea1887b5c
35203 F20101208_AABNRZ james_m_Page_097.QC.jpg
7160116309a80f68a42288a846b7258b
9759869a2befe6945ac1a88a645f1d3ac4161ba5
F20101208_AABMQA james_m_Page_019.tif
ce53e7aa59186002ae771ea76b2ce217
fe6bad9fa21bb3c08a4b900bfce79bd7bb3128c8
F20101208_AABMPM james_m_Page_005.tif
8f52f0bddc5eeb644c574a7abba9db5a
3d277b8bc58acfc05b9ab4bcea6f69b225d6ef58
120616 F20101208_AABMOX james_m_Page_163.jp2
912571f6020f2c129a850ebf78749223
9000a7239a4d0939e845c43027ff6800d3110e92
22940 F20101208_AABNTD james_m_Page_047.QC.jpg
d8fc95c5a0b47e0f9d7376cac6bb6a52
de402f2fae02ad3b2bf1a61d7d88eb7f5e02c40c
36211 F20101208_AABNSP james_m_Page_017.QC.jpg
cdf5f83cdb564fac16568c45e3caccf8
37a557d7ab325b4b77daba8c9b6ef3fca2ecc706
F20101208_AABMQB james_m_Page_020.tif
9b874e59f45c4675c2a04ed441ac21bb
89357d5607d07e3c978fa114d11464f6a23c9c0f
F20101208_AABMPN james_m_Page_006.tif
da0522caeefe8fec695377247b97aaa1
14e49e74989de1e74e1e34052ade7333b234cd64
133019 F20101208_AABMOY james_m_Page_164.jp2
f8b5745995bca142a15c85da08e93361
ec28b14e098507f32aa768c6b3f8c30b2fccdc8a
17499 F20101208_AABNTE james_m_Page_048.QC.jpg
7a3eb979cff068c889779457e5a82484
5c3457d890c9decaee68efb4ef2dc07cccbcc18e
8869 F20101208_AABNSQ james_m_Page_018thm.jpg
e5028ef2fa57b29ad8eaa29a596ba5c6
fd98cac15ecbcb8d7a38ca08a0737a8a2d3c4bdd
F20101208_AABMQC james_m_Page_021.tif
eefc833872b10a86312b132bc6420eea
e567c34e15c95cf099560fd7b61c36d4588bd87c
F20101208_AABMPO james_m_Page_007.tif
9cdc2cbf27a077b53795c46a2feeaadd
0418d93506b6e8ad4dbc43401deb39175074e812
126109 F20101208_AABMOZ james_m_Page_165.jp2
82a6636a4bda276866e1d236f9243cef
113bd5255df4b328d94e2648db590fa148251a55
5706 F20101208_AABNTF james_m_Page_050thm.jpg
a948a6657c4b49986569fb799cb03715
3c14dc4a04b3bfd061a464e9e6ad5268988f64f8
8725 F20101208_AABNSR james_m_Page_020thm.jpg
8a6490726784adfc9e0b5329b77e512c
9133703d44a3e204c87192442eac8dd76710b3b1
F20101208_AABMQD james_m_Page_022.tif
ca32dc7ab803527cf83e0a6c3f6ee16b
6ed99ec6a3b09d104b3f9ceb6f03282f3d2fcec2
F20101208_AABMPP james_m_Page_008.tif
08d68bb450f34e5610a78d8aa1919d19
17a08731a0c7f376836e8567b2db907f0378acb0
15992 F20101208_AABNTG james_m_Page_052.QC.jpg
10f21b0272c1df40383925afc068ef31
8f627bf4f76da43863ffd2d220bdf42095143671
34719 F20101208_AABNSS james_m_Page_020.QC.jpg
3f64cbc350b50ed050905777719e531e
f5719542292e440afff168a285932b37389c0e81
F20101208_AABMQE james_m_Page_023.tif
9513d1b6c6299ed58db772f5be23e0ff
c4c7fe46527c7d1d9c70f723c45fdc7ff8bc420b
F20101208_AABMPQ james_m_Page_009.tif
715c2517961c8d268a1567d55f31332e
d35b567c9be0076e4f019a5d2ad1037908debf4e
8721 F20101208_AABNTH james_m_Page_053.QC.jpg
a0b06a42d7d4b15349bc16491820ae29
da134be4aa553904f5942eda635ccf03a2d6c26e
F20101208_AABMQF james_m_Page_024.tif
6a2a3d7ce1f1ccce28a78195ebd800d8
b90b2198b983a0dd6868d3c03a0df55da7e7fab6
17151 F20101208_AABNTI james_m_Page_054.QC.jpg
9caaa3c46f2fe328a5aa3561ba33bbd5
acd3420ba64285b22a204606d0f1d0946faa9a5a
8253 F20101208_AABNST james_m_Page_022thm.jpg
b193980609f5e658e8f53bfdf2d8a252
9676923003bc815108a65c816ca48aee8dc74e89
F20101208_AABMPR james_m_Page_010.tif
3e2cf9bc1bd1e0bcd05895ce05ff49cb
778e5fa652cf292c00c4bff2b8fd264264a53fcd
F20101208_AABMQG james_m_Page_025.tif
f0a3fbbca7075b3cecbc88cb991ce178
25284eca7c353aaeb2e3c0768bbe38e39cbef84a
5312 F20101208_AABNTJ james_m_Page_055thm.jpg
bb4ee309be4ff5e9b56096550c53aa2e
9ff910d9fe3f9694bae9455ee10a64db99e8128c
8397 F20101208_AABNSU james_m_Page_024thm.jpg
f0e97af95e18bf85d6226594b61e779c
601b77d176dfcb80942202ae5a742c72a070bbe9
F20101208_AABMPS james_m_Page_011.tif
3440179d09eab7da52bbdc877d9d78c3
71ad41e99462fb7e72a7e81d321abdf9abcd8562
F20101208_AABMQH james_m_Page_026.tif
822997eaf30882aac5b5b05f9d15bb41
e2b77b3752b1c1ca559a2ed8a5287c1ff1079047
5744 F20101208_AABNTK james_m_Page_057thm.jpg
08bd4c9c0e9b3b277f7bf29f751455a0
cb34ee4afba52a1c534d40eec3bf86e97629ce4a
8871 F20101208_AABNSV james_m_Page_026thm.jpg
987dde524dcd0556184c760909bc81e8
ac0f0d374381c2edc8ed9bc62709d380f70b2eeb
F20101208_AABMPT james_m_Page_012.tif
4390cb7b64cd27e8ef1570779d738867
7b7e655eb865aac3b28ce8a08ba7100b517b49a6
F20101208_AABMQI james_m_Page_027.tif
767c6c2864a681c4b5913611f37baf19
f61ba405bb0cdedc997981faac20cc26fabd1f35
18962 F20101208_AABNTL james_m_Page_058.QC.jpg
882082b3d7447c8b418ed77de3317a1f
4c713499af7f4a9b51d83f82b0211ccfb5df303b
31257 F20101208_AABNSW james_m_Page_035.QC.jpg
19e255fb0f0df21db32afb8fa533780f
ffdf66fcdea8fb0bdf262cf00f75ed72aae5a2d6
F20101208_AABMPU james_m_Page_013.tif
5646a2302e6227b4cb25b3cc64575699
89eefffc80d7c977826cce76f3673bebb2106e26
F20101208_AABMQJ james_m_Page_028.tif
b7846d006795d016088bf4d5ae1dbef9
2596c528be5a32f1c3d3110ce681b0beb079c78a
9093 F20101208_AABNUA james_m_Page_083thm.jpg
842f57fab55f8dfdb95b1f42a88399af
8202cdc3ca5306eb7a101ee9df2be89489b5b1d3
33267 F20101208_AABNTM james_m_Page_059.QC.jpg
7124f4623174a5822dcb46b049cfc846
f9897afaa06b02880407dda4716496f0378cfa60
8777 F20101208_AABNSX james_m_Page_036thm.jpg
34dd87f78ceff4623a45e73f3636cb58
526663e8e0a38533e8b20ea1379af30b2a592a1e
F20101208_AABMPV james_m_Page_014.tif
6c2ab964f97a5a3d242a5719ee02306b
e319ad3baa19b314a81f0e8c2c3150dd57529e7f
F20101208_AABMQK james_m_Page_029.tif
56663dd873a8504fb6038c32b9ab853c
a2e70a6d015d72918762220760d3ae5abcbeb97c
29613 F20101208_AABNUB james_m_Page_083.QC.jpg
63203a59e27908f519da909fa791bd85
5f4827f110f88b8257d3001b4db4a03240dc9064
8022 F20101208_AABNTN james_m_Page_063thm.jpg
73bfb72c52b58b3382266678cfe00012
556fd736e731eef0ca988672ddcc138549b60634
34791 F20101208_AABNSY james_m_Page_038.QC.jpg
3582b48c87774cf04e8cafa8fcbff2c6
19b6fad7db4d339d3422262223506dc3d03f9b8b
F20101208_AABMPW james_m_Page_015.tif
daa9ea9ba48678157ef26b3d7606e85d
f0d1ee839b2c07e21b6fc4b327046a8d2aee75ea
F20101208_AABMQL james_m_Page_030.tif
5f14b1d699e3d9558f891b298c7ce7f2
9cca3ae8e251cb043a668c1364339680df9b6cfa
9556 F20101208_AABNUC james_m_Page_084.QC.jpg
1548265224264c4e76d52a04d9d8fcb8
38b0fbdbf67bb57e3e8b79a8d7df42c839774319
38002 F20101208_AABNTO james_m_Page_067.QC.jpg
02a083ec8e9f96117443edcd518e64f1
74df9faeaf5987c358e1ffe71c3c3fc3edd3f831
8301 F20101208_AABNSZ james_m_Page_042thm.jpg
230c9f868f4e27f50396cce53bb784e4
910fceda42b84f7e5986d9fd7125c3a65b0624cd
F20101208_AABMPX james_m_Page_016.tif
77bb718900d3073df2c3f649320ba50f
caa52202dd117a101978234da5a2e87e1b508fc2
F20101208_AABMRA james_m_Page_045.tif
c50d8e6816bf86aecd7f303849167f12
f3805a040419c5a930cadab8f5127598f50085f3
F20101208_AABMQM james_m_Page_031.tif
2c94c79f5950e69ea46715cab8c8478e
79ef1e0b817588293014a00066b641267def49da
9830 F20101208_AABNUD james_m_Page_086.QC.jpg
a741a056de7577301fdd5b607f5a20b5
a5b3007d46e55022388b6deec6ec841ff6be2457
8147 F20101208_AABNTP james_m_Page_068thm.jpg
cad736db4f2d055039cae7a50535c387
673a1ce6de81d2d7f08c0211d450841b2e1702fa
F20101208_AABMPY james_m_Page_017.tif
be0ccc4e7d50ff057951cd65ae27c7c6
16282aa61f9991c27293506ef3554b0517a51de4
F20101208_AABMRB james_m_Page_046.tif
cdde8037f603b2aad308fd268f2f6bbd
daca1eebef94562f4ea1f04c8f4cb3cd9b0c6b0b
F20101208_AABMQN james_m_Page_032.tif
6a48bfbe7f70cd91d631dffbf4f52e82
db0709d6f7efadb73d6d11babc8c42992ce0099e
3983 F20101208_AABNUE james_m_Page_087thm.jpg
e17ab8006f139c8b7782b32a95ead392
a56c99418c952709ab5bffd92eb4831b6cf56e88
8946 F20101208_AABNTQ james_m_Page_070thm.jpg
fb81c156be2326156d3aaa4b517e15ba
4caace9501851dea6f288cb273cdcc4ba6474ae9
F20101208_AABMPZ james_m_Page_018.tif
8d712570f586cdf8847d6f217751187d
61592a385ae0b72bda2aaa29c4627470c6e23994
F20101208_AABMRC james_m_Page_047.tif
4675017c923912f84564921689e5a9c9
dceb4d1ab631f90549e2bbe12ab748d4359c0c63
F20101208_AABMQO james_m_Page_033.tif
ce2eeddc0813580b9da63dba98e7bf1f
72e78e15ed9abd7b5cb1c29bdcc09b8d758e62dc
4171 F20101208_AABNUF james_m_Page_088thm.jpg
7dc9309243cb2aa2219802e32dfe36ca
e11e63c91ff392ae6de693c79ff7baec688b12ee
8294 F20101208_AABNTR james_m_Page_072thm.jpg
6bc2a1fa12836a5227e0cb442c078da1
99a53e4e807a003834012db15dd694846491bf23
F20101208_AABMRD james_m_Page_048.tif
838a06e9d97a0daef3b3df26f92685e9
9b5609fc319cd33c3f3654e4dc4a2ba72593ca91
F20101208_AABMQP james_m_Page_034.tif
4ea8183517547d84e43d295a0e586284
dad3c6ae462289380b2a12084e33799c8ca0338b
13818 F20101208_AABNUG james_m_Page_088.QC.jpg
8711d00036ce6864240d728a7e5fc18b
b30a35b9609cb6442742ce440bf7e5034bf7d7a9
34836 F20101208_AABNTS james_m_Page_073.QC.jpg
bf75fd7bf0b85def8270811840cba32b
cb4bae6f037a3cf421cbfc3702760374f18e281d
F20101208_AABMRE james_m_Page_049.tif
828ecff8cf10fd7d451298e33851344e
800a5b2a2128f685905679aaf3cdffe21eb9b686
F20101208_AABMQQ james_m_Page_035.tif
f4d7e038aada09ca42146faf50f2e39f
0bea0e246f5e91bdda75d6d26fcb870d4f4d3329
7013 F20101208_AABNUH james_m_Page_090thm.jpg
e2b1009ed7e741354b59dd369f64c1fa
6af979487c8fb0123ee0006e88383c0775d14564
8574 F20101208_AABNTT james_m_Page_074thm.jpg
bf8265ad27f34215ffa446d8fddab753
f171a34bd2430017c5f03b9ed76df43001f52c98
F20101208_AABMRF james_m_Page_050.tif
e74a05923de3f8d14bfd2dbae75b955c
837f58793b606227a87ca8542838bd6fb0cbc69d
F20101208_AABMQR james_m_Page_036.tif
62b58569d45301254b709943f9dc12a3
338f274f84d7ccf937689a8bbb220e442504daff
1516 F20101208_AABNUI james_m_Page_093thm.jpg
83c10cdec0a842440cb699aef41c9fda
e88a7ff421588dfef397b4185c673f1073911fd7
F20101208_AABMRG james_m_Page_051.tif
f946231aca9a5c962f971ccf957dc7d9
bf330334effb27a4e7f35d458afa183102891ac1
34393 F20101208_AABNUJ james_m_Page_095.QC.jpg
0609adec27a6359ac0e75a8ba6da188c
5338641b3a42129c9a39a06444078c646db576b3
9028 F20101208_AABNTU james_m_Page_077thm.jpg
e958eb73f4ec403660fb43d9c3bfdb41
43c67b8401d2c5fe46e41c83a9f9393e2d59df97
F20101208_AABMRH james_m_Page_052.tif
0ddbf30e26bfc82ebe3c9dbe00b9eb72
38d049e5be907b7c199b798fabaa4d5191b827b9
F20101208_AABMQS james_m_Page_037.tif
bb49641dae8010d740405d7941ab8195
58a69e500ace1f549a7a0c36d0ec373d62383f2c
8342 F20101208_AABNUK james_m_Page_096thm.jpg
0f8d71ee8be308a2c4bf137716d10dc7
4a1eb8570653857c6553f50c04f39dd18f8acee3
34950 F20101208_AABNTV james_m_Page_078.QC.jpg
fe71eea2f5ebbeab73a4e7dc20a02a25
3c96f29f6fad4088aece706550347892569b882c
F20101208_AABMRI james_m_Page_053.tif
a03673d30c41b6a983d8e87bf0e4d6e9
7592ce51fffc66b2fdc50ca7c8623c7955680932
F20101208_AABMQT james_m_Page_038.tif
f8c14ebb8fe93fc70746920ddd1f9b5c
39655e24099764a768adb81ac37ba15571b8f514
8704 F20101208_AABNUL james_m_Page_099thm.jpg
9890cb0fc3af98d92b7344bb10be0ff1
8d3b74d751ad59e98a53f73349a2a0a0c2efbf40
3581 F20101208_AABNTW james_m_Page_080thm.jpg
9abd687dc3f8a64f83c7ec02e78857db
830157e919fda0bd9588386ff1019a78c986849b
F20101208_AABMRJ james_m_Page_054.tif
555668db13a4b29ae19670dc371c70c0
bd4f28ca28a393dc4f4446a954f9a949ee865410
F20101208_AABMQU james_m_Page_039.tif
fe0339091419a7946e2baaba56fa2adb
eee3c0eaf17293e9b6aa6f8085080fac2061628b
34040 F20101208_AABNVA james_m_Page_118.QC.jpg
e940901322ab7dfece4fd68ba511ea64
5ef27400e4667e897ba914daf0b173be74507708
8533 F20101208_AABNUM james_m_Page_100thm.jpg
5770aa368a261666dcd59e55103d85d0
51cfdf4c6139d88db96245faa4b45b6de15625b6
11542 F20101208_AABNTX james_m_Page_080.QC.jpg
f77d0c6aeeb33510992512d7ab917606
d088ebf210fb89c980679bd9a545b05a0f841549
F20101208_AABMRK james_m_Page_055.tif
5d58857e1edc71763dcb0eef6ef9d597
b54a47e0669f63b90439cec6e87a4a3764a78f0d
F20101208_AABMQV james_m_Page_040.tif
8c2a8423d00711acd3b692750f48a9b7
5ed6f3c5f521bad04ac15bb1f23fdd04cb6b4180
8800 F20101208_AABNVB james_m_Page_119thm.jpg
0b35cca2ad93eac09bc083c921faab62
8a2b18a7b764f3fff6c1b700abfb9e4b4b52c06d
34077 F20101208_AABNUN james_m_Page_100.QC.jpg
86d1a6ca25180f6ddd173d9f2b4180b5
7443808660c6541cc0f7a84e68aed96788c96ead
3512 F20101208_AABNTY james_m_Page_081thm.jpg
e9ec3801c201a4586501b59a22164e16
78c7e8bf75bb0fca2223a3894db2b19eb89e2a79
F20101208_AABMRL james_m_Page_056.tif
7a1afd8a374de88ed597c8cfb3a93c2f
06502b13acba2924d112ea8aff0de238b014d506
F20101208_AABMQW james_m_Page_041.tif
104839fc07a309b0a0d543afad0ab7b4
4b32e3c647181cb6e4e65a3f31046d0a18f66e70
37921 F20101208_AABNVC james_m_Page_125.QC.jpg
8a639ecc2f033f6a6470f47182c27f7c
440dceddc7ad29e7228dda577b17b0c472c30e6b
36584 F20101208_AABNUO james_m_Page_101.QC.jpg
a9d98a047c8b109280738a8373580766
64de534c5764122db4b0aeb9477da9a392592170
13088 F20101208_AABNTZ james_m_Page_081.QC.jpg
c37be8418a7c553289873fcdf323e9e2
74e50f7de0f4cf4ceb927d8188653fbdfaed72ef
F20101208_AABMSA james_m_Page_072.tif
fbe4e1116b9d3218483ec41194ea036a
49ee6c7962229701e1a7fae51bdc6f29fa370a98
F20101208_AABMRM james_m_Page_057.tif
1444cf45589bd9f7ac411fb06995edd5
0ea40d630258f247a779891e4f3f792304e916c5
F20101208_AABMQX james_m_Page_042.tif
796540591396338eb636c363ca4aecce
cacf53adf6b44c91e9b4bedecfd787ce0179a45c
35953 F20101208_AABNVD james_m_Page_126.QC.jpg
b9c32b77fd0ef6fa701539daa500fd6b
fd5d177bf8a2ca894d947425c4806af5627cde0f
34773 F20101208_AABNUP james_m_Page_103.QC.jpg
a7661215ca894b0d47b00bd4f8cf1050
1eebc7ad0139453e3c550fe588096bd66d0d68f3
F20101208_AABMSB james_m_Page_073.tif
e42e1312a0d0e3a7d46d343c538bccd9
7021d1024ea2c2ea97957ec985812a429f910496
F20101208_AABMRN james_m_Page_058.tif
9a21b35e05d72017626ce3d4adf0ed33
98525c09072ed82602eec08cee59f5e5b7b36490
F20101208_AABMQY james_m_Page_043.tif
cd6c55ac9f61f7cae441f42886042a88
25c1ce099bb8f5e6f4bb94078a298b7d86d34313
39276 F20101208_AABNVE james_m_Page_128.QC.jpg
e9b545dceff21fe3e526a3b8a46f3dba
7bfc320b08cb9c94555aef53bba1f427f1f5e0ca
F20101208_AABNUQ james_m_Page_104thm.jpg
8ef9014e848b16762f5e552439a886c2
77532698ff0d674d4778372c4319cc036106a07f
F20101208_AABMSC james_m_Page_074.tif
58e75d6c65c514f38e88e566e8042fec
a5a10c77ab50082bb2436a10854dc4510d54429d
F20101208_AABMRO james_m_Page_059.tif
1c2223c4c59586cc19b966c312cf7f29
4e0e95a942a142ddee18f1eb6dff52192a705338
F20101208_AABMQZ james_m_Page_044.tif
d3f908761c6ad9a5ebcf0a6c5831f2f8
5415a95780c8710cf9ece3426751545d27c15626
9200 F20101208_AABNVF james_m_Page_129thm.jpg
8abb77dea217e99b5f54c65c286ab00b
c142decdc7780b7602372c1e6745f2668122599d
3029 F20101208_AABNUR james_m_Page_108thm.jpg
52fd05cebb5661cd1da1ec5d0c006d3f
fd0294e94fa3d00ab93382fb394e719c6dada174
F20101208_AABMSD james_m_Page_075.tif
bc43d7c2cf34e79bc4df9068a14786d7
ab27ab5762b515f79a490eb38e56157b16d5323e
F20101208_AABMRP james_m_Page_060.tif
a85d877449a9603970fb47e9ddd830ae
becc4e82a780c3a4a7dbf4026c1d6147a2eed50a
8922 F20101208_AABNVG james_m_Page_130thm.jpg
8bf552ba6e5671b56d4c507b06f90a59
ab59195f90784df17bfaaf926e2adbed3b0ad188
21982 F20101208_AABNUS james_m_Page_111.QC.jpg
c28b524c3ddbc6d1e7c98d2f60438f1a
a9d893a322d91770e07aabb97978575f6319ca07
F20101208_AABMSE james_m_Page_077.tif
7a84935e200bee6d2f409b4e25b1d69e
277d8507183d61c1b6818f45c93f93d2d472e93e
F20101208_AABMRQ james_m_Page_061.tif
b82403f2e8dce99d3bff6abf35346348
6d9bfb6fbe5d44ce57973a33c7d05901840dd902
5339 F20101208_AABNVH james_m_Page_133.QC.jpg
5a91060e032d15198c9e37aca7c30ddb
66527ab6928f99829f27735f83a17b20c8520c1d
14773 F20101208_AABNUT james_m_Page_112.QC.jpg
a836e03233e009ebf0378ad817f1a48b
5fb30371bc592fd241631caebf2a6ff6af4187b6
F20101208_AABMSF james_m_Page_078.tif
3e20af1a9529bff34863080ca9d45ee5
e7437cc34b9cc7703f83d0ec176408a549b07219
F20101208_AABMRR james_m_Page_062.tif
192c1a298e567cd529f858365e359206
70a66f083f94395d45362ddbb40e368730668196
4262 F20101208_AABNVI james_m_Page_135thm.jpg
cc9bf45d8f752f953b47bb23ee5a1b34
f2ab90646b7c2cfb9a34f8abbf9bb3084b058fc2
17871 F20101208_AABNUU james_m_Page_113.QC.jpg
c736286c5f8a949b3baf912772b5a6a1
10d9d7cc3015258aa64f01dd4a63dac6e04bf24a
F20101208_AABMSG james_m_Page_079.tif
745fd4cecacdf3009d7d3ab040672b33
43ae12c923f84c130da528bc3c59f266b7cfd0eb
F20101208_AABMRS james_m_Page_063.tif
b82d4282f2f3c7f46486f9814c41cc1c
459807cbdd85a7bb0214bb72af0bac68244f9752
9183 F20101208_AABNVJ james_m_Page_138.QC.jpg
995f2b570d4dd57610266c6a4b6d644b
51f51f5bd4cc83757a7196d9aaf367d2900ec002
F20101208_AABMSH james_m_Page_080.tif
220f401bfa2a38749a1aee9d1bd7d13d
ad51391681d20fbc8697070d11a48f5e6bb4037d
4226 F20101208_AABNVK james_m_Page_140thm.jpg
7508ade027bdc3b7e0046d2fbb3d49fe
0b7e4091f401f1879af38bfb63ffec02a4edc5d9
4096 F20101208_AABNUV james_m_Page_115thm.jpg
fbd65a422b9e2dc5fe3eae20621247ae
2b6b666401e162597ced18b5825d982e565646fd
F20101208_AABMSI james_m_Page_081.tif
3c83fbcceca90f336a4f0aa71f33fbe0
2b082e647122ce1eea7b2b4e37784b1e51829cdd
F20101208_AABMRT james_m_Page_064.tif
dc84a3971427c2098bceb606fce47e8b
9e8a1044378b551f38fb490f9988e317df5d56e5
3499 F20101208_AABNVL james_m_Page_141thm.jpg
05f805d87557b543bf0cbdf3fb213d2d
038532dee26d95f3e6f0749f6bedc2adc6ee8919
13906 F20101208_AABNUW james_m_Page_115.QC.jpg
67b23223fa42bdf4a1d364d066410b43
77802e3b50c82fdc22286d1fecb1f6a8288b895c
F20101208_AABMSJ james_m_Page_082.tif
9285da45a06ed49b9fd0f49913f82c38
b4c44d5fa1ca1682bc090ffe26bdebd22f18d0c8
F20101208_AABMRU james_m_Page_066.tif
9e6105b94aeb6d2dd5025075f6802206
af139cfb3786bc20c19357355ef89eac622362df
36212 F20101208_AABNWA james_m_Page_167.QC.jpg
578da2e3bb637a0d3f74e0dfe3fe2c85
c61a85db5dbf3470adbabeb544e66afbc3fbf253
13524 F20101208_AABNVM james_m_Page_142.QC.jpg
e0fbc7b27e2d19b444a05bb79c2393a7
5dc420b33531395ed4665917c52f4c66260d2bae
4628 F20101208_AABNUX james_m_Page_116thm.jpg
f5d61f0e662c0f53110f87d44276d710
356e0c4bc93f36844bb8b4fb6d9f7b045610b49c
F20101208_AABMSK james_m_Page_083.tif
420578fa847a52107ea83c11202e2178
0e21a43ac9d18cac1e67e48123aa4e579243ae12
F20101208_AABMRV james_m_Page_067.tif
fc8df062443398f94791a39305da8137
e68c2b65af337aab1dd5ea6700f79d9ea13e0049
9608 F20101208_AABNWB james_m_Page_168thm.jpg
4dc666e0e4d1cbc8081384232dcf2fc9
1473cf7359fc0c74a07d03cb9fc30a28f82f07c9
3155 F20101208_AABNVN james_m_Page_143thm.jpg
8f2b896d8567308d689f34e5c300d758
3c24e3ec660d9d91dd9d85d8753c110ceaaa5eaa
34229 F20101208_AABNUY james_m_Page_117.QC.jpg
1c997e29cd0ec2d2d073449b546eb761
d02f069a6dcfe9d120bb1523c599dbcb6d4520e5
F20101208_AABMSL james_m_Page_084.tif
7f18313d1a3621318c9ff02dea58efbf
d7457ac327270bbf863a430c9f878092e4f89f5d
F20101208_AABMRW james_m_Page_068.tif
7ba9a5fbf8ef7d47fed412a565404798
039e90124ae00da022cb03518a056fc2857f732b
F20101208_AABNWC james_m_Page_169thm.jpg
da56a09bfa16a13cd989bc23c72af320
44a306adbf078d71ab5aa57b44397d91f59e8721
4422 F20101208_AABNVO james_m_Page_144thm.jpg
2c102049720af5260d97509f02bba974
682fd18bcbe85a986e6a664c5cd166c93e3a6974
8782 F20101208_AABNUZ james_m_Page_118thm.jpg
2700e1408272137168a5fbb413217382
00d7b94967ae02d0e0de30756dbbbc56e454903e
F20101208_AABMSM james_m_Page_085.tif
586d83630f649b102d59a2c6f0f9e774
52e896b26d14a6de2259f9c164678b07ad0f5a1a
F20101208_AABMRX james_m_Page_069.tif
8f29ef4419fe4a65856384ea5da2fac6
3360c6ee4ccfee43b4110e4c80c811efa3c307d3
F20101208_AABMTA james_m_Page_099.tif
11131e55305588701b945ef48362a647
131f76e2a517a8bc7d4b65b68e75103c6d774e2c
38085 F20101208_AABNWD james_m_Page_169.QC.jpg
631afd81697999476b0e11abe5718cfe
3cb426df6fb4632d87d5dbe95780ba664b607aac
8310 F20101208_AABNVP james_m_Page_146thm.jpg
76c1c6ca5bd7e95b6cbf059d2df24b6f
8d367ba4eb36c769e14073f1c9b74d54139b2625
F20101208_AABMSN james_m_Page_086.tif
cee22e712a7be7408bfe1e154c659ba4
4d8f34cd0ef6decb8fc8dbd66120cfed30af04fa
F20101208_AABMRY james_m_Page_070.tif
4ad559afc536bbefd5b4dadce55375f4
adb7a2de3dc4c6bb49a11465121072bcc7f51661
F20101208_AABMTB james_m_Page_100.tif
5c005726ef6ac5c276fb2ea2a5db4680
d24e585645f0256fe7c1c6120341c43cd24888f0
8851 F20101208_AABNWE james_m_Page_171thm.jpg
a88a1c979e86561b305bc8c3f8af91b4
869b6a920e81f10d602793971904df45ff38eae1
8220 F20101208_AABNVQ james_m_Page_147thm.jpg
0a614f8e0d97caf2af4fb7caf016b3c0
b035d0a8f934ffcb8723201b67f8d7abcedd13e1
F20101208_AABMSO james_m_Page_087.tif
75323d466f45c108610eb082e30467c8
e91f36148eb41143e0c0ec335df4935ce68ee379
F20101208_AABMRZ james_m_Page_071.tif
de32ba5ea1e7d8681bc2ba971993e1c8
636371c4432abf0460832accbe4192c14788918d
F20101208_AABMTC james_m_Page_101.tif
8420cffc0865f5b7d4af908fcbfcfae3
6073d8e38f0f5cfc0434a9aa56ed64495476dec0
13268 F20101208_AABNWF james_m_Page_173.QC.jpg
bb9f7134f0178ea4c457f25d0582f4fc
c6426b8128c82263c65704adfd90683a5275c940
8553 F20101208_AABNVR james_m_Page_149thm.jpg
564be3718bb3a1b46d52d780da0281a4
bb9384532a395125f34e198710ca0dcb953720f9
F20101208_AABMSP james_m_Page_088.tif
e62dbbad35a1553d3115a84bc7605e0e
5e4e6897a3c5172964b20a584b4ea1f1fdc52f5b
F20101208_AABMTD james_m_Page_102.tif
2ffd53c087cc675447981ac3b98b0c50
138945b22ff11e66895b178af587a9025a5c8bb0
35328 F20101208_AABNVS james_m_Page_150.QC.jpg
fedcf3b65e0804c883ed681257856ac2
0fb7df8a3a70e78b42325eb9839bd0cbecf43129
F20101208_AABMSQ james_m_Page_089.tif
486e751a571f9784892de2712fb96598
7ddb8c2cdce95fe07f06abd690fdfd5ec015c633
F20101208_AABMTE james_m_Page_103.tif
71d71b01b2afa69911ca6c19b278777c
851550e5a6bc25437f4f76ccb8687cb92211e317
8763 F20101208_AABNVT james_m_Page_153thm.jpg
ece33a19d61e7bc30b99a0c2f05153f8
b7541c144ec089563c75dc90988162e408ce4586
F20101208_AABMSR james_m_Page_090.tif
0b2a2be138962306201f6b66426f86be
e64a411c6fffd47a2ae364c734f393c67bd54c3a
F20101208_AABMTF james_m_Page_104.tif
ac40497c0afe4b1bbfbfb1dd75b9ffe5
8ae7aafd5e74c2b4e9458b4a18048e6996df107d
35976 F20101208_AABNVU james_m_Page_153.QC.jpg
b8ad5c9d1999e03d043cfab5f799ec33
147a4baa888653a32cc156a40856de6e578339ee
F20101208_AABMSS james_m_Page_091.tif
6dcd1e10ba1560ebb16c03fce1275493
07044a665192fbb90bf7e2f683ceba390bfdaaf7
F20101208_AABMTG james_m_Page_105.tif
b16e2535539c8977b3c6927d5398d4f4
6a47817d113d5358bd4d9f2c405bc2b2b9a3d748
35017 F20101208_AABNVV james_m_Page_155.QC.jpg
2b05f5b57ce25eb4407e427298139a41
75f0d9ece9ae775f8b33141a9b84b1dc95e55f2e
F20101208_AABMST james_m_Page_092.tif
e23298cf402205dbe4bf0bbeef2a924e
1b59ee2abbc8b0a0561720c8370b97a67d116f2a
F20101208_AABMTH james_m_Page_106.tif
47ef4c42541e86f927a4d8abfb6d7219
aa4af08e4e0eefbac3cdc695b943eb8159bd0aeb
F20101208_AABMTI james_m_Page_107.tif
6efcb1f32d8926ad91a77788536a3b2c
9821f2fb24c74120c46372bb6e430700f3171df8
22462 F20101208_AABNVW james_m_Page_156.QC.jpg
77f68e1eb464175e8e9e84d1c4613557
0e408cf686ae7605c022c5592c4e2da358e2c584
F20101208_AABMSU james_m_Page_093.tif
2032544c916ca8ce2ce86b58475b200c
3b237dcb780f8adcf0dc214268a180ae732b7cf6
F20101208_AABMTJ james_m_Page_108.tif
4fa988e46b98d33e6877fcd8261ed844
3c9ceaed6fa2e9bfebe5b4c3edef10e1e4e426f9



PAGE 1

1 MOLECULAR INTERACTIONS IN SURFACTANT SOLUTIONS: FROM MICELLES TO MICROEMULSIONS By MONICA A. JAMES-SMITH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

PAGE 2

2 Copyright 2006 by Monica A. James-Smith

PAGE 3

3 To my parents who have been my #1 supporters since October 17, 1977.

PAGE 4

4 ACKNOWLEDGMENTS I thank my Almighty Heavenly Father for allo wing me to make it to this point and for seeing me through every obstacle that arose. I am forever grateful to my husband, Rod, for all of his support, love and encouragement. I sincerely thank my parents, Dan and Elaine James, for always believing in me, for their constant pray ers, and for always providing the right words when the journey seemed difficult. I would like to thank Melanie, Dan, Chris, and Bruce for knowing how to make me feel lik e I can accomplish anything. I owe a huge debt of gratitude to my best friend, Brandi Chestang, who has been there to answer every phone call and has cheered me on all my life. I am also gr eatly appreciative to all of my other friends, family, and loved ones. I must also extend my sincerest appreciati on to my in-laws who have taken me in as a family member and provided tremendous support as I have pursued this degree. I am forever grateful to Dr Dinesh O. Shah for being a mentor, an advisor, and a confidant, for providing me with the highest caliber of guidanc e and for always pushing me towards greatness. I would like to extend sincerest thanks to Dr s. Brij Moudgil, Manoj Varshney, Yakov Rabinovich, Anuj Chauhan, Oscar Crisalle Ranga Narayanan, Donn Dennis, and Richard Dickenson for stimulating conversation and insigh tful comments and suggestions. I also would like to thank all of my colleagues who have been of great assistance throughout me years here at UF. Last, but definitely not least, I would like to thank Pastor Kevin W. Thorpe and the Faith Baptist Church family for showing me that my fam ily is bigger than I think and for helping me to make it through this journey.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............12 CHAPTER 1 LITERATURE REVIEW.......................................................................................................14 1.1 Micelles................................................................................................................... ..........14 1.1.1 Introduction............................................................................................................14 1.1.2 Dynamic Nature of Micellar Solutions...................................................................18 1.2 Macroemulsions............................................................................................................. ...24 1.2.1 Emulsion Droplet Size............................................................................................26 1.2.2 Viscosity of Emulsions...........................................................................................28 1.2.3 Determination of Emulsion Type (O/W or W/O)...................................................28 1.2.4 Emulsion Stability..................................................................................................29 1.2.4.1 Coalescence..................................................................................................29 1.2.4.2 Charge stabilization: Th e electrical double layer.........................................30 1.2.4.3 Phase inversion in emulsions.......................................................................31 1.2.4.4 Emulsion creaming.......................................................................................31 1.2.5 Demulsification......................................................................................................32 1.2.6 Surfactant Selection for Emulsification..................................................................33 1.2.7 Applications of Emulsions.....................................................................................34 1.3 Microemulsions............................................................................................................. ...35 1.3.1 Formation of Microemulsions................................................................................41 1.3.2 Applications of Microemulsions............................................................................42 1.3.3 Nano-emulsions......................................................................................................42 2 A NOVEL METHOD TO ELUCIDATE TH E PRESENCE OF SUB-MICELLAR AGGREGATES IN SURFACTANT SOLUTIONS..............................................................61 2.1 Introduction............................................................................................................... ........61 2.2 Experimental Procedure....................................................................................................63 2.2.1 Materials................................................................................................................ .63 2.2.2 Ultracentrifugation.................................................................................................63 2.2.3 Two-Phase Dye Transfer (Methylen e Blue Complexation) and UV-Vis Analysis....................................................................................................................... .64 2.2.4 Foamability.............................................................................................................64 2.2.5 Fabric Wetting........................................................................................................65 2.2.6 Dynamic Surface Tension......................................................................................65

PAGE 6

6 2.3 Results and Discussion.....................................................................................................65 2.3.1 SDS Surfactant Solutions.......................................................................................65 2.3.2 Effect of Counter-Ions on Sub-Mi cellar Aggregate Concentration.......................73 2.3.3 Importance of Sub-Micellar Aggreg ates in Technological Processes....................75 2.3.3.1 Foaming........................................................................................................76 2.3.3.2 Fabric wetting...............................................................................................77 2.3.3.3 Dynamic surface tension..............................................................................77 2.4 Conclusions................................................................................................................ .......78 3 DETERMINATION OF DRUG AND FATTY ACID BINDING CAPACITY TO PLURONIC F127 IN MICROEMULSI ONS FOR DETOXIFICATION..............................94 3.1 Introduction............................................................................................................... ........94 3.2 Experimental Procedure....................................................................................................96 3.2.1 Materials................................................................................................................ .96 3.2.2 Microemulsion Preparation....................................................................................96 3.2.3 Turbidity Analysis..................................................................................................96 3.2.4 Dynamic Surface Tension......................................................................................97 3.2.5 Foamability.............................................................................................................97 3.2.6 Fabric Wetting........................................................................................................98 3.2.7 Surface Tension......................................................................................................98 3.3 Results and Discussion.....................................................................................................98 3.3.1 Effect of Sodium Caprylate Concentr ation on Drug and Fatty Acid Binding to Microemulsions............................................................................................................98 3.3.2 Determination of Free Fatty Acid by Dynamic Processes...................................101 3.3.3 Effect of Fatty Acid Chain Lengt h on Drug and Fatty Acid Binging to Microemulsions..........................................................................................................104 3.3.4 Effect of the Number of Ethylene Oxide (EO) and Propylene Oxide (PO) Groups of Pluronics on Fatty Acid and Drug Binding..............................................105 3.4 Conclusions................................................................................................................ .....106 4 A Novel Method to Quantify the Amount of Surf actant at the Oil/Water Interface and to Determine Total Interfaci al Area of Emulsions....................................................................117 4.1 Introduction............................................................................................................... ......117 4.2 Experimental Procedure..................................................................................................119 4.3 Results and Discussion...................................................................................................121 4.3.1 Effect of Surfactant Concentration on Partitioning to the Oil/Water Interface....121 4.3.2 Effect of Alkyl Sulfate Chain Le ngth on Partitioning to the Oil/Water Interface.....................................................................................................................125 4.3.3 Effect of Oil Chain Length on SDS Pa rtitioning to the Oil/Water Interface........128 4.3.4 Effect of Alcohol Chain Length on SDS Partitioning to the Oil/Water Interface.....................................................................................................................130 4.3.5 Effect of Oil Phase Volume Fraction on SDS Partitioning to the Oil/Water Interface.....................................................................................................................131 4.4 Conclusions................................................................................................................ .....131

PAGE 7

7 5 SUMMARY AND RECOMMENDATIO NS FOR FUTURE WORK................................146 5.1 Micelles................................................................................................................... ........146 5.1.1 Summary...............................................................................................................146 5.1.2 Future Work..........................................................................................................147 5.2 Microemulsions............................................................................................................. .149 5.2.1 Summary...............................................................................................................149 5.2.2 Future Work..........................................................................................................151 5.3 Macroemulsions............................................................................................................. .152 5.3.1 Summary...............................................................................................................152 5.3.2 Future Work..........................................................................................................155 APPENDIX A GIBBS ADSORPTION EQUATION AND AREA PER SURFACTANT MOLECULE DETERMINATION.............................................................................................................157 B CALCULATION OF TOTAL INTERFACIAL AREA FROM FILTRATION RESULTS........................................................................................................................ .....160 C CALCULATION OF TOTAL INTERF ACIAL AREA FROM DROPLET SIZE RESULTS........................................................................................................................ .....161 LIST OF REFERENCES.............................................................................................................162 BIOGRAPHICAL SKETCH.......................................................................................................173

PAGE 8

8 LIST OF TABLES Table page 1-1: Summary of methods used to produce emulsions..............................................................54 1-2: Common tests for determining emulsion type (W/O or O/W)44........................................55 1-3: Types of breakdown pro cesses occurring in emulsions.....................................................56 1-4. Factors influencing th e stability of emulsions...................................................................57 1-5. Parameters that affect phase inversio n in emulsion and the effect they have....................58 1-6. Commonly used physical methods of demulsification......................................................59 1-7. A summary of HLB range s and their application..............................................................59 1-8 Microemulsions vs. Nano-emulsions.................................................................................60 2-1. Dimensionless dynamic surface tension ( ) of different counter-ions of dodecyl sulfates (50 mM) at a bubble life time of 50 msec (from ref 33)........................................93 3-1. Effect of fatty acid chain length on maximum binding...................................................116 3-2. Effect of # of PO groups on maximum binding...............................................................116 3-3. Effect of # of EO groups on maximum binding..............................................................116 4-1. Effect of SDS concentration on total inte rfacial area 1% (v/v) hexadecane-in-water emulsions...................................................................................................................... ...144 4-2. Area per molecule values at the hexadecane/water interface for alkyl sufates and total interfacial area as a functi on of alkyl sulfate chain length......................................144 4-3. Effect of oil chain length on total interfacial area (TIA).................................................145 4-4. Effect of alcohol chain lengt h on total interfaci al area (TIA)..........................................145

PAGE 9

9 LIST OF FIGURES Figure page 1-1. Schematic diagram of a surfactant molecule, micelle, and reverse micelle......................48 1-2. Properties of surfactant solutions showing abrupt change at the solution critical micelle concentration (cmc)...............................................................................................48 1-3. Schematic design of micellar solution...............................................................................49 1-4. Schematic diagram of the f our major micellar structures..................................................49 1-5. Mechanisms for the two characteristic relaxation times for a micelle...............................50 1-6. Typical size distribution curve of aggregates in a micellar solution.................................50 1-7. Schematic of sodium counter-ion cloud around SDS spherical micelle........................51 1-8. Schematic diagram of the adsorption of surfactant monomers from the bulk to the oil/water interface during emulsion formation..................................................................51 1-9. The emulsion droplet size in the hexadecane/SDS solution system..................................51 1-10. Schematic depiction of the Stern-Graham model of the electrical double layer...............52 1-11. Schematic diagram of an o il-in-water (O/W) microemulsion...........................................52 1-12. Thermodynamic explanation for behavior of macroemulsions and microemulsions........53 2-1. Schematic diagram of the ultracentrifugation process.......................................................80 2-2. Size distribution curves of a ggregates in a micellar solution............................................81 2-3. Schematic diagrams of surfactant solutions, filtration of solutions, and plot of filtrate concentration as a function of total surfactant concentration............................................82 2-4. Schematic representation of the two possi ble reaction paths for the formation of micelles....................................................................................................................... .......84 2-5. Filtration of SDS through 10,000 MW CO ultracentrifuge tubes.......................................85 2-6. Tailoring of micellar stability............................................................................................85 2-7. Filtrate of SDS+C12TAB through 10,000 MWCO ultracentrifuge tubes.........................86 2-8. Filtration of SDS alone or SDS + C12X (X = OH or TAB) through 10,000 MWCO ultracentrifuge tubes.......................................................................................................... .87

PAGE 10

10 2-9. SDS concentration in the filtrate for 80:20 SDS:C12TAB systems after filtration through 3,000 and 10,000 MWCO tubes, as compared to pure SDS solutions (50 mM)............................................................................................................................ ........88 2-10. Filtrate surfactant concentrations for 25 mM lithium dodecyl sulfate (LiDS), sodium dodecyl sulfate (NaDS), and cesium dodecyl sulfate (C sDS) and 12.5 mM magnesium dodecyl sulfate (Mg(DS)2).............................................................................89 2-11. Schematic depiction of foam column................................................................................90 2-12. Foamability of SDS mi cellar solution and SDS + C12X mixed micellar solutions...........90 2-13. Wetting time of 1in2 strips of 50:50 cotton:polyester blend fabric....................................91 2-14. Dynamic surface tension of solutions of 50 mM SDS and 50 mM SDS + 12.5 mM C12TAB............................................................................................................................ ..92 3-1. Amitriptyline Hydrochloride, MW = 313.9.....................................................................109 3-2. Titration of microemulsions with 0.2 M AMT................................................................109 3-3. Titration of mixed micelles and microemulsion systems................................................110 3-4. Schematic diagram of turbidity in various solutions.......................................................111 3-5. Titration of microemulsion sy stems with AMT ([SC] = 25 -125 mM)...........................112 3-6. Properties of Plur onic F127 microemulsion....................................................................113 3-7. Binding of SC and drug to F127......................................................................................114 3-8. Schematic depiction of microemulsion droplet...............................................................115 4-1. Schematic depiction of filtration of oil-in-water emulsion through nanoporous filter membrane....................................................................................................................... ..134 4-2. Effect of total SDS concentrati on on SDS concentration in the filtrate..........................135 4-3. Effect of SDS concentration on mean dr oplet diameter of 1% (v/v) hexadecane-inwater emulsions...............................................................................................................136 4-4. Master diagram showing the changes in emulsion characteristics with increasing SDS concentration...........................................................................................................137 4-5. Amount of alkyl sulfate surfactant that pa rtitions to the interf ace as a function of chain length................................................................................................................... ...138 4-6. Mean droplet size of hexadecane-in-wat er emulsions as a function of alkyl sulfate chain length. Droplet size was de termined by light scattering........................................139

PAGE 11

11 4-7. Effect of oil chain length on the amount of SDS that partitions to the interface.............140 4-8. Schematic depiction of emulsion droplets.......................................................................141 4-9. Effect of alcohol chain length on the am ount of SDS that partit ions to the interface and mean droplet size.......................................................................................................142 4-10. Effect of oil phase (hexadecane) volume fraction on partitioning of SDS to the oil/water interface............................................................................................................143

PAGE 12

12 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MOLECULAR INTERACTIONS IN SURFACTANT SOLUTIONS: FROM MICELLES TO MICROEMULSIONS By Monica A. James-Smith December 2006 Chair: Dinesh O. Shah Co-chair: Brij M. Moudgil Major Department: Chemical Engineering To effectively use surfactants for various applications, one must have a basic understanding of the molecular in teractions occurring within su rfactant solutions. Micelles, microemulsions, and macroemulsions are three of the most commonly investigated surfactant containing systems. For many years, these systems ha ve been studied in light of their structure, properties, and applications. In this dissertation, we investigat e these systems using filtration through nanoporous membranes to understand the role of monomers and sub-micellar aggregates in controlling their properties. It is commonly accepted that micellar solu tions consist of three surfactant species; adsorbed monomers at the air/ water interface, monomers disper sed in the aqueous phase, and micellar aggregates. Using f iltration through nanoporous membranes, we found evidence suggesting that another species, sub-micellar aggr egates, exists, in significant concentration, in micellar solutions. This is a potentially revolutio nary finding that provides for a more accurate picture of micellar solutions and yields insigh t to the mechanism by which micellar stability affects dynamic processes.

PAGE 13

13 The presence of monomers and sub-micellar aggr egates in microemulsions is important in determining the efficacy of microemulsions for drug binding. We used filtration through nanoporous membranes and turbidity analysis to delineate the dr ug-fatty acid monomer interactions. The results showed that one Pluronic F127 molecule binds a maximum of eleven sodium caprylate fatty acid molecules and twelve Amitriptyline drug molecules. We were also able to determine that the initia l driving force for Amitriptyline upt ake is electrostatic attraction. Filtration through nanoporous membranes is idea lly suited to determine an unresolved issue of surfactant portioning at the oil/w ater interface and in the bulk solution in macroemulsions. We were able to approximate th e total interfacial area (TIA) of the emulsions and showed that this calculation produced valu es approximately two orders-of-magnitude larger than the area calculated using mean droplet sized from light scatte ring analysis. Possible explanations for this difference are discussed.

PAGE 14

14 CHAPTER 1 LITERATURE REVIEW Surfactants, or surfaceactive agents, are used to enhan ce the quality of products used in every aspect of life; from food to cosmetics, from oil recovery to detergency, and even from pharmaceutics to chemical mechanical polishing of silicon wafers. Given these many diverse applications, it becomes critical to have a suffi cient understanding of the molecular interactions that occur within surfactant solutions in order to effectively manipulate them for a specific use. Three invisible compartments in a surfactant solution are an adsorbed film, monomers, and micelles made up of surfactant molecules.1 Surfactant molecules are continually exchanging among these three compartments, and hence surfactant solutions are in dynamic equilibrium. For technological processes, such as foaming, emul sification, and wetting, surfactant molecules are driven to the new surfaces and it becomes necessa ry for micelles to disintegrate and provide monomers to these surfaces. In such rapid pro cesses, micelle stability could become a ratelimiting step.2 The following is the detailed review of mice lles, microemulsions, and macroemulsions. 1.1 Micelles 1.1.1 Introduction A surfactant, or sur f aceact ive ag ent is defined as a substance that adsorbs onto surfaces or interfaces of solutions to lower the su rface or interfacial te nsion of the system.3 The magnitude of the lowering of the surface or interfacial tension depe nds on the surfactant structure, concentration, and the physico-chemical conditions of the solution (e.g. pH, salt concentration, temperature, pressure, etc.).3 Surfactants are typically amphiphatic species, meaning that they are made up of a hydrophobic component, referred to as the tail, and a hydrophilic component, referred to as the hea d group (see Figure 1-1). When placed in

PAGE 15

15 solution, surfactant molecules tend to orient in such a way as to minimize the interactions of the hydrophobic tail with water in aq ueous solutions, or to minimize the interactions of the hydrophilic head with oil in or ganic solvents. This leads to adsorption of the surfactant molecules onto surfaces or at in terfaces, and above a certain concen tration, known as the critical micelle concentration or cmc, surfactants form aggregates known as micelles. When placed into aqueous solutions, surfactant molecules will form spherical aggregates at the cmc where the hydrophobic tails are pointed inward and removed fr om interaction with water molecules by the hydrophilic head groups as shown in Figure 1-1. When placed into organic solutions, surfactant molecules will form reverse micelles with the hydrophobic tails pointed outward (see Figure 11). When the critical micellar concentration, or cmc, is reached, many of the physical properties of the surfactant solution in water sh ow an abrupt change as shown in Figure 1-2. Some of these properties include the surface tension, osmotic pre ssure, electrical conductivity, and solubilization. The cmc is a measure of th e free monomer concentr ation in surfactant solutions at a given temperature, pressure, and composition. Mcbain4 first investigated the unusual behavior of fatty acid salts in dilute aq ueous solution at the cmc in the 1910s and 1920s and was followed by Hartley5, 6 in the 1930s. Other evidence fo r surfactant aggr egation into micelles was obtained from vapor pressure measurements and the solubility of organic molecules in water. The formation of colloidal-sized clusters of individual surfacta nt molecules in solution is known as micellization. McBain first suggested that micelles are spheri cal in shape. However, the first concrete model for spherical micelles is attributed to Hartley.6 It is currently accepted in the field that above the cmc, a typical surfactant solution cons ists of three major sp ecies: 1) surfactant

PAGE 16

16 molecules dispersed as monomers in the aqueous phase, 2) aggregates in the form of micelles, and 3) adsorbed films at the air/liquid interf ace (see Figure 1-3). The surfactant is in dynamic equilibrium between these states, implying that the rates of adsorp tion and desorption are equal. Thus, at a given temperature, pressure, and conc entration, the number of monomers adsorbed at the air/water interface, the numbe r of monomers in the bulk phas e, and the number of micelles present in solution is fixed under equilibrium conditions. The concentration of monomers and micelles changes with equilibrium conditions such as pressure, temperature, or surfactant and salt concentration. The micellization process is primarily an entropy-driven process.7, 8 When surfactants are dissolved in water, the hydr ophobic tail disrupts the hydrogen-bonde d structure of water thereby increasing the free energy of the system. As prev iously mentioned, for this reason surfactant molecules will concentrate at in terfaces so that their hydrophobi c tail groups are removed or directed away from the water minimizing the free energy of the solution. The formation of micelles is yet another means that the system will use to reduce its free energy. The orientation of the hydrophobic tails in the inte rior of the micelle decreases their interaction with water molecules. However, the surfactant molecules th at are confined in the micelle conceivably experience some loss of freedom. In addition, in th e case of ionic surfactant s, the molecules that are present in the micellar aggregate may experi ence electrostatic repulsion from other similarly charged surfactant molecules. These forces in crease the free energy of the system and oppose micellization. Hence, micelle formation depends on the force balance between the factors favoring micellization (Van der Waals and hydr ophobic forces) and those opposing it (kinetic energy of the molecules, electrostatic repul sion, and chemical poten tial factor due to concentration gradient for micelles and monomer).9

PAGE 17

17 The number of surfactant molecules that make up a micelle is known as the aggregation number. This number can be determined through the use of NMR self-diffusion coefficients,10 small-angle neutron scattering,11, 12 freezing point and vapor pressu re methods, osmotic pressure, and fluorescent probes. The surf actant structure plays a signifi cant role in the aggregation number of the micelles formed by a give surfacta nt. The aggregation number tends to increase with increasing chain length of the hydrophobic tail and decrease w ith increasing size (i.e., cross sectional area) of the hydrophilic head group. The aggregation number is also affected by the nature of the aqueous phase. For example, the ad dition of neutral electrol ytes to ionic surfactant solutions leads to an increase in the aggregation number. This is due to a phenomenon known as salting out by which the effective amount of water that is available to solubilize the surfactant molecules is decreased because it is used up to accommodate the ions (a more favorable interaction). Increasing the temper ature of an ionic surfactant so lution typically leads to a small decrease in aggregation number which can be owed to an increas e in the size of the head group as a result of thermal agitation as well as the incr ease in the kinetic energy of the surfactant and solvent molecules. When the temperature is increased in nonionic surfactant solutions (particularly of the polyoxyethylene variety), th e aggregation number slowly increases until the cloud point of the surfactant is reached. The cloud point is the temperature at which the solution begins to exhibit turbidity due to the dehydration of the polyoxyethylene head groups. Factors that increase the aggrega tion number tend to decrease the cmc. It is also important to mention that the cmc values of nonionic surfactants are much lower than the cmc values for ionic surfactants. In the past couple of decades considerable in terest has been generated in self-assembled surfactant aggregates such as cylindrical, lamella r, and reverse micelles due to the ability of

PAGE 18

18 surfactant association structure to mimic biological structures.13 Enzymes, for example, are proteins that speed up (i.e., catal yze) chemical reactions by providi ng sites for a substrate to fit into to form a reactive intermediate. The highly e fficient and specific catalytic effect of enzymes makes their investigation an inte resting area of biomedical and detergent research (as enzymes are often added to laundry deterg ents to improve performance).14, 15 Likewise, cell membranes perform a variety of functions in cellular bioc hemical and physiological processes. Surfactant structures can be used as model systems to mimic both enzymes and membranes. Micelles and reversed micelles now play an increasingly importa nt role in catalysis and separation processes in engineering and environmental science and technology.16-18 A theory of micellar structure, based upon the geometry of various micellar shapes and the space occupied by the hydrophilic and hydrophobic groups of the surfactant molecules, has been developed by Israelachvili et al.19 In aqueous systems, for exampl e, surfactants with bulky or loosely packed head groups a nd long, thin hydrophobic tails tend to form spherical micelles, while those with short, bulky tail groups and small, close-packed head groups tend to form lamellar or cylindrical micelles. At concentrations slightly above the cmc, micelles are considered to be of spherical shape.20 Changes in temperature, surfactant concentration, or additives in the solution may cha nge the size, shape, aggregati on number, and stability of the micelles. There are four major types of micelles: 1) spherical micelles which generally have an aggregation number less than 100 molecules, 2) cylindrical or rod-like micelles, 3) lamellar micelles, and 4) vesicles. These four structures are shown in Figure 1-4. 1.1.2 Dynamic Nature of Micellar Solutions Although micelles are often shown as static stru ctures in solution, they are in fact quite dynamic in nature, constantly breaking and reform ing in solution. As stated above, there is a

PAGE 19

19 dynamic equilibrium between the surfactant monome rs in the bulk, the surfactant molecules in the micelle, and molecules that are adsorbed at the interface. There ar e two characteristic time scales of relevance in micellar dynamics.21, 22 The first is a fast relaxation time, referred to as 1, which is a measure of the time that it takes for one surfactant molecule to go in or to come out of the micelle. The second is a slow relaxation time, referred to as 2, which is a measure of the time that it takes for one micelle to completely disintegrate or to completely form. The fast relaxation time is generally on the order of microseconds, whereas the slow relaxation time is on the order of milliseconds to minutes. The rate of disi ntegration of a micelle is equal to the rate of micellar formation. Thus in a micellar solution, if X number of micelles are broken down per second, X number of new micelles are formed so mewhere else in the solution during the same time. If this requirement is not obeyed, then the number of micelles in th e solution would change as a function of time. The fast and slow relaxa tion processes are illust rated in Figure 1-5. The lifetime of a micelle can be a pproximated to be equal to n 2 where n is the aggregation number of the micelle23. Thus for a typical solution of sodi um dodecyl sulfate (SDS) at 200 mM, the 2 is 78 seconds and n is 65. Thus, n 2 is equal to 7.6 to 8.6 minutes. Therefore, when comparing the lifetime of micelles (n 2) at 10 mM and 200 mM, it is 65 msecs to 8.6 minutes, respectively. There are many methods that are used to measure micellar relaxation time. The stoppedflow method24 involves the rapid dilution of a micellar solution containi ng an oil soluble dye and monitoring of the dye adsorbance as a function of time. The temperature-jump25 and pressurejump26 methods involve analysis of the electrical conductiv ity of the system af ter perturbation of the temperature or pressure of the system. Ultrasonic absorption27 is yet another method to measure the kinetics of micelles.

PAGE 20

20 Micellar kinetics may be manipulated by changi ng physical properties of the solution, such as temperature, pressure, and concentration, or by placing additives into the surfactant solution. Leung and Shah28 have shown that the addition of shor t chain alcohols (methanol to pentanol) leads to the destabilization of s odium dodecyl sulfate (SDS) micelle s and subsequently leads to a decrease in the 2 value. Patist and Shah29, 30 later showed that the addition of longer chain alcohols such as dodecanol or the addition of oppositely charged surfactants, such as dodecyltrimethylammonium bromide (C12TAB) to SDS solutions leads to marked increases in the 2 values, indicating enhanced stability of mixed micelles. A 25 mM SDS solution has a 2 value of 1 millisecond, but the addition of dodecanol or C12TAB increases the 2 value to 230 msec or 2000 msec, respectively. During the 1970s, Aniansson and Wall discovered the existence of the two (fast and slow) relaxation processes and developed a model of the kinetic process of micelle formation and disintegration.21 The first major assumption of this model was that the free surfactant monomers are assumed to be completely dissociated and the size distribution of the aggregates in a surfactant solution is assumed to follow the be havior that is shown in Figure 1-6 where C ( An) denotes the total concentrati on of aggregates containing n monomers and is a function of temperature, pressure, and to tal surfactant concentration. The second major assumption Aniansson and Wall made was that the association and dissociation of micelles is a step wise process involving the entr y and departure of one monomer at a time from the micelle. Thus, there is a series of equilibriums, , A1 nn n (1-1) where An represents an aggregate containing n monomers, and kn + and kn are the forward and reverse rate constants for a gi ven step, respectively. As a resu lt, when the equilibrium of a nk nk

PAGE 21

21 surfactant solution is perturbed (e.g., by temperatur e or pressure), the excess surfactant has to move through regions of different aggregation nu mbers (Region II of Figure 1-6). According to equation (1-1), this occurs in steps that are ve ry small compared to the distance in aggregate space traveled. Therefore, the process will have th e characteristics of a flowing system, which is important because it allows the kinetics of the abstract process of mice lle aggregation to be studied in terms of the more familiar phenomena of heat and material flow. Initially, micellar kinetics was analyzed on the basis of a heat conductance problem. It was later viewed in light of a mass transfer di ffusion problem, based on a model of mass flow through a tube having two wide ends that are connected by a narrow section. This model is analogous to the two high concentration regi ons (Regions I and III ) of Figure 1-6 being connected by the low concentration region (Region II). In this mass transfer diffusion model, the rate limiting step to equilibration between the two wi de ends is considered to be the diffusion of materials through the narrow sec tion of the tube. Analogously, th e low concentration region of transient intermediate sub-micellar aggregate stat es (Region II) that surfactant monomers must pass through (i.e., A2, A3, A4 ,An-1 in Equation (1-1)) when a micelle is formed (Region III), or disintegrated (Region 1) into free monomers is the rate-limiting step in the formation or disintegration of a micelle. A ssuming the aggregation number n to be a continuous variable and applying the above analogy to mass transfer, An iansson and Wall derived the expression for the fast relaxation process cmc cmc C a with a n k 1 12 2 1 (1-2) where is the half-width of the di stribution curve of micellar si zes (assumed to be Gaussian, Figure 1-6), kis the stepwise dissociation rate consta nt, which is assumed to be independent of n in the micellar region, C is the to tal surfactant concentration, and cmc is the critical micelle

PAGE 22

22 concentration. Equation (1-2) predic ts a linear relationship between 1/ 1 and the total surfactant concentration, which is in agreement with pressure-jump and sound absorption experiments.31, 32 It is obvious that as the total surfactant concentration increases, the number of micelles increases also, resulting in a decrease in intermicellar di stance. Hence, the time required for a monomer to collide with a micelle is shorte r at higher surfactant concentra tion. The length of the surfactants hydrocarbon tail affects the magnitude of 1 (i.e., the shorter the chain length, the faster the relaxation time). This is because the longer th e chain length, the greater the Van der Waals attractive forces will be between the chains of neighboring surfactant molecule. This will lead to more stable, tighter packed mice lles with increasing chain length. Using the same analogy of diffusion through a vari able-width tube as described above, an expression for the slow relaxation time, 2, was derived and simplified to 1 2 2 21 1 a n R cmc n (1-3) where R is a term which may be visualized as the resistance to flow through the narrow region (i.e., Region II in Figure 1-6) connecting the monomers to the micelles and is given by 211n n n n nA k R (1-4) where n is the aggregation number of some colloidal aggregate and An is the equilibrium concentration of aggregates of order n The dependence of 1/ 2 upon ionic strength, concentration, and temperature has been interpreted in terms of their effect on R Interestingly, the two relaxation times can be used to calculate two important parameters of a micellar solution: 1) the residence time of a surfact ant monomer in a micelle and 2) the average lifetime or stability of micelles33-36. The residence time of a surfactant monomer in a micelle is equal to n/k-, where n

PAGE 23

23 is the mean aggregation number and kis the dissociation rate constant of a monomer from a micelle. The average micellar lifetime Tm is given by 23 2 2 21 n a n na Tm (1-5) When the concentration of surfactant is much greater than cmc, the micellar lifetime is approximately equal to n2. Although first derived for nonionic surfactants, th e results of Aniansson and Walls theory on micellar aggregation kinetics were compared primarily with experiments on ionic systems, simply because it was much easier to detect the relaxation times in ionic systems than in nonionic systems. Even so, the agreement between theory and experiment was, in general, satisfactory in the regime of low surfactant concentrations.37 At higher concentrations, however, the theory did not match experimental results.38 As previously stated, e quation (1-3) predicts that 2 should increase with in creasing surfactant concentration. However, it has been reported that for some ionic surfactant systems 2 first increases, passes through a maximum, and then decreases again.39-41 This behavior of 2 in ionic micellar solutions is not predicted in the AnianssonWall model. Kahlweit and coworker s, using their own temperature-jump and pressure-jump results22, 42, 43 concluded that in ionic surfactant systems at high concentration, the reaction path for the formation of micelles must be different than that at low concentration. Therefore, the following model was proposed explaining the occurren ce of a maximum in 2. Ionic micelles, including sub-micellar aggregates can be considered charged particles. When ionic surfactant molecules such as SDS are adde d to water, the surfactant molecules dissociate into negatively charged dodecyl sulfate molecule s and their positively charged sodium counter-

PAGE 24

24 ions. These counter-ions are pr esent in solution as a cloud su rrounding the negatively charged micelle (see Figure 1-7). It is believed that at low surfactan t counter-ion concentration, the micelles are stable with respect to coagulation du e to repulsive electrostatic forces. Consequently they can grow only by stepwise incorporation of monomers according to Equation (1-1) above. As more and more surfactant is added into the system, the counter-i on concentration also increases, which compresses the electrical double layer and reduces char ge repulsion, allowing the micelles to come closer to each other so th at attractive dispersion fo rces (i.e., Van der Waals forces) lead to a reversible fusi onfission coagulation according to i l k A A Ai l k (1-6) Therefore, Kahlweit suggested that there are essentially two possible pathways for micelle formation in ionic surfactant solu tions: 1) formation as a result of step-wise incorporation of monomers, and 2) formation as a result of submicellar aggregates coming together. The same idea holds true for the dissociation of micelle s by two possible pathways. His model proposed that the first pathway was dominant for low su rfactant concentrations, where electrostatic repulsion would be high between droplets. Th e second pathway would become dominant for higher surfactant concentrations, where the counte r-ion concentration is high and repulsion is minimized. 1.2 Macroemulsions It is a commonly known fact that oil and water do not mix. However, emulsifying agents, typically surfactants can be adde d to a mixture of oil and water to promote the dispersion of one phase in the other in the form of droplets. Over the years, em ulsions have been defined in a variety of ways. For the purpose of this dissertation, emulsions will be defined as thermodynamically unstable, heterogeneous systems, consisting of at least one immiscible liquid

PAGE 25

25 intimately dispersed in another in the form of droplets, whose diameters are generally in the range of 1 100 m.44 There are two main types of emulsi ons: oil-in-water emulsions, in which oil droplets are dispersed in a continuous water phase, or water-i n-oil emulsions, in which water droplets are dispersed in a continuous oil phase. The most fundamental thermodynamic property of any interface is the interfacial free energy, or interfacial tension. The interfacial free energy is the amount of work necessary to create a given interface. The inte rfacial free energy per unit area is a measure of the interfacial tension between two phases. A high value of inte rfacial tension implies that the two phases are highly dissimilar in nature. Ther e are many methods available to measure the interfacial tension between two liquids including the Du Noy ring method, Wilhelmy plate method, drop-weight or drop-volume method, pendant drop method, spinning drop method, and Sessile drop method.3 Emulsification involves the generation of a large total interfacial area. Considering that the two phases in emulsions are not miscible, in orde r to generate this large interfacial area, the interfacial tension must be lowered signifi cantly according to the following equation: W = A (1-7) where W is the work done on an interface, is the interfacial tension, and A is the change in interfacial area associat ed with the work W. According to Equation (1-7), when a constant amount of work is applied to generate an interface, A will be large if is small, and thus the interface will expand significantly to form smaller emulsion droplets. As previously mentioned, the primary means by which the interfacial tension is lowered is through the addition of emulsifying agents, usua lly surfactants. The surf actant molecule also plays a second role in emulsions which is to st abilize the interface for a ti me against coalescence with other droplets and concomitant phase separation.

PAGE 26

26 A large number of methods have been develope d to provide the energy needed to achieve complete emulsification in a given system. Thes e methods can be broken up into a number of classes. Table 1-1 summarizes methods and apparatus used to produce emulsions, including a characterization of each method.36, 44-46 Some of these methods are almost exclusively used in laboratory settings (i.e., bench sc ale), such as 1, 4d, 8b, 9, 10 and 12. For large-scale production of emulsions, methods 4b, 5, 7, and 8a are us ed. There are often times when two methods are combined, such as 3 and 7 or 4 and 7. 1.2.1 Emulsion Droplet Size Emulsions are classified as either wate r-in-oil (W/O) or oil-in-water (O/W) depending on which phase is continuous and wh ich is dispersed. The dispersed phase in emulsions, whether oil or water, is usually compos ed of spherical droplets within the continuous phase. These droplets may be nearly monodisperse in terms of droplet size; or they may have a wide size distribution depe nding on seve ral factors.44 In most cases, the wider the size distribution, the less stable is the emulsion. In other words, emulsions with a more uniform size distribution tend to remain stable for longer tim e while those with wide size distribution will usually undergo Ostwald ri pening, a phenomenon where larger dr oplets grow at the expense of smaller droplets.45 In general, emulsions with a narrow size dist ribution and a small mean droplet size tend to exhibit a greater emulsion stability, all other things being equal.44 The change in the size distribution wi th time reflects the kinetics of coalescence in emulsions. Depending on the surfac tant that is used to stabilize the emulsion, emulsions can have lifetimes ranging from hours to as long as a few years.44 In general, emulsions exhibiting a higher yield stress tend to show higher em ulsion stability and shelf life.44 Emulsion droplet size is also related to the method of preparation that is employed to generate the emulsion. This is a result of the re lationship between interfac ial area and work that

PAGE 27

27 is done on the system, according to Equation (1-7 ). As can be seen in this equation, if the interfacial tension is constant with time, the change in interfacial area is directly proportional to the amount of work that is pu t into the system. Some emulsi on preparation methods provide more energy (work) than others, and thereby lead to smaller droplets and higher interfacial area. Micellar stability is another factor that affects the droplet size of emulsions.46 The ability of the surfactant molecules to adsorb rapidly from the bulk to the droplet interface in an emulsion determines the dynamic inte rfacial tension, which is related to th e total interfacial area and therefore the droplet size. In emulsion formation, as the large total interfacial area is being generated by dispersion of oil phase, additional monomers have to be pr ovided to this newly created interface by disintegration of micelles. If the micelles are ve ry stable, flux of surfactant monomers to the interface of droplets will be low, resulting in a higher interfacial tension at the droplet surface and a large droplet size will occur as predicted by Equation 1-7. The process of monomer diffusion from the bulk to the oil/water interface is illustrated in Figure 1-8. A considerable amount of work has been done on characte rizing micellar stability in surfactant solutions. It has been shown that more stable mi celles provide less monomer flux to the oil/water interface, wh ich results in a higher interfacial tension and he nce, a larger droplet size.46 In order for the droplet size in an emulsion to be small, a short-lived (or labile) micelle is desired, since it will supply monomer to the oil/w ater interface with ease. This is illustrated in for emulsions of hexadecane/sodium dodecyl sulfate (SDS) solutions in Figure 1-9. The droplet size is largest for the most stable micelle, which is known to be at the 200 mM concentration for SDS solutions.37, 41 This relationship between mice lle stability an d droplet size has also been verified for cesium dodecyl sulfate solutions which forms the most stable micelle when compared to micelles of sodium dodecyl sulfate and lithium dodecyl sulfate.47 Therefore,

PAGE 28

28 knowledge of the surfactant micellar stability as a function of concentration is necessary to predict the droplet. 1.2.2 Viscosity of Emulsions The viscosity, or resistance to flow, of emulsi ons could be considered as one of their most important properties. This is true from both a pr actical and a theoretical viewpoint. In a practical sense, certain cosmetic or even food emulsions are only desirable at a sp ecific viscosity (e.g. lotions, milk, salad dressings, etc.). Manipulatio n of emulsion viscosity to achieve the desired product specifications is not a tr ivial matter. From a theoretical perspective, the viscosity measurements can be used to provide insightful information about the st ructure and possibly the stability of an emulsion. The overall emulsion stability is a ffected by the following factors:44, 48 Viscosity of the external phase Concentration (i.e., volume frac tion) of the internal phase Viscosity of the internal phase Nature of the emulsifiers Surface viscoelasticity of th e interfacial film formed at the oil/water interface Droplet size distribution 1.2.3 Determination of Emulsi on Type (O/W or W/O) Emulsions consist of a disperse d phase and a continuous phase. Most often the dispersed phase is present as spherical dr oplets within the continuous phas e. Most emulsions follow the Bancroft49, 50 rule for emulsions, which states that th e phase in which the surfactant is most soluble becomes the continuous phase in an emulsion. There are several methods commonly used to determine which phase is continuous and which phase is dispersed. These methods utilize the property differences between oil a nd water to determine phases. The methods are listed in Table 1-2.

PAGE 29

29 1.2.4 Emulsion Stability As mentioned previously, one of the most impo rtant parameters in emulsification processes is emulsion stability. For example, milk is a natural emulsion of the O/W type in the food industry. If the stability of milk was only a week or two, the milk would have to be shaken vigorously before pouring. Ho wever, in this case nature ha s provided us with a stable emulsion. Another common example is shampoo, anot her emulsion. It would be inconvenient if the shampoo were not a stable emulsion, since shaking would be necessary. There are also cases where it is necessary to break down unwanted, naturally occurring stable emulsions. Such examples are the W/O type emulsions which bui ld up in oil storage tanks, or the O/W type emulsions that arise in effluent waters. So it becomes necessary to understand how to develop an em ulsion system to be stable or unstable, depending on the n eeds of the industry. To unders tand emulsion stability, it is important be cognizant that there are five t ypes of breakdown processes which can occur in emulsions. These are listed in Table 1-3, along with factors that influence that type of breakdown.51 1.2.4.1 Coalescence When two oil drops approach each other, a th in film of the continuous water phase is trapped between the drops. The beha vior of the thin film determines the degree of stability of the emulsion, and the rate of thinning of the f ilm determines the time required for the two drops to coalesce (i.e., coalescence rate). When the film has thinned to a critical thickness, it ruptures, and the two drops unite or coal esce to form one larger drop.52, 53 The rate of film thinning depends on the surface viscosity of the surfactant film adsorbed at th e oil/water interface. The film may drain evenly or unevenly depending on the in terfacial tension gradie nt due to adsorbed surfactant.54

PAGE 30

30 The factors that influence the rate of film thinning between droplets therefore influence the emulsion stability. A summary of all of the factors influencing coalescence of droplets is given in Table 1-4.55 1.2.4.2 Charge stabilization: The electrical double layer Breakdown of an emulsion can occur due to elect rostatic attraction be tween droplets in the dispersed phase. The attraction can be induced if it is desirable to breakdown an emulsion, or attraction can be eliminated if it is desirable to maintain a stable emulsion. To understand the concept of charge stabilization, it is necessary to understand the nature of the electrical double layer that surrounds the droplets and the factors that influence it. The electrical double layer refers to the volume around the emulsion droplets that is infl uenced by the charge on the droplet, if any. This volume can be broken down into two distinct regions, and this is illustrated in Figure 1-10. The electrical double layer is co mposed of two layers; the firs t is known as the Stern plane and is characterized by a linear drop in electrical potential, and the second is referred to as the shear plane and it is characterized by an expone ntial drop in potential. The presence of counter ions on the surface effectively neutralizes some of the surface charge on the droplet. These counter ions may be introduced in the sy stem by the presence of an electrolyte. The electrical double layer defi nes the region of influence of a droplet caused by the surface charge. If the double layer thickness, also known as the Debye length, is large for a pair of similarly charged droplets, the droplets will be electrostatically repelled. If an electrolyte is added to the system to decrease the double layer thickness, the repulsion will decrease and may be low enough that the droplets can get close en ough to be attracted to one another. This phenomenon would result in destabilization of the emulsion. Therefore, in an emulsion in which

PAGE 31

31 the droplets are charged, any a dditive or parameter change whic h influences the electrical double layer thickness will influence th e stability of the emulsion. 1.2.4.3 Phase inversion in emulsions Phase inversion can occur in emulsions due to a number of factors. For a given emulsifier concentration, the viscosity of an emulsion gr adually increases as the phase volume of the dispersed phase is increased. However, at a certain critical volume fraction c, there is a sudden decrease in viscosity, which corresponds to the point at which the emulsion inverts. c was found to increase with increasing emulsifier concentration.56 The sudden decrease in viscosity is due to the sudden reduction in disperse d phase volume fraction. Often c is in the range of 0.74, so that upon inversion, the dispersed phase volume fracti on reduces from 0.74 to 0.26, thus reducing the viscosity significantly. c should theoretically be in the range of 0.74 for spheres of equal radii to be at the maximum packing,57 but c=0.99 was found for paraffin oil/aqueous surfactant solutions58 and c=0.25 was found for olive oil/water emulsions.59 The phase inversion of emulsions can be brought about by several parameter changes, listed in Table 1-5. 1.2.4.4 Emulsion creaming As described previously, "creaming" is a spec ial case of emulsion instability that occurs when there is no change in droplet size or size distribution, but buildup of an equilibrium droplet concentration within the emulsion occurs Creaming is not so mu ch a breaking of an emulsion as it is a separation into two emulsion s, one of which is richer in the dispersed phase, the other poorer, than the original emulsion.44 The more concentrated emulsion is referred to as the cream. The creaming phenomenon can result from an external force field, such as gravitational, centrifugal, or electrosta tic. In most cases creaming is undesirable, as in pharmaceutical products and agricultural sprays wh ere the product must first be shaken in order

PAGE 32

32 to uniformly disperse the droplets again. In so me cases, however, creaming is desirable, such as in the separation of isop rene droplets from water in the rubber latex industry. Regardless, it is important to understand the physical parameters that affect the creaming, or sedimentation rate. 1.2.5 Demulsification As discussed previously in this section, it is not always desirable to have a stable emulsion. Often an emulsion is present in a system in wh ich it is undesirable. One example is the presence of aqueous emulsion droplets dispersed in crude oil. Crude oil is always associated with water or brine in oil reservoirs and also contains natural emulsifying agents, such as resins and asphaltenes. These emulsifying agents form a thick, visc ous interfacial film around the water droplets, resulting in a very stable emulsion. Therefore, demulsification is very important in the crude oil industry. Many physical methods have been developed for demulsification, depending on the industrial application. These methods are described in Table 1-6. A wide variety of chemical additives for demu lsification have been developed in recent years. These additives are all relatively hi gh molecular weight polymers capable of being adsorbed at the O/W interface and displacing the film. The primary advantage of these additives is that they can be added to the system even before emulsion formation, so that they act as inhibitors. In the petroleum industry, demulsifiers ha ve been considered for breaking the common fuel oil emulsions. In this area, chemical demulsif iers that have been investigated include ultrahigh molecular weight polyoxiranes60 and micellar solutions cont aining petroleum sulfonates, electrolytes, and cosurfactants.61

PAGE 33

33 It is evident that there are ma ny methods for demulsification. The nature of the emulsion to be separated is the key factor in determini ng which method(s) is best for each particular demulsification problem. 1.2.6 Surfactant Selection for Emulsification Often the selection of surfactants in the prep aration of either O/W or W/O emulsions is made on an empirical basis. However, in 1949, Griffin62 introduced a semi-empirical scale for selecting an appropriate surfactant or blend of surfactants. This scale, termed the hydrophilelipophile balance (HLB), is based on the re lative percentage of hydrophilic to hydrophobic groups in the surfactant molecules and ranges from 1 to 40. An HLB of 1 repres ents a surfactant that is highly oil-soluble and an HLB of 40 represents a highly water-soluble surfactant. Surfactants with a low HLB number normally fo rm W/O emulsions, whereas those with a high HLB number often form O/W emulsions.63 A summary of the HLB range required for various purposes is given in Table 1-7. The calculation of the HLB number for a given surfactant, as developed by Griffin,62 is quite laborious and requires a number of trial and error procedures. Simplification methods were later developed by Griffin62 that applied to certain surfactants. Davies63 developed a method for calculating the HLB values of surfactants directly from their chemical formulas, using empirically determined numbers. The HLB number can also be determined experi mental through several correlations that have been developed. These correlations relate the HLB number to such parameters as the cloud point,64 water titration va lue for polyhydric alcohol esters,65 and the heat of hydration of ethoxylated surfactants.66 Another method that may be used to select a su rfactant suitable for forming an emulsion is by using the phase inversion temperature (PIT) method. The phase inversion temperature (PIT) is the

PAGE 34

34 temperature at which an emulsion experiences phase i nversion, as described in a previous section. The PIT of non-ionic emulsifiers has been shown to be in fluenced by the surfactant HLB number, so the PIT can be used similarly to the HLB number in selecting an emulsifier.59 The primary distinction is that the PIT is a characteristic property of the emulsion, not of the emulsifying agent.59 Due to this, the PIT includes the effect of additives on the solvent, the effe ct of mixed emulsifiers or mixed oils, etc. In other words, the HLB number is actually a function of al l of these properties, but only the PIT completely analyzes a given emulsion system. The PIT method is useful because the PIT is a measurable property which is related to the HLB number A summary of effects of PIT a nd droplet stability from different investigations is given below:59 The size of emulsion droplets depends on the temperature and the HLB of emulsifiers The droplets are less stable towa rd coalescence cl ose to the PIT Relatively stable O/W emulsions are obtained wh en the PIT of the sy stem is some 20 to 65C higher than the storage temperature A stable emulsion is obtained by rapi d cooling after formation at the PIT The optimum stability of an emulsion is rela tively insensitive to changes of HLB value or PIT of the emulsifier, but instability is very sensitive to the PIT of the system The stability agains t coalescence increases ma rkedly as the molar ma ss of the lipophilic or hydrophilic groups increased When the distribution of hydrophilic chains is broad, the cloud point is lower and PIT is higher than when there is a narrow size distribution. The PIT can be measured by the following methods: 1) direct visual assessment,67 2) conductivity measurement,34, 68, 69 3) Differential Thermal Analysis (DTA) or Differential Scanning Calorimetry (DSC),70 and 4) viscosity measurement.71, 72 Both the HUB and PIT methods for selecting an emulsifier in a system have been wi dely used and adapted to meet industry needs. 1.2.7 Applications of Emulsions Emulsions are desirable for many different a pplications because they provide a system having a large interfacial area. Historically, cosmetic emulsions are the oldest class of manufactured emulsions.44 Emulsions are desirable for cosmetic applications because: 1) they increase the rate and extent of penetration into the skin, 2) they open up the possibility of

PAGE 35

35 applying both waterand oil-sol uble ingredients simultaneously (e .g. deodorants), and 3) they provide for efficient cleansing. Emulsions are also widely used for pharmaceutic al applications in the form of creams or ointments and as drug delivery vehicles. They are also ideal for use as polishes (e.g. furniture polishes, floor waxes, etc.) paints, and agricu ltural sprays. Many foods are manufactured in the form of emulsions including ma yonnaise, salad dressings, milk, and margarine. Another industry where emulsion technology is important is the as phalt industry when the principal requirement is the production of water-repellent su rfaces. Emulsions are also used as polymerization vehicles to aid in the production of high polymer ic materials such as plastics, synthetic fibers, and synthetic rubbers. These are just a few of th e many applications of emulsions. 1.3 Microemulsions A microemulsion is a thermodynamically stab le, isotropic dispersion of oil and water containing domains of nanometer dimensions stabil ized by an interfacial film of surface-active agent(s).73 The term microemulsion originated from Jack H. Schulman and coworkers in 1959,74 although Hoar and Schulman originally desc ribed water-in-oil microemulsions, which they referred to as transparent water-in-oil dispersions, in 1943.75 As implied above, microemulsions may be of the oil-in-water (O /W) (see Figure 1-11) or the water-in-oil (W/O) type depending on conditions of the system and system components. According to Bancroft,49, 50, 76 phase volume ratios are less important in the determination of the microemulsion type that will be form ed (i.e., W/O or O/W) than the surfactant characteristics (e.g. HLB). As previously menti oned, the Bancroft rule states that whichever phase the surfactant has a greater affinity for will typically be the continuous phase.

PAGE 36

36 The creation of a microemulsion entails the ge neration of a huge inte rfacial area, which, according to the following equation,77 requires a significant lowering of the interfacial tension (usually << 1 mN/m):78 W = A (1-7) where W is the work performed, is the surface or interfacial tens ion at the air/water or oil/water interface and A is the change in surface or interfacial area. This ultr a-low interfacial tension in spontaneously formed microemulsio ns is achieved by the incorporati on of surfactant(s) (typically a surfactant + a cosurfactant, especia lly when ionic surfactants are used).17 Figure 1-12 shows the thermodynamic explanation for the behavior of macroand microemulsions. As can be concluded from the graph, there is an optimum ra dius for microemulsion systems where the free energy of dispersion becomes negative, there by making the microemulsion stable and its formation energetically favorable.79 Schulman and others first noticed microemuls ion systems in 1943 when they observed that the addition of a medium chain-le ngth alcohol made a coarse macr oemulsion that was stabilized by an ionic surfactant become transparent.75 Even then, Hoar and Schulman recognized the important role of a very low interfacial tens ion in causing spontaneous emulsification of the added water in oil.75 They concluded that the role of the alcohol is as a stabilizer against the repulsive electrostatic forces that the ionic surfactant head groups would experience. Schulman and others used a variety of expe rimental techniques (e.g. X-ray diffraction,74 ultra-centrifugation,80 light scattering,74 viscosimetry,81 and nuclear magnetic resonance (NMR)82, 83) to elucidate some of the characteristic s of these microemulsion systems following the groundbreaking work of Schulman and Hoar. Th ese studies were instrumental in providing them with information about the structure, size, and interfacial film beha vior of microemulsions.

PAGE 37

37 They were able to determine the size of the droplets and they found th at the presence of the alcohol within the system led to greater interfacial fluidity. Later, in 1967, Prince74 proposed a theory that the forma tion of microemulsions was due to the negative interfacial tension that results from high su rface pressure of the film. Prince explained this negative interfaci al tension based on the depressi on of the interfacial tension between the oil and water phase that occurs when surfactant is added. Th e principle behind this theory is described by the series of equations that follow. The surf ace pressure of th e film at the air/water interface, aw, is defined as:17, 74 s o aw (1-8) where o is the surface tension of the pur e surface (without surfactant) and s is the surface tension of the surface with surfactant. In the case where oil is the second phase (oil/water system), the surface pressure of the surf actant film at the oi l/water interface, ow, can be defined as: s w o o w o ow/ / (1-9) where ( o/w)o is the interfacial tension of the pure oil/water interface (i.e., in the absence of surfactant) and ( o/w)s is the interfacial tension of the oil/water interface in the presence of surfactant film. Rearrangemen t of Equation (1-9) gives: ow o w o s w o / / (1-10) Based on Equation (1-9), for a surfactant film that can generate a very high surface pressure ( ow), the interfacial tension of the surf actant film at the oil/water interface ( o/w)s becomes negative. This is only a transient phenomenon because generation of a negative

PAGE 38

38 interfacial tension leads to a ne gative free energy of formation of the emulsion, which is an unstable situation. This is illustrated by the following equation: Gform = A T Sconfig (1-11) where A is the increase in interfacial area, Sconfig is the configurationa l entropy of the droplets of the liquid that are formed and T is the absolute temperature.84 The negative interfacial tension accounts for the spontaneous incr ease in interfacial area that occurs in the formation of microemulsions. When a transient (unstable) ne gative interfacial tensi on is experienced, the system will seek to stabilize by spontaneously ge nerating new interfacial area, thereby raising the interfacial tension back to acceptabl e, stable limits. As previously mentioned, in order to form microemulsions, it is required that the concentrat ion of surfactants be greater than that required to reduce the oil/water interfacial tension to zero and to cover the total interfacial area of all dispersed droplets. The transient negative interfacial tension that is generated facilitates the spontaneous break-up of droplets. The nature of the thermodynamic stability of microemulsions has long been studied. The stability can be attributed to the fact that the interfacial tension is low enough that the increase in interfacial energy accompanying di spersion of one phase in the other is outweighed by the free energy decrease that is associated with the entropy of dispersion.79 Furthermore, the free energy decrease that accompanies adsorption of surf actant molecules from a bulk phase favors the existence of a large interfacial area and hence pl ays a major role in stabilizing microemulsions.79 One must also understand the role of cosurf actants in microemulsion formulations. The addition of a short-chain alcohol to a surfactant soluti on to enhance microemulsion formation has long been practiced.85 This cosurfactant (short-chain alcohol) serves to 1) fluidize the interface, 2) decrease interfacial viscosity, 3) destroy the lamellar liquid crys talline structures, 4) provide

PAGE 39

39 additional interfacial area, 5) re duce electrical repulsi on between droplets and also between polar head groups of surfactants by acting as charge screeners and decreasing surface charge density, and 6) induce the appropriate curvature changes.85 In 1972, Gerbacia and Rosano86 investigated the forma tion and stabilization of microemulsions, with emphasis on the role of th e cosurfactant (in this case, pentanol). They suggested that a critical aspect of the mechanism of formation of microemulsions involves the diffusion of the pentanol through the interface. This process has been found to be a necessary, but not sufficient condition for micr oemulsification. In essence, th e alcohol transiently lowers the interfacial tension to zero as it diffuses thr ough the interface, thereby inciting the spontaneous dispersion of one phase into the other. The surf actant then acts to stabil ize the system against coagulation and coalescence. NMR data and calc ulation of free energies of adsorption of the pentanol into the interface have proven that a strong associat ion between the surfactant and cosurfactant is not necessary for microemulsion formation.86 The stability of the phases of surfactant in microemulsions results from the competition between entropic and elastic c ontributions to the free energy.87 The two intrinsic parameters that ultimately affect the structure of the aggregat es existing in solution are the mean bending modulus, and the Gaussian bending modulus, The bending energy, Fb, is directly related to the mean bending modulus and the Gaussian bending modulus, as can be seen below: 2 1 2 0 2 12 2 1 C C C C C Fb (1-12) where C1 and C2 are the local principal curvatur es of the surfactant layer and C0 is the spontaneous curvature. Bellocq88 states that the contribution of the bending energy to the total free energy is a crucial determining factor in the type and characteris tic size of the structure. In Equation (1-12) above, the first term represents the amount of energy needed to bend a unit area

PAGE 40

40 of interface by a unit curvature am ount, and the second term is im portant to the change of the membrane topology and the phase transition.88 The spontaneous curvature, C0, is determined by the nature of the interactions between the su rface-active molecules; i.e., the competition in packing of the polar heads and the hydrocar bon tails of the surfact ant. If the dominant interactions are between the polar heads, then the surfactant orientation will be concave to water and a water-in-oil microemulsion will be formed, whereas, if the dominant interactions are between the hydrocarbon tails, th e surfactant orientation will be convex to water and an oil-inwater microemulsion will be formed. The addition of cosurfactan t (short-chain alcohol) can have a profound effect on the curvature. Bellocq reported that there is a ve ry efficient lowering of the bending constant, of the surfactant film with the dilution of the system with short-chain alcohols.88 This lowering of was attributed to thinning of the interface, and this attributio n was confirmed with deuterium solid-state NMR. The chain length of the cosurf actant (alcohol) was found to be critical in microemulsion formation. The chain length determines what types of phases (bicontinuous, lamellar, sponge, vesicle, etc.) will be of importance. These phases pl ay an important role in the type, structure and size of the microemulsion that will be formed.89 In simplistic terms, a short-chain cosurfactant acts to prevent the formation of or destroy lame llar liquid crystals. Since there is a significant difference between the surfactant ch ain length and the short-chain alcohol, there is a tail wagging effect due to the fact that the excess hydrocarbon tails have more freedom to disrupt molecular packing through conformational diso rder, increased tail motion, a nd penetration and/or buckling of the chain into the monolayer.90 This motion of the hydrocarbon tail prevents or disrupts

PAGE 41

41 ordering of the molecules and therein prevents fo rmation of or destroys lamellar liquid crystals and enhances microemulsion formation. It must be noted that cosurfactant addition to microemulsion-forming systems is typically only applicable for ionic surfactant systems. Microemulsions that incorporate non-ionic surfactants may be formed without the need for cosurfactant additi on; especially in the case of non-ionic surfactants of the polyeth ylene oxide adducts (POE). This is because these surfactants are composed of a homologous series of varying composition and molecular weight,17 which serves the same purpose of enhanc ing the interfacial film fluidity. The important factor in the formation of these types of microemulsions is te mperature, because this class of materials is solubilized in water by means of hydrogen bonding between the water molecules and the POE chain. Hydrogen bonding is a temperature-se nsitive phenomenon, which decreases with increasing temperature. Therefore, the temper ature conditions under which a microemulsion is formed are important to the type of microemu lsion that is formed. Above a characteristic temperature, which is commonly known as the phase inversion temperature (PIT),91 the nonionic surfactant changes its affinity from the wa ter phase to the oil phase. Below the PIT, O/W microemulsions will be formed and above th e PIT, W/O microemulsions will be formed. 1.3.1 Formation of Microemulsions Now that the some of the basic principles of microemulsions have been discussed, it is easier to understand what conditi ons must be met for microemulsion formation and why they are required. There are three major factors that must be considered in order to form microemulsions.59 First, given the importance of achieving an ultra-low surface tension at the oil/water interface, surfactants must be carefully chosen so that this may be accomplished. Secondly, there must be a large enough concentratio n of surfactants to stabilize the newly formed interface such

PAGE 42

42 that phase separation does not occur. It must be mentioned that th e type, structure and characteristics (e.g. Hydrophilic-Lipophilic Balan ce (HLB), degree of ioni zation, etc.) of the surfactants may potentially play a major role in determining just how high the surfactant concentration needs to be to stabilize the in terface. The third required condition for forming microemulsions is that the interface must be flui d (flexible) enough to faci litate the spontaneous formation of micro-droplets with a small radi us of curvature (50 500 ). That is where cosurfactant structure can become very important. 1.3.2 Applications of Microemulsions Microemulsions have a range of industrial app lications. They are useful in technologies such as enhanced oil recovery,92 pharmaceuticals,93 cosmetics,94, 95 food sciences,96 and detergency.97, 98 Microemulsions have been extensively studied in regards to their use as drug delivery vehicles, and are now gaining attention as possible mediums for use in detoxification of blood to remove free drug from th e blood stream of overdose patients.99 Drugs that have significant hydrophobic functiona lity have been shown to partition into the corona (area at the interface) and/or interior core of O/W microemulsions.99 It is generally accepted that this is largely due to hydrophobic interactions. Recall th at the formation of microemulsions leads to the generation of a large in terfacial area. It is believed that this large interfacial area faci litates the uptake of relatively large amounts of drug into the microemulsion in a time efficient manner, thereby significantly decreasing the bulk drug concentration. 1.3.3 Nano-emulsions As early as 1943, Dr. T. P. Hoar and J. H. Schulman75 discovered that he could prepare transparent water-in-oil dispersi ons of nanometer size, which displayed stable thermodynamic characteristics (i.e., the water droplets remained st ably dispersed indefinitely if left unperturbed, with respect to temperature, pressure, or co mpositional conditions). Anomalous systems could

PAGE 43

43 also be prepared in which oil droplets are disper sed in water. Later, other scientists, including Dr. Stig Friberg,89 found that they could develop transpar ent nanometer size dispersions of one medium within another continuous medium, whic h were not thermodynamically stable, but were kinetically stable (i.e., given time, there will be a breakdown in the stability of the dispersion that will lead to phase separation. Such systems have come to be referred to as nano-emulsions (thermodynamically unstable systems). Thus, both Schulmans microe mulsions and nanoemulsions start in the nanometer range, but th e microemulsions are thermodynamically stable and maintain the same size whereas the nano-em ulsions coalescence and display an increase in size with time, which ultimately causes phase separation. In order to sufficiently comprehend the di fference between microemulsions and nanoemulsions, there must be clarification of the nom enclature of the two. As previously mentioned microemulsions is a misleading title consideri ng that their average diameter ranges from 10 100 nm. Light scattering and X-ray analysis have indicated that microemulsions are, in fact, coarse mixtures as opposed to molecular dispersions.78 Nano-emulsions lie within the same size range, but may have diameters that considerably exceed the size of a microemulsion droplet (20500 nm). The emphasis must be placed on the fact that nano-emulsions are in fact emulsions of nano-size, meaning that they are kinetically st able, (thermodynamically unstable) heterogeneous systems in which one immiscible liquid is disper sed as droplets in anot her liquid (as emulsions are defined). Although they are on similar size scales, mi croemulsions and nano-emulsions have markedly different characteristics (see Table 1-8). Nano-emulsions are formally defined as thermodynamically unstable, generally opaque, submicron-sized (20 500 nm) systems that are

PAGE 44

44 stable against sedimentation and creaming.100 They may have the appearance of microemulsions, but they do not necessarily require as much su rfactant concentration in their preparation.100 As early as 1981, Rosano et al.86 found that certain microemulsion systems, which they termed unstable microemulsions, displayed significantly different characteristics from traditional microemulsions. These systems were dependent upon the order of addition of components (i.e., the mixing protocol) and their formation was contingent upon having a large enough concentration gradient to allow diffusion of amphipathic materials across the oil/water interface. El-Aasser et al.101 performed studies of a miniemulsi fication process, which produced O/W miniemulsions (another term for what we refer to as nano-emulsions in this paper) having an average droplet diameter in the size range of 100 400 nm. The miniemulsions that they produced were generated by means of a mixed em ulsifier protocol, which was comprised of a mixture of ionic surfactant and long-chain fatty acid in concentrations of 1-3 % by weight in the oil phase. They stated four distinct and si gnificant differentiati ng aspects between the necessary conditions of preparation for mi niemulsification systems and traditional microemulsion systems, which may also be produced by mixed emulsifier systems: 1) Concentration of the mixed emulsifier system : only 1 wt% (with respect to the oil phase) is sufficient for the formation a nd stabilization of miniemulsions, whereas microemulsions typically require 15-30 wt %. 2) Droplet size : their miniemulsion droplets range from 100-400 nm in diameter, as opposed to microemulsions, which ra nge from 10-100 nm in diameter.

PAGE 45

45 3) Chain length of the cosurfactant (fa tty alcohols or acids in this case) : miniemulsions require at least a 12-carbon chain length, whereas microemulsions can be prepared with alcohols of shorter chain lengths. 4) Order of mixing of the ingr edients (mixing protocol) : successful production of miniemulsions requires that the ionic surf actant and the fatty alcohol be initially mixed in the water phase for 30 minutes to 1 hour at a temperature above the melting point of the fatty alcohol, prior to the addition of the oil phase, whereas order of mixing does not affect microemulsion formation. El-Aasser et al.101 performed an array of experiments to help them to better understand the miniemulsification process. They found that the process was a spontaneous phenomenon based on results yielded by both dynamic and static sp inning drop experiments. The oddity in this discovery lay in the fact that the measured interfacial tensions were unexpectedly high, ranging from 5 to 15 dynes/cm. They attributed this fi nding to the formation of emulsion droplets by diffusion of the oil (in this case, styrene) from drops into the adjacent liquid crystal structure of the mixed emulsifier system. They also stated that the presence of mixed emulsifier liquid crystals, which was confirmed by birefringe nce observations, significantly improved the emulsification process and led to greater emulsion stability. They concluded that the mechanism of formation of miniemulsions wa s by swelling of the mixed emulsi fier liquid crystals by oil (in this case, toluene). This swelling of the liquid crystalline structure led to its break-up or subdivision to form small emulsion droplets, which were stabilized by the adsorption of the mixed emulsifier complex at the oil-water interface.101 Although emulsion stability is generally known to increase with droplet surface charge, the miniemulsions that El-Aasser prepared displayed contradictory behavior; th eir stability increase

PAGE 46

46 corresponded to a reduction in su rface charge. These results sugge sted that the steric component of stabilization was the dominating factor.101 The mechanism that has been attributed to nano-emulsion breakdown in a ternary system of brine, oil, and non-ionic su rfactant is a 3-stage process.102 The first and last stages of the droplet growth process were found to be due to Ostwald ripening, whereas the droplet size distribution of the second stage became too broad compared to the expected theoretical distribution (as predicted by the Lifshitz-Slezov-Wagner theory) to be due to Ostwald ripening. Katsumoto et al.102 made this assessment after plotting the cube of the z-average radius, rz, as a function of time, and obtaining a linear relations hip. In addition, thei r group found that the volume of bound water on miniemulsion droplets plays an important ro le in obtaining a homogeneous miniemulsion.32 The storage temperature of th e miniemulsion solution and the molecular weight of the surfactant were determined to significantly affect the systems stability: as storage temperature is decreased, the rate of coalescence increases, and as the molecular weight of the surfactant is increased, the rate of coalescence decreases. The former finding is due to an increase in surface tensi on because non-ionic surfactants become more water-soluble as temperature is decreased. The latter finding is due to steric effects that become predominant as the surfactant size is increased. Nano-emulsions may be formed by a few diff erent experimental methods: condensation,103 low-energy emulsification met hods involving phase inversion,100, 104 or by high energy input during emulsification.105 Forgiarini et al.100 reinforced the concept that was proposed by ElAasser101 concerning the importance of the mixing pr otocol in the formation of nano-emulsions. They found that they only obtained dispersions of nanometer droplet size when the nano-

PAGE 47

47 emulsion was formed via stepwise addition of water to a solution of the surfactant in oil. If other methods of addition were used, th e droplet size ra nged from 6 10 m. Izquierdo et al. 104 analyzed the formation and stabili ty of nano-emulsions that were prepared by the phase inversion te mperature (PIT) method, in which emulsions were formed at a temperature near the PIT and then rapi dly cooled to room temperature (25 C) by immersion in an ice bath. They proposed that a change in the interfaci al curvature is crit ical to nano-emulsion formation, and that the presence of lamella r liquid crystals was probably a necessary, but insufficient requirement for pr eparation of nano-emulsions.100 They concluded that the key factor for nano-emulsion formation should be credited to the kinetics of the emulsification process. Nano-emulsions have possible applica tions as drug delivery vehicles,105, 106 in drug targeting, as reaction media for polymerizati on, in personal care and cosmetics, and in agrochemicals. It is also plau sible that nanoemulsions may be used in most industries where extraction will ultimately be required. This idea is based on the premise that the nanoemulsions may be designed so that their st ability characteristics will coincide with the requirements of the application.

PAGE 48

48 Figure 1-1. Schematic diagram of a surfact ant molecule, micelle, and reverse micelle. Figure 1-2. Properties of surfactant solutions showing abrupt change at the solution critical micelle concentration (cmc). Hydrophobic head Hydrophilic head Micelle Reverse micelle Surfactant molecule

PAGE 49

49 Figure 1-3. Schematic design of micellar solution showing the three major species that are in dynamic equilibrium: 1) monomers, 2) micelles, 3) adsorbed film. Figure 1-4. Schematic diagram of the four majo r micellar structures: A) spherical micelle, B) cylindrical, rod-like micelles, C) unilamellar vesicle, D) lamellar micelle. Micelle Monomer Adsorbed film Water A B C D

PAGE 50

50 Figure 1-5. Mechanisms for the two characteristic rela xation times for a micelle in a surfactant solution, 1 and 2, above cmc. Figure 1-6. Typical size di stribution curve of aggregates in a micellar solution according to the Aniansson-Wall model of stepwise micella r association. Region (I) corresponds to monomers and oligomers; Region (III) to abundant micelles with a Gaussian distribution around the mean aggregation number; and Region (II) to the connecting wire (heat transfer analogy) or tube (ma ss transfer analogy) between Regions (I) and (III). + + Fast relaxation time, microseconds Slow relaxation time, milliseconds to minutes + I II III Aggregation number, n C (An) 2 n

PAGE 51

51 Figure 1-7. Schematic of sodium counter-i on cloud around SDS spherical micelle. Figure 1-8. Schematic diagram of the adsorption of surfactant monomers from the bulk to the oil/water interface during emulsion formation Figure 1-9. The emulsion droplet size in th e hexadecane/SDS solution system after 30 s emulsification at: (A) 50 mM, (B) 100 mM, (C) 200 mM, (D ) 300 mM, and (E) 400 mM Na + Na + Na + Na + Na + Na + Na + Na + Na + Na +

PAGE 52

52 Figure 1-10. Schematic depiction of the Stern-Graham model of the electr ical double layer. Figure 1-11. Schematic diagram of an oil-in-water (O/W) microemulsion + + + + + + + + + + + + + + + + + + + distance shear plane Counter Ion Co-Ion Stern plane Charged surface WATE R OIL

PAGE 53

53 Figure 1-12. Thermodynamic explanation for beha vior of macroemulsions and microemulsions R* Droplet radius G of dispersionMacroemulsions (IFT = 1 mN/m) Microemulsions (R* = 10-100 nm, IFT 10-3 mN/m) + 0

PAGE 54

54 Table 1-1: Summary of methods used to produce emulsions Method Related to Method Drops mainly disrupted bya Energy densityb Mode of Operationc Restrictionsd 1. Shaking 4a CD L B N 2. Pipe Flow a. Laminar 5 V L-M C V b. Turbulent 4a T L-M C N 3. Injection lOa L C 4. Stirring a. Simple stirrer l,2b T,V L B,C b. Rotor-stator (5) T.V M-H B,C c. Scraper 5 V L-M B,C V d. Vibrator 8a ? L B,C N 5. Colloid Mill 2a,4c,6 V M-H C V 6. Ball and roller mills 5 V M B(C) V 1. High-press. Homogenizer (2b) T,C,V H C N 4d C,T M-H C W 8. Ultrasonic a. Vibrating knife b. Magneto-striction C M-H B,C W 9. Electrical lOb Elec. Charge M B(C) Several 10. Aerosol to liquid a. Mechanica l 3 L -M B C # b. Electrical 9 M B,C Several 11. Foaming or boiling Spreading L-M (W) 12. Condensation Several a V=viscous forces in laminar flow, T=turbulence, C=cavitation b L=low, M=moderate, H=high c B=batch and C=continuous d The continuous phase should be V=vi scous, N=not too viscous, W=aqueous

PAGE 55

55 Table 1-2: Common tests for determining emulsion type (W/O or O/W)44 Name Description Dilution Test Without shaking, a drop of oil is placed in the emulsion. If W/O type, the a dded oil dissolves rapidly in the emulsion and disappears. If O/W type, the added oil floats on top of the wa ter continuous emulsion. Electrical Conductivity Test Electrical conductivity is measured. If W/O, conductivity is low (like oil) If O/W, conductivity is high (like water). Viscosity Test Viscosity is measured while a few drops of water are added to the emulsion. If W/O type, added water increases viscosity by adding more droplets to the dispersed phase. If O/W type, added water decreases viscosity (or shows little change ) by slightly diluting the continuous phase. Refractive Index Test A parallel beam is shined through an emulsion of low turbidity. If the beam converges, an O/W type emulsion is present. If it diverges, it is W/O type. This is due to the relative refractive indices of oil and water.107 Dye Solubilization Test Without shaking, a few drops of water soluble dye are added. If O/W type emulsion, the dye dissolves rapidly in the continuous phase, changing color. If W/O type, the dye dissolves very slowly in the dispersed phase, and most will sink through the emulsion. Filter Paper Test Filter paper is dipped into the emulsion. If O/W type, paper turns pink immediatel y from being wetted. If W/O type, paper stays dry (blue) for a long time since the continuous phase does not wet it.

PAGE 56

56 Table 1-3: Types of breakdown pr ocesses occurring in emulsions Breakdown Type Description No change in droplet size (or size distribution) Buildup of an equilibrium dr oplet concentration gradient within the emulsion. This phenomenon results from external force fields, usually gravitation, cent rifugal, or electrostatic, acting on the system. "Creaming" is a special case in which the droplets collect in a concentrated layer at the top of an emulsion. No change in basic droplet size, but with buildup of aggregates of droplets in the emulsion This process is called "flocculation" and results from the existence of attractive fo rces between the droplets. Flocculated droplets in an aggregate coalesce to form larger droplets This process also occurs wh en creaming or sedimentation results in a close-packed array of droplets and these droplets coales ce. The limiting state is the complete separation of the emulsion into two immiscible bulk liquids. Average droplet size increases due to the two liquids forming the emulsion being not totally immiscible This process does not invo lve actual coalescence of droplets, but rather the transfer of dispersed phase across continuous phase after solubization occurs. If the emulsion is polydispe rse, larger droplets will form at the expense of smaller droplets due to the difference in chemical potential for differe nt size droplets (Ostwald ripening). In principle, th e system will tend to an equilibrium state in whic h all the droplets have combined and are one large dr oplet, or a separated phase. Emulsion type inverts from W/O to O/W type. This is a questionable "breakdown" process since essentially another emulsion is formed. The inversion process can be brought about by numerous parameter changes, which will be discussed in detail later in this section.

PAGE 57

57 Table 1-4. Factors influenci ng the stability of emulsions Factor Description of Effect Physical nature of the interfacial film For mechanical stability, a surfact ant film with strong lateral intermolecular forces and high film elasticity is desired. A mixture of two or more surfactants is pr eferred over a simple surfactant (i.e., lauryl alcohol + sodium lauryl sulfate). Electrical barrier Significant only in O/W type emul sions, because of conductivity in continuous phase. In the case of n onionic emulsifying agents, charge may arise due to adsorption of ions from the aqueous phase. The repulsion or attraction can be influenced by changing th e thickness of the double layer, which is described below. Viscosity of the continuous phase or of emulsions A higher viscosity reduces the diffusion coefficient of the dispersed droplets, resulting in reduced frequency of collision and lesser coalescence. Viscosity can be increased by adding natural or synthetic thickening agents. Viscosity also increases as the number of droplets increases; so many emulsions are more stable in concentrated form than when diluted. Size distribution of dispersed droplets Uniform size distribution is more stable than an emulsion with the same average droplet size but ha ving a wider size distribution. Phase volume ratio As volume of dispersed phase increases, stability decreases Phase inversion can occur if dispersed phase volume is increased enough. Temperature Usually, as temperature increases, emulsion stability decreases because of increased frequency of co llision. Steric barrier Addition of polymer that adsorbs at interface can influence stability. Polymer chains can prevent coalescence due to bulkiness, but they can also enhance flocculation and decrease stability.

PAGE 58

58 Table 1-5. Parameters that affect phase inve rsion in emulsion and the effect they have. Parameter Effect on phase inversion Order of phase addition W O + emulsifier W/O O W + emulsifier O/W Nature of emulsifier Bancroft's Rule Making the em ulsifier more oil soluble tends to produce a W/O emulsion and viceversa Phase volume ratio Oil/Water Ratio increased in an O/W emulsion W/O emulsion and vice-versa, as described above in the text Phase in which emulsifying agent is dissolve d If surfactant can be dissolved at leas t partially in either water or oil Bancroft's Rule If surfac tant is dissolved in water O/W emulsion Temperature Depends on the surfactant and its temperature dependence. If emulsion is O/W type with polyoxye thylenated nonionic surfactant, phase inverts to W/O with increase in temperature due to increased hydrophobicity of the surfactant. Addition of electrolytes Strong electrolytes (polyvalent Ca ) added to O/W (stabilized by ionic surfactant) inversion to a W/O type Because of decrease in double layer thickness around oil droplets, droplets coalesce and become the continuous phase.

PAGE 59

59 Table 1-6. Commonly used physical methods of demulsification Method Desription Centrifugal separation108 Basic centrifugation techniques are applied to separate emulsions of either O/W or W/O type. Centrifugal methods are advantageous when: 1. Viscosity of continuous phase is not too high 2. Droplets are above a certain minimum size dependent on the viscosity 3. Density difference between continuous and dispersed phase is low Gravitational settling Inexpensive, simple process in which time is the only parameter Only useful for larger size droplets, and usually used only after one of the other methods has formed large droplets Filter coalescence, Ultrafiltration Filter with large surface area is used to collect droplets until enough droplets combine to form a large droplet, which breaks and moves downstream Wetting of the fibers by the coalescing dispersed phase is desirable Fiber bed thickness is not a factor, as coalescence occurs on the front face of the fibers only Gently shaking or stirring More efficient than gravitational settling, but not very efficient for small drops Ultrasonic vibrations Low intensity of vibration is necessary for coalescence of droplets High intensity would cause loosely held floccules to separate Granular bed floatation Emulsion passes through a granular bed which coalesces droplets May be either gravity or pressure driven process Common filters include anthracite coal, sand, and gravel May have a combination of beds with different filter material at each stage Electrically induced Widely used on large industrial scale due to low cost of electricity Works on both O/W and W/O type emulsions For O/W, droplets move towards electrode of opposite charge and coalesce For W/O, electric field indu ces a charge on the droplets which causes them to collide and eventua lly coalesce at the oppositely charged electrode Since O/W has a high conductivity continuous phase, ch arge dissipates rapidly but droplets attract due to rapid travel of charge through the medium For W/O with a low conductivity continous phase, droplets hold charge for a long period of time which allows time for droplets to travel to the electrode In gene ral, W/O type emulsion coalescence is faster than O/W coalescence Heating Heating above 70 C will rapidly break most emulsions Coalescence is increased at higher temperatures due to diffusion of droplets Freezing Freezing of water droplets in a W/O emulsi on will cause ice to form, which expands and breaks the film + oil envelope. Requir es repeated freezing and thawing due to elasticity of oil envelope Generally un economical due to apparatus for repeated freezing and thawing Table 1-7. A summary of HLB ranges and their application HLB Range Application 3 to 6 W/O emulsifie r 7 to 9 Wettin g a g en t 8 to 18 O/W emulsifie r 13 to 15 Deter g en t 15 to 18 Solubilize r

PAGE 60

60 Table 1-8. Microemulsions vs. Nano-emulsions. Characteristic Microemulsions Nano-emulsions Stability Thermodynamically stable Thermodynamically unstable Droplet size 10 100 nm 20 500 nm Surfactant Concentration Usually require 10 30 wt% surfactant Can be formed with 4 8 wt % surfactant Formation Independent of mixing protocol Depends on mixing protocol

PAGE 61

61 CHAPTER 2 A NOVEL METHOD TO ELUCIDATE TH E PRESENCE OF SUB-MICELLAR AGGREGATES IN SURFACTANT SOLUTIONS 2.1 Introduction It is generally believed that surfactant molecu les in micellar solutions exist in equilibrium in three states: 1) as surfactant molecules that ar e adsorbed at the interface, 2) as monomers that are dispersed in the aqueous phase, and 3) as micellar aggregates.1 Little, if any, attention has been given to sub-micellar aggregates, which ma y in fact be a signifi cant fourth state of existence, particularly in micellar solutions with short relaxation times (< 100 msec). Given that micellar solutions are used in va rious technological and biological processes, one must consider the potential impact of the often overlooked sub-mice llar aggregates. It has been shown that the monomeric fo rm of surfactants disp lays significantly diffe rent properties in solution when compared to micelles, especially when considering the disruption of biomembrane structures and when using micelle s for solubilization of proteins.109, 110 Midura and co-workers111 illustrated how one might manipul ate the relative concentrations of monomeric versus micellar form s of various surfactants. They have shown that the monomeric concentration can be determined by filtering th e surfactant solution through ultracentrifuge tubes having a nanoporous membrane with a nominal mol ecular weight cutoff that is smaller than the combined molecular weight of the surfactants in the micellar aggregate (see schematic diagram in Figure 2-1). They have also shown that the cr itical micelle concentra tion (cmc) of a surfactant can be determined by means of ultrafiltration.111 However, they did not acknowledge the contribution of sub-micellar aggregates, which will conceivably play a role in such processes as biomembrane disruption (hemolysis), solubi lization of hydrophobic molecules in aqueous solutions and in drug delivery via micelles.

PAGE 62

62 When one considers the dynamic nature of micelle s, it is conceptually obvious that at any given time, there must be sub-micellar aggregat es present in solution in significantly large quantities, particularly if mi celles break and form rapidly (i .e. relaxation times less than 100 msec). If micelles were infinitely stable, then one would only see the exis tence of monomers and micelles in the solution. But if the micelles become more and more unstable, the concentration of sub-micellar aggregates must increa se in the solution. In order to prove this hypothesis, we have performed ultrafiltration experi ments of sodium dodecyl sulf ate (SDS) surfactant solutions followed by analysis of the filtrate by th e dye complexation method to determine the concentration of SDS. We have taken ultracentr ifugation tubes having a 10,000 molecular weight cutoff (i.e. molecules or aggregates that exceed th is molecular weight, aggregates greater than 34 molecules, will not pass through the filter), a nd measured the filtrate concentration for SDS concentrations ranging from 1 mM to 100 mM. Gi ven that SDS has a molecular weight of 288.34 grams/mole and an aggregation number of ~65 molecules/micelle, micelles definitely will not pass through the ultracentrifugation membrane filter. If the notion that micelles exist in only three equilibrium states is true, then one would expect the concentration of SDS in the filtrate to increase as a function of total SDS concentration up to the critical micelle concentration (cmc), and then for the SDS concentr ation in the filtrate to remain constant with respect to increasing to tal SDS concentration beyond the cmc. If sub-micellar aggregates are a significant entity (i.e. a fourth equilibrium stat e) in the micellar phenomena, one might expect that as the total concentration of SDS exceeds th e cmc, the concentration of SDS in the filtrate will increase, albeit at a different slope (lesser) than the pre-cmc slope due to the presence of sub-micellar aggregates that are made up of 34 molecules or less (whi ch may pass through the 10,000 MWCO ultracentrifugation filter membranes).

PAGE 63

63 Here, we present the results of our findings which support the exis tence of sub-micellar aggregates and allude to their significance in technological and bi ological processes. To take our hypothesis one step further, we examine the effect of micellar dynamics on sub-micellar aggregate concentration by stabiliz ing SDS micelles with dodecanol (C12OH) and dodecyltrimethylammonium bromide (C12TAB), (as shown by their relaxation times2), and by changing the counter-ion of the dodecyl sulfate. 2.2 Experimental Procedure 2.2.1 Materials Ultrapure sodium dodecyl sulfat e (SDS) from MP Biomedicals, Inc. (Solon, OH) was used as received. The following chemicals were al so used without furt her purification: ndodecyltrimethylammonium bromide (C12TAB) from Tokyo Kasei Kogyo Co. (Tokyo, Japan) and 1-dodecanol (C12OH) from Acros Organics (New Jers ey). Double distilled, deionized Millipure water was used for all solutions. 2.2.2 Ultracentrifugation Centricon YM-3 and YM-10 ultracentrifuga tion filter tubes, having a 3,000 and a 10,000 molecular weight cutoff (MWCO), respectively, were purchased from Fisher Scientific. Two milliliters of SDS solutions of various concentr ations (ranging from 1 mM to 50 mM) were placed into the top portion of the ultracentrifugation tubes and subsequently centrifuged at ~2900g (10,000 MWCO) or ~4500g (3,000 MWCO) for approximately 10 minutes so that less than 10% of the total solution volume was collected as filtrate. All samples were centrifuged in a bench top IEC Clinical Centrifuge (Damon/IEC Di vision, Needham Hts, Mass). The filtrate was collected in the bottom attachment and diluted to the micromolar concentration regime for analysis by a slightly modi fied dye complexation method112 and compared to a previously prepared calibration curve.

PAGE 64

64 2.2.3 Two-Phase Dye Transfer (Methylene Bl ue Complexation) and UV-Vis Analysis Methylene blue dye, chloroform, sodium phos phate monobasic, and sulfuric acid were purchased from Fisher Scientific. Methylene blue reagent was prepared using these materials according to standard preparation procedure.113 Two milliliters of methylene blue reagent were added to two milliliters of the diluted filtrate from the ultrafiltration experiments. Two milliliters of chloroform were added and the solution wa s shaken on a Vortex mixer for approximately thirty seconds. Any SDS that was in the filtrate complexed through electros tatic interaction with the positively charged methylene blue, became o il soluble, and thereby partitioned into the chloroform organic phase. The so lution was allowed to phase separate and the organic phase was removed and placed into a separate test tube. Th is process was repeated two more times and the organic phase was then analyzed by UV-Vi sible spectrometry at 652 nm and the SDS concentration was determined upon comparison with a calibration curve. A Hewlett Packard HP 8453 UV-vis spectrometer was used for all UV-Vis analysis. SDS solutions were also pr epared with 1-dodecanol (C12OH) or n-dodecyltrimethylammonium bromide (C12TAB) at various SDS:C12X (X= OH or TAB) ratios. These solutions also underwent ultrafiltration, dye complexation, and subsequent UV-Vis analysis to determine the SDS concentration in the f iltrate. It must be noted that the dye complexation method was specific to SDS; therefor e the presence of any C12TAB or C12OH in the filtrate did not interfere with the detection of SDS to any significant degree. 2.2.4 Foamability Ten milliliter samples of 25 mM SDS, 25 mM SDS + 6.25 mM C12OH, and 50 mM SDS + 6.25 mM C12TAB were placed into 100-mL graduated cy linders and capped. Each cylinder was vigorously shaken 10 times by hand and the volume of the foam is recorded immediately after shaking. Each solution is tested at least five ti mes and the reproducibility is better than 2 ml.

PAGE 65

65 2.2.5 Fabric Wetting A commercially gained 50:50 cotton: polyester blend fabric of 1 in.2 was placed on the surface of pure water (control) or surfactant solution at 25 C. The surfactant solutions that were used were 50 mM SDS a nd 50 mM SDS + 12.5 mM C12TAB. The water or surfactant solution displaces air in the cotton surface by a wetting process and when sufficient air has been displaced, the fabric starts sinking. The reside nce time of fabric on the surface of the solution before it was completely immersed was measured as wetting time in this study. This wetting time in each solution was measured at least 5 times. 2.2.6 Dynamic Surface Tension Dynamic surface tension was measured usi ng the maximum bubble pressure technique. The pressure required to form a new bubble in solu tion is measured by a pressure transducer, and the reading is transmitted to an oscilloscope. For these experiments, the dynamic surface tension was measured for micellar solutions consis ting of 50 mM SDS and 50 mM SDS + 12.5 mM C12TAB. All dynamic surface tension measurements were taken usi ng a 22 gauge needle tip with a gas flow rate of 3 cm3/min (which corresponds to 6 to 13 bubbles per second or approximately 77 to 167 msec per bubble residence time at the needle tip). This flow rate was chosen because at higher low rates the nitrogen gas forms a continuous jet in the su rfactant solution at the needle tip. At lower flow rates, the results are si milar to equilibrium surface tension results. 2.3 Results and Discussion 2.3.1 SDS Surfactant Solutions During the 1970s, Aniansson and coworkers di scovered the existen ce of two (fast and slow) relaxation processes and deve loped a model of the kinetic pr ocess of micelle formation and disintegration.21 The first major assumption of this model was that the free surfactant monomers are assumed to be completely dissociated and the size distribution of the aggregates in a

PAGE 66

66 surfactant solution is assumed to follow the behavior that is shown by the solid line, A, in Figure 2, where monomers and micelles are the predomin ant species. The curve shown by the dashed line, B, in Figure 2-2 represents a micellar so lution where a substantial fraction of surfactant exists as sub-micellar aggregates in the soluti on. This representation suggests that the submicellar region (Region II) is larger and that the actual micellar region (Region III) is broader as compared to the generally accepte d model for micellar solutions. Anianssons model suggests that the only im portant contributing species in micellar solutions are the micelles themselves and m onomeric forms of the surfactant as shown schematically by Figure 2-3A. Over the years ma ny researchers have based their thermodynamic models of micelles on such a perspective114 (i.e. monomers and micelle s as two main species in solution). We propose here that sub-micellar aggregates are another major component of micellar solutions as shown by Figure 2-3B. If one were to filter either solution from Figure 2-3A or 2-3B through a nanoporous filter with pore size smaller than the size of micelles, then in the case of Figure 2-3A, there would only be monomers in the filtrate as reflected by Figure 2-3C; whereas, in the case of Figure 2-3B, there would be bot h monomers and sub-mice llar aggregates in the filtrate as reflected by Figure 2-3D. If the surfactant concentration in the filtrate were plotted as a function of the total surfactant concentration, in the first case, one would expect for the surfactant concentration in the filtrate to increase proportiona lly to the tota l surfactant concentration up to the cmc, and then the surfacta nt concentration in the filtrate would remain the same irrespective of the total surfactant c oncentration as shown by Figure 2-3E. If the second case were true, one would expect the same initial behavior (i.e increase of surfactant concentration in the filtrate proportional to to tal surfactant concentration up to the cmc), but

PAGE 67

67 beyond the cmc, the filtrate concentration should c ontinue to increase with a different slope due to the fact that sub-micellar aggregates (but not the micelle s) can pass through the pores (see Figures 2-3D and 2-3F). In Figure 2-3F, the plot suggests that the monome ric contribution to the filtrate surfactant concentration is represented by the region from point Q to point P, whereas the sub-micellar aggregate contribu tion is represented by the regi on from point R to point Q. The concept of sub-micellar aggregates c ontributing to micelle formation has been considered previously by Zana39 and by Kahlweit,22, 42 but only for very high (~ 25 times the cmc) concentrations. It has been well established that micelles have two characteristic relaxation times associated with them: a fast relaxation time (referred to as 1), which represents the time that it takes for one surfactant monomer to diffuse into or out of a micelle, and a slow relaxation time (referred to as 2), which represents the time that it ta kes for a single micelle to fully break down or to fully form. According to Ania nssons model, the slow relaxation time, 2, should increase with increasing surfactant concentration.21 However, it has been reported that for some ionic surfactants, such as SDS, 2 first increases, passes through a maximum, and then decreases again.38, 39, 41 This behavior in the slow relaxation process of ionic micelles is not predicted in the AnianssonWall model. Kahlweit and coworkers, using their own T -jump and p-jump results,22, 42, 43 concluded that in ionic surfac tant systems at high concentra tion, the reaction path for the formation of micelles must be different than that at low concentration. Based on this conclusion, they came up with a new model for micelle formation (Figure 2-4). This model is based upon the principle th at ionic micelles, including sub-micellar aggregates, can be considered as charged particle s. When ionic surfactant molecules such as SDS are added to water, the surfactant molecules di ssociate into negatively charged dodecyl sulfate molecules and their positively charged counter-i ons. These counter-ions are present in solution

PAGE 68

68 as a cloud surrounding the negatively charge d micelle. At low surfactant counter-ion concentration, the micelles are stable with respec t to coagulation due to repulsive electrostatic forces. Consequently they can grow only by step wise incorporation of monomers according to Anianssons model. As more and more surfacta nt is added into the system, the counter-ion concentration also increases, which compresse s the electrical double layer around the micelles and reduces charge repulsion, allowing the micelle s to come closer to each other so that attractive dispersion forces (i.e., Van der Waal s forces) lead to a re versible fusionfission coagulation according to Ak + Al Ai k + l = I (2-1) where k and l are classes of sub-micellar aggregates. Kahlweit42 then represented the micelle formation reaction path by two parallel resistors, R1 and R2 (Figure 4b), and compared the formation of micelles to the discharge of a capacitor through two parallel resistors,115 so that the change in the monomer concentr ation with time was given by 1 2122ln 11 dA dt (2-2) where 21 refers to the reaction path way corresponding to the stepwi se formation of micelles by addition of one monomer at a time, and 22 refers to the reaction pathway which corresponds to the merging of sub-micellar aggr egates to form micelles. At low surfactant concentration, and hence at correspondingly low counter-ion concentration, R2 is very high due to electr ostatic repulsion between sub-micellar aggregates, so stepwise aggregation dominates and R1 determines the rate of micelle formation. As the surfactant concentration is incr eased, the counter-ion c oncentration also increases, and hence, R1 increases as R2 decreases. The concentration where R1 equals R2 is the point where 1 = 2 passes through a minimum and 2 is highest (for the SDS micelle, this occurs at 200 mM).116 If the

PAGE 69

69 counter-ion concentration is still further increased, R1 becomes so high that R2 determines the rate of micelle formation according to the re action mechanism in Equation 2-1). Kahlweits model suggests that the concentr ation of sub-micellar aggregates in micellar solutions only becomes significantly large at very high (25 X cmc or above 200 mM) surfactant (and counterion) concentrations. Here, we pr opose that sub-micellar aggregates are present in relatively large concentrations even at lower concentrations (3 -4 times the cmc). For example, in a 25 mM SDS solution, the sub-micellar aggreg ates account for ~ 11-12 mM SDS. In order to determine if sub-micellar aggreg ates are indeed a significant component in micellar equilibrium, SDS solutions were prepar ed at concentrations below and above the reported cmc value.117 Upon ultrafiltration and anal ysis of SDS in the filtrate, we have found that the SDS concentration in the filtrate increas es nearly proportionally to the total SDS concentration up to the cmc value. However, beyond the cmc, the SDS concentration does not remain constant but continues to increase with a d ecrease in the slope of the curve (Figure 2-5). Since the SDS concentration in the filtrate does not remain constant beyond the cmc (as would have been expected if there were no submicellar aggregates in the system), we have concluded that the increase in SDS concentration in the filtrate must be due to the presence of sub-micellar aggregates that are made up of fewer than 35 SDS molecules. Th is graph is striking, as it suggests that in a 50 mM SDS system, give n a cmc of ~ 8 mM, more than one-third of the surfactant molecules (~ 17-18 mM) are in the form of sub-micellar aggregates of less than 35 molecules. This tells us that sub-micellar aggregates represent a significant portion of the micellar solutions. This is the first conclusive evidence that sub-micellar aggregates represent a significant portion of micellar solutions and th ereby, cannot be ignored. Such a finding has potential significance in applications related to flux of oil soluble drugs with respect to

PAGE 70

70 controlled drug delivery (sub-mice llar aggregates would carry a la rge percentage of the drug to the target organs), hemolysis of red blood cells (a system with a large sub-micellar aggregate population would cause much more hemolysis than a system that has few to no sub-micellar aggregates), and even possibly in predicting the anti-microbial effi ciency of a given solution that consists of surface active bactericidal agents. In order to further prove our hypothesis, we d ecided to make the SDS micelles more stable by adding C12OH or C12TAB in various mole fractions. The Shah research group has previously shown that one may tailor micellar stability by the addition of long chai n alcohols or oppositely charged surfactants2, 28 (such as alkyltrimethylammonium bromides, commonly referred to as CnTABs) as shown in Figure 2-42 and that the stability is especi ally enhanced when the additives have the same chain length as the SDS.30 The long chain alcohols enhance micellar stability through charge shielding and the long chai n TABs enhance micellar stability through electrostatic interactions betw een their positively charged head group and the negatively charged head group of SDS. We have not iced that the addition of C12TAB tends to better stabilize the micelles when compared with C12OH. Given this previous knowledge, we began by adding C12TAB in increasing mole fractions to the SDS system to see if the concentration of SDS in the filtrate decreases. As shown in Figure 2-7, the filtrate concentrati on does indeed decrease for C12TAB mole fractions up to 20 mole% and subsequently levels off. It is import ant to note that by adding up to 20 mole % C12TAB, the concentration of SDS in the filtrate was reduc ed from 26 mM to ~3 mM. This is an amazing finding which suggests that the addition of the C12TAB made the micelles so stable that only 3 mM SDS was free, presumably as SDS monome rs, to pass through the filter (i.e. the SDS+C12TAB micelles are behaving somewh at like rigid spheres).

PAGE 71

71 If this is the case, then if we reproduce Figure 2-5, but w ith the addition of 20 mole % C12TAB and C12OH for all SDS concentrations, then one would expect that the curve beyond the cmc should shift towards zero slope, w ith SDS having the steepest slope, C12TAB having the near zero slope, and C12OH falling in between the two curves. As can be seen by Figure 2-8, the addition of 20 mole % of C12OH and C12TAB did indeed decrease the slope beyond the cmc, with C12TAB leading to a virtuall y zero slope line! This confirms our hypothesis, but in order to completely eliminate any possible doubt, we decided to perform one more critical experiment. If there are indeed no sub-micellar aggregates in the 80:20 SDS:C12TAB system, then if these solutions we re filtered through a filter that has an even smaller molecular weight cut-off (MWCO) than 10,000, one would expect that the SDS concentrations in the filtrate for a given SDS/C12TAB system should be the same irrespective of the MWCO. We ran the samples in 3,000 MWCO where aggregates w ith less than 11 SDS molecules can pass through the pores of the me mbrane tubes at ~ 4500g for 10 minutes and plotted the results together w ith the 10,000 MWCO results and as seen in Figure 9, the SDS filtrate concentrations in the two different filter sizes were approximately the same in all cases where C12TAB was added. In the case of 50 mM SDS (with no added C12TAB), as expected, there is a significant difference between the fi ltrate concentration fr om the 3,000 MWCO tubes versus the 10,000 MWCO tubes. This sugge sts that in mixed systems of SDS and C12TAB, there are no aggregates larger than 11 molecules, wh ereas in pure SDS micella r solutions, there are aggregates at least up to 34 molecules and po ssibly having even higher aggregation numbers. Various other physical properties of SD S systems, including osmotic pressure116 and conductivity,120 have been shown to increase proportiona lly to the total SDS concentration up to the cmc and subsequently display a change in slope just as seen in our ul trafiltration studies. We

PAGE 72

72 believe that the presence of sub-micellar aggreg ates contributes signifi cantly to the deviation from ideality that is observed in the osmotic pr essure of SDS as reflected in the work of Amos and coworkers.116 We are also convinced that the rise in conductivity beyond the cmc is partially due to the increasing concentration of sub-mice llar aggregates in addi tion to the number of micelles formed. Micelles have also been used throughout the years as a vehicle for carrying out various reactions18 and it has been shown that the reactio n rates are dependent upon the micellar stability.118 We believe that the underlying factor here is the contribu tion of sub-micellar aggregates, which may very well solubilize some of the reactants and thereby significantly influence the reaction kinetics. Therefore, this is yet another application of micelles where the presence of sub-micellar aggreg ates must be considered when utilizing various surfactant systems. Sodium dodecyl sulfate was used as the chosen surfactant for these studies because it is an extensively studied surfactant.46, 119-121 However, Midura111 illustrated that this phenomenon of increasing filtrate concentration beyond the cmc holds true for at least two other surfactant systems: Triton X-100 and Chaps ((3-[( 3-cholamidopropyl)-dimethylammonio]-1propanesulfate). This shows that the presence of sub-micellar a ggregates is not limited only to ionic surfactant, but that they ar e present in non-ionic surfactant systems as well. Based on these findings, care must be taken when modeling su rfactant micellar solutions and when designing micellar solutions for usage in controlled drug delivery, anti-microbial solutions, for solubilization and denaturing of proteins,111 and when considering the hemolytic activity of a given surfactant system. These findings also prov ide great insight into the actual mechanism of

PAGE 73

73 micelle relaxation in surfactant solutions and allows for further correlation to various technological processes, such as foaming, de tergency, fabric wetting, and emulsification. 2.3.2 Effect of Counter-Ions on Sub-M icellar Aggregate Concentration Another interesting aspe ct of micellar solutions is the e ffect of counter-ions on the micellar stability. Pandey et. al .125 have shown how counter-ions affect surface and foaming properties of dodecyl sulfates. They have illustrated how cha nging the counter-ion in dodecyl sulfates from sodium to lithium, cesium or magnesium leads to distinct differences in the corresponding dynamic surface tension of the solutions. Dynami c surface tension is a measure of the actual surface tension of the interface at a specific point in time where new liquid/liquid or gas/liquid interfaces are rapidly being genera ted in a surfactant solution. Dynamic surface tension directly reflects the surfactant concentra tion at the interface at that point in time, and hence the availability of monomers and sub-micellar aggregates to diffuse to and stabilize the newly created interfaces such as those created in the ge neration of foams and emulsions. As such, it is heavily dependent upon micellar st ability in that a more unsta ble micelle will provide more monomers and sub-micellar aggregates to diffuse to the interface. Dyna mic surface tension can be measured by the maximum bubble pressure method126 and can be represented by using the parameter which normalizes the dynamic surface ac tivity with respect to the equilibrium surface activity as follows: /deqweq (2-3) where d is the dynamic surface tension, eq is the equilibrium surface tension measured by the Wilhelmy plate method, and w is the surface tension of pure water at 25C. The value of = 0 (or d = eq) indicates that the surfacta nt adsorption under dynamic c ondition is the same as that under equilibrium conditions and the micelles are labile and the monomers are diffusing fast,

PAGE 74

74 whereas = 1 ( d = w) indicates no surfactant is present at the interface under the dynamic conditions existing during the bubbling process impl ying either the presen ce of relatively stable micelles or monomers with high characteristic diffusion time. Pandey et. al 122 measured the dynamic surface tensio n of four dodecyl sulfate solutions (having lithium, sodium, cesium, and magnes ium as counter-ions) and reported the parameter as shown in Table 2-1 below. The parameter values are lower and similar for lithium dodecyl sulfate (LiDS) and sodium dodecyl sulfate (referre d to here as NaDS to distinguish the counterion), while they are higher for cesium dodecyl sulfate (CsDS) and magnesium dodecyl sulfate (Mg(DS)2), suggesting a higher dynamic surface ac tivity of LiDS and NaDS. This finding suggests that the LiDS and NaDS have a lowe r micellar stability, while the CsDS and Mg(DS)2 have a higher micellar stability. Readers may refer to reference 33 for a more detailed explanation of the factors res ponsible for the counter-ion eff ects on micellar stability. Pandey et. al found that the trends in dynamic surface tensi on correlated well to the foamability behavior in these systems as well. The Shah research group has shown that more stable micelles tend to have low foamability, but high foam stability.2 The stability of foam depe nds on how quickly liquid is drained from the foam lamellae.90 Nikolov and Wasan123 extensively studied the micellar structure inside the thin liquid film of the foam lamellae and they showed that the drainage of the liquid film can be explained by a layer-by-layer th inning of ordered structures of micelles inside the film. This structured phenomenon is a re flection of the micellar e ffective volume fraction, stability, interaction, and polydispersity. Based on these studies of counter-ion effects on micellar stability, we decided to determine the relative concentrations of the sub-micella r aggregates in LiDS, NaDS, CsDS, and Mg(DS)2.

PAGE 75

75 We prepared 25 mM solutions of the LiDS, NaDS, and CsDS and a 12.5 mM solution of the Mg(DS)2. The Mg(DS)2 was prepared at half the concentrat ion of the other solutions because it has two dodecyl sulfate chains in every mol ecule. After centrifugation in the 10,000 MWCO filter tubes, we measured the filtrate surfact ant concentration by the dye complexation method. We were pleased to discover that the trends in filtrate concentrations relative to counter-ion correlated well with the dynamic surface tens ion values reported by Pandey et al.122 (see Figure 2-10). Figure 2-10 clearly shows that the filtrate surfactant concentr ations are significantly higher for LiDS and NaDS than they are for CsDS and Mg(DS)2. This behavior gives further credence to our speculation that the presence of sub-micella r aggregates is directly linked to the micellar stability of a given surfactant system. 2.3.3 Importance of Sub-Micellar Aggre gates in Technological Processes The Shah research group has shown significan t evidence correlating micellar stability to various technological processes including foamability,124, foam stability,2 emulsion droplet size,46 fabric wetting,125 and detergency.126 The effect of mi cellar stability on these processes was explained on the basis of the micelles ability to break and supply monomers to the bulk that can adsorb at the interfaces that are created in each application. For example, in the case of foamability, a less stable (more labile) micelle will break rapidly, giving up its monomers to stabilize the foam against inst antaneous breakdown. However, this explanation has been met with a bit of skepticism over the years because the timescale of micellar breakdown (milliseconds) is so much shorter than the timescale of these processes. This argument does have merit and until now, there was no sufficient explanation for how micellar stability extended to effect technological applications. We have shown here the intimate relationship between micellar stability and sub-micellar aggregates. It follows that the operating molecular mechanism in the

PAGE 76

76 dependence of the aforementioned t echnological processes on micellar stability is directly related to the flux of not only monomers, but also to a larger extent, to the flux of sub-micellar aggregates to the newly created interfaces in each of the processes. The more labile the micelle, the higher is the concentration of sub-micellar aggr egates and higher is the flux of the monomers to the interface. 2.3.3.1 Foaming A foam is a coarse dispersion of a gas in a li quid with the gas making up most of the phase volume and with the liquid in thin sheet s, lamellae between the gas bubbles.127 Foamability, the degree to which a surfactant solution is able to generate foam, is a relevant property for many industrial applications, in cluding detergency, food processing, and mineral floatation. One of the major factors that affect foamability is the ability of the stabilizing agent (surfactant in this case) to adsorb at the newly created air/water inte rface to prevent immediat e breakdown of the foam.63 Therefore, foamability is highly dependent upon the concentration of monomors and submicellar aggregates which can r eadily provide the monomers to the interface. Figure 2-11 shows a schematic diagram of the lamellae of a foam. In 1991 Oh and Shah124 showed that foamability is influe nced by the average lifetime of a micelle. We have shown that the sub-micellar aggr egate concentration is very high in solutions of SDS alone, lower in solutions of SDS + C12OH, and almost non-existent in solutions of SDS + C12TAB. After measuring the foamability of each of these systems (25 mM SDS, 25 mM SDS + 6.25 mM C12OH, and 25 mM SDS + 6.25 mM C12TAB) we found that the SDS system, which has the highest concentration of sub-micellar aggregates, also displayed the greatest foamability as shown in Figure 2-12. The foamability decreas es with increasing micellar stability, with the SDS + C12OH micellar solution generating less foam than the SDS solution, and the SDS + C12TAB micellar solution generating the least amount of foam. Since the SDS + C12TAB

PAGE 77

77 solution had few, if any, sub-micellar aggregates only free monomers were available to adsorb at the air/water interf ace of the foam. The free monomer conc entration is very low in this solution, and as such, the foamability is low. 2.3.3.2 Fabric wetting Fabric, or textile, wetting is another proce ss where the presence of sub-micellar aggregates is important. Due to the large surface area of fabric s, equilibrium conditions are rarely attained in the time allowed for wetting in practical processes.3 As such, the kinetics of surfactant adsorption at the solid/liquid interface of the fabric is a co ntrolling parameter in fabric wetting. If there is a high concentration of surfactant th at is available to adsorb at the interface, making the fabric more water-wettable, the wetti ng time will be short. The hydr ophobic tails of the surfactant molecules adsorb onto the fabric and lower the inte rfacial tension so that water can penetrate into the interstitial spaces of the fabric weave. Th erefore, one would expect a solution of SDS (50 mM), which has a high sub-micellar aggregate concen tration, to have a faster wetting rate than a solution of 50 mM SDS + 12.5 mM C12TAB. Wetting experiments were performed on these systems, and as expected the fastest wetting time was found for the SDS solution, as can be seen in Figure 2-13. The fabric wetting time was al so measured in pure water as a control. 2.3.3.3 Dynamic surface tension Dynamic surface tension is a measure of the ab ility of surfactant mol ecules to adsorb at newly created interfaces under dynamic conditions Dynamic surface tension can be measured by determining the maximum bubble pressure when ga s is bubbled through a surfactant solution at a specific flow rate. When there is a significan t concentration of mono mers and sub-micellar aggregates, the dynamic surface tension is low becau se the flux of monomers to the interface is high. The dynamic surface tension was found to in crease with increasing micellar stability so that a solution of 50 mM SDS exhibited a lowe r dynamic surface tension than a mixed micellar

PAGE 78

78 system of 50 mM SDS + 12.5 mM C12TAB as shown in Figure 2-14. This is an interesting finding because the equilibrium surface tension of an SDS solution will always be lower than that of an SDS/C12TAB mixed solution. Therefore, one must take care when choosing surfactant solutions for various dynamic processes, because if the micelles are too stable, there will be a low concentration of sub-micellar aggregates, an d the solution will not be able to effectively lower the surface or in terfacial tension. 2.4 Conclusions Here, we have presented the first conclusive evidence of sub-micellar aggregates as a significant component in micellar solutions. The following conclusions can be drawn based upon the results reported here: 1. The generally accepted notion that surfact ant solutions consist of only three compartments, namely, adsorbed film at air/ water interface, monomers, and micelles, is incorrect and we have shown that sub-micella r aggregates are a significant entity in surfactant solutions making up as much as one-third of the surfactant concentration for example, as in 50 mM SDS. 2. We have shown here that micellar dynamics are intimately linked to the presence of submicellar aggregates (i.e. the more stable a mi celle is, the less is the concentration of submicellar aggregates in the system) and th at by stabilizing SDS micelles through the addition of dodecanol (C12OH) or dodecyltrimethylammonium bromide (C12TAB), we can effectively eliminate sub-micellar aggr egates and reduce the monomeric surfactant concentration to values as low as 3 mM. 3. It is known that counter-ions affect micella r stability. We have shown that counter-ions also affect the concentration of sub-micellar aggregates in dodecyl sulfate systems (i.e. counter-ions such as Mg2+ and Cs+, which enhance micellar stability, were shown to have lower concentrations of surfactant in the filtrate, whereas, Li + and Na+, which form relatively unstable micellar systems, have hi gher concentrations of surfactant in the filtrate) which correlates well with previ ously reported dynamic surface tension data.122 4. We have shown that sub-micellar aggregates, or micellar fragments, exist in the micellar solution even at low concentrations, such as 25 100 mM. 5. We have determined that it is the presence of sub-micellar aggregates that provides the missing link in our understanding of the e ffect of micellar stability on technological

PAGE 79

79 processes. We have shown that when ther e is a high concentra tion of sub-micellar aggregates, the foamability is the highest, the fabric wetting rate is the fastest, and the dynamic surface tension is the lowest.

PAGE 80

80 Figure 2-1. Schematic diagram of the ultracentrifug ation process. Sample is placed in top portion of tube and the tube is centrifuged at ~2900g (for 10,000 MWCO ultracentrifuge tubes) or ~4500g (for 3,000 MWCO ultracentrifuge tubes) for 10 minutes so that less than 10% of the solution volume is collected as filtrate. Porous filter membrane Filtrate lar g e aggregates are excluded Force = 2900 g Axis of Rotation Sample solution with large aggregates

PAGE 81

81 Figure 2-2. Size distribution curves of aggreg ates in a micellar solution. The solid curve represents a typical size distribution cu rve of aggregates in a micellar solution according to the Aniansson-Wall model of stepwise micellar association. Region (I) corresponds to monomers and oligomers; Region (III) to abundant micelles with a Gaussian distribution around the mean a ggregation number; a nd Region (II) to the connecting wire (heat transfer analogy) or tube (mass transfer analogy) between Regions (I) and (III). The dashed curve re presents our hypothesized size distribution for a micellar solution containing a signi ficant concentration of sub-micellar aggregates. B A I II III Aggregation number, n C (An) 2 n

PAGE 82

82 Figure 2-3. Schematic diagrams of surfactant solutio ns, filtration of solutions, and plot of filtrate concentration as a function of total surfactant concentration. A) Schematic diagram of surfactant solution above cmc consisting of three components; adsorbed film of surfactant molecules, surfactant monomers, and micelles; B) Schematic diagram of surfactant solution above cmc consisting of four components; adsorbed film of surfactant molecules, surfactant monomers, sub-micellar aggregates and micelles; C) Schematic illustration of filtration of so lution from Figure 3A where only surfactant monomers are present in the filtrate and micelles were not able to pass through the filter; D) Schematic illustration of filtration of solution from Figure 3B where surfactant monomers and smaller sub-micellar aggregates are present in the filtrate and micelles and larger sub-micellar aggr egates were not able to pass through the filter; E) Expected results of a plot of surfactant concentration in the filtrate versus total surfactant concentratio n for a system like the one shown in Figure 3A. F) Expected results of a plot of surfactant concentration in the filtrate versus total surfactant concentration for a system like the one shown in Figure 3B. The monomer contribution to the filtrate is represented by the region from point Q to point P and the sub-micellar aggregate contribution to the filtrate surfactant concentration is represented by the region from point R to point Q.

PAGE 83

83 Micelle Figure 2-3A Figure 2-3B Mon omer Sub-micellar aggregate Figure 2-3C Figure 2-3D Filter Adsorbed film Total [Surf] Total [Surf] Surf. conc. in filtrate cmc cmc Figure 2-3E Figure 2-3F P Q R Surf. conc. in filtrate

PAGE 84

84 Figure 2-4. Schematic representation of the two possible reaction paths for the formation of micelles (a) and the corresp onding resistances (b): (1) fo rmation by incorporation of monomers and (2) formation by reverse coagulation of sub-micellar aggregates.

PAGE 85

85 Figure 2-5. Filtration of SDS through 10,000 MWCO u ltracentrifuge tubes for ~10 minutes at 2900*g. The point at which the slope chan ges is considered the critical micelle concentration (cmc). The dotted line is the result that one would have expected if there were no sub-micellar aggregates present in the system. Figure 2-6. Tailoring of micellar stability by the addition of 1-dodecanol (C12OH) or ndodecyltrimethylammonium bromide (C12TAB). Conc. SDS in filtrate (mM) 0 5 10 15 20 25 30 0 10 20 30 40 50 60total [SDS] (mM)

PAGE 86

86 Figure 2-7. Filtrate of SDS+C12TAB through 10,000 MWCO ultracentrifuge tubes for ~10 minutes at 2900g. The C12TAB mole fr action was increased from 5 mole% to 25 mole%. The total SDS concentration is fixed at 50 mM. Conc. SDS in filtrate (mM) 0 5 10 15 20 25 30 0 5 10 15 20 25 30 mole % C12TAB in solnTotal [SDS] = 50 mMSamples run in 10,000 MWCO Centrifuge tubes at ~2900g for 10 minutes

PAGE 87

87 Figure 2-8. Filtration of SDS alone or SDS + C12X (X = OH or TAB) through 10,000 MWCO ultracentrifuge tubes for ~10 minutes at 2900g. The C12OH and C12TAB were added at a molar ratio of 80:20 SDS: C12X (i.e. the concentrati on of C12OH or C12TAB in each system is 20 mole % of the total surfactant concentration (SDS+C12X)). 0 5 10 15 20 25 30 35 40 0 20 40 60 80 100 120 total [SDS] (mM)Conc. SDS in filtrate (mM) SDS alone 80:20 SDS:C12TAB mixed micelles 80:20 SDS:C12OH Samples run in 10,000 MWCO Centrifuge tubes at ~2900g for 10 minutes

PAGE 88

88 Figure 2-9. SDS concentration in the filtrate for 80:20 SDS:C12TAB systems after filtration through 3,000 and 10,000 MWCO tubes, as compared to pure SDS solutions (50 mM). Samples in 3,000 MWCO ultracentrif uge tubes were centrifuged at ~ 4500g and samples in 10,000 MWCO ultracentrifuge tubes were centrifuged at ~ 2900g for 10 minutes. [ 0 5 10 15 20 25 30 7.5mMSDS/1.875mM C12TAB 10mMSDS/2.5mM C12TAB 25mMSDS/6.25 mM C12TAB 50mMSDS/12.5mM C12TAB 50mMSDSConc. SDS in filtrate (mM) 3000 MWCO 10,000 MWCO

PAGE 89

89 0 2 4 6 8 10 12 14 16 18 20 LiDSNaDSCsDSMg(DS)2Filtrate Concentration (mM)Total Concentrat ion = 25 mM for all samples except Mg(DS)2, which has a total co ncentration = 12.5 mM Figure 2-10. Filtrate surfactant concentra tions for 25 mM lithium dodecyl sulfate (LiDS), sodium dodecyl sulfate (NaDS), and cesium dodecyl sulfate (CsD S) and 12.5 mM magnesium dodecyl sulfate (Mg(DS)2).

PAGE 90

90 Figure 2-11. Schematic depiction of foam colu mn generated by passing air through a surfactant solution (left) and magnified view of foam lamella, thin sheet of surfactant solution between adjacent air bubbles (right). Figure 2-12. Foamability of SDS micellar solution and SDS + C12X mixed micellar solutions (X = OH or TAB) Monomers and sub-micellar aggregates Foam Lamella 0 10 20 30 40 50 60 70 80 90 10025 mM SDS25mMSDS/6.25mM C12OH 25mMSDS/6.25mM C12TABFoamability (mL)High conc. sub-micellar aggs. High foamability Low conc. sub-micellar aggs. Low foamability

PAGE 91

91 Figure 2-13. Wetting time of 1in2 strips of 50:50 cotton:polyester blend fabric in pure water, 50 mM SDS, and 50 mM SDS + 12.5 mM C12TAB. 0 10 20 30 40 50 60 70water50 mM SDS50 mM SDS/12.5 C12TAB SolutionFabric Wetting Time (sec)High conc. sub-micellar aggs. Low Fabric wetting time Low conc. sub-micellar aggs. High Fabric wetting time

PAGE 92

92 0 5 10 15 20 25 30 35 40 45 50 50 mM SDS50 mM SDS + 12.5 mM C12TAB Surfactant SolutionDynamic Surface Tension (mN/m) 22 gauge needle 6 13 bubbles/sec Air flowrate = 3 cc/min Figure 2-14. Dynamic surface tension of solutio ns of 50 mM SDS a nd 50 mM SDS + 12.5 mM C12TAB.

PAGE 93

93 Table 2-1. Dimensionless dynamic surface tension ( ) of different counter-ions of dodecyl sulfates (50 mM) at a bubble li fetime of 50 msec (from ref 33). Ion parameter Li+ 0.138 Na+ 0.131 Cs+ 0.202 Mg++ 0.353

PAGE 94

94 CHAPTER 3 DETERMINATION OF DRUG AND FATTY AC ID BINDING CAPACITY TO PLURONIC F127 IN MICROEMULSIONS FOR DETOXIFICATION 3.1 Introduction Drug overdose incidences are a common a nd problematic occurrence both nationally and globally. Many life-threatening drugs do not have specific pharmacological antidotes to reverse the toxic effects that resu lt when an overdose occurs.99 Attempts are currently underway to develop procedures to detoxify bloo d in a timely and efficient manner.128-130 Therefore, the development of an effective methodology for the removal of free drug from the blood of an overdosed patient in a timely manner (less than 15 minutes) is critically important. In the past few years, efforts have been underway to use nanoparticulate systems to accomplish this task. Microemulsions are one of the systems that are cu rrently under investigati on. Upon injection of a biocompatible, nontoxic microemulsion in the bloo d of an overdosed person, the microemulsion, having extremely high interfacial area, can effectiv ely adsorb and solubili ze drug molecules, and thereby quickly decrease the concentration of fr ee drug molecules in the blood. However, in order to fully grasp their function as toxicity re versal agents, one must understand the molecular mechanism of drug uptake and be able to dete rmine and manipulate the contributing interfacial forces. Preliminary results from pH studies have led us to believe that electrostatic forces can play a significant role in adsorption of drug onto the microemulsion. Amitriptyline Hydrochloride, shown in Figure 3-1, is an antidepressant and as of yet, there is no efficient method to reverse the effects of an overdose in a pati ent; therefore it is the target drug for the experiments reported here. Amitriptyline has a pKa of approximately 9.4 so that at physiological pH (~ 7.4), it will be positively charged and can thereby interact through electrostatics with a negatively charged microemulsion. These microemulsions are composed of Pluronic F127, Ethyl Butyrate, and

PAGE 95

95 Sodium Caprylate fatty acid (which gives the negative charge) and are prepared in Phosphate Buffered Saline at pH 7.4. The objective of this study is to develop a better understand ing of the important interactions that occur between the microemulsion and the drug. We have shown, through turbidity analysis expe riments, that there is a linear relationship between the Amitripty line Hydrochloride solubilizati on capacity (i.e. the amount of Amitriptyline that the microemulsion can acco mmodate before turbidity occurs) of the microemulsions and Pluronic surfactant co ncentration up to a certain Pluronic F127 concentration. Above that critical Pluronic F127 concentration, further titration with Amitriptyline never yields turbidity. We have also seen that turbidity is not observed in systems that do not have sodium caprylate present. Based on these findings we have concluded that at the critical Pluronic concentration, there is no longer any free (unassociated) sodium caprylate molecules in the bulk phase, presumably due to bi nding of all fatty acid molecules with Pluronic molecules. Therefore, we are able to determin e how many molecules of sodium caprylate and Amitriptyline are associat ed with each Pluronic molecule. Each Pluronic F127 molecule can associate with approximately eleven molecules of sodium caprylate and twelve molecules of Amitriptyline at the critical concentration (i.e. th ere appears to be a nearly 1:1 association of sodium caprylate to Amitriptyline). This yields further credence to ultrafiltration studies that we have done as a function of pH which show that electrostatic interactions are important in Amitriptyline binding to microemulsions produce d by Pluronic F127 and fatty acid soap. The findings of this study will provide substantial information regarding the mechanism of reduction of overdosed drugs and will allow us to approximate the uptake capacity of a particular microemulsion system.

PAGE 96

96 3.2 Experimental Procedure 3.2.1 Materials. Pluronic surfactants were obtained from BASF Inc. (Mount Olive, NJ). Pluronic was used as a nonionic surfactant co mposed of a symmetric triblock copolymer of propylene oxide (PO) and ethylene oxide (EO) The polypropylene oxide block was sandwiched between the more hydrophilic poly(ethylene oxide) blocks. The block copolymer was denoted by (EO)x(PO)y(EO)x, where x and y are the number of units of EO and PO, respectively. Amitriptyline Hydrochloride, sodium caprylate sodium decanoate, and sodium dodecanoate were purchased from the Sigma Chemical Co. (S t. Louis, MO). Ethyl butyrate was purchased from ACROS Organics (New Jersey). Sodium phosphate monobasic, sodium phosphate dibasic, sodium chloride, and potassium chloride which were used to prepare the phosphate buffered saline were purchased from Fisher Scientific In c. (Suwanee, GA). Double distilled, deionized Millipure water was used for all solutions. 3.2.2 Microemulsion Preparation Oil-in-water microemulsions were prepared by first solubilizing the appropriate c oncentration (3 9 mM) of Plur onic F127 surfactant in phosphate buffered saline at pH 7.4 (physiological pH). So dium caprylate (fatty acid surfactant) was then added to this Pluronic solution in concentrations ranging from 25 100 mM. Lastly, ethyl butyrate (oil) was added to the solution and the system was stirred until it became clear. The ethyl butyrate concentration was fixed at 110 mM for all experiments in which microemulsions were used. The microemulsions were subsequently allowed to equilibrate for at least one day prior to use. 3.2.3 Turbidity Analysis. Micelles, mixed micelles and microemulsions were prepared with varying compositions of Pluronic F127, and/ or Sodium Caprylate, and/or Ethyl butyrate. The aqueous phase was phosphate buffered saline (PBS) with a pH ~ 7.4. Ten milliliters of the micelle or microemulsion sample was placed into a vial. The solution was titrated with 0.2 M

PAGE 97

97 Amitriptyline (prepared in PBS) until the onset of turbidity was observed visually. The systems were sensitive enough that the transition from cl ear to turbid was very sharp (i.e. occurring over a change in volume of 50 microliters or less). In some systems, prior to the system reaching turbidity, upon each incremental addition of Am itriptyline, the solutions would exhibit a momentary cloudiness, but gently swirling would lead to a return in clarity. During titration, if the initial cloudiness was not observed upon the additions of Amitriptylin e, copious amounts of drug was added to that system; if turbidity was not observed, then the system was categorized as one where turbidity would never occur. 3.2.4 Dynamic Surface Tension. Dynamic surface tension was measured using the maximum bubble pressure technique The pressure required to fo rm a new bubble in solution is measured by a pressure transducer, and the readi ng is transmitted to an oscilloscope. For these experiments, the dynamic surface tension was measured for microemulsions consisting of fixed sodium caprylate (100 mM) and ethyl butyrat e (110 mM) concentrations and increasing concentrations of Pluronic F127. All dynamic su rface tension measurements were taken using an 18 gauge needle tip with a gas flow rate of 5 cm3/min (which corresponds to 3 to 10 bubbles per second or approximately 100 to 333 msec per bubble residence time at the needle tip). We chose this flow rate because at higher low rates the nitrogen gas forms a continuous jet in the surfactant solution at the needle tip. At lower flow rates, the results are similar to equilibrium surface tension results. 3.2.5 Foamability. Twenty milliliter samples of microemulsions consisting of fixed sodium caprylate (100 mM) and ethyl butyrat e (110 mM) concentrations and increasing concentrations of Pluronic F127 were placed into 100-mL graduated cylinders and capped. Each cylinder was vigorously shaken 10 times by ha nd and the volume of the foam is recorded

PAGE 98

98 immediately after shaking. Each solution is tested at least three times and the reproducibility is better than 2 ml. 3.2.6 Fabric Wetting. A commercially gained cotton fabric of 1 in.2 was placed on the surface of microemulsion solution at 25C. The microemulsions used consisted of fixed sodium caprylate (100 mM) and ethyl butyrate (110 mM) concentrations and increasing concentrations of Pluronic F127. The surfactant solution displaces air in the cotton surface by a wetting process and when sufficient air has been displaced, the co tton starts sinking. The residence time of cotton fabric on the surface of the solution before it wa s completely immersed was measured as wetting time in this study. This wetting time in each mi croemulsion solution was measured at least 3 times. 3.2.7 Surface Tension. Surface tension measurements were carried out to determine the critical micelle concentration (cmc) using the Wilhelmy plate method. In this method, the plate is lowered into surfactant solutions of known con centrations and the corresponding output from a gram-force sensor holding the plate is sent to a transducer and then to a voltage readout. The system was calibrated using two known solutions (water at 72.5 mN/m and acetone at 23 mN/m). The platinum plate was heated between each reading to clean off anything that may have adsorbed onto the plate. 3.3 Results and Discussion 3.3.1 Effect of Sodium Capryl ate Concentration on Drug and Fatty Acid Binding to Microemulsions As previously reported,99 we have taken a systematic ap proach to design a biocompatible microemulsion system that would effectively redu ce the free concentration of target drugs in the blood. This microemulsion system is composed of Pluronic F127 as the surfactant, sodium caprylate (SC) fatty acid as the co-surfactant, ethy l butyrate (EB) as the oil phase and is prepared

PAGE 99

99 in a phosphate buffered saline solution at pH 7.4. Given that Pluronic F127 is a block copolymer and sodium caprylate is a co-surfactant, if we can understand the nature of the polymersurfactant interactions in this microemulsion, then we can have a better understanding of the structure of the microemulsion and the molecu lar mechanism of uptake of the drug. For many years now, polymer-surfactant interactions have been studied extensively in relation to various interfacial processes.84, 131-137 One of the methods of analyzing polymer-surfactant interactions is through titration studies.54, 68 Here, we take various microemulsion compositions and titrate them with concentrated Amitriptyline solutions to turbid ity. We are using the results of these studies to determine the pertinent stoichiometric ratios in our optimal microemulsion formulations. For our initial titration studies we took the various microemulsion components and titrated them individually. So our first titrations were of sodium caprylate (SC) solutions in phosphate buffered saline (PBS) (pH 7.4). In this study, we found that at 100 mM SC, turbidity occurred when 1 molecule of AMT was adde d for every 100 molecules of SC. Next, we titrated systems cont aining only Pluronic F127 in PBS (pH 7.4) with 0.2 M AMT. In these systems we found that turbidity was never obtained, irrespective of how much AMT was added to the system. Then we added the ethyl butyrate oil to the Pluronic F127 systems and titrated these solutions with 0.2 M AM T. Once again, turbidity was never obtained. Finally, we added our last component, the sodium caprylate fatty acid, to the system and found that upon titration with 0.2 M AMT, turbidity was seen in these systems. For these systems, the sodium caprylate concentration was held fixed at 100 mM, the ethyl butyrate concentration was held fixed at 110 mM, and the Pluronic F127 co ncentration was varied from 3 mM to 9 mM. One of the interesting observations that we noticed in these titrations was that turbidity was achieved

PAGE 100

100 for every Pluronic F127 concentration up to 8 mM. Above 8 mM F127, turbidity was never achieved (see Figure 3-2). Our next experiment involved titration of Pluronic F127 and sodium caprylate mixed micellar systems (i.e. no oil is present). In this case, the sodium caprylate concentration was held fixed at 100 mM and the F127 concentration was varied from 1 mM to 9 mM. We were somewhat surprised to see that th e lack of oil in these systems di d not seem to affect the amount of AMT needed to induce turbidity (i.e. the gr aph for the mixed micellar system is nearly the same as that of the microemulsion system (see Figure 3-3). These experiments provided us with two im portant insights. First, turbidity is only observed in the systems where the sodium caprylate is present. Based on this finding, we can conclude that the turbidity is arising from AMT forming a comple x with the SC. Secondly, in the systems where Pluronic F127 is present with SC turbidity is observed up to some critical F127 concentration, above which turbidity is no long er observed. Based on this finding, we can conclude that the critical F127 c oncentration is the concentration at which no more SC exists as free monomers in the bulk solution and that the turbidity is a result of the AMT complexing with the free SC in the bulk. Figure 3-4 provides a schematic illustration of this hypothesis. In order to test our hypothesis we did th e turbidity experiments for various sodium caprylate concentrations. If our hypothesis is co rrect, we would expect for the critical F127 concentration to decrease proportionally to the decr ease in SC concentration. As can be seen in Figure 3-5, the decrease in the cr itical concentration of F127 is i ndeed nearly proportional to the decrease in the SC concentration. The critical F127 concentration is never reached in the system where the SC concentration is 125 mM because above a F127 concentration of 9 mM, the solution becomes a gel.

PAGE 101

101 3.3.2 Determination of Free Fatty Acid by Dynamic Processes We also decided to test our hypothesis by analyzing various characteristics of the microemulsion system that would be sensitive to the presence of bulk surfactant. We tested the dynamic surface tension, fabric wetting time, and the foamability of the microemulsion systems having fixed SC and EB concentration (S C = 100 mM and EB = 110 mM) and varying concentrations of F127 (F127 = 3 9 mM). Dynamic surface tension (DST) is a measure of th e availability of surf ace active species to partition to a newly created surface and stabiliz e it. Dynamic surface tension is an important quantity in any process in which a new gas/liquid or liquid/liquid interface is rapidly generated.2 A high DST value is reflective of the fact that any surface active species that are present in the system are not readily available to diffuse to the newly created interface, whereas a low DST reflects that the species can quickly diffuse to the surface. Therefore, we would expect that as the F127 concentration is increased, the amount of free SC in the bul k decreases, and as such, the DST value should increase. Fabric wetting time is a measure of the time that it takes for a fabric to be completely wetted by a liquid. For a hydrophobic fabric, this time could be relatively long, depending upon the weight of the fabric. There are three forces at play in this s ituation: hydrophobicity, buoyancy, and gravity. The hydrophobic and the buoyancy forces oppose the fabric sinking into the solution, whereas the gravity promotes the fabric to sink into th e solution. The hydrophobic force can be minimized if surface active species are present in th e solution to adsorb onto the fabric and make it hydrophilic. In this situa tion, the hydrophobic tail of the surfact ant adsorbs onto the fabric and the hydrophilic head is ex posed to the solution, hence making the fabric appear to be hydrophilic. Based on this knowl edge, the fabric wetting time can be used as a relative measure of the amount of free sodium caprylate in the microe mulsion solution. One

PAGE 102

102 would expect that as the F127 concentration is increased, the SC concentr ation decreases and the fabric wetting time increases. Foamability is a measure of the relative ability of a solution to generate foam. As foam is being formed a new air/liquid interface is gene rated. If this new ai r/liquid interface can be rapidly stabilized (i.e. if surf actant molecules are available to rapidly diffuse to the interface) then the foam volume will be high. This soluti on would be considered as one having a high foamability. If no surfactan t is available to stabilize this newl y created interface, as the foam is generated, it will almost instantaneously collapse and the foam volume will be very low. This solution would be considered to have a low foamability. Given this information, one would expect that as the F127 concentration is increas ed, the SC concentration decreases and as such the foamability should decrease. Figure 3-6 shows how the experimental re sults of DST, fabric wetting time, and foamability support our hypothesis. One point to note is that as the F127 concentration is increased, the viscosity of the solution also in creases. The viscosity would also effect each of these processes (DST, fabric wetting time, and foamability), but the fact that each of these properties exhibit a distinct change in their curv e around 8 mM supports the fact that there must be some change in the system around this concentr ation range. This is the point at which there is no free SC in the bulk solution. Now that we have some confidence that ou r hypothesis is correct, we can plot the concentration of F127 at which turbidity is no lo nger observed versus th e SC concentration, and determine the optimal binding ratio of F127 to SC. We can also pl ot the concentration of F127 at which turbidity is no longer observed versus the AMT concentration and determine the optimal binding ratio of F127 to AMT. Figure 3-7 shows the results of these plots. As can be seen in the

PAGE 103

103 figure, the optimal binding ratio of SC and F127 is approximately 11 molecules of SC for every 1 molecule of F127, and the optimal binding ratio of AMT and F127 is approximately 12 molecules of AMT for every 1 molecule of F127. When plotting the mmoles of AMT for turbidity versus the mmoles of bound SC, as shown in the inset of Figure 3-7, we see that there is nearly a 1:1 association between the two molecules. This suggests that the electrostatic interaction between the negatively charged SC and the positively charged AMT plays a significant role in the AMT binding to the microe mulsion. Ultrafiltration experiments have also been performed in which the drug is introduced to the microemulsion system, filtered through nanoporous membrane filters, and the AMT concen tration in the filtrate is subsequently measured. We observed that the AMT extraction by the microemulsion is very high at pH values that are equal to or lower than the pKa of AM T (9.4) and is very low above the pKa. This verifies that electrostatic forces are indeed th e predominant force in the initial uptake of AMT into the microemulsion, thereby giving us further proof of the molecular mechanism of AMT uptake by Pluronic microemulsions. The findings of this research suggest that microemulsions of Pluronic F127 + SC + EB are effective for binding of Amitriptyline. However, one might venture to ask why a microemulsion is necessary (i.e. why cant a micellar or mixe d micellar solution be used). We have shown, through this study that the SC is necessary to pr ovide the charge at the interface so that the AMT will be attracted to the droplet. Testing of these systems has also been done on isolated guinea pig hearts to measure their relative eff ectiveness as toxicity reversal mediums.138 These tests have shown that the oil, EB, is necessary to keep the drug molecu les sequestered (i.e. to prevent the drug molecules from be re-released into the blood). Therefore, a most effective system for

PAGE 104

104 detoxification should contain the Pluronic F127, which can be used in high enough concentrations to form microemulsions without becoming toxic, EB, and SC. 3.3.3 Effect of Fatty Acid Chain Length on Drug and Fatty Acid Binging to Microemulsions The ultimate goal of this research is to develop a biocompatible, biodegradable microemulsion that well efficiently and effec tively reduce the free drug concentration in blood. Now that it has been determined that the initial driving force for Amitriptyline uptake is largely due to electrostatic interactions, if the charge density on the oil droplets in the microemulsions can be increased, than we can effectively increase the drug uptake. One possible method to increase the charge density coul d be to increase the chain le ngth of the fatty acid. This hypothesis is based on the notion that in creasing the chain length will increase the hydrophobicity of the fatty acid and thereby incr ease the driving force for partition to the oil/water interface. It is a well kn own fact that increasing surfactant (fatty acid in this case) chain length decreases the cmc because the surfactant affinity to remain as monomers in water decreases.139 This decrease in cmc is a reflection of the surfactants surface activity and suggests that there will be greater partitioni ng of longer chain length surfac tants to the air/water interface. This concept can be extended to apply to the partitioning at the oi l/water interface also. Microemulsions of Pluronic F127 (1 8 mM), ethyl butyrate (110 mM), and phosphate buffered saline (pH 7.4) with sodium decanoate or sodium dodecanoate as the fatty acid (15 100 mM) were prepared. These microemulsions of varying compositions were titrated with 0.2M Amitriptyline to turbidity. The results were analyzed to determine if increasing the fatty acid chain length increased its binding to the Pluronic F127. To our su rprise, increasing the fatty acid chain length in fact acted to decrease the bindin g to F127 as shown in Table 3-1. As mentioned above, with increasing chain length, the cmc d ecreases. The cmc values of the fatty acids in phosphate buffered saline were determined to be 225 mM, 50 mM, and 10 mM for C8, C10, and

PAGE 105

105 C12 fatty acid soaps, respectively. The decrease in cmc with increasing fatty acid chain length also corresponds to a decrease in the monomer concentration. This means that there will be fewer monomers available to partition into the crevices of the Pluronic F127 co vered interface. Figure 3-8 shows a schematic of our microemulsion drople One can see that the Pluronic molecules are unique surfactant molecules, having hydrophili c tails, instead of hydrophobic tails. These hydrophilic tails are the ethylene oxide groups of the block co polymer. Since the Pluronic tail protrudes out into the water, the fatty acid needs to be in monomer form to penetrate in between these flailing tails. Therefore, the decrease in monomer concentration with increasing fatty acid chain length could be the major reason why less fatty acid is bound to the F127. 3.3.4 Effect of the Number of Ethylene Oxide (EO) and Propylene Oxide (PO) Groups of Pluronics on Fatty Acid and Drug Binding Pluronic is the trade name (BASF) of a se ries of symmetric tr i-block copolymers of propylene oxide (PO) and ethylene oxide (E O). The polypropylene oxide block, PPO is sandwiched between two polyethylene oxide (P EO) blocks. PEO blocks are hydrophilic while PPO, is hydrophobic block in the Pluronic molecule. Being amphiphilic in nature, Pluronic surfactants form aggregates in aqueous solution and forms micelles.140-142 The block copolymer is denoted by the symbol (EO)x(PO)y(EO)x, where x and y are the number of units of EO and PO respectively. Depending on number of EO and PO units, various types of Pluronics are available commercially with molecular weight ranging fr om 1100-14600 and the weight fraction of the hydrophilic PEO block ranging between 0.1 and 0.8. In the Pluronics that were used for this study the alphabetical designation explains the physical form of the product: 'P' for pastes, 'F' for solid forms. The first digit (two digits in a three-digit number) in the numerical designation, multiplied by 300, indicates the approximate mol ecular weight of the hydrophobe. The last digit, when multiplied by 10, indicates the approximate ethylene oxide content in the molecules.

PAGE 106

106 For this study, we first held the number of EO groups constant and varied the number of PO groups. Pluronics F68, F88, and F108 were used to analyze the effect of increasing hydrophobicity on binding. Microemulsions of Pl uronic, sodium caprylate, ethyl butyrate and Phosphate buffered saline were prepared and titrat ed to turbidity with 0.2 M Amitriptyline. After a series of titrations, the maxi mum binding ratios of sodium capry late and Amitriptyline to each Pluronic was determined and the values are listed in Table 3-2. It is evident from this table that increasing the hydrophobicity of the Pluronic increases the binding of sodium caprylate and Amitriptyline. Increasing hydrophobic ity will increase the number of binding sites for the fatty acid to bind to. This leads to greater binding of Amitriptyline. Next, the number of PO groups was held co nstant and the number of EO groups was varied. Pluronics P85, F87, and F88 were used for this study. The maximum binding ratios that were determined are listed in Table 3-3. It is evident that there is no apparent trend in binding with increasing hydrophilicity. One observation that must be noted is that Pluronic F88, which is the most hydrophilic of the three Pluronics, has the lowest binding to both SC and AMT. This can be explained based on the idea that as the number of EO groups is increased, the hydrophilic tail which is protruding out into the aqueous phase is getting l onger. The thermal motion of this tail will increase with increasing length and this flailing of the tail could make it more difficult for the sodium caprylate to easily diffuse between the tails to reach the interface. These studies provide us with valuable insigh t into how to design an optimally effective microemulsion for detoxification. It is intuitiv e that the microemulsion that will provide the greatest binding will be very hydrophobic and moderately hydrophilic in its structure. 3.4 Conclusions Here, we have shown how one can use turbidity analysis to gain valuable insight to the molecular structure of Pluronic F127-based mi croemulsion systems. We have shown that the

PAGE 107

107 uptake of Amitriptyline (AMT) by the microemuls ion increases with F127 concentration. We also found that the addition of AMT to the Plur onic F127 microemulsions produced turbidity in all systems up to a critical F127 concentration. Ab ove this critical concentration, turbidity is no longer observed irrespective of how much AMT is added. Other titration experiments proved that turbidity would only occur when there was free Sodium Caprylate (SC) fatty acid soap for the AMT to interact with. Based on these find ings, we concluded that above the critical F127 concentration, there is no longer any free SC available for the AMT to interact with. This allowed us to determine the optimal binding ratios of SC to Pluronic F127 and of AMT to F127. These ratios were found to be approximately 11 molecules of SC per molecule of F127 and approximately 12 molecules of AMT per molecule of F127. We also found the ratio of SC to AMT to be very nearly 1:1 which indicates that electrostatic intera ctions play a major role in the uptake of AMT by the microemulsions due to the negatively charged SC interacting with the positively charged AMT. We also performed foamability, fabric wettin g, and dynamic surface tension studies to correlate with our theory that above a critical F127 concentration there are no longer any free SC molecules present in the bulk. These experiments exhibited a lower foamability, longer fabric wetting time, and higher surface tension at th e critical F127 concentration. These findings support our turbidity analysis conclusions because they indicate that th ere is less surfactant monomer to partition to the interf aces that are present in these experiments (i.e. air/water for foamability, solid/liquid for fabric wetting, and air/water for dynamic surface tension). Lastly, we looked at methods to improve the uptake of Amitriptyline through the manipulation of the fatty acid chain length and th e number or EO and PO groups in Pluronic surfactants. We determined that increasing the fatty acid chain length does not increase binding

PAGE 108

108 and it was concluded that the decreasing amount of monomers that are available to penetrate the Pluronic molecule due to a decrease in cmc w ith increasing chain length The hydrophobicity of Pluronic surfactants directly affects binding of fa tty acid and drug to the microemulsion (i.e. the greater the degree of hydrophobic ity, the greater the binding). Th e hydrophilicity did not appear to directly affect the binding. However, a larger degree of hydr ophilicity does seem to inhibit binding somewhat. This research has provided us with valuable information to prepare an optimal microemulsion for detoxification purposes.

PAGE 109

109 Figure 3-1. Amitriptyline Hydrochloride, MW = 313.9 (pH 7.4); Microemulsion composition : SC, EB, F127, PBS 0 20 40 60 80 100 120 140 0246810 Pluronic F127 Concentration (mM)[AMT] for turbidity, mMMacroemulsion Microemulsion [SC] = 100 mM [EB] = 110 mM Figure 3-2. Titration of microemulsions with 0. 2 M AMT. The solution is clear below the curve and turbid above the curve. N C H3 C H3 H + Cl-

PAGE 110

110 pH 7.40 20 40 60 80 100 120 140 0246810 mmoles Pluronic F127mmoles AMT Mixed Micelles MicroemulsionCLEAR TURBID [SC] = 100 mM [EB] = 110 mM (only in microemulsion) Figure 3-3. Titration of mixed mi celles and microemulsion systems. The solution is clear in the area below the curve and turbid in the area above the curve.

PAGE 111

111 Figure 3-4. Schematic diagram of turbidity in vari ous solutions. Surfactants with black tails and white head groups represent the sodium cap rylate. Red tri-ring structure represents the Amitriptyline drug. Brown coils repr esent the Pluronic F127. The orange agglomerate system represents the comp lex that is formed between the free (monomeric) sodium caprylate in the bulk and the Amitriptyline. This complex formation causes the turbidity to appear in the system. No Turbidity Soln remains clear Turbidity SC salt alone Excess free SC no excess free SC Turbidity A B C Increasing Pluronic F127 Conc. Drug + SC Aggregate Pluronic + SC + drug complex

PAGE 112

112 (pH 7.4); Microemulsion composition : SC, EB, F127, PBS0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 00.010.020.030.040.050.060.070.080.090.1 mmoles Pluronic F127mmoles AMT for turbidty [SC] = 25 mM [SC] = 50 mM [SC] = 75 mM [SC] = 100 mM [SC] = 125 mM[EB] = 110 mMCLEARTURBID Figure 3-5. Titration of microemulsion systems w ith AMT ([SC] = 25 -125 mM). The solution is clear in the area below the curve and turbid in the area above the curve.

PAGE 113

113 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70345678910 [F127] (mM)Foamability; mmoles AMT ; Wetting Time40 42 44 46 48 50 52 54Dynamic Surface Tension (mN/m) mmoles Amitriptyline for turbidity Foamability, mL Wetting time Dynamic Surface Tension, mN/m [SC] = 100 mM [EB] = 110 mM pH = 7.4 Figure 3-6. Properties of Pluronic F127 microemulsion. Grap h of mmoles of AMT for turbidity, foamability, and fabric wetting time (all show n on left y-axis), and dynamic surface tension (shown on right yaxis) of microemulsion systems with SC concentration fixed at 100 mM, EB concentration fixe d at 110 mM and with varied F127 concentration.

PAGE 114

114 Figure 3-7. Binding of SC and drug to F127. A) Optimal binding ratio of SC to F127 in microemulsions (shown by blue points) a nd optimal binding ratio of AMT to F127 in microemulsions (shown by red points). B) Inset shows binding ratio of SC to AMT. mmoles F127 0.000.020.040.060.080.10 mmoles SC 0.2 0.4 0.6 0.8 1.0 1.2 mmoles AMT 0.0 0.2 0.4 0.6 0.8 1.0 1.2 SC per F127 AMT per F127 y = 10.748x + 0.1682 R 2 = 0.9888 SC/F127 y = 11.944x + 0.1174 R 2 = 0.9868 AMT/F127 mmoles SC 0.20.40.60.81.01.2 mmoles AMT 0.0 0.2 0.4 0.6 0.8 1.0 1.2 y = 1.112x 0.07 R 2 = 0.9993A B

PAGE 115

115 Figure 3-8. Schematic depicti on of microemulsion droplet. = Ethyl butyrate oil molecule = Polypropylene oxide portion of Pluronic (hydrophobic) = Polyethylene oxide portion of Pluronic (hydrophilic tails) = Fatty acid surfactant molecule Legend

PAGE 116

116 Table 3-1. Effect of fatty acid chain length on ma ximum binding of fatty acid to Pluronic F127 and of Amitriptyline to F127 Chain length of fatty acid # fatty acid molecules / molecule F127 # molecules AMT / molecule Pluronic F127 8 10.748 11.944 10 8.3773 10.1 12 8.2881 7.827 Table 3-2. Effect of # of PO groups on maximum binding of s odium caprylate and Amitriptyline to Pluronic microemulsions Pluronic molecules SC / molecule Pluronic molecules AMT / molecule Pluronic F68 2.81.8 F88 4.65.5 F108 8.68.1 Table 3-3. Effect of # of EO groups on maxi mum binding of sodium caprylate and Amitriptyline to Pluronic microemulsions Pluronic molecules SC / molecule Pluronic molecules AMT / molecule Pluronic P85 8.39.4 F87 1012.2 F88 4.65.5

PAGE 117

117 CHAPTER 4 A NOVEL METHOD TO QUANTIFY THE AMOUNT OF SURFACTANT AT THE OIL/WATER INTERFACE AND TO DETERM INE TOTAL INTERFACIAL AREA OF EMULSIONS 4.1 Introduction Macroemulsions, which we will refer to simply as emulsions, are generally defined as a heterogeneous systems of one liquid dispersed in an other in droplet form w ith droplets sizes that typically range from 1 100 m in diameter and with each droplet having a monolayer of surfactant or emulsifier molecules as a coating.89 In most instances, the two liquid phases are immiscible, chemically unreactive, and the sy stems that they form are thermodynamically unstable.46 Emulsions may consist of oil droplets di spersed in a continuous water phase (oil-inwater) or of water droplets dispersed in a cont inuous oil phase (water-in-oil). The type of emulsion that is formed depends on many factor s including the oil:water ratio, the molecular structure and concentration of the surfactant, the presence or absence of electrolytes, temperature, and pressure. Emulsion stability is dependan t upon a variety of factors incl uding the physical nature of the interfacial film, the presence of electrostatic or steric barriers on the droplet, the viscosity of the continuous phase, the droplet size dist ribution, oil:water ratio, and temperature.3 Emulsion stability is also closely related to the molecular packing of surfactant that adsorbs at the interface. When more surfactant adsorbs at the interface, the interfacial tension is decreased, and the surfactant molecules act as a barr ier delaying the coalescence of dr oplets by electrostatic and/or steric repulsion. Emulsions typically require the input of energy into the system for their formation. The energy that is input into the system acts to di sperse one liquid into the continuous phase as droplets, which correspondingly increases the in terfacial area significantly. The size of the

PAGE 118

118 interfacial that is genera ted is directly related to the amount of energy that is input into the system is determined by the Equation 1-7.77 W = A (1-7) where is the interfacial tension and A is the change in interfacial area. For a given amount of work, the only way to increase the interfacial area would be to decrease the interfacial tension. Therefore, if one were to manipulate the surfac tant (by changing the ch ain length) so that it would have a greater desire to pa rtition to the interface, then the interfacial area should increase, as represented by smaller droplet sizes. Other possible methods to manipulate the interfacial area include changing the oil chain length or introduci ng additives to the oil phase. In this manuscript, we will investigate the effect that these variables have on surfactant partitioning to the interface. Emulsion have a wide variety of applicati ons including cosmetics, pharmaceutics, foods, paints, polishes, pesticides, and metal cutting oils. Due to this multitude of applications, emulsions have been extensively investigated over the years, and as such their molecular structure is relatively well understood. However, to our knowledge, there is no method available to quantitatively determine the amount of surfactant that reside s at the interface of the emulsion droplet relative to the bulk surfactant concentration. Here, we present, for the first time, a method to quantify the amount of sulfate surfactant, primarily sodium dodecyl sulfate (S DS), that partitions to the oi l/water interface of oil-in-water emulsions of SDS, alkane oils, and water. We simply filter the emulsion, by centrifugation, through nanoporous filters having a 30,000 mo lecular weight cutoff (MWCO). The filtrate is then analyzed to determine the surfactant concentr ation. This concentration is considered to be the bulk surfactant concentration and the remainin g surfactant is assumed to be adsorbed at the oil/water interface as SDS does no t partition into oil. Assuming th at the space occupied by each

PAGE 119

119 individual surfactant molecule at the oil/water interface is very close to area/molecule of the surfactant determined from Gibbs Adsoprtion is otherm at the oil/water interface, we can calculate the total interfacial area (TIA) by multiplying the area/molecu le by the number of molecules at the oil/water interface as determin ed from the ultracentrifugation and subsequent filtrate analysis of SDS. 4.2 Experimental Procedure Materials. Ultrapure sodium dodecyl sulfate (SDS) from MP Bi omedicals, Inc. (Solon, OH) was used as received. Sodium octyl sulfate, sodium decyl sulfate, and tetradecyl sodium sulfate were purchased from Sigm a Aldrich and used without furt her purification. The following chemicals were also used without further pu rification: hexane, octane, decane, dodecane, tridecane, tetradecane, pentadecan e, and hexadecane. 1-Octanol, 1-decanol, 1-dodecanol (Acros Organics (New Jersey)), 1-tetradecanol, 1-he xadecanol, and 1-octadecanol were used as purchased. Double distilled, deionized Mil lipure water was used for all solutions. To investigate the effect of surfactant co ncentration on partitioning at the oil/water interface, oil-in-water emulsions were prepared by placing 1% (v/v) hexadecane into solutions of SDS at concentrations ranging from 8 mM to 20 0 mM. The solution was then mixed on a Vortex hand mixer (Fisher Vortex Genie 2) at the highest setting (8) for one minute. For the other studies (i.e. effect of surfactant chain length and effect of oil chain length) three-component emulsions (water + oil + surfactant) were prepared by taking 50 mM solutions of surfactant solution (such as SDS) and subsequen tly adding 1% (v/v) of the oil phase. In the study of the oil phase volume fraction, three-co mponent emulsions were prepared by taking 50 mM solutions of SDS and subsequently adding from 1% to 25% (v/v) of hexadecane. In each case, the emulsion solution that had been prep ared was then mixed on a Vortex hand mixer (Fisher Vortex Genie 2) at the highest setting (8) for one minute. The four-component emulsions

PAGE 120

120 (water + oil + alcohol + surfactant), 10% v/v of C8 to C12 alcohols and 10% w/v of C14 to C18 alcohols were added to the hexadecane oil phase and equilibrated. Gentle heating was necessary to dissolve the C16 and C18 alcohols, but they were cooled to within 2-3 degrees of room temperature before being added to the 50 mM SDS. The solutions were then mixed on the Vortex mixer at setting 8 for 1 minute. Centricon YM-30 ultracentrifugation filter tube s, having a 30,000 molecular weight cutoff (MWCO) were purchased from Fisher Scientific. Two milliliters of the emulsion solution were placed into the top portion of th e ultracentrifugation tubes and subsequently centrifuged at ~900g for approximately 25 minutes so that less than 10 % of the total solution volume was collected as filtrate. All samples were centrifuged in a bench top IEC Clinical Centrifuge (Damon/IEC Division, Needham Hts, Mass). The filtrate was co llected in the bottom attachment and diluted to the micromolar concentration regime for analysis by a slightly modified dye complexation method112 and compared to a previously prepared calibration curve. Methylene blue dye, chloroform, sodium phosphate monobasic, and sulfuric acid were purchased from Fisher Scientific. Methylene blue reagent was prepared according to standard preparation procedure.113 Two milliliters of methylene blue r eagent were added to two milliliters of the diluted filtrate from the ultrafiltration ex periments. Two milliliters of chloroform were added and the solution was shaken on a Vortex mixer for approximately thirty seconds. Any SDS that was in the filtrate complexed through electr ostatic interaction with the positively charged methylene blue became oil soluble, and thereby partitioned into the chloroform organic phase. The solution was allowed to phase separate and the organic phase was removed and placed into a separate test tube. This process was repeated two more times and the organic phase was then analyzed by UV-Visible spectrometry at 652 nm and the SDS concentration was determined

PAGE 121

121 upon comparison with a calibration curve. A He wlett Packard HP 8453 UV-Vis spectrometer was used for all UV-Vis analysis. Droplet size analysis was performed on a Brookhaven ZetaPlus Size Analyzer. The refractive index of the dispersed phase is input into the software. The emulsion sample was diluted by 10% with water, immediately placed in to a cuvet and inserted into the instrument. The droplet diameter is determined by means of light scattering. 4.3 Results and Discussion 4.3.1 Effect of Surfactant Concentration on Partitioning to the Oil/Water Interface As previously mentioned, emulsions have been thoroughly studied throughout the years and there is quite a bit of gene ral knowledge available regardin g methods to manipulate emulsion size and stability. According to Equation 1-7 above, the only method by which to increase interfacial area for a give n amount of work is by decreasing th e interfacial tension. The primary use of surfactants is to reduce the surface and/or interfacial tension of a given solution. This property of a surfactant is direct ly dependent upon its ability to partition to the interface and replace the solvent molecules, which is reflected by the surface excess, This relationship between surface or interfacial tension and surf ace excess is reflected by the Gibbs equation,143 as follows: i i id d (4-1) where d is the change in surface or inte rfacial tension of the solvent, i is the surface excess concentration of any component of the system, and di is the change in chemical potential of any component of the system. Over the years various efforts have been made to investigate adsorption of surfactants at oil/water interfaces.144 However, these efforts have been limited by the lack of suitable

PAGE 122

122 experimental techniques. Staples145 investigated the adsorption of mixed surfactant at the oil/water interface through the use of small-angle neutron scattering (SANS) in combination with hydrogen/deuterium isotopic substitution. While these studies have been insightful in many regards, as of yet, there is no method available to quantify the amount of su rfactant that partitions to the oil/water interf ace of emulsions. We have devel oped a novel ultrafiltration methodology, illustrated in Figure 4-1, that will allow us to quantitatively determine how much surfactant partitions to the oil/water in terface and how much remains in the bulk (aqueous) phase. We first investigated the effect of surfactan t concentration on partitioning to the oil/water interface. Emulsion solutions of 1% hexadecane in increasing concentrations of SDS were prepared and filtered through the 30,000 MWCO filters (see Figure 4-1). Since SDS has an aggregation number of ~ 65 mol ecules/micelle and a molecular weight of 288, we know that any micelles (MW of SDS micelle = 18 ,720) that may be present in the emulsion should freely pass through the 30,000 MWCO filters. As shown in Figure 4-2, the concentration of SDS in the filtrate increases linearly with increasing total SDS concentration. Since the 30,000 MWCO filters allow micelles to pass through into the filtrate, the SDS concentration that is not in the filtrate must be present at the oil/water in terface. The slope of the line represents the fraction of SDS that is in the filtrate. The take home message here is that in emulsions of 1% hexadecane in SDS solutions, a pproximately 40% of the surfactant resides at the oil/water interface of the emulsion droplets and the remain ing 60% remains in water in this concentration range. After subtracting the SDS concentration in the filtrate from the total SDS concentration, one will see that the amount of SDS at the interface also incr eases with increasing total SDS concentration. This implies that the total inte rfacial area increases w ith increasing total SDS

PAGE 123

123 concentration, which should make droplet size sma ller as we have a fixed volume of oil in the system. To confirm this, we measured the drop let size of 1% hexadecane-in-water emulsions with increasing SDS concentration. As can be se en in Figure 4-3 the drop let size decreases with increasing SDS concentration. This correlates well with the increasing amount of SDS at the hexadecane/water interface as the total SDS concen tration is increased. The decrease in mean droplet diameter with increasing SDS concentration means that there must be an increase in the number of oil droplets and hen ce in the total interfacial area. The results of these experiments raise an interesting observati on. Equation 1-7, W = A suggests that for a given interfaci al tension and a given work valu e, the total in terfacial area should be constant. Given that we are at SDS con centrations that are sign ificantly higher than the cmc value, one might suspect that the interfacial tension would not cha nge significantly with increasing SDS concentration. However, the dr oplet size results suggest that the interfacial tension must be changing significantly. This fact made us re-evaluate Equation 1-7 and we realized that we were attempting to apply an eq uilibrium equation to a dynamic situation. As the emulsion is being generated, it is the dynamic in terfacial tension that is important in determining the total interfacial area that can be stabilized by the surfactan t. Therefore, it would be more accurate under dynamic conditions fo r Equation 1-7 to be rewritten as: f i fAdt d dWt t 0 (4-2) where the first integral runs from time, t0 to time, tf = tfinal which represents the time that it takes to generate the emulsion and the second integral represents the dynamic interfacial tension (d ) and runs from initial ( i) to final ( f).

PAGE 124

124 Given that we now have a method to measure how much surfactant is at the interface of these emulsion droplets, we can now approximat e the total interfacial area by multiplying the area per molecule of each surfact ant by the total number of molecules at the interface. The area/molecule at the interface can be calculated using the Gibbs adsorption isotherm:146 C d d RT ln 2 1 (4-3) where is the concentration of surfactant at the interface, R is the ideal gas constant, T is the temperature, is the interfacial tension at the oil/water interface, and C is the initial concentration of surfactant in the bulk solutio n. The area/molecule at the interface can be calculated by dividing the total interfacial area by the total number of surfactant molecules at the interface. An area per molecule of 50 2/molecule at the oil/water interface was used as reported in literature8 and as suggested by the findings of Rehfeld.147 The total interfacial area (TIA) values were calculated from the results of the filtration experiments and compared to values which were calculated from the m easured droplet size. In order to determine the TIA from the mean droplet size, the following series of equatio ns had to be solved. First, the volume of a single drop can be determined from: 33 4 r V (4-4) where r = radius of droplet as determined from light scattering. Dividing the total volume of oil by the volume of a droplet as given by Eq. 4-4 w ill give the total number of droplets, N. The total interfacial area is then simply calculated from: 24 r N TIA (4-5) where r is the measured droplet size fr om light scattering measurements.

PAGE 125

125 As can be seen from Table 4-1, the TIA that is calculated based on the filtration experiment is two orders of magnitude higher than the TIA from the droplet size measurement. It must be noted that the droplet size (from the dynamic light scatte ring results) that is used to determine the TIA is in fact a mean droplet size and that there is indeed a size distribution associated with each of these emulsions. Ther efore, the TIA calculated from light scattering measurements only provides an approximation of the TIA that will be sufficient for making order of magnitude comparisons in contrast to direct determination of surfactan t at the interface using filtration through nanoporous membranes. One other possible flaw of dynamic light scattering is that in an emulsion with a broa d size distribution, larger drop lets would dominate the light scattering and smaller are ignored.148 On the contrary, filtration through nanoporous membranes accounts for surfactant lost by adsorption on small as well as large droplets. The magnitude of the difference in TIA for the two methods is si gnificantly large. Give n that the 30,000 MWCO filters retain nearly all droplets larger than appr oximately 5-6 nm, the TIA that is calculated from ultrafiltration method is significantly more accura te than the value that can be deduced from droplet size measurements using light scattering. From this methodology we are able to determin e that for increasing co ncentration of SDS, the total amount of SDS at the interface increases, the droplet si ze decreases, the number of droplets increases, the total interfacial area (as m easured from the filtrati on data) increases. The total interfacial area (as measured from light sc attering droplet si ze results) also increases as shown in the master diagram shown in Figure 4-4. which correlates the trends among all of these properties of emulsions. 4.3.2 Effect of Alkyl Sulfate Chain Length on Partitioning to the Oil/Water Interface One might intuitively think that increasing the chain length of the surfactant would increase the surface excess (decreas e interfacial tension) due to th e fact that increasing the chain

PAGE 126

126 length magnifies the unfavorable interaction between the surfactant chain and water. However, it has been shown that increasing the surfactan t chain length beyond 10 carbon atoms has very little effect on the surface excess at the heptane/water interface.3 This is probably due to the fact that the cross-sectional area of th e sulfate head group reaches to its smallest size for oils greater than C10 carbon chain length. While these effect s are known at the heptane/water interface, to the best of our knowledge, they have yet to be investigated at the hexadecane/water interface. We decided to investigate the effect of incr easing surfactant chain length on adsorption at the hexadecane/water interface because hexadecane is a more hydrophobic substance than heptane and could possibly show a more pronounced effect on adsorption than that of the heptane/water case. We prepared emulsions of 1% hexadecane in 50 mM solutions of sodium octyl sulfate (C8SO4), sodium decyl sulfate (C10SO4), sodium dodecyl sulfate or SDS (C12SO4), and sodium tetradecyl sulfate (C14SO4). We then filtered these solu tions (by centrifuga tion) through a nanoporous membrane filter having a molecular we ight cutoff (MWCO) of 30,000. The filtrate concentration was determined by the me thylene blue dye complexation method113 and used to determine the amount of surfactant that had partit ioned to the interface. It was assumed that all surfactant that did not pass through the filter was at the oil/water in terface. Hexadecane was also chosen as the oil phase because the surfactant ha s minimal, if any, solubility in it and will therefore reside only at the inte rface and in bulk solution. Figure 4-5 shows the results of this analysis. Figure 4-5 shows that the partitioning of surfactant to the interface increases with increasing alkyl sulfate chain length. This s hows that when the oil phase is a long chain hydrocarbon (C16), the adsorption effects ar e more pronounced than when a shorter hydrocarbon, such as heptane (C7), is the oil phase. The droplet si ze of each of these emulsions

PAGE 127

127 (1% hexadecane in 50 mM alkyl sulfate) was th en measured to determine if our method of assessing partitioning of surfactant to the interfac e is reasonable. Droplet sizes were determined by light scattering measurements and, as can be seen in Figure 4-6, correlate well with the partitioning results. The partitioning to the interface was least for C8SO4, which resulted in the highest interfacial tension, and hence, the la rgest droplet size. However, in the case of C14SO4, the partitioning was the highest, and consequentia lly the droplet size was the smallest. In each surfactant case, the drop let size distribution was mono-modal (i .e. there was a si ngle peak, albeit somewhat broad). Next, we determined the total interfacial area as a function of surfactant chain length by the same method that was used in the previous section. The area per molecule at the hexadecane/water interface that was used fo r each surfactant is given in Table 4-2. The total interfacial area (TIA) values were calculated fr om the results of the filtration experiments and compared to values which were calculated from th e measured droplet size. Just as our previous results (on the effect of surfact ant concentration) showed, once again the TIA values that were calculated from the filtration studies are approximate ly two orders of magnitude greater than the values which were calculated from the droplet size measurements. One way to verify that the results that we ha ve obtained from our filtration experiments are accurate is to compare the ratios between the TIA values for C12 and C14 sulfate that were calculated from our results to the TIA values calc ulated from the mean droplet size given by light scattering. As shown in Table 4.2 the TIA for em ulsions prepared from C12 and C14 sulfate was 97,837.6 and 140,899.7 cm2/L, respectively, as calculated from the filtration results. In this case the TIA of the C14 sulfate emulsion is approximat ely 1.9 times greater than the TIA of the C12 sulfate emulsion. Table 4.2 also gives TIA valu es for the C12 and C14 sulfate emulsions as

PAGE 128

128 515.8 and 908.3 cm2/L, respectively, as calcu lated from the mean droplet size given by light scattering. In this case, the TIA of the C14 su lfate emulsion is approximately 1.8 times greater than the TIA of the C12 sulfate emulsion. Give n that these two methods (filtration and light scattering) were totally independent of each other and yet the ratios of the TIA values of the C12 and C14 sulfate emulsions are near ly the same (1.9 vs. 1.8) for both methods, we can conclude that our filtration method is providing reasonably accurate results. 4.3.3 Effect of Oil Chain Length on SDS Partitioning to the Oil/Water Interface Next, we decided to investigate the effect of increasing the chain leng th of alkane oils on adsorption of surfactant at the oil/water interface. We prepared emulsions of 1% alkane oil (octane to hexadecane) in 50 mM solutions of SDS (C12SO4). We then filtered these solutions (by centrifugation) through a 30,000 MWCO nanopor ous membrane filter and determined the SDS concentration in the filtrate. Figure 4-7 shows the results of this analysis. It is hypothesized that increas ing the chain length of the o il in an analogous series of alkane oils will increase the driving force fo r SDS surfactant to par tition to the oil/water interface. This would lead to a greater surface excess (lower interfacial tension). We employed our methodology to test this hypothesi s and found that when the chain length of the oil is shorter than the chain length of the surf actant (SDS), increasing the oil ch ain length from C8 to C12 in fact, acts to decrease the surfactant partitioning (see Figure 4-7). When the chain lengths of the oil and surfactant are equal, there is a minimum in surfactant adsorption at the interface, and as the oil chain length is increased be yond C12, the partitioning of SD S to the interface increases. For the oil chain lengths that are less than or equal to 12 carbons, the partitioning of SDS decreases with increasing chain length, which is in agreement with the findings of Hallworth and Carless.149, 150 They found that for the shor ter oil chain lengths (< C12) the adsorption of SDS at the oil/ water interface was the gr eatest for the shortest chain length (hexane). We believe that

PAGE 129

129 this phenomenon may be due to solubility of the SDS in shorter chain length oils. The minimum in partitioning that is seen when dodecane (C12) is the oil phase is due to a chain length compatibility effect which leads to penetration of the oil molecules in to the space between the hydrophobic tails of SDS molecules as depicted by Figure 4-8. If oils with chain lengths different from C12 penetrate the interfacial space, the differen ce in the chain lengths between the oil and SDS (C12SO4) would lead to tail wagging30 which would lead to less effective molecular packing at the interface. This would be unf avorable, so the longer-chain oils remain more in the interior of the droplet and the interface is free for the SDS to partition. Therefore, less SDS is present at the interface when dodecane is the oil phase as shown in Figure 4-8. In other words, maximum penetration of oil in th e interfacial monolayer occurs when the oil and the su rfactant molecules have the same chain length. Chain length compatibility is a phenomenon that can exhibit its effect s in foam stability,120 evaporation of water through monolayers,151 micellar stability,30 and even in changes in the area per molecule of surfactants.151 Over the years it has been show n that chain length compatibility between the oil phase and the su rfactant can also play a majo r role in boundary lubrication,152 solubilization of water into water-in-oil microemulsions28, 120, 153 and in emulsion formation and stabilization.154 The increase in SDS adsorption with incr easing chain length beyond C12 oil (dodecane) reflects the findings of McClements and coworkers.155 They showed that the Ostwald ripening rates decreased with increasing oil chain length fo r 5 wt % oil-in-water emulsions stabilized with 20 mM SDS. Ostwald ripening is a destabilization process in emulsions by which larger emulsion droplets grow at the e xpense of the smaller droplets.51 This suggests that with increasing chain length, the emulsions become mo re stable against coalescence, which could be

PAGE 130

130 due to the adsorption of more surfactant at the interface whic h will create greater Coulombic repulsion between adjacent droplets In order to further clarify how oil chain length affects SDS partitioning to the interface, th e emulsion droplet size was measured and plotted beside the partitioning results in Figure 4-7. The plot shows th at the largest particle size is obtained for the case when the oil phase is dodecan e (C12), which also corresponds to the lowest partitioning of SDS at the interface. Also, for C12 to C16, the partitioning increases and the droplet size decreases. Total interfacial area calculations were once again two orders of magnitude higher when calculated from partitioning (filtration) results than when calculated from droplet size measurements. The values for TIA are given in Table 4-3. 4.3.4 Effect of Alcohol Chain Le ngth on SDS Partitioning to the Oil/Water Interface Another possible method by which to manipulat e interfacial tension and emulsion droplet size is by the addition of long-ch ain alcohols into the oil phase. Long-chain alcohols were chosen as additives because they are soluble in the he xadecane and can provide charge shielding when adsorbed at the interface between SDS molecules. Ten (10) % solutions (v/v % for C8OH to C16OH and w/v % for C18OH) of alcohol in hexadecane were prepared and 1% (v/v) of this solution was added (as the oil phase) into 50 mM solutions of SDS. The samples were filtered a nd the filtrate was analyzed. The results showed that there is a chain length compatibil ity effect present when dodecanol (C12OH) is added to hexadecane (see Figure 4-9). The dodecanol and SDS combination are able to form a tightly packed interface and allows for maximum adsorpti on of SDS as compared to other alcohol chain lengths. When the chain lengths are the same, the Van der Waals interactions between the hydrophobic tails are maximized.41, 90 The other alcohol chain le ngths are not effective at enhancing SDS adsorption at the interface because th e difference in the chain lengths leads to tail

PAGE 131

131 wagging. It has been reported90 that as the difference in chai n lengths of mixed surfactants increases, the spacing between adj acent surfactant molecules incr eases due to disorder produced in the chain by the thermal motion of the term inal segments. Although th ese changes are very small, it has been shown that they have a very large effect upon the interfacial and bulk properties of the solutions (e.g., foamability, fo am stability, surface tension, surface viscosity, contact angle, bubble size, fluid displacement in porous media, and microemulsion stability).90, 120, 125, 126, 154 The emulsion droplet size is also the smallest when dodecanol is the added alcohol as can be seen in Figure 4-9. The total inte rfacial area values are given in Table 4-4. 4.3.5 Effect of Oil Phase Volume Fraction on SDS Partitioning to th e Oil/Water Interface Our next experiment involved increasing the oil phase volume fraction from 1% to 25% to determine its effect on SDS partitioning to the oi l/water interface. We found that the partitioning of SDS showed no change with increasing volume fraction as s hown in Figure 4-10. This makes perfect sense because Equation 1-7 suggests that for a given amount of wo rk, if the interfacial tension does not change (which it will not because the surfac tant and the oil phase are not changing), then there will be no ch ange in the interfacial area. If there is no change in the interfacial area then the amount of SDS that is adsorbed at the interface also should not change. Because the amount of surfactant adsorbed at the interface of emulsion droplets remained the same, it clearly establishes that the same am ount of work done in producing emulsions will produce the same total interfacial area irrespec tive of the relative volume fraction of oil and water. 4.4 Conclusions We have, for the first time, developed a met hod to quantitatively determine the amount of surfactant that partitions to the oil/water interface and effectively used these results to calculate the total interfacial area of a given oil-in-water emulsion. By using nanop orous filter membranes

PAGE 132

132 having a 30,000 molecular weight cutoff (MWCO) through which emulsion droplets could not pass, we were able to determine the free concen tration of sodium dodecyl sulfate (SDS) (i.e. the concentration that was not at th e interface of the oil droplets). We have assumed that the amount of surfactant that is not in the filtrate is at the oil/water interface. The following conclusions can be drawn as a result of the work done here: 1. The partitioning of surfactant to the interface increases linearly with increasing total surfactant concentration. By filter ing the samples thro ugh the 30,000 MWCO filters, we were able to ensure that micelles would pass through the filter and thereby determine the amount of surfactant th at is present at the oil/water interface. We determined that for solutions of 1% (v/v) hexadecane in SDS, approximately 40% of the surfactant in the solutions is pr esent at the oil/water interface. We have also shown that mean droplet size decreas es with increasing SDS concentration as expected based on the filtration results 2. The chain length of the surfactant does significantly effect its partitioning to the interface when the oil phase is sufficien tly hydrophobic, as is the case with hexadecane. The partitioning increases with increasing surfactant chain length and this increase is reflected by decreasing droplet size with increasing chain length. 3. When the total interfacial ar ea is calculated based upon the partitioning (filtration) experiments, the value is significantly more accurate (2 orders of magnitude higher) than when it is calculated from droplet size measurements obtained by dynamic light scattering. 4. The partitioning of SDS to the oil interf ace is highly dependent upon the oil phase chain length. When the oil chain length is equal to or less than C12, the partitioning decreases with increasing oil chain length. This has been attributed to the SDS being able to solubilize into shorter chai n length oils. When the chain length is greater than C12, the partit ioning increases with increas ing chain length. This has been attributed to the inability of the longer chain oils to penetrate into the interfacial film. The increased partiti oning at the interface is reflected by the smaller droplet size for oils higher than C12 decreasing with increasing oil chain length. 5. When a small amount of alcohol (of varying chain length) is added to the oil phase (hexadecane) the maximum partitioning of SDS to the interface is observed when the alcohol is dodecanol (i.e. when the ch ain lengths of SDS and alcohol are the same, even though the oil was hexadecane). The chain length compatibility is a major contributing factor to the enhanced adsorption of SDS. The other alcohols (C8OH C10OH and C14OH C18OH) did not effect the partitioning of SDS (i.e.

PAGE 133

133 the amount of SDS at the interface was the same as when no alcohol was added to the hexadecane). 6. Increasing the volume fraction of the oil pha se has no effect on the partitioning of SDS to the oil/water interface, as woul d be expected from Equation 1-7, W= A.

PAGE 134

134 Figure 4-1. Schematic depiction of filtration of oil-in-water emulsi on through nanoporous filter membrane. Emulsion droplets are retained in the filtrand (top portion) and only surfactant monomers and mi celles are small enough to pass through the filter pores. Nanoporous filter membrane Filtrate emulsion droplets excluded; only monomers and micelles pass through filter Force = 900 g Axis of Rotation Sam p le solution Oil-in-water emulsion droplet Surfactant monomer Micelle

PAGE 135

135 y = 0.5999x 0.5011 R2 = 0.99640 20 40 60 80 100 120 1400255075100125150175200225 Total [SDS] (mM)Amount of SDS in filtrate, mM YM30 Linear (YM30)1% (v/v) Hexadecane in SDS emulsions Filtration through 30,000 MWCO Micelles pass through Figure 4-2. Effect of total SDS concentration on SDS concentration in the filtrate after filtration of 1% (v/v) hexadecane-in-water em ulsions through 30,000 (YM30) molecular weight cutoff (MWCO) filters. Sample s were centrifuged at 900g for 25 minutes. Schematic inset depicts filtration thr ough 30,000 MWCO filter with micelles in filtrate (bottom portion).

PAGE 136

136 0 200 400 600 800 1000 1200 1400 1600 0255075100125150175200225 [SDS], mMMean droplet diameter, nm1% (v/v) hexadecane-in water emulsions Figure 4-3. Effect of SDS concentration on mean droplet diameter of 1% (v/v) hexadecane-inwater emulsions.

PAGE 137

137 Figure 4-4. Master diagram show ing the changes in emulsion char acteristics with increasing SDS concentration. 0 200 SDS concentration Droplet size Amount of SDS at interface # of droplets TIA from droplet diamete r TIA from filtration results

PAGE 138

138 Alkyl Sulfate Chain Length C8C10C12C14 Amount of Surfactant at Interface, mM 0 10 20 30 40 50 Samples run in 30,000 MWCO tubes at 900g for 25 min Initial [Surfactant] = 50 mM in each sample Volume fraction hexadecane = 1% (v/v) Figure 4-5. Amount of alkyl sulfat e surfactant that partitions to the interface as a function of chain length.

PAGE 139

139 Figure 4-6. Mean droplet size of hexadecane-in-water emulsions as a function of alkyl sulfate chain length. Droplet size was determined by light scattering. Alkyl Sulfate Chain Length C8C10C12C14 Mean Droplet Diameter, nm 0 500 1000 1500 2000 2500 3000 3500 [Surfactant] = 50 mM in each sample Volume fraction hexadecane = 1% (v/v)

PAGE 140

140 Figure 4-7. Effect of oil chain length on the amo unt of SDS that partitio ns to the interface and mean droplet size of oil-in-water emulsions. Oil Chain Length C8C10C12C13C14C15C16 Amount of SDS at the Interface, mM 0 5 10 15 20 25 30 Mean droplet Diameter, nm 0 1000 2000 3000 4000 SDS at interface Mean droplet diameter Samples run in 30,000 MWCO tubes at 3000 rpm (900g) for 25 min Initial [SDS] = 50 mM in each sample Volume fraction alkanes = 1% (v/v) Samples stirred on vortex hand-shaker for 1 minute at level 8

PAGE 141

141 Figure 4-8. Schematic depiction of emulsion droplets coated with A) SDS alone with oil molecules more towards the interior of the droplet or B) SDS with dodecane penetrating the interfacial space between SD S molecules and forming a more rigid, interfacial film. SDS molecule Long-chain oil (< or > C12) Dodecane (C12) molecule penetrating interface A B

PAGE 142

142 Figure 4-9. Effect of alcohol ch ain length on the amount of SDS th at partitions to the interface and mean droplet size of oil-in-water emulsions with added alcohol. alcohol chain length no alcoholC8OHC10OHC12OHC14OHC16OHC18OH Amount of SDS at the Interface, mM 0 10 20 30 40 Mean droplet diameter, nm 0 1000 2000 3000 4000 5000 [SDS] at interface, mM Mean droplet diameter, nm Samples run in 30,000 MWCO tubes at 900g for 25 min Initial [SDS] = 50 mM in each sample Volume fraction hexadecane + 10% (v/v or w/v) alcohol = 1% (v/v) Samples stirred on vortex hand-shaker for 1 minute at level 8

PAGE 143

143 Figure 4-10. Effect of oil phase (hexadecane) volume fraction on partitioning of SDS to the oil/water interface. Vol % Hexadecane -5051015202530 Amount of SDS at interface, mM 0 10 20 30 40 Samples run in 30,000 MWCO tubes at 3000 rpm (900g) for 25 min Initial [SDS] = 50 mM in each sample Samples stirred on vortex hand-shaker for 1 minute at level 8

PAGE 144

144 Table 4-1. Effect of SDS concentration on total inte rfacial area 1% (v/v) hexadecane-in-water emulsions. SDS concentration, mM Total Interfacial Area (cm2/L) [from filtration results] Total Interfacial Area (cm2/L) [from light scattering droplet size] 8 1.831E+07 4.776E+05 50 5.982E+07 8.06E+05 100 1.088E+08 9.390E+05 200 2.461E+08 1.624E+06 Table 4-2. Area per molecule values at the hexa decane/water interface for alkyl sufates and total interfacial area as a function of alkyl sulfate chain length. All values taken at room temperature except for C8SO4, which was heated to around 40C in order to obtain a reproducible measurement. Alkyl sulfate Area per molecule, 2 Total Interfacial Area (cm2/L) [from filtration results] Total Interfacial Area (cm2/L) [from light scattering droplet size] C8SO4 80 11923.2 207.9 C10SO4 75 90116.3 457.8 C12SO4 70 97837.6 515.8 C14SO4 50 140899.7 908.3

PAGE 145

145 Table 4-3. Effect of oil chain length on total interfacial area (TIA). TIA is calculated from filtration results and from droplet size measurements. Oil chain Length Total Interfacial Area (cm2/L) [from filtration results] Total Interfacial Area (cm2/L) [from light scattering droplet size] C8 36261.1 582.5 C10 27853.3 201.4 C12 18366.2 185.5 C13 45767.0 216.4 C14 53326.4 329.7 C15 55626.3 377.7 C16 68486.3 515.8 Table 4-4. Effect of alcohol chain length on to tal interfacial area (TIA ) when 10% alcohol is added into hexadecane and used as the oil phase. The emulsions contained 1% of this oil phase. TIA is calculated from filtration results and from droplet size measurements. Alcohol chain Length Total Interfacial Area (cm2/L) [from filtration results] Total Interfacial Area (cm2/L) [from light scattering droplet size] C8OH 76181.1 178.1 C10OH 91125.0 508.5 C12OH 132133.8 868.2 C14OH 95764.8 309.8 C16OH 82746.2 405.0 C18OH 81063.7 308.8

PAGE 146

146 CHAPTER 5 SUMMARY AND RECOMMENDATIONS FOR FUTURE WORK 5.1 Micelles 5.1.1 Summary Micellar solutions are generally regarded as consisting of monomers and micelles. When one considers the dynamic nature of micelles, it is conceptually obvious that at any given time, there must be sub-micellar aggregates present in solution in significantly large quantities, particularly if micelles break and form rapidly (i.e. relaxation times less than 100 msec). If micelles were infinitely stable, then one would only see the existence of monomers and micelles in the solution. But if the micelles become more and more unstable, the concentration of submicellar aggregates must increase in the solu tion. This hypothesis was tested by using the ultrafiltration method. Solutions of sodium dodecyl sulf ate (SDS) at concentrations below and above the critical micelle concentration (cmc) we re filtered through nanoporous membrane filters having a molecular weight cutoff (MWCO) of 10,000. This filtration process allows monomers to pass through the pores and will retain micelles. The filtrate concentr ation of SDS was measured for each sample and plotted against the total surfactan t concentration. The filtrate concentration was essentially equal to the total SDS concentration up to the cmc. Since no micelles were yet present in solution, all monomers passed through the pores ). However, when the cmc was reached, the filtrate concentration continued to increase with increasing total concentration but at a different (more gradual) slope. If monomers and micelles are the only two species in micellar solutions and micelles are not allowed to pass through the filte r pores, than one would expect for the slope beyond the cmc to be horizontal (i.e. no change in filtrate concentrati on beyond cmc). Since the filtrate concentration continues to increase beyond the cmc, there must be some other species

PAGE 147

147 present in the solution that are small enough to pass through the pores. These species are submicellar aggregates. It is intuitive that the con centration of sub-micellar aggreg ates should be related to the stability of the micelles. We stabilized the SDS micelles by addi ng 20 % dodecanol and dodecyltrimethylammonium bromide (C12TAB). When the dodecanol was added, the slope beyond the cmc decreased significantly. Howeve r, it still was not horizontal. When the C12TAB was added, the slope became essentially horizont al indicating that this mixed micellar system consisted almost solely of monomers and micelle s and that we had effectively eliminated the presence of sub-micellar aggregates from the sy stem. This finding provi des significant insight into the role of micellar stability in dynamic processes as it is the presence of sub-micellar aggregates that is responsible for controlli ng the dynamics in many t echnological applications including foaming, fabric wetting, dyna mic surface tension, and emulsification. 5.1.2 Future Work 1. There is still a need for another method to prove the existence of sub-micellar aggregates in surfactant solutions. While dynamic light scattering could be useful for accomplishing this goal for larger surfact ants, it is not effective for surfactants as small as SDS because the sub-micellar aggregates are too small to detect. Another possible method could be to use of Gel Permeation Chromatography (GPC) at low temperature. This could be feasible because in GPC, the heavier entities would pass through the column more slowly. Therefore, one might expect to see three distinct regions in a 50 mM SDS solution, for example: monomers, sub-micellar aggregates, and whole micelles. However in an SDS + C12TAB mixed system, there would only be two regions: monomers and whole micelles. One other method that should be attempted is Electr on Spin Resonance. In this method, a probe would be placed into the SDS system and into the SDS + C12TAB mixed micelle system and the system could be observed to see if the presence of submicellar aggregates could be detected in the SDS system. 2. The findings of this research also bring to light the need to model these surfactant systems taking care to account for the pr esence of sub-micellar aggregates. One could model the 50 mM SDS system and compare it to a model of the 50 mM SDS:12.5 mM C12TAB system.

PAGE 148

148 3. One could also look at the effect of making micelles more labile, through the addition of short chain alcohols, on the presence of sub-micellar aggregates. The Shah research group has shown that th e addition of short-chain alcohols, C3OH C8OH, act to destabilize micellar solutions of SDS. Since we have been able to correlate micellar stability with sub-mice llar aggregate concentration, one might expect that if a short-chain al cohol is added to an 80:20 SDS:C12TAB mixed micellar solution (above cmc) the micelle s would become unstable and the submicellar aggregate concentration would in crease. This study should be done as a function of surfactant concen tration and as a function of alcohol concentration. 4. Considering that for the 80:20 SDS:C12TAB mixed system the sub-micellar aggregate concentration does not change be yond the cmc, it would be interesting to take three SDS:C12TAB concentrations at this ratio (10 mM SDS:2.5 mM C12TAB, 25 mM SDS:6.25 mM C12TAB, and 50 mM SDS:12.5 mM C12TAB) and measure the micellar stability by pressure jump or stopped flow method. Since the submicellar aggregate concentration is fixed, and with increasing concentration you are only increasing the number of micelles, one would suspect that the micellar stability should not change as a function of concentration. 5. The effect of sub-micellar aggregates s hould be investigated more thoroughly in relation to various technological proce sses. Foamability, foam stability, dynamic surface tension, detergency, and fabric wetti ng are a few applications that could be looked at in more detail. Gi ven our previous results, one would expect that for the system with the highest sub-micellar a ggregate concentration there would be a maximum foamability, minimum foam stability (due to less stable micelles to prevent the lamellae from collapsing) minimum dynamic surface tension, and maximum fabric wetting rate. The detergen cy would be interes ting to investigate because if the micelles are ve ry stable (low sub-micellar aggregate concentration) than they can better solubilize more oil-so luble soils than a very labile (high submicellar aggregate concentration). On th e other hand, a system with a high concentration of sub-micellar aggregates could possibly rem ove a stain faster because the sub-micellar aggregates, whic h have a faster diffusion time than an intact micelle, could participate in the removal of the soil. 6. One could prepare an 80:20 mixture of SDS:C12TAB and filter the solution through a 10,000 MWCO ultrafiltration tube. Th en instead of determining the SDS concentration in the filtrate, they could measure the C12TAB concentration in the filtrate either by some specific colorimetric technique or by NMR analysis of the filtrate. This would provide a conclusive method to determine how many molecules of C12TAB associate with each molecule of SDS. As of now, there is no other methodology available to make this determination.

PAGE 149

149 5.2 Microemulsions 5.2.1 Summary Microemulsions are often even more attr active than emulsions for many industrial applications because they have much smalle r droplets which corresponds to much greater interfacial area. One of the mo re recent applications of microemulsions is for use as a detoxification vehicle to reduce the free drug concentration in th e blood of an overdose patient. In order to successfully accomplish this goal of using microemulsions for detoxification, one must be able to understand and manipulate the mol ecular interactions that are occurring at the interface of these microemulsion droplets. One method to analyze drug binding to the mi croemulsion consists of titrating the microemulsion with concentrated drug until turb idity is reach. The point where turbidity becomes apparent is important a nd gives us valuable information about the binding of both fatty acid and drug molecules to the microemulsion. Microemulsions of Pluronic F127 (a triblock ethylene oxide propylene oxide c opolymer), sodium caprylate fa tty acid, ethyl butyrate, and phosphate buffered saline at pH 7.4 were prepared at various compositions and titrated to turbidity. We have shown that there is a linear relationship between the Amitriptyline Hydrochloride solubilization capacity (i.e. the amount of Amitriptyline th at the microemulsion can accommodate before turbidity occurs) of the microemulsions and Pluronic surfactant concentration up to a certain Pluronic F127 concen tration. Above that cr itical Pluronic F127 concentration, further titration with Amitriptyline never yields turbidity. We have also seen that turbidity is not observed in systems that do not have sodium caprylate present. Based on these findings we have concluded that at the critical Pluronic concentra tion, there is no longer any free (unassociated) sodium caprylate molecules in th e bulk phase, presumably due to binding of all fatty acid molecules with Pluroni c molecules. Therefore, we ar e able to determine how many

PAGE 150

150 molecules of sodium caprylate and Amitriptyline are associated with each Pluronic molecule. After extensive experimentation and analysis it was determined that each Pluronic F127 molecule can bind eleven molecules of sodium caprylate and twelve molecules of Amitriptyline drug maximum. This finding gives us information about the charge densit y at the interface of each droplet and suggests that the initial mechan ism of drug uptake is driven by electrostatic interaction between the positively charged Amitriptyline and the negatively charged sodium caprylate. This will also allow us to appr oximate the uptake capacity of a particular microemulsion system. Our next goal was to find ways to enhance the uptake of drug by the microemulsion. Since the initial driving force for uptake is due to el ectrostatic interactions, we first investigated methods to increase the charge density of the drop lets. By increasing the fatty acid chain length, the driving force for adsorption at the oil/wate r interface should increase. However, we found that the binding of fatty acid to Pluronic F127 de creased with increasing ch ain length. This is due to the lowering of the cmc with increasing ch ain length, which results in a lower monomer concentration. Monomers are need ed to partition betw een the ethylene oxide tails of the F127 to adsorb at the interface. If there are not as many monomers available, the increased driving force to adsorb at the oil/wa ter interface with increasing chai n length becomes a non-factor. The results of these studies also suggested that sub-mi cellar aggregates can play a significant role in the binding of drug molecules. The Pluronic surfactant was the ne xt factor to be manipulated. In this study we evaluated the effect of the number of ethylene oxide (EO) and propylene oxide (PO) groups (or the effect of hydrophilicity and hydrophobicity) on the binding of fatty acid and drug to the Pluronic. We found that increasing the degree of hydrophobicity of the Pluronic increases the binding of fatty

PAGE 151

151 acid and drug to the microemulsion. Increasing hydrophobicity will increase the driving force for the Pluronic to adsorb at the oil/water interface, and as such will lower the surface tension more and lead to the generation of more interfacial area. When more inte rfacial area is generated, more droplets will develop which will require more adsorption of sodi um caprylate. This also would lead to greater binding of Amitr iptyline. Increasing th e hydrophilicity did not appear to have a direct effect on the binding of fatty acid or drug molecules. Howe ver, it was found that the least binding occurred when the degree of hydrophilicity was the greatest (i.e. when there were the most number of EO groups). This is due to incr easing thermal vibrations of the EO tails as the number of EO groups is increase d. When this occurs, it beco mes more difficult for fatty acid molecules to diffuse between these flai ling tails to adsorb at the interface. 5.2.2 Future Work 1. One extension of this work could be to verify the existence of a fatty acid/F127 complex (1:11 ratio). This could be a ccomplished by preparing microemulsions with excess fatty acid concentration (so th at there is free fatty acid in the bulk phase) and subsequently filtering the mi croemulsion solution through a nanoporous membrane to determine the amount of free fatty acid (i.e. concentration of fatty acid in the filtrate). A further proof woul d be to prepare a microemulsion solution at the critical F127 concentr ation (where no fatty acid is free in the bulk phase) and add excess drug (i.e. higher concentration of drug than the microemulsion can bind as predicted by the turbidity experiments). This solution should be filtered and the amitriptyline concentration should be determined by HPLC and compared to the value that was predicted from the turbidity experiments. 2. All of the experiments in this chapter were carried out at pH 7.4. At this pH, there was a 1:1 association of fa tty acid and amitriptyline. This work should be extended to other pH values such as pH 4 and pH 10 to see if the ratio of association changes. At pH 4, the fatty acid would b ecome more unionized and at pH 10, the drug would become more unionized. Ther efore, one would expect that the association between the fatty acid and th e amitriptyline would deviate from 1:1. 3. One could also perform dialysis experiment s to investigate the re-release of drug from the microemulsions after bein g bound as a function of microemulsion composition. In this study, microemuls ions should be formed and a fixed concentration of drug should be added to th e solutions (so that the microemulsion is nearly saturated with drug). This solution should then be placed into a dialysis bag and the bag should be placed into a fresh buffer solution or a fresh microemulsion

PAGE 152

152 solution (with no drug). Samples should be taken from the external solution at regular time intervals and the concentration of drug in the samples should be measured. 5.3 Macroemulsions 5.3.1 Summary Emulsions are used for a wide range of i ndustrial applications including cosmetics, pharmaceutics, detergency, paints, and foods. The major reason why emulsions are so attractive for such applications is due to the large interfacial area that they possess. The interfacial area that is generated by the formation of an emulsion is di rectly related to the ability of the emulsifier (often surfactants) to adsorb at the oil/wa ter interface and lower th e interfacial tension. Emulsions have been extensively investigated over the years, and as such their molecular structure is relatively well understood. Howeve r, to the best of our knowledge, there is no method available to quantitatively determine the am ount of surfactant that resides at the interface of the emulsion droplet relative to the bulk su rfactant concentration. We have developed a methodology to accomplish this through the novel use of filtration through nanoporous membrane filters. In this methodology we prepare emulsions of 1% hexadecane-in-water with varying SDS concentration (8 mM to 200 mM) and f ilter them through a 30,000 MWCO nanoporous membrane filter. Emulsion droplets are to large to pass through the pores of this filter, so only monomers and micelles can pass through. Given th is fact, the filtrate is analyzed and the concentration of surfactant in the filtrate is subt racted from the total surfactant concentration to determine how much surfactant is at the oil/water interface of emulsion droplets. Knowing the area per molecule of the surfactan t, we are able to approximate the total interfacial area (TIA) by multiplying the number of surfactant molecules at the interface by the area per molecule. We also measure the droplet size of each emulsion by dyna mic light scattering to correlate it with the

PAGE 153

153 results of the filtration experiments. According to W = A, for a given amount of work, if the interfacial tension is lowered, the total interfacial area must increase. The findings were rather intere sting for our first study on the effect of SDS concentration on partitioning to the oil/water interface; the fraction of the total SDS concentration that partitioned to the oil/water inte rface remained constant with in creasing SDS concentration at 40 %. In other words, for any given SDS concentr ation, 40% of the SDS would partition to the oil/water interface and the remaining 60% would remain in the bulk water phase. There was a linear increase in the amount of SDS that partitioned to the in terface with increasing total SDS concentration. The droplet size was measured fo r these emulsions and as expected, the droplet size decreased with increasing SDS concentration. This is due to the fact that as the SDS concentration is increasing, the dynamic interfacial tension is d ecreasing, and following Equation 1-7, the total interfacial area incr eases (i.e. droplet size decreases). We also determined the TIA from the filtration results and compared it to the TIA that was calculated from the droplet size measurements. The TIA that was calculated from th e filtration results was found to be two orders of magnitude greater than the TIA from the dr oplet size measurements for each emulsion. This finding has been attributed to the fact that th e droplet size that is reported by dynamic light scattering is a mean droplet size and it is feasib le that since larger droplets scatter more light, they may dominate and smaller, nano-sized, droplets may be overlooked. These nano-droplets that are overlooked would account for a large interfacial area, so the value that is calculated from light scattering will always be much less than that of the filtration results. Therefore, filtration of emulsions through nanoporous filters and subseque nt determination of the amount of surfactant at the interface provides a nove l and effective method by which to approximate the total interfacial area.

PAGE 154

154 Next, we used our method to determine the eff ect of the chain length of alkyl sulfates on surfactant partitioning to the interface. One perc ent (1 %) solutions of hexadecane-in-water emulsions were prepared with alkyl sulfates (C8SO4 C14SO4) as the emulsifier and filter the emulsion through a 30,000 MWCO filter. Through thes e studies we were able to determine that for long chain oils, such as hexadecane, the ad sorption of surfactant at the oil/water interface increases with increasing chain length of surfactant. Correspondi ngly, the droplet size was the smallest for the longest chain length (C14SO4) and the largest for the shortest chain length (C8SO4). Once again, the TIA values that were cal culated from the filtration results were approximately two orders of magnitude greater th an the values that were calculated from drop size. The adsorption of surfactant (SDS ) at the interface was also ex amined as a function of oil chain length, chain length of al cohol added to the oil phase, a nd oil phase volume fraction. For oil chain lengths up to twelve carbons (octan e to dodecane), the partitioning of SDS to the interface decreased with increasing oil chain length This finding was attributed to solubilization of SDS into the oil phase of s horter chain lengths. For chain lengths greater than C12, the partitioning of SDS increased with increasing oi l chain length. The droplet size measurements reflected these results as the droplet size was the largest for dodecane (which had the least adsorption of SDS at the interface) and it decreas ed from C12 down to C8 and it also decreased from C12 up to C16 oil. The reason for this behavi or is that as the oil chain length increases, the oil phase becomes more hydrophobic and the SDS has a greater driving force to partition to the interface. This was shown to be true through me asurements of the inte rfacial tension at the oil/pure water interface and at the oil/SDS solu tion interface. The difference between the two interfacial tension valu es was calculated for each oil chai n length and was found to be the

PAGE 155

155 greatest for C16, indicating that ad sorption of SDS is the greatest in this case. Another important factor for the increase in partitioning of SDS from C12 to C16 oil is the resist to desorption of SDS as the chain length of the oil increases. The longe r the chain length th e more likely the SDS is to remain at the interface longer, preventing coalescence and maintaining the smaller droplet size. Small amounts of long chain alcohols (C8OH to C18OH) were added to hexadecane and used as the oil phase for the generation of an emulsion with SDS. The gr eatest adsorption of SDS at the interface occurred when dodecanol was the alcohol. This is due to chain length compatibility (i.e. the SDS and the dodecanol both have twelve carbons in their chain) which allows for tighter packing of SDS at the interface. The other alcohol s did not appear to effect the partitioning of SDS to the interf ace because SDS cannot pack as tightly due to tail wagging that would occur as a result of the difference in chain lengths. The oil phase volume fraction did not have any effect on the amount of SDS that partitions to the oil/water interface. This is in agreement with the equation, W = A, because for a given amount of work, if the in terfacial tension is fixed, the inte rfacial area will not change either. Therefore, regardless of how much oil is placed into the system, since the interfacial tension does not change, the total area that is generated remains the same, and as such the amount of SDS that adsorbs at the interface will remain the same. 5.3.2 Future Work 1. This work could be further extended by inve stigation of the effect of the method of emulsification (i.e. work input into the system) on surfactant partitioning. Emulsions could be generated by sonicat ion, passing the solution through a tiny orifice, and by shaking. These methods of emulsion formation could be compared internally (e.g. effect of sonication at different sonication levels) and externally (e.g. comparison of emulsion formed by s onication versus an emulsion formed by shaking). One would expect to find distinct differences in the amount of interfacial

PAGE 156

156 area that is generated by th ese differing methods, and ther efore, the partitioning of surfactant to the interface should be effected. 2. Another interesting extension of this research could be to investigate surfactant partitioning in systems that undergo spontan eous emulsification. Because systems that undergo spontaneous emulsification can typically produce droplets in the nanometer size range, one might expect that these systems would typically generate much more interfacial area and therefore more surfactant would partition to the oil/water interface of these systems as oppos ed to an emulsion that is generated by another method (other than spontaneous emulsification). 3. When the oil chain length was varied, ther e was a minimum in SDS partitioning to the interface when dodecane was the oil. This was attributed to chain length compatibility which leads to dodecane mol ecules penetrating into the interfacial space. This can be proven by preparing so lutions of dodecane/water/SDS at various SDS concentrations and determining the area per molecule of SDS from Gibbs adsorption isotherm. Gibbs adsorption make s no assumptions about penetration of oil into the interfacial film. It assumes that only surfactant is at the interface. If the dodecane is in fact penetrating into the interfacial space, then the SDS area per molecule that would be calculated from the Gibbs adsorption isotherm would be much greater than the area per molecule that the Gibbs adsorption isotherm gives when no dodecane is present. This experiment should also be carried out with octane and with hexadecane as the oil pha ses to show that they do not penetrate into the interfacial space as much as dodecane does.

PAGE 157

157 APPENDIX A GIBBS ADSORPTION EQUATION AND AREA PER SURFACTANT MOLECULE DETERMINATION The total energy (E) of a system containing surfactant can be thought as a summation of three individual energies; one each for the bulk phases (say surfactant solution and vapor) and one interface. Thus, a small reversible change in the energy (dE) of the system can be written as: dE = dE + dE + dE (A.1) where subscript represents the interface region and subscript represent the two bulk regions. The energy changes in bulk phases and in terface can then be expanded as functions of system variables: dV P dn dS T dEi i i ) ( (A.2) dV P dn dS T dEi i i ) ( (A.3) dA dn dS T dEi i i ) ( (A.4) where S is entropy, T is temperature, i is the chemical potential of species i ni is the number of moles of i P is pressure, V is volume, A is area, and is the surface or interfacial tension. Note that in equation (A.4), the bulk work term P dV is replaced with the surface work term dA The interfacial component of internal energy ( E) is related to the interfaci al component of Gibbs free energy ( G) by the defining relation,

PAGE 158

158 G = E + A T S (A.5) which in turn is related to the chemical potential and number of moles by, i i in G (A.6) Substituting equation (A.6) into (A.5), differen tiating the resulting equation and, eliminating with equation (A.4), one obtains, i i id n d A dT S 0 (A.7) At constant temperature, for a surfactant (solut e, 1) in water (solvent, 2) the above equation simplifies to, 2 2 1 1 d A n d A n d (A.8) Equation (A.8) can be simplified further by a pr oper selection of the actual location of the interface. Normally, the location is chosen to be where the net adsorption of water (n2 /A) is zero and the equation (A.8) reduces to, 1 1 d A n d (A.9) For dilute surfactant solutions (< 10-2 mM), C T Roln1 1 (A.10) and the equation (A.9) becomes, A n C d d RT1ln 1 (A.11) which is the most widely used form of Gi bbs adsorption equation fo r non-ionic surfactants. Ionic surfactants are treated as electrolytes. For a fully diss ociated surfactant of the 1:1 electrolyte (A+B-) type, equation (A.9) becomes,

PAGE 159

159 ) ln ln (1 1 B B A AC d A n C d A n RT d (A.12) On applying electro neutrality cond itions, equation (A.12) simplifies to, A n C d d RTA A 1ln 2 1 (A.13) which is found to be correct for most A+Btype ionic surfactants as well as Gemini surfactants which are two hydrocarbon tailed surfactants join ed together by a linker having two anions. The slope of the surface tension plot against the natural log of surfact ant concentrations in regions preceding the cmc enables one to calculate area/surfact ant molecule at the interface. Equation (A.13) has been used for calculating the area/mo lecule for the alkyl sulfates in Table 3-1

PAGE 160

160 APPENDIX B CALCULATION OF TOTAL INTERFACIAL AREA FROM FILTRATION RESULTS An emulsion of 1% (v/v %) hexadecane wa s formed in a solution of 100 mM SDS and then it was filtered through a 30,000 MWCO ultrafiltration tube at 900g for 25 minutes. The filtrate was analyzed to determine the c oncentration of SDS. Once the average SDS concentration in the filtrate, [SDS]filtrate, was determined, the total interfacial area (TIA) was calculated by the following series of equations. [SDS]interface = [SDS]total [SDS]filtrate The average filtrate concentration for the 100 mM SDS emulsion was found to be 63.51 mM SDS. From this data, the SDS concentratio n at the interface can be calculated to be: [SDS]interface = 100 mM 63.51 mM = 36.49 mM To determine the number of moles of SDS that are at the interface in a 1 liter solution, the following equation was used: Moles of SDS at interface = (36.49 mM X 1 L)/1000 = 3.649 X 10-2 moles SDS The number of molecules of SDS at the inte rface can be determined by multiplication of the moles by Avogadros number: Molecules of SDS at interface = (3.649 X 10-2)(6.022 X 1023) = 2.197 X1022 molecules SDS This number of molecules of SDS at the inte rface can then be multiplied by the area per molecule of SDS, 50 2/molecule and converted to cm2/L. TIA = (2.197 X 1022 molecules SDS)(502/molecule)(1 X 10-16 cm2/2) = 1.09 X 108 cm2/L

PAGE 161

161 APPENDIX C CALCULATION OF TOTAL INTERFACIA L AREA FROM DROPLET SIZE RESULTS An emulsion of 1% (v/v %) hexadecane wa s formed in a solution of 100 mM SDS. The droplet size was then determined. The total interfacial area (TIA) was calculated from the average droplet size, Davg, by using the following series of equations. First, volume of a single droplet, VSD, was determine by: VSD (cm3) = (4/3) ((Davg/2)/10000000)3 The Davg is given in nanometers, so it was divided by 1 X 107 to convert it into cm. For 100 mM SDS emulsion, the Davg was found to be 639 nm, so: VSD = (4/3) ((639 nm/2)/10000000)3 = 1.366 X 10-13 cm3 Next the number of droplets in 1 L can be calculated. # droplets = 1000 mL/(1.366 X 10-13 mL) = 7.321 X 1015 droplets in 1 L Now the TIA can be calculated as follows: TIA = 4 ((Davg/2)/10000000)2(# droplets) TIA = 4 ((639/2)/10000000)2(7.321 X 1015) = 9.39 X 105 cm2/L

PAGE 162

162 LIST OF REFERENCES 1. Debye, P., Ann. N. Y. Acad. Sci., 1949, 51, 575. 2. Patist, A.; Kanicky, J. R.; Shukla, P. K.; Shah, D. O., Importance of micellar kinetics in relation to technological processes. J. Colloid Interface Sci. 2002, 245, (1), 1-15. 3. Rosen, M. J., Surfactants and Interfacial Phenomena 3rd ed.; John Wiley & Sons: Hoboken, New Jersey, 2004. 4. McBain, J. W.; Salmon, C. S., Colloidal electrolytes. Soap solutions and their constitution. J. Am. Chem. Soc. 1920, 42, 426. 5. Hartley, G. S.; Collie, B.; Samis, C. S., Transport numbers of paraffin-chain salts in aqueous solution. I. Measurement of tr ansport numbers of cetylpyridinium and cetyltrimethylammonium bromides and their interpretation in terms of micelle formation, with some data also for cetanesulfonic acid. Trans. Faraday Soc. 1936, 32, 795-815. 6. Hartley, G. S., Aqueous Solutions of Paraffin-Chain Salts Paris, 1936. 7. Hiemenz, P. C., and Rajagopalan, R., Principles of Colloid and Surface Chemistry 3rd ed.; Marcel Dekker, Inc.: New York, 1997. 8. Kong, L.; Beattie, J. K.; Hunter, R. J ., Electroacoustic study of n-hexadecane/water emulsions. Aust. J. Chem. 2001, 54, (8), 503-511. 9. Tanford, C., The Hydrophobic Effect. The Formati on of Micelles and Biological Membranes John Wiley & Sons: New York, 1980. 10. Nilsson, P. G.; Lindman, B., Water self -diffusion in non-ionic surfactant solutions hydration and obstruction effects. J. Phys. Chem. 1983, 87, (23), 4756-4761. 11. Cebula, D. J.; Ottewill, R. H., Ne utron-scattering studies on micelles of dodecylhexaoxyethylene glycol monoether. Colloid Polym. Sci. 1982, 260, (12), 1118-1120. 12. Triolo, R.; Hayter, J. B.; Magid, L. J.; J ohnson, J. S., Small-angle neutron scattering from H2O/D2O solutions of a sodium alkylbenzenesul fonate having a branched alkyl group. J. Chem. Phys. 1983, 79, (4), 1977-1980. 13. Bergethon, P. R., and Simons, E. R., Biophysical Chemistry: Mo lecules to Membranes Springer-Verlag: New York, 1990. 14. Gloxhuber, C., and Kunstler, K., Anionic Surfactants: Biochemistry, Toxicology, Dermatology 2nd ed.; Mercel Dekker, Inc.: New York, 1992. 15. Van Ee, J. H., Misset, O., and Baas, E. J., Enzymes in Detergency Mercel Dekker, Inc.: New York, 1997.

PAGE 163

163 16. Fendler, J., Catalysis in Micellar and Macromolecular Systems Academic Press: New York, 1975. 17. Myers, D., Surfaces, Interfaces, and Colloid s: Principles & Applications 2nd ed.; John Wiley & Sons: New York, 1999; p 253-292. 18. Chen, M.; Gratzel, M.; Thomas, J. K., Phot ochemical reactions in micelles of biological importance. Chem. Phys. Lett. 1974, 24, (1), 65-68. 19. Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W., Theory of self-assembly of hydrocarbon amphiphiles into micelles and bilayers. J. Chem. Soc. Faraday Trans. II 1976, 72, 1525-1568. 20. Laughlin, R. G., Equilibrium vesicles: Fact or fiction? Colloids Surf., A 1997, 128, (1-3), 27-38. 21. Aniansson, E. A. G.; Wall, S. N., On th e kinetics of step-wise micelle association. J. Phys. Chem. 1974, 78, (10), 1024-1030. 22. Kahlweit, M.; Teubner, M., On the kineti cs of micellization in aqueous solutions. Adv. Colloid Interface Sci. 1980, 13, (1-2), 1-64. 23. Aniansson, G. E. A., The mean lifetime of a micelle. Prog. Colloid Polym. Sci. 1985, 70, 2-5. 24. Lang, J.; Auborn, J. J.; Eyring, E. M., Kinetics of octylpheny l polyoxyethylene alcohol micelle dissociation by a stopped-flow technique. J. Colloid Interface Sci. 1972, 41, (3), 484490. 25. Kresheck, G. C.; Hamori, E.; Davenpor, G.; Scheraga, H. A., Determination of dissociation rate of dodecylpyr idinium iodide micelles by a te mperature-jump technique. J. Am. Chem. Soc. 1966, 88, (2), 246. 26. Mijnlief, P.; Ditmarsc, R., Rate of micelle formation of sodium alkyl sulphates in water. Nature 1965, 208, (5013), 889. 27. Yasunaga, T.; Oguri, H.; Miura, M., Acoustic study on kinetics for dissociationrecombination reaction between micelle and co unterion in sodium dodecyl sulfate solution. J. Colloid Interface Sci. 1967, 23, (3), 352. 28. Leung, R.; Shah, D. O., Dynamic properties of micellar solutions .1. Effects of shortchain alcohols and polymer s on micellar stability. J. Colloid Interface Sci. 1986, 113, (2), 484499. 29. Patist, A.; Kanicky, J. R.; Shukla, P. K.; Shah, D. O., Importance of micellar kinetics in relation to technological processes. Journal of Colloid and Interface Science 2002, 245, (1), 115.

PAGE 164

164 30. Patist, A.; Chhabra, V.; Pagidipati, R.; Sh ah, R.; Shah, D. O., Effect of chain length compatibility on micellar stability in sodium dodecyl sulfate/alkyltrim ethylammonium bromide solutions. Langmuir 1997, 13, (3), 432-434. 31. Aniansson, E. A. G.; Wall, S. N., A corre ction and improvement of "On the kinetics of step-wise micelle association" by E. A. G. Aniansson and S. N. Wall. J. Phys. Chem. 1975, 79, (8), 857-858. 32. Rassing, J.; Sams, P. J.; Wynjones, E ., Kinetics of micellization from ultrasonic relaxation studies. J. Chem.Soc. Faraday Trans. II 1974, 70, (7), 1247-1258. 33. Lang, J.; Zana, R., In Chemical Relaxation Methods in Surfactants in SolutionNew Methods of Investigation Zana, R., Ed. Dekker: New York, 1987. 34. Attwood, D.; Florence, A. T., In Surfactant Systems Chapman & Hall: London, 1983. 35. Muller, N., In Solution Chemistry of Surfactants Mittal, K. L., Ed. Plenum: New York and London, 1979; Vol. 1. 36. Gormally, J.; Gettings, W. J.; Wyn-Jones, E., In Molecular Interactions Ratajczak, H.; Orville-Thomas, W. J., Eds. Wile y: New York, 1980; Vol. 2, p 143. 37. Aniansson, E. A. G.; Wall, S. N.; Almgre n, M.; Hoffmann, H.; Kielmann, I.; Ulbricht, W.; Zana, R.; Lang, J.; Tondre, C., Theory of kinetics of micellar equi libria and quantitative interpretation of chemical relaxation studies of micellar solutions of ionic surfactants. J. Phys. Chem. 1976, 80, (9), 905-922. 38. Lessner, E.; Teubner, M.; Kahlweit, M., Rela xation experiments in aqueous solutions of ionic micelles. 1. Theory and experiments on the system H2O-sodium tetradecyl sulfate-NaClO4. J. Phys. Chem. 1981, 85, (11), 1529-1536. 39. Lang, J.; Zana, R.; Bauer, R.; Hoffmann, H. ; Ulbricht, W., Chemical relaxation studies of micellar equilibria. J. Phys. Chem. 1975, 79, (3), 276-283. 40. Hall, D. G., Micellization ki netics of ionic surfactants. J. Chem. Soc. Faraday Trans. II 1981, (77), 1973-2006. 41. Inoue, T.; Shibuya, Y.; Shimozawa, R., Chem ical relaxation studies in micellar solutions of sodium alkyl sulfates. J. Colloid Interface Sci. 1978, 65, (2), 370-379. 42. Kahlweit, M., Kinetics of formation of association colloids. J. Colloid Interface Sci. 1982, 90, (1), 92-99. 43. Herrmann, C. U.; Kahlweit, M., Kinetics of micellization of Triton X-100 in aqueous solutions. J. Phys. Chem. 1980, 84, (12), 1536-1540. 44. Becher, P., Emulsions: Theory and Practice 2nd ed.; Reinhold Publishing Corporation: New York, 1965.

PAGE 165

165 45. Kabalnov, A. S.; Pertzov, A. V.; Shchuki n, E. D., Ostwald ripening in emulsions .1. Direct observations of Ostw ald ripening in emulsions. J. Colloid Interface Sci. 1987, 118, (2), 590-597. 46. Oh, S. G.; Jobalia, M.; Shah, D. O., The e ffect of micellar lifetime on the droplet size in emulsions. J. Colloid Interface Sci. 1993, 156, (2), 511-514. 47. Oh, S. G.; Shah, D. O., Effect of counter ions on the interfacial tension and emulsion droplet size in the oil-wa ter dodecyl sulfate system. J. Phys. Chem. 1993, 97, (2), 284-286. 48. Sutheim, G. M., Introduction to Emulsions Chemical Publishing Co., Inc.: Brooklyn, N. Y., 1947. 49. Bancroft, W. D., The theory of emulsification, V. J. Phys. Chem. 1913, 17, 501. 50. Bancroft, W. D., The theory of emulsification, VI. J. Phys. Chem. 1915, 19, 275. 51. Tadros, T.; Vincent, B., Emulsion Stability. In Encyclopedia of Emulsion Technology: Basic Theory Becher, P., Ed. Marcel Dekker, Inc.: New York and Basel, 1983; Vol. 1, p 129. 52. Menon, V. B.; Wasan, D. T., Encyclopedia of Emulsion Technology Marcel Dekker, Inc: New York, 1983; Vol. 2. 53. Barreleiro, P. C. A.; Olofsson, G.; Brown, W.; Edwards, K.; Bonassi, N. M.; Feitosa, E., Interaction of octaethylene glycol n-dodecyl monoether with dioctadecyldimethylammonium bromide and chloride vesicles. Langmuir 2002, 18, (4), 1024-1029. 54. Wu, X. Y.; Pelton, R. H.; Tam, K. C. ; Woods, D. R.; Hamielec, A. E., Poly(NIsopropylacrylamide) .1. Interact ions with sodium dodecyl-sulfate measured by conductivity. J. Polym. Sci., Part A: Polym. Chem. 1993, 31, (4), 957-962. 55. Liem, A. J. S.; Woods, D. R., Review of Coalescence Phenomena 1974; Vol. 70. 56. Ross, S., Emulsion control. J. Soc. Cosmetic Chem. 1955, 6, 184-192. 57. Ostwald, W., Emulsions. Wilmersdorf. Z. Chem. Ind. Kolloide 1910, 8, 103-109. 58. Pickering, S. U., Emulsions. J. Chem. Soc. Faraday Trans. 1907, 91, 2001-2021. 59. Shinoda, K.; Nakagawa, T., Colloidal Surfactants: Some Physicochemical Properties Academic Press: New York, 1963. 60. Mange, F. E.; Buriks, R. S.; Fauke, A. R. Demulsification with ultrahigh-molecularweight polyoxiranes. 3617571, 19711102, 1971. 61. Webb, T. O. Use of micellar solution as a hydrocarbon-water emulsion \"breaker\". 3554289, 19710112, 1971.

PAGE 166

166 62. Griffin, W. C., Calculation of HLB values of non-ionic Surfactants. J. Soc. Cosmetic Chem. 1954, 5, 249-355. 63. Davies, J. T.; Rideal, E. K., Interfacial Phenomena 2nd ed.; Academic Press: New York and London, 1963. 64. Greenwald, H. L.; Brown, G. L.; Finema n, M. N., Determination of the hydrophilehipophile character of surface active agen ts and oils by a water titration. Anal. Chem. 1956, 28, (11), 1693-1697. 65. Racz, I.; Orban, E., Calorimetric dete rmination of hydrophile-lipophile balance of surface-active substances. J. Colloid Sci. 1965, 20, (2), 99. 66. Esumi, K.; Miyazaki, M.; Arai, T.; Koid e, Y., Mixed micellar properties of cationic gemini surfactants and a nonionic surfactant. Colloids Surf., A 1998, 135, (1-3), 117-122. 67. Bhatnagar, S. S., Studies in emulsions. I. A new method for determining the inversion of phases. J. Chem. Soc., Trans. 1920, 117, 542-552. 68. Aoki, K.; Hori, J.; Sakurai, K., Interaction between surface active ag ents and proteins .3. Precipitation curve of the system sodium dodecy l sulfate-egg albumin at various pHs and the determination of the concentration of pr otein by the titration using surfactant. Bull. Chem. Soc. Jpn. 1956, 29, (7), 758-761. 69. Shimamoto, T., Studies on the emulsion I. Phase inversion by ho mogenizer processing. Jpn. J Pharmacol. 1962, 82, 1237-1240. 70. Matsumot, S.; Sherman, P., A DTA technique for identifying phase inversion temperature of O/W emulsions. J. Colloid Interface Sci. 1970, 33, (2), 294. 71. Lin, T. J.; Lambrechts, J. C., Effect of initial surfactant location on emulsion phase inversion. J. Soc. Cosmetic Chem. 1969, 20, (3), 185-198. 72. Sunderla, V.; Enever, R. P., Influence of formulation variables on phase inversion temperatures of emulsions as determin ed by a programmed viscometric technique. J. Pharm. Pharmacol. 1972, 24, (10), 804. 73. Lindman, B.; Friberg, S. E., Microe mulsionsA Historical Overview. In Handbook of Microemulsion Science and Technology Kumar, P.; Mittal, K., Eds. Marcel Dekker, Inc: New York, 1999; p 1. 74. Schulman, J. H.; Stoeckenius, W.; Prince, L. M., Mechanism of formation and structure of micro emulsions by electron microscopy. J. Phys. Chem. 1959, 63, (10), 1677-1680. 75. Hoar, T. P.; Schulman, J. H., Transparent water-in-oil dispersions: the oleopathic hydromicelle. Nature (London, U. K.) 1943, 152, 102-103.

PAGE 167

167 76. Ruckenstein, E., Thermodynamic insights on macroemulsion stability. Adv. Colloid Interface Sci. 1999, 79, (1), 59-76. 77. Walstra, P., Formation of Emulsions. In Encyclopedia of Emulsion Technology Becher, P., Ed. Dekker: New York, 1983; Vol. 1, pp 57-128. 78. Kegel, W. K.; Overbeek, T. G.; Lekkerkerker, H. N. W., Thermodynamics of Microemulsions I. In Handbook of Microemulsion Science and Technology Kumar, P.; Mittal, K., Eds. Marcel Dekker, Inc: New York, 1999; pp 13-44. 79. Ruckenstein, E.; Chi, J. C ., Stability of microemulsions. J. Chem. Soc. Faraday Trans. II 1975, 71, 1690-1707. 80. Bowcott, J. E.; Schulman, J. H., Emulsions Control of droplet si ze and phase continuity in transparent oil-water dispersions stabilized with soap and alcohol. Zeitschrift Fur Elektrochemie 1955, 59, (4), 283-290. 81. Cooke, C. E.; Schulman, J. H., Surface Chemistry Munksgaard: Copenhagen, Denmark, 1965; p 231-235. 82. Sears, D. F.; Schulman, J. H., Influence of water structures on surface pressure surface potential + area of soap monolayers of lithium sodium potassium + calcium. J. Phys. Chem. 1964, 68, (12), 3529. 83. Zlochowe, I.; Schulman, J. H., A study of molecular interactions and mobility at liquid/liquid interfaces by NMR spectroscopy. J. Colloid Interface Sci. 1967, 24, (1), 115. 84. Somasundaran, P.; Lee, L. T., Polymer-surf actant interactions in flotation of quartz. Sep. Sci. Technol. 1981, 16, (10), 1475-1490. 85. Shah, D. O., Microemulsions and their Technological Applications. In Surfactants: Principles and Applications (Short Course on Surfactants) Gainesville, 2004; p Chapter 8. 86. Gerbacia, W.; Rosano, H. L., Microemu lsions Formation and stabilization. J. Colloid Interface Sci. 1973, 44, (2), 242-248. 87. Gelbart, W. M.; Ben-Shaul, A.; Roux, D., Micelles, Membranes, Microemulsions and Monolayers Springer-Verlag: New York, 1994. 88. Bellocq, A. M., Handbook of Microemulsion Science and Technology Marcel Dekker, Inc.: New York, 1999; p 139-184. 89. Shinoda, K.; Friberg, S., Emulsions and Solubilization Wiley: New York, 1986. 90. Shiao, S. Y.; Chhabra, V.; Patist, A.; Free, M. L.; Huibers, P. D. T.; Gregory, A.; Patel, S.; Shah, D. O., Chain length compatibility eff ects in mixed surfactant systems for technological applications. Adv. Colloid Interface Sci. 1998, 74, 1-29.

PAGE 168

168 91. Brooks, J. T.; Cates, M. E., The role of added polymer in dilute lamellar surfactant phases. J. Chem. Phys. 1993, 99, (7), 5467-5480. 92. Pillai, V.; Kanicky, J. R.; Shah, D. O., A pplications of Microemulsions in Enhanced Oil Recovery. In Handbook of Microemulsi on Science and Technology Kumar, P.; Mittal, K. L., Eds. Marcel Dekker Inc.: New York, 1999; pp 743-754. 93. Malmsten, M., Microemulsions in Pharmaceuticals. In Handbook of Microemulsion Science and Technology Kumar, P.; Mittal, K. L., Eds. Ma rcel Dekker, Inc: New York, 1999; pp 755-772. 94. Myers, D., Physical Properties of Surfactants Used in Cosmetics. In Surfactants in Cosmetics Rieger, M. M.; Rhein, L. D., Eds. Marc el Dekker, Inc.: New York, 1985; Vol. 68, pp 29-82. 95. Patist, A.; Bhagwat, S. S.; Penfield, K. W.; Aikens, P.; Shah, D. O., On the measurement of critical micelle concentrations of pur e and technical-grade nonionic surfactants. J. Surfactants Deterg. 2000, 3, (1), 53-58. 96. Engstrm, S.; Larsson, K., Microemulsions in Foods. In Handbook of Microemulsion Science and Technology Kumar, P.; Mittal, K., Eds. Marc el Dekker, Inc.: New York, 1999; pp 789-796. 97. Drfler, H., Application of Microemulsions in Textile Cleaning Us ing Model Detergency Tests. In Handbook of Microemulsion Science and Technology Kumar, P.; Mittal, K., Eds. Marcel Dekker, Inc.: New York, 1999; pp 811-832. 98. Lpez-Montilla, J. C.; James, M. A.; Oscar, D. C.; Shah, D. O., Surfactants and protocols to induce spontaneous emulsifi cation and enhance detergency. J. Surfactants Deterg. 2005, 8, (1), 45-53. 99. Varshney, M.; Morey, T. E.; Shah, D. O.; Flint, J. A.; Moudgil, B. M.; Seubert, C. N.; Dennis, D. M., Pluronic microemu lsions as nanoreservoirs for extraction of bupivacaine from normal saline. J. Am. Chem. Soc. 2004, 126, (16), 5108-5112. 100. Forgiarini, A.; Esquena, J.; Gonzalez, C. ; Solans, C., Formation of nano-emulsions by low-energy emulsification methods at constant temperature. Langmuir 2001, 17, (7), 2076-2083. 101. Elaasser, M. S.; Lack, C. D.; Vanderhoff, J. W.; Fowkes, F. M., The miniemulsification process Different form of spontaneous emulsification. Colloids Surf. 1988, 29, (1), 103-118. 102. Katsumoto, Y.; Ushiki, H.; Mendiboure, B. ; Graciaa, A.; Lachaise, J., Evolutionary behaviour of miniemulsion phases: II. Grow th mechanism of miniemulsion droplets. J. Phys.: Condens. Matter 2000, 12, (15), 3569-3583. 103. Forster, T., Principles of Emulsion Formation. In Surfactants in Cosmetics Rieger, M. M.; Rhein, L. D., Eds. Marcel Dekker Inc.: New York, 1997; Vol. 68, pp 105-125.

PAGE 169

169 104. Izquierdo, P.; Esquena, J.; Tadros, T. F.; De deren, C.; Garcia, M. J.; Azemar, N.; Solans, C., Formation and stability of nano-emulsions pr epared using the phase inversion temperature method. Langmuir 2002, 18, (1), 26-30. 105. Benita, S.; Levy, M. Y., Submicron emulsions as colloidal drug carriers for intravenous administration Comprehensive physicochemical characterization. J. Pharm. Sci. 1993, 82, (11), 1069-1079. 106. Klang, S.; Benita, S., Submicron Emulsions in Drug Targeting and Delivery Harwood Academic Publishers: The Netherlands, 1998; p 119-172. 107. Bennett, H.; Bishop, J. L.; Wulfringhoff, M. F., Practical Emulsions Chemical Publishing Co.: Brooklyn, New York, 1968. 108. Mittal, K. L.; Vold, R. D., Effect of in itial concentration of emulsifying agents on ultracentrifugal stability of oil-in-water emulsions. J. Am. Oil Chem. Soc. 1972, 49, (9), 527. 109. Helenius, A.; McCaslin, D. R.; Fries, E.; Tanford, C., Properties of detergents. Methods Enzym. Biomembr., G 1979, 56, 734-749. 110. le Maire, M.; Champeil, P.; Moller, J. V ., Interaction of membrane proteins and lipids with solubilizing detergents. Biochim. Biophys. Acta, Biomembr. 2000, 1508, (1-2), 86-111. 111. Midura, R. J.; Yanagishita, M., Chaotr opic solvents increase the critical micellar concentrations of detergents. Anal. Biochem. 1995, 228, (2), 318-322. 112. Ray, A.; Reynolds, J. A.; Polet, H.; Stei nhar.J, Binding of large organic anions and neutral molecules by native bovine serum albumin. Biochemistry 1966, 5, (8), 2606. 113. Rump, H. H., Laboratory Manual for Examination of Water, Waste Water and Soil Wiley-VCH Verlag: Weinheim, Germany, 1999. 114. Amos, D. A.; Markels, J. H.; Lynn, S.; Ra dke, C. J., Osmotic pressure and interparticle interactions in ionic micellar surfactant solutions. J. Phys. Chem. B 1998, 102, (15), 2739-2753. 115. Halliday, D.; Resnick, R.; Walker, J., Fundamentals of Physics 4th ed.; Wiley: New York, 1993. 116. Lessner, E.; Teubner, M.; Kahlweit, M., Rela xation experiments in aqueous-solutions of ionic micelles. 2. Experiments on the system H2O-NaDS-NaClO4 and their theoretical interpretation. J. Phys. Chem. 1981, 85, (21), 3167-3175. 117. Mukerjee, P.; Mysels, K. J., Critical Micelle Concentration in Aqueous Surfactant Systems National Bureau of Standards, NS RDS-NBS 36: Washington, D.C., 1971. 118. Cramer, L. R.; Berg, J. C., Effect of micelles on kinetics of Cannizzaro reaction. J. Phys. Chem. 1968, 72, (10), 3686.

PAGE 170

170 119. Palla, B. J.; Shah, D. O., Stabilization of high ionic strength slur ries using surfactant mixtures: Molecular factors that determine optimal stability. J. Colloid Interface Sci. 2002, 256, (1), 143-152. 120. Sharma, M. K.; Shah, D. O.; Brigham, W. E., Correlation of chai n-length compatibility and surface-properties of mixed foaming agents w ith fluid displacement efficiency and effective air mobility in porous-media. Ind. Eng. Chem. Res. 1984, 23, (2), 213-220. 121. Jha, B. K.; Patist, A.; Shah, D. O., Effect of antifoaming agents on the micellar stability and foamability of sodium dodecyl sulfate solutions. Langmuir 1999, 15, (9), 3042-3044. 122. Pandey, S.; Bagwe, R. P.; Shah, D. O., Effect of counterions on surface and foaming properties of dodecyl sulfate. J. Colloid Interface Sci. 2003, 267, (1), 160-166. 123. Nikolov, A. D.; Wasan, D. T., Ordered mice lle structuring in thin-films formed from anionic surfactant soluti ons. 1. Experimental. J. Colloid Interface Sci. 1989, 133, (1), 1-12. 124. Oh, S. G.; Shah, D. O., Relationship between micellar lifetime and foamability of sodium dodecyl sulfate and sodium dodecy l sulfate/1-hexanol mixtures. Langmuir 1991, 7, (7), 13161318. 125. Chattopadhyay, A. K.; Ghaicha, L.; Oh, S. G.; Shah, D. O., Salt effects on monolayers and their contribution to surface viscosity. J. Phys. Chem. 1992, 96, (15), 6509-6513. 126. Oh, S. G.; Shah, D. O., The effect of mi cellar lifetime on the rate of solubilization and detergency in sodium dodecyl sulfate solutions. J. Am. Oil Chem. Soc. 1993, 70, (7), 673-678. 127. Boys, C. V., Soap Bubbles Macmillan: London and New York, 1924. 128. Fallon, M. S.; Chauhan, A., Seque stration of amitriptyline by liposomes. J. Colloid Interface Sci. 2006, 300, (1), 7-19. 129. Barry, J. D.; Durkovich, D. W.; Williams, S. R., Vasopressin treatment for cyclic antidepressant overdose. J. Emergency Med. 2006, 31, (1), 65-68. 130. Underhill, R. S.; Jovanovic, A. V.; Carino, S. R.; Varshney, M.; Shah, D. O.; Dennis, D. M.; Morey, T. E.; Duran, R. S., Oil-filled silica nanocapsules for lipophilic drug uptake: Implications for drug detoxification therapy. Chem. Mater. 2002, 14, (12), 4919-4925. 131. Piculell, L.; Lindman, B., Association a nd segregation in aqueous polymer/polymer, polymer/surfactant, and surfactant/surfactan t mixtures Similarities and differences. Adv. Colloid Interface Sci. 1992, 41, 149-178. 132. Moudgil, B. M. The Role of Polymer-Surfact ant Interactions in Interfacial Processes". Columbia University, Columbia, 1981. 133. Allen, G., Binding of sodium dodecyl-sulf ate to bovine serum-al bumin at high binding ratios. Biochem. J. 1974, 137, (3), 575-578.

PAGE 171

171 134. Strauss, G.; Strauss, U. P., The binding of sodium dodecyl sulfate by serum albumin in the absence of added electrolyte. J. Phys. Chem. 1958, 62, (10), 1321-1324. 135. Argillier, J. F.; Ramachandran, R.; Ha rris, W. C.; Tirrell, M., Polymer surfactant interactions studied with the surface force apparatus. J. Colloid Interface Sci. 1991, 146, (1), 242-250. 136. Goddard, E. D., Polymer/surfactant interac tion Its relevance to detergent systems. J. Am. Oil Chem. Soc. 1994, 71, (1), 1-16. 137. Regismond, S. T. A.; Winnik, F. M.; Godda rd, E. D., Stabilization of aqueous foams by polymer/surfactant systems: effect of surfactant chain length. Colloids Surf., A 1998, 141, (2), 165-171. 138. Varshney, M.; Dennis, D.; Morey, T.; Shah D. O., Microemulsions for Preventing and Treating Tricyclic Drug Overdose Toxicities. Proc. Natl. Acad. Sci. U. S. A. to be submitted. 139. Rosen, M. J., Surfactants and Interfacial Phenomena 3rd ed.; John Wiley & Sons: Hoboken, New Jersey, 2004; p 121-137. 140. Zhou, Z.; Chu, B., Phase-behavior a nd association properties of poly(oxypropylene)poly(oxyethylene)-poly(oxypropyl ene) triblock copolymer in aqueous-solution. Macromolecules 1994, 27, (8), 2025-2033. 141. Nagarajan, R., Solubilization of hydrocarbons and resulting aggregate shape transitions in aqueous solutions of Pluronic (R) (PEO-PPO-PEO) block copolymers. Colloids Surf., B 1999, 16, (1-4), 55-72. 142. Jain, N. J.; Aswal, V. K.; Goyal, P. S.; Bahadur, P., Salt induced micellization and micelle structures of PEO/PPO/PEO bl ock copolymers in aqueous solution. Colloids Surf., A 2000, 173, (1-3), 85-94. 143. Adamson, A. W., Physical Chemistry of Surfaces 4th ed.; John Wiley & Sons: New York, 1982; p Chapter 3. 144. Cosgrove, T.; Phipps, J. S.; Richardson, R. M., Neutron reflection from a liquid/liquid interface. Colloids Surf. 1992, 62, (3), 199-206. 145. Staples, E.; Penfold, J.; Tucker, I., Adso rption of mixed surfactants at the oil-water interface. J. Phys. Chem. B 2000, 104, (3), 606-614. 146. Adamson, A. W., Physical Chemistry of Surfaces 4th Ed. ed.; John Wiley & Sons: New York, 1982. 147. Rehfeld, S., Adsorption of sodium d odecyl sulfate at various hydrocarbon-water interfaces. J. Phys. Chem. 1967, 71, (5), 738.

PAGE 172

172 148. van deHulst, H. C., Light Scattering by Small Particles Dover Publications Ltd.: New York, 1981. 149. Hallwort, G. W.; Carless, J. E., Stabilization of oil-in-wat er emulsions by alkyl sulfates Influence of nature of oil on stability. J. Pharm. Pharmacol. 1972, 24, P71. 150. Hallwort, G. W.; Carless, J. E., Stabilization of oil-in-wat er emulsions by alkyl sulfates effect of a long-chain alcohol. J. Pharm. Pharmacol. 1973, 25, P87-P95. 151. Chiang, M. Y.; Chan, K. S.; Shah, D. O., A correlation of interfacial charge with variousinterfacial properties in relation to o il recovery efficiency during waterflooding. J. Can. Pet. Tech. 1978, 17, (4), 1-8. 152. Cameron, A.; Crouch, R. F., Intera ction of hydrocarbon and surface-active agent Nature (London, U. K.) 1963, 198, (4879), 475-476. 153. Bansal, V. K.; OConnell, J. P.; Shah, D. O., Influence of Alkyl Chain Length Compatibility on Microemulsion Structure and Solubilization. J. Colloid Interface Sci. 1980, 75, (2), 462-475. 154. Chattopadhyay, A. K.; Shah, D. O.; Ghaicha, L., Double-Tailed Surfactants and Their Chain-Length Compatibility in Water-in-Oil Emulsions. Langmuir 1992, 8, (1), 27-30. 155. Weiss, J.; Herrmann, N.; McClements, D. J., Ostwald ripening of hydrocarbon emulsion droplets in surfactant solutions. Langmuir 1999, 15, (20), 6652-6657.

PAGE 173

173 BIOGRAPHICAL SKETCH Monica A. James-Smith is a native of Albany, GA. She graduated as the valedictorian of Dougherty Comprehensive High School in 1996. Moni ca is an alumnus of Tuskegee University where she completed her Bachelor of Science degr ee in chemical engineering in 2000. She is currently a Ph.D. candidate at th e University of Florida in the Chemical Engineering Department where she is working with Dr. Dinesh O. Sh ah on Molecular Interactions in Surfactant Solutions: From Micelles to Microemulsions. Monica plans to graduate in December 2006 and subsequently pursue a post-doctoral position. Her ultimate goal is to become a full professor at a Research I institution.


Permanent Link: http://ufdc.ufl.edu/UFE0017540/00001

Material Information

Title: Molecular Interactions in Surfactant Solutions: From Micelles to Microemulsions
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0017540:00001

Permanent Link: http://ufdc.ufl.edu/UFE0017540/00001

Material Information

Title: Molecular Interactions in Surfactant Solutions: From Micelles to Microemulsions
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0017540:00001


This item has the following downloads:


Full Text





MOLECULAR INTTERACTIONS


IN SURFACTANT SOLUTIONS: FROM MICELLES TO
MICROEMULSIONS


By

MONICA A. JAMES-SMITH


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2006

































Copyright 2006

by

Monica A. James-Smith






























To my parents

who have been my #1 supporters since October 17, 1977.









ACKNOWLEDGMENTS

I thank my Almighty Heavenly Father for allowing me to make it to this point and for

seeing me through every obstacle that arose. I am forever grateful to my husband, Rod, for all of

his support, love and encouragement. I sincerely thank my parents, Dan and Elaine James, for

always believing in me, for their constant prayers, and for always providing the right words

when the journey seemed difficult. I would like to thank Melanie, Dan, Chris, and Bruce for

knowing how to make me feel like I can accomplish anything. I owe a huge debt of gratitude to

my best friend, Brandi Chestang, who has been there to answer every phone call and has cheered

me on all my life. I am also greatly appreciative to all of my other friends, family, and loved

ones. I must also extend my sincerest appreciation to my in-laws who have taken me in as a

family member and provided tremendous support as I have pursued this degree.

I am forever grateful to Dr. Dinesh O. Shah for being a mentor, an advisor, and a

confidant, for providing me with the highest caliber of guidance and for always pushing me

towards greatness. I would like to extend sincerest thanks to Drs. Brij Moudgil, Manoj Varshney,

Yakov Rabinovich, Anuj Chauhan, Oscar Crisalle, Ranga Narayanan, Donn Dennis, and Richard

Dickenson for stimulating conversation and insightful comments and suggestions. I also would

like to thank all of my colleagues who have been of great assistance throughout me years here at

UF .

Last, but definitely not least, I would like to thank Pastor Kevin W. Thorpe and the Faith

Baptist Church family for showing me that my family is bigger than I think and for helping me to

make it through this journey.












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ........._..__..... ._._ ...............8....


LIST OF FIGURES .............. ...............9.....


AB S TRAC T ............._. .......... ..............._ 12...


CHAPTER


1 LITERATURE REVIEW ................. ...............14...............


1.1 Micelles............... ...............14
1.1.1 Introduction ................ .. ......... ..........1
1. 1.2 Dynami c Nature of Mi cellar Soluti ons ............_...... ..............1
1.2 M acroemulsions................ ............2
1.2.1 Emulsion Droplet Size............... ...............26..
1.2.2 Viscosity of Emulsions.............__ ..........__ .........__ ...........2
1.2.3 Determination of Emulsion Type (O/W or W/O) ................. ................. ......28
1.2.4 Emulsion Stability .............. ...............29....
1.2.4. 1 Coalescence ........................ ._ .. ..... .__ ............2
1.2.4.2 Charge stabilization: The electrical double layer............. ..._.........__ ...30
1.2.4.3 Phase inversion in emulsions .............. ...............31....
1.2.4.4 Emulsion creaming............... ...............3
1.2.5 Demulsification ............... ........ .. .............3
1.2.6 Surfactant Selection for Emulsification ....._ .....___ ..........._ ........3
1.2.7 Applications of Emulsions .............. ...............34....
1.3 Microemulsions .............. .... ...............35.
1.3.1 Formation of Microemulsions .............. ...............41..._.__ ...
1.3.2 Applications of Microemulsions .............. ...............42....
1.3.3 Nano-emulsions ............ ...... .___ ...............42...


2 A NOVEL METHOD TO ELUCIDATE THE PRESENCE OF SUB-MICELLAR
AGGREGATES IN SURFACTANT SOLUTIONS .............. ...............61....


2. 1 Introducti on ................. ...............61........... ...
2.2 Experimental Procedure............... ...............6
2.2. 1 M ateri al s ................ ...............63..............
2.2.2 Ultracentrifugation ...................... .. .... .. .. ..... .......6
2.2.3 Two-Phase Dye Transfer (Methylene Blue Complexation) and UV-Vis
Analy si s............... ...............64
2.2.4 Foamability ................. ...............64.......... ......
2.2.5 Fabric W getting ................. ...............65................
2.2.6 Dynamic Surface Tension .............. ...............65....











2.3 Results and Discussion .............. ...............65....
2.3.1 SDS Surfactant Solutions .............. ... .... ... .... ... ............6
2.3.2 Effect of Counter-lons on Sub-Micellar Aggregate Concentration .......................73
2.3.3 Importance of Sub-Micellar Aggregates in Technological Processes..................75
2.3.3.1 Foaming ............ ...... ._ __ ...............76....
2.3.3.2 Fabric wetting............... ...............77
2.3.3.3 Dynamic surface tension .............. ...............77....
2.4 Conclusions............... ..............7


3 DETERMINATION OF DRUG AND FATTY ACID BINDING CAPACITY TO
PLURONIC Fl127 IN MICROEMUL SINS FOR DETOXIFICATION. ................... ..........94


3 .1 Introducti on ................. ...............94........... ...
3.2 Experimental Procedure............... ...............9
3.2.1 Material s. .............. ..... ...............96.
3.2.2 Microemulsion Preparation .............. ...............96....
3.2.3 Turbidity Analysis ................ ...............96................
3.2.4 Dynamic Surface Tension .............. ...............97....
3.2.5 Foamability ................. ...............97.......... .....
3.2.6 Fabric W getting ................. ...............98.......... .....
3.2.7 Surface Tension ................. ...............98................
3.3 Results and Discussion ................. .. ........... ... .... .... ........9
3.3.1 Effect of Sodium Caprylate Concentration on Drug and Fatty Acid Binding to
Mi croemul si ons ............... .... .... ... .... ..... ... .. ...............98.
3.3.2 Determination of Free Fatty Acid by Dynamic Processes .............. ... ................101
3.3.3 Effect of Fatty Acid Chain Length on Drug and Fatty Acid Binging to
M i croem ul si ons ............... ... ........ ....... ..... ........ ... ...... .. ............ 0
3.3.4 Effect of the Number of Ethylene Oxide (EO) and Propylene Oxide (PO)
Groups of Pluronics on Fatty Acid and Drug Binding ................. ................. ... 105
3.4 Conclusions............... ..............10


4 A Novel Method to Quantify the Amount of Surfactant at the Oil/Water Interface and to
Determine Total Interfacial Area of Emulsions ................. .........____...... 117___ ...


4. 1 Introducti on .................. ...............117._____....
4.2 Experimental Procedure .....___................. ...............119 ....
4.3 Results and Discussion ................. ........_ ._ ........ ........ ..... ........ .........12
4.3.1 Effect of Surfactant Concentration on Partitioning to the Oil/Water Interface....121
4.3.2 Effect of Alkyl Sulfate Chain Length on Partitioning to the Oil/Water
Interface ........... .. .......................... ......................12
4.3.3 Effect of Oil Chain Length on SDS Partitioning to the Oil/Water Interface........ 128
4.3.4 Effect of Alcohol Chain Length on SDS Partitioning to the Oil/Water
Interface ............ ... .. ........................................ 1
4.3.5 Effect of Oil Phase Volume Fraction on SDS Partitioning to the Oil/Water
Interface .............. ...............13 1...
4.4 Conclusions.............. .............13












5 SUMMARY AND RECOMMENDATIONS FOR FUTURE WORK ............... .... .........._.146


5.1 Micelles............... ...............14
5.1.1 Summary............... ...............146
5.1.2 Future W ork. ........._.. ..... ._ ._ ...............147..
5.2 M icroemulsions .............. ...............149....
5.2.1 Summary............... ...............149
5.2.2 Future W ork ................. ...............15. 1..............
5.3 M acroemul sions............... ............15
5.3.1 Summary............... ...............152
5.3.2 Future W ork ................. ...............155........... ...


APPENDIX


A GIBBS ADSORPTION EQUATION AND AREA PER SURFACTANT MOLECULE
DETERMINATION ................. ...............157......... ......


B CALCULATION OF TOTAL INTERFACIAL AREA FROM FILTRATION
RE SULT S ................. ...............160......... ......


C CALCULATION OF TOTAL INTERFACIAL AREA FROM DROPLET SIZE
RE SULT S ................. ...............161......... ......


LIST OF REFERENCES ................. ...............162................


BIOGRAPHICAL SKETCH ................. ...............173......... ......










LIST OF TABLES


Table page

1-1: Summary of methods used to produce emulsions ................. ...............54........... .

1-2: Common tests for determining emulsion type (W/O or O/W)44....... ..............~~~~~~~~~~~~~.55

1-3: Types of breakdown processes occurring in emulsions ................. .........................56

1-4. Factors influencing the stability of emulsions .............. ...............57....

1-5. Parameters that affect phase inversion in emulsion and the effect they have. ................... 58

1-6. Commonly used physical methods of demulsification ................ .......... ...............59

1-7. A summary of HLB ranges and their application .....___._ .... ... .__ ......._._.......5

1-8. Microemulsions vs. Nano-emulsions. ......___ ........__ ...._ ............6

2-1. Dimensionless dynamic surface tension (6) of different counter-ions of dodecyl
sulfates (50 mM) at a bubble lifetime of 50 msec (from ref 33) ................. ................. .93

3-1. Effect of fatty acid chain length on maximum binding ................. .......... .............1 16

3-2. Effect of # of PO groups on maximum binding ................. ...............116............

3-3. Effect of # of EO groups on maximum binding ................. ...............116............

4-1. Effect of SDS concentration on total interfacial area 1% (v/v) hexadecane-in-water
em ulsions. ............. ...............144....

4-2. Area per molecule values at the hexadecane/water interface for alkyl sufates and
total interfacial area as a function of alkyl sulfate chain length .............. ....................144

4-3. Effect of oil chain length on total interfacial area (TIA) .............. ......................145

4-4. Effect of alcohol chain length on total interfacial area (TIA) ................. ............... ....145










LIST OF FIGURES


Figure page

1-1. Schematic diagram of a surfactant molecule, micelle, and reverse micelle. .....................48

1-2. Properties of surfactant solutions showing abrupt change at the solution critical
micelle concentration (cmc) ................. ...............48........... ....

1-3. Schematic design of micellar solution. ................ .............. ......... ........ .....49

1-4. Schematic diagram of the four maj or micellar structures ................. .......................49

1-5. Mechanisms for the two characteristic relaxation times for a micelle............... ................50

1-6. Typical size distribution curve of aggregates in a micellar solution .............. .................50

1-7. Schematic of sodium counter-ion "cloud" around SDS spherical micelle. .......................51

1-8. Schematic diagram of the adsorption of surfactant monomers from the bulk to the
oil/water interface during emulsion formation .............. ...............51....

1-9. The emulsion droplet size in the hexadecane/SDS solution system .............. .................51

1-10. Schematic depiction of the Stern-Graham model of the electrical double layer. ..............52

1-11. Schematic diagram of an oil-in-water (O/W) microemulsion .............. .....................5

1-12. Thermodynamic explanation for behavior of macroemulsions and microemulsions ........53

2-1. Schematic diagram of the ultracentrifugation process ......____ ... ......_ ..............80

2-2. Size distribution curves of aggregates in a micellar solution .............. ....................8

2-3. Schematic diagrams of surfactant solutions, filtration of solutions, and plot of filtrate
concentration as a function of total surfactant concentration. ................ ............... .....82

2-4. Schematic representation of the two possible reaction paths for the formation of
m icelles. ............. ...............84.....

2-5. Filtration of SDS through 10,000 MWCO ultracentrifuge tubes. ...........__.................85

2-6. Tailoring of micellar stability. ...........__.....___ ........ ......... ...............85

2-7. Filtrate of SDS+C12TAB through 10,000 MWCO ultracentrifuge tubes .......................86

2-8. Filtration of SDS alone or SDS + C12X (X = OH or TAB) through 10,000 MWCO
ultracentrifuge tubes............... ...............87.











2-9. SDS concentration in the filtrate for 80:20 SDS:C12TAB systems after filtration
through 3,000 and 10,000 MWCO tubes, as compared to pure SDS solutions (50
mM ) ................. ...............88.................

2-10. Filtrate surfactant concentrations for 25 mM lithium dodecyl sulfate (LiDS), sodium
dodecyl sulfate (NaDS), and cesium dodecyl sulfate (CsDS) and 12.5 mM
magnesium dodecyl sulfate (Mg(DS)2). ............. ...............89.....

2-11. Schematic depiction of foam column ................ ............. ......... ........ .......90

2-12. Foamability of SDS micellar solution and SDS + C12X mixed micellar solutions ...........90

2-13. Wetting time of lin2 Strips of 50:50 cotton:polyester blend fabric............... .................9

2-14. Dynamic surface tension of solutions of 50 mM SDS and 50 mM SDS + 12.5 mM
C 12TAB .............. .. ...............92................

3-1. Amitriptyline Hydrochloride, MW = 313.9............... ...............109.

3-2. Titration of microemulsions with 0.2 M AMT ..........._ ..... ..__ ......__ .........0

3-3. Titration of mixed micelles and microemulsion systems. ................ ............ .........1 10

3-4. Schematic diagram of turbidity in various solutions ................. ......... .................11 1

3-5. Titration of microemulsion systems with AMT ([SC] = 25 -125 mM) ................... ........1 12

3-6. Properties of Pluronic F l27 microemulsion. ................ ...............113........... ..

3-7. Binding of SC and drug to Fl27. ................ ...............114........... .

3-8. Schematic depiction of microemulsion droplet. ........... ..... .__ ..........__......11

4-1. Schematic depiction of filtration of oil-in-water emulsion through nanoporous filter
membrane ...... ................. ...............134......

4-2. Effect of total SDS concentration on SDS concentration in the filtrate ................... .......13 5

4-3. Effect of SDS concentration on mean droplet diameter of 1% (v/v) hexadecane-in-
water emulsions. ............. ...............136....

4-4. Master diagram showing the changes in emulsion characteristics with increasing
SDS concentration.. ............ ...............137.....

4-5. Amount of alkyl sulfate surfactant that partitions to the interface as a function of
chain length ....._ ................. ........__ _..........13

4-6. Mean droplet size of hexadecane-in-water emulsions as a function of alkyl sulfate
chain length. Droplet size was determined by light scattering. ............. ....................13











4-7. Effect of oil chain length on the amount of SDS that partitions to the interface. ............140

4-8. Schematic depiction of emulsion droplets ................ ...............141.....__._...

4-9. Effect of alcohol chain length on the amount of SDS that partitions to the interface
and mean droplet size............... ...............142.

4-10. Effect of oil phase (hexadecane) volume fraction on partitioning of SDS to the
oil/water interface. ............ .............143......









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

MOLECULAR INTTERACTIONS INT SURFACTANT SOLUTIONS: FROM MICELLES TO
MICROEMULSIONS

By

Monica A. James-Smith

December 2006

Chair: Dinesh O. Shah
Co-chair: Brij M. Moudgil
Major Department: Chemical Engineering

To effectively use surfactants for various applications, one must have a basic

understanding of the molecular interactions occurring within surfactant solutions. Micelles,

microemulsions, and macroemulsions are three of the most commonly investigated surfactant

containing systems. For many years, these systems have been studied in light of their structure,

properties, and applications. In this dissertation, we investigate these systems using filtration

through nanoporous membranes to understand the role of monomers and sub-micellar aggregates

in controlling their properties.

It is commonly accepted that micellar solutions consist of three surfactant species;

adsorbed monomers at the air/water interface, monomers dispersed in the aqueous phase, and

micellar aggregates. Using filtration through nanoporous membranes, we found evidence

suggesting that another species, sub-micellar aggregates, exists, in significant concentration, in

micellar solutions. This is a potentially revolutionary finding that provides for a more accurate

picture of micellar solutions and yields insight to the mechanism by which micellar stability

affects dynamic processes.










The presence of monomers and sub-micellar aggregates in microemulsions is important in

determining the efficacy of microemulsions for drug binding. We used filtration through

nanoporous membranes and turbidity analysis to delineate the drug-fatty acid monomer

interactions. The results showed that one Pluronic F l27 molecule binds a maximum of eleven

sodium caprylate fatty acid molecules and twelve Amitriptyline drug molecules. We were also

able to determine that the initial driving force for Amitriptyline uptake is electrostatic attraction.

Filtration through nanoporous membranes is ideally suited to determine an unresolved

issue of surfactant portioning at the oil/water interface and in the bulk solution in

macroemulsions. We were able to approximate the total interfacial area (TIA) of the emulsions

and showed that this calculation produced values approximately two orders-of-magnitude larger

than the area calculated using mean droplet sized from light scattering analysis. Possible

explanations for this difference are discussed.









CHAPTER 1
LITERATURE REVIEW

Surfactants, or "surface-active agents," are used to enhance the quality of products used in

every aspect of life; from food to cosmetics, from oil recovery to detergency, and even from

pharmaceutics to chemical mechanical polishing of silicon wafers. Given these many diverse

applications, it becomes critical to have a sufficient understanding of the molecular interactions

that occur within surfactant solutions in order to effectively manipulate them for a specific use.

Three invisible compartments in a surfactant solution are an adsorbed film, monomers, and

micelles made up of surfactant molecules.l Surfactant molecules are continually exchanging

among these three compartments, and hence surfactant solutions are in dynamic equilibrium. For

technological processes, such as foaming, emulsification, and wetting, surfactant molecules are

driven to the new surfaces and it becomes necessary for micelles to disintegrate and provide

monomers to these surfaces. In such rapid processes, micelle stability could become a rate-

limiting step.2

The following is the detailed review of micelles, microemulsions, and macroemulsions.

1.1 Micelles

1.1.1 Introduction

A surfactant, or surface-active agent, is defined as a substance that adsorbs onto surfaces

or interfaces of solutions to lower the surface or interfacial tension of the system.3 The

magnitude of the lowering of the surface or interfacial tension depends on the surfactant

structure, concentration, and the physico-chemical conditions of the solution (e.g. pH, salt

concentration, temperature, pressure, etc.).3 Surfactants are typically amphiphatic species,

meaning that they are made up of a hydrophobic component, referred to as the "tail," and a

hydrophilic component, referred to as the "head" group (see Figure 1-1). When placed in









solution, surfactant molecules tend to orient in such a way as to minimize the interactions of the

hydrophobic "tail" with water in aqueous solutions, or to minimize the interactions of the

hydrophilic "head" with oil in organic solvents. This leads to adsorption of the surfactant

molecules onto surfaces or at interfaces, and above a certain concentration, known as the critical

micelle concentration or "cmc," surfactants form aggregates known as micelles. When placed

into aqueous solutions, surfactant molecules will form spherical aggregates at the cmc where the

hydrophobic tails are pointed inward and removed from interaction with water molecules by the

hydrophilic head groups as shown in Figure 1-1. When placed into organic solutions, surfactant

molecules will form reverse micelles with the hydrophobic tails pointed outward (see Figure 1-



When the critical micellar concentration, or cmc, is reached, many of the physical

properties of the surfactant solution in water show an abrupt change as shown in Figure 1-2.

Some of these properties include the surface tension, osmotic pressure, electrical conductivity,

and solubilization. The cmc is a measure of the free monomer concentration in surfactant

solutions at a given temperature, pressure, and composition. Mcbain4 fifSt investigated the

unusual behavior of fatty acid salts in dilute aqueous solution at the cmc in the 1910s and 1920s

and was followed by Hartley5, 6 in the 1930s. Other evidence for surfactant aggregation into

micelles was obtained from vapor pressure measurements and the solubility of organic molecules

in water. The formation of colloidal-sized clusters of individual surfactant molecules in solution

is known as micellization.

McBain first suggested that micelles are spherical in shape. However, the first concrete

model for spherical micelles is attributed to Hartley.6 It is currently accepted in the field that

above the cmc, a typical surfactant solution consists of three major species: 1) surfactant









molecules dispersed as monomers in the aqueous phase, 2) aggregates in the form of micelles,

and 3) adsorbed films at the air/liquid interface (see Figure 1-3). The surfactant is in dynamic

equilibrium between these states, implying that the rates of adsorption and desorption are equal.

Thus, at a given temperature, pressure, and concentration, the number of monomers adsorbed at

the air/water interface, the number of monomers in the bulk phase, and the number of micelles

present in solution is fixed under equilibrium conditions. The concentration of monomers and

micelles changes with equilibrium conditions such as pressure, temperature, or surfactant and

salt concentration.

The micellization process is primarily an entropy-driven process.7" When surfactants are

dissolved in water, the hydrophobic tail disrupts the hydrogen-bonded structure of water thereby

increasing the free energy of the system. As previously mentioned, for this reason surfactant

molecules will concentrate at interfaces so that their hydrophobic tail groups are removed or

directed away from the water minimizing the free energy of the solution. The formation of

micelles is yet another means that the system will use to reduce its free energy. The orientation

of the hydrophobic tails in the interior of the micelle decreases their interaction with water

molecules. However, the surfactant molecules that are confined in the micelle conceivably

experience some loss of freedom. In addition, in the case of ionic surfactants, the molecules that

are present in the micellar aggregate may experience electrostatic repulsion from other similarly

charged surfactant molecules. These forces increase the free energy of the system and oppose

micellization. Hence, micelle formation depends on the force balance between the factors

favoring micellization (Van der Waals and hydrophobic forces) and those opposing it (kinetic

energy of the molecules, electrostatic repulsion, and chemical potential factor due to

concentration gradient for micelles and monomer).9









The number of surfactant molecules that make up a micelle is known as the aggregation

number. This number can be determined through the use of NMR self-diffusion coefficients,'o

small-angle neutron scattering,11, 12 freezing point and vapor pressure methods, osmotic pressure,

and fluorescent probes. The surfactant structure plays a significant role in the aggregation

number of the micelles formed by a give surfactant. The aggregation number tends to increase

with increasing chain length of the hydrophobic tail and decrease with increasing size (i.e., cross

sectional area) of the hydrophilic head group. The aggregation number is also affected by the

nature of the aqueous phase. For example, the addition of neutral electrolytes to ionic surfactant

solutions leads to an increase in the aggregation number. This is due to a phenomenon known as

"salting out" by which the effective amount of water that is available to solubilize the surfactant

molecules is decreased because it is used up to accommodate the ions (a more favorable

interaction). Increasing the temperature of an ionic surfactant solution typically leads to a small

decrease in aggregation number which can be owed to an increase in the size of the head group

as a result of thermal agitation as well as the increase in the kinetic energy of the surfactant and

solvent molecules. When the temperature is increased in nonionic surfactant solutions

(particularly of the polyoxyethylene variety), the aggregation number slowly increases until the

"cloud point" of the surfactant is reached. The cloud point is the temperature at which the

solution begins to exhibit turbidity due to the dehydration of the polyoxyethylene head groups.

Factors that increase the aggregation number tend to decrease the cmc. It is also important to

mention that the cmc values of nonionic surfactants are much lower than the cmc values for ionic

surfactants .

In the past couple of decades considerable interest has been generated in self-assembled

surfactant aggregates such as cylindrical, lamellar, and reverse micelles due to the ability of









surfactant association structure to mimic biological structures.13 Enzymes, for example, are

proteins that speed up (i.e., catalyze) chemical reactions by providing sites for a substrate to fit

into to form a reactive intermediate. The highly efficient and specific catalytic effect of enzymes

makes their investigation an interesting area of biomedical and detergent research (as enzymes

are often added to laundry detergents to improve performance).14, 15 Likewise, cell membranes

perform a variety of functions in cellular biochemical and physiological processes. Surfactant

structures can be used as model systems to mimic both enzymes and membranes. Micelles and

reversed micelles now play an increasingly important role in catalysis and separation processes

in engineering and environmental science and technology.16-18

A theory of micellar structure, based upon the geometry of various micellar shapes and the

space occupied by the hydrophilic and hydrophobic groups of the surfactant molecules, has been

developed by Israelachvili et all 9 In aqueous systems, for example, surfactants with bulky or

loosely packed head groups and long, thin hydrophobic tails tend to form spherical micelles,

while those with short, bulky tail groups and small, close-packed head groups tend to form

lamellar or cylindrical micelles. At concentrations slightly above the cmc, micelles are

considered to be of spherical shape.20 Changes in temperature, surfactant concentration, or

additives in the solution may change the size, shape, aggregation number, and stability of the

micelles.

There are four maj or types of micelles: 1) spherical micelles which generally have an

aggregation number less than 100 molecules, 2) cylindrical or rod-like micelles, 3) lamellar

micelles, and 4) vesicles. These four structures are shown in Figure 1-4.

1.1.2 Dynamic Nature of Micellar Solutions

Although micelles are often shown as static structures in solution, they are in fact quite

dynamic in nature, constantly breaking and reforming in solution. As stated above, there is a









dynamic equilibrium between the surfactant monomers in the bulk, the surfactant molecules in

the micelle, and molecules that are adsorbed at the interface. There are two characteristic time

scales of relevance in micellar dynamics.21, 22 The first is a fast relaxation time, referred to as zl,

which is a measure of the time that it takes for one surfactant molecule to go in or to come out of

the micelle. The second is a slow relaxation time, referred to as z2, which is a measure of the

time that it takes for one micelle to completely disintegrate or to completely form. The fast

relaxation time is generally on the order of microseconds, whereas the slow relaxation time is on

the order of milliseconds to minutes. The rate of disintegration of a micelle is equal to the rate of

micellar formation. Thus in a micellar solution, if X number of micelles are broken down per

second, X number of new micelles are formed somewhere else in the solution during the same

time. If this requirement is not obeyed, then the number of micelles in the solution would change

as a function of time. The fast and slow relaxation processes are illustrated in Figure 1-5. The

lifetime of a micelle can be approximated to be equal to nz2 where n is the aggregation number of

the micelle23. Thus for a typical solution of sodium dodecyl sulfate (SDS) at 200 mM, the z2 is 7-

8 seconds and n is 65. Thus, nz2 is equal to 7.6 to 8.6 minutes. Therefore, when comparing the

lifetime of micelles (nz2) at 10 mM and 200 mM, it is 65 msecs to 8.6 minutes, respectively.

There are many methods that are used to measure micellar relaxation time. The stopped-

flow method24 inVOlves the rapid dilution of a micellar solution containing an oil soluble dye and

monitoring of the dye adsorbance as a function of time. The temperature-jump25 and pressure-

jump26 methods involve analysis of the electrical conductivity of the system after perturbation of

the temperature or pressure of the system. Ultrasonic absorption27 is yet another method to

measure the kinetics of micelles.









Micellar kinetics may be manipulated by changing physical properties of the solution, such

as temperature, pressure, and concentration, or by placing additives into the surfactant solution.

Leung and Shah28 have shown that the addition of short chain alcohols (methanol to pentanol)

leads to the destabilization of sodium dodecyl sulfate (SDS) micelles and subsequently leads to a

decrease in the z2 value. Patist and Shah29 30 later showed that the addition of longer chain

alcohols such as dodecanol or the addition of oppositely charged surfactants, such as

dodecyltrimethylammonium bromide (C12TAB) to SDS solutions leads to marked increases in

the z2 ValUeS, indicating enhanced stability of mixed micelles. A 25 mM SDS solution has a z2

value of 1 millisecond, but the addition of dodecanol or C12TAB increases the z2 value to 230

msec or 2000 msec, respectively.

During the 1970s, Aniansson and Wall discovered the existence of the two (fast and slow)

relaxation processes and developed a model of the kinetic process of micelle formation and

disintegration.21 The first maj or assumption of this model was that the free surfactant monomers

are assumed to be completely dissociated and the size distribution of the aggregates in a

surfactant solution is assumed to follow the behavior that is shown in Figure 1-6 where C(A,,)

denotes the total concentration of aggregates containing n monomers and is a function of

temperature, pressure, and total surfactant concentration.

The second maj or assumption Aniansson and Wall made was that the association and

dissociation of micelles is a stepwise process involving the entry and departure of one monomer

at a time from the micelle. Thus, there is a series of equilibriums,

A, + A,,_ z A,, n = 2,3,4,..., (1-1)

where An represents an aggregate containing n monomers, and kn' and kn- are the forward and

reverse rate constants for a given step, respectively. As a result, when the equilibrium of a










surfactant solution is perturbed (e.g., by temperature or pressure), the excess surfactant has to

move through regions of different aggregation numbers (Region II of Figure 1-6). According to

equation (1-1), this occurs in steps that are very small compared to the distance in aggregate

space traveled. Therefore, the process will have the characteristics of a flowing system, which is

important because it allows the kinetics of the abstract process of micelle aggregation to be

studied in terms of the more familiar phenomena of heat and material flow.

Initially, micellar kinetics was analyzed on the basis of a heat conductance problem. It was

later viewed in light of a mass transfer diffusion problem, based on a model of mass flow

through a tube having two wide ends that are connected by a narrow section. This model is

analogous to the two high concentration regions (Regions I and III) of Figure 1-6 being

connected by the low concentration region (Region II). In this mass transfer diffusion model, the

rate limiting step to equilibration between the two wide ends is considered to be the diffusion of

materials through the narrow section of the tube. Analogously, the low concentration region of

transient intermediate sub-micellar aggregate states (Region II) that surfactant monomers must

pass through (i.e., Az, A3, A4, An-1 in Equation (1-1)) when a micelle is formed (Region III), or

disintegrated (Region 1) into free monomers is the rate-limiting step in the formation or

disintegration of a micelle. Assuming the aggregation number n to be a continuous variable and

applying the above analogy to mass transfer, Aniansson and Wall derived the expression for the

fast relaxation process

1 k cr C cmc
= 1+- 0 itha (1-2)
Z1 CT ICHIC

where %~ is the half-width of the distribution curve of micellar sizes (assumed to be Gaussian,

Figure 1-6), k- is the stepwise dissociation rate constant, which is assumed to be independent of

n in the micellar region, C is the total surfactant concentration, and cmc is the critical micelle









concentration. Equation (1-2) predicts a linear relationship between 1/zl and the total surfactant

concentration, which is in agreement with pressure-jump and sound absorption experiments.31, 32

It is obvious that as the total surfactant concentration increases, the number of micelles increases

also, resulting in a decrease in intermicellar distance. Hence, the time required for a monomer to

collide with a micelle is shorter at higher surfactant concentration. The length of the surfactant' s

hydrocarbon tail affects the magnitude of zl (i.e., the shorter the chain length, the faster the

relaxation time). This is because the longer the chain length, the greater the Van der Waals

attractive forces will be between the chains of neighboring surfactant molecule. This will lead to

more stable, tighter packed micelles with increasing chain length.

Using the same analogy of diffusion through a variable-width tube as described above, an

expression for the slow relaxation time, z2, WAS derived and simplified to

1 n2 2 -
1+-a (1-3)
v2 cmc x R n

where R is a term which may be visualized as the resistance to flow through the narrow region

(i.e., Region II in Figure 1-6) connecting the monomers to the micelles and is given by


R = i[ (1-4)
n-n-, k A

where n is the aggregation number of some colloidal aggregate and An is the equilibrium

concentration of aggregates of order n. The dependence of 1/z2 upon ionic strength,

concentration, and temperature has been interpreted in terms of their effect on R. Interestingly,

the two relaxation times can be used to calculate two important parameters of a micellar solution:

1) the residence time of a surfactant monomer in a micelle and 2) the average lifetime or stability

of micelles33-36. The residence time of a surfactant monomer in a micelle is equal to aI F., where n









is the mean aggregation number and k- is the dissociation rate constant of a monomer from a

micelle. The average micellar lifetime T,, is given by 23



T,, = z, naz= n r, (1-5)
1+ -a


When the concentration of surfactant is much greater than cmc, the micellar lifetime is

approximately equal to n T2-

Although first derived for nonionic surfactants, the results of Aniansson and Wall's theory

on micellar aggregation kinetics were compared primarily with experiments on ionic systems,

simply because it was much easier to detect the relaxation times in ionic systems than in

nonionic systems. Even so, the agreement between theory and experiment was, in general,

satisfactory in the regime of low surfactant concentrations.37 At higher concentrations, however,

the theory did not match experimental results.38 As previously stated, equation (1-3) predicts that

T2 Should increase with increasing surfactant concentration. However, it has been reported that

for some ionic surfactant systemS T2 fifSt increases, passes through a maximum, and then

decreases again.39-41 This behavior of r2 in ionic micellar solutions is not predicted in the

Aniansson-Wall model. Kahlweit and coworkers, using their own temperature-jump and

pressure-jump results22, 42' 43 COncluded that in ionic surfactant systems at high concentration, the

reaction path for the formation of micelles must be different than that at low concentration.

Therefore, the following model was proposed explaining the occurrence of a maximum in T2-

Ionic micelles, including sub-micellar aggregates, can be considered charged particles. When

ionic surfactant molecules such as SDS are added to water, the surfactant molecules dissociate

into negatively charged dodecyl sulfate molecules and their positively charged sodium counter-










ions. These counter-ions are present in solution as a cloud surrounding the negatively charged

micelle (see Figure 1-7). It is believed that at low surfactant counter-ion concentration, the

micelles are stable with respect to coagulation due to repulsive electrostatic forces. Consequently

they can grow only by stepwise incorporation of monomers according to Equation (1-1) above.

As more and more surfactant is added into the system, the counter-ion concentration also

increases, which compresses the electrical double layer and reduces charge repulsion, allowing

the micelles to come closer to each other so that attractive dispersion forces (i.e., Van der Waals

forces) lead to a reversible fusion-fission coagulation according to

Ak + A, A, k +1=i (1-6)

Therefore, Kahlweit suggested that there are essentially two possible pathways for micelle

formation in ionic surfactant solutions: 1) formation as a result of step-wise incorporation of

monomers, and 2) formation as a result of sub-micellar aggregates coming together. The same

idea holds true for the dissociation of micelles by two possible pathways. His model proposed

that the first pathway was dominant for low surfactant concentrations, where electrostatic

repulsion would be high between droplets. The second pathway would become dominant for

higher surfactant concentrations, where the counter-ion concentration is high and repulsion is

minimized.

1.2 Macroemulsions

It is a commonly known fact that oil and water do not mix. However, emulsifying agents,

typically surfactants can be added to a mixture of oil and water to promote the dispersion of one

phase in the other in the form of droplets. Over the years, emulsions have been defined in a

variety of ways. For the purpose of this dissertation, emulsions will be defined as

thermodynamically unstable, heterogeneous systems, consisting of at least one immiscible liquid









intimately dispersed in another in the form of droplets, whose diameters are generally in the

range of 1 100 Clm.44 There are two main types of emulsions: oil-in-water emulsions, in which

oil droplets are dispersed in a continuous water phase, or water-in-oil emulsions, in which water

droplets are dispersed in a continuous oil phase.

The most fundamental thermodynamic property of any interface is the interfacial free

energy, or interfacial tension. The interfacial free energy is the amount of work necessary to

create a given interface. The interfacial free energy per unit area is a measure of the interfacial

tension between two phases. A high value of interfacial tension implies that the two phases are

highly dissimilar in nature. There are many methods available to measure the interfacial tension

between two liquids including the Du Notty ring method, Wilhelmy plate method, drop-weight

or drop-volume method, pendant drop method, spinning drop method, and Sessile drop method.3

Emulsification involves the generation of a large total interfacial area. Considering that the

two phases in emulsions are not miscible, in order to generate this large interfacial area, the

interfacial tension must be lowered significantly according to the following equation:

W = y *A (1-7)

where W is the work done on an interface, y is the interfacial tension, and AA is the change in

interfacial area associated with the work W. According to Equation (1-7), when a constant

amount of work is applied to generate an interface, AA will be large if y is small, and thus the

interface will expand significantly to form smaller emulsion droplets.

As previously mentioned, the primary means by which the interfacial tension is lowered is

through the addition of emulsifying agents, usually surfactants. The surfactant molecule also

plays a second role in emulsions which is to stabilize the interface for a time against coalescence

with other droplets and concomitant phase separation.









A large number of methods have been developed to provide the energy needed to achieve

complete emulsification in a given system. These methods can be broken up into a number of

classes. Table 1-1 summarizes methods and apparatus used to produce emulsions, including a

characterization of each method.36, 44-46 Some of these methods are almost exclusively used in

laboratory settings (i.e., bench scale), such as 1, 4d, 8b, 9, 10 and 12. For large-scale production

of emulsions, methods 4b, 5, 7, and 8a are used. There are often times when two methods are

combined, such as 3 and 7 or 4 and 7.

1.2.1 Emulsion Droplet Size

Emulsions are classified as either water-in-oil (W/O) or oil-in-water (O/W)

depending on which phase is continuous and which is dispersed. The dispersed phase in

emulsions, whether oil or water, is usually composed of spherical droplets within the continuous

phase. These droplets may be nearly monodisperse in terms of droplet size; or they may have a

wide size distribution depending on several factors.44 In mOst cases, the wider the size

distribution, the less stable is the emulsion. In other words, emulsions with a more uniform size

distribution tend to remain stable for longer time while those with wide size distribution will

usually undergo Ostwald ripening, a phenomenon where larger droplets grow at the expense of

smaller droplets.45 In general, emulsions with a narrow size distribution and a small mean

droplet size tend to exhibit a greater emulsion stability, all other things being equal.44 The

change in the size distribution with time reflects the kinetics of coalescence in emulsions.

Depending on the surfactant that is used to stabilize the emulsion, emulsions can have lifetimes

ranging from hours to as long as a few years.44 In general, emulsions exhibiting a higher yield

stress tend to show higher emulsion stability and shelf life.44

Emulsion droplet size is also related to the method of preparation that is employed to

generate the emulsion. This is a result of the relationship between interfacial area and work that









is done on the system, according to Equation (1-7). As can be seen in this equation, if the

interfacial tension is constant with time, the change in interfacial area is directly proportional to

the amount of work that is put into the system. Some emulsion preparation methods provide

more energy (work) than others, and thereby lead to smaller droplets and higher interfacial area.

Micellar stability is another factor that affects the droplet size of emulsions.46 The ability

of the surfactant molecules to adsorb rapidly from the bulk to the droplet interface in an

emulsion determines the dynamic interfacial tension, which is related to the total interfacial area

and therefore the droplet size. In emulsion formation, as the large total interfacial area is being

generated by dispersion of oil phase, additional monomers have to be provided to this newly

created interface by disintegration of micelles. If the micelles are very stable, flux of surfactant

monomers to the interface of droplets will be low, resulting in a higher interfacial tension at the

droplet surface and a large droplet size will occur as predicted by Equation 1-7. The process of

monomer diffusion from the bulk to the oil/water interface is illustrated in Figure 1-8.

A considerable amount of work has been done on characterizing micellar stability in

surfactant solutions. It has been shown that more stable micelles provide less monomer flux to

the oil/water interface, which results in a higher interfacial tension and hence, a larger droplet

size.46 In Order for the droplet size in an emulsion to be small, a short-lived (or labile) micelle is

desired, since it will supply monomer to the oil/water interface with ease. This is illustrated in

for emulsions of hexadecane/sodium dodecyl sulfate (SDS) solutions in Figure 1-9.

The droplet size is largest for the most stable micelle, which is known to be at the 200 mM

concentration for SDS solutions.37, 41 This relationship between micelle stability and droplet size

has also been verified for cesium dodecyl sulfate solutions which forms the most stable micelle

when compared to micelles of sodium dodecyl sulfate and lithium dodecyl sulfate.47 Therefore,









knowledge of the surfactant micellar stability as a function of concentration is necessary to

predict the droplet.

1.2.2 Viscosity of Emulsions

The viscosity, or resistance to flow, of emulsions could be considered as one of their most

important properties. This is true from both a practical and a theoretical viewpoint. In a practical

sense, certain cosmetic or even food emulsions are only desirable at a specific viscosity (e.g.

lotions, milk, salad dressings, etc.). Manipulation of emulsion viscosity to achieve the desired

product specifications is not a trivial matter. From a theoretical perspective, the viscosity

measurements can be used to provide insightful information about the structure and possibly the

stability of an emulsion. The overall emulsion stability is affected by the following factors:44, 48

Viscosity of the external phase
Concentration (i.e., volume fraction) of the internal phase
Viscosity of the internal phase
Nature of the emulsifiers
Surface viscoelasticity of the interfacial film formed at the oil/water interface
Droplet size distribution
1.2.3 Determination of Emulsion Type (O/W or W/O)

Emulsions consist of a dispersed phase and a continuous phase. Most often the dispersed

phase is present as spherical droplets within the continuous phase. Most emulsions follow the

Bancroft49, 50 Tule for emulsions, which states that the phase in which the surfactant is most

soluble becomes the continuous phase in an emulsion. There are several methods commonly

used to determine which phase is continuous and which phase is dispersed. These methods

utilize the property differences between oil and water to determine phases. The methods are

listed in Table 1-2.










1.2.4 Emulsion Stability

As mentioned previously, one of the most important parameters in emulsifieation processes

is emulsion stability. For example, milk is a natural emulsion of the O/W type in the food

industry. If the stability of milk was only a week or two, the milk would have to be shaken

vigorously before pouring. However, in this case nature has provided us with a stable

emulsion. Another common example is shampoo, another emulsion. It would be inconvenient if

the shampoo were not a stable emulsion, since shaking would be necessary. There are also cases

where it is necessary to break down unwanted, naturally occurring stable emulsions. Such

examples are the W/O type emulsions which build up in oil storage tanks, or the O/W type

emulsions that arise in effluent waters.

So it becomes necessary to understand how to develop an emulsion system to be stable or

unstable, depending on the needs of the industry. To understand emulsion stability, it is

important be cognizant that there are Hyve types of breakdown processes which can occur in

emulsions. These are listed in Table 1-3, along with factors that influence that type of

breakdown."

1.2.4.1 Coalescence

When two oil drops approach each other, a thin film of the continuous water phase is

trapped between the drops. The behavior of the thin film determines the degree of stability of

the emulsion, and the rate of thinning of the film determines the time required for the two drops

to coalesce (i.e., coalescence rate). When the fi1m has thinned to a critical thickness, it ruptures,

and the two drops unite or coalesce to form one larger drop.52, 53 The rate of film thinning

depends on the surface viscosity of the surfactant film adsorbed at the oil/water interface. The film

may drain evenly or unevenly depending on the interfacial tension gradient due to adsorbed

surfactant.54









The factors that influence the rate of film thinning between droplets therefore influence the

emulsion stability. A summary of all of the factors influencing coalescence of droplets is given in

Table 1-4.5

1.2.4.2 Charge stabilization: The electrical double layer

Breakdown of an emulsion can occur due to electrostatic attraction between droplets in the

dispersed phase. The attraction can be induced if it is desirable to breakdown an emulsion, or

attraction can be eliminated if it is desirable to maintain a stable emulsion. To understand the

concept of charge stabilization, it is necessary to understand the nature of the electrical double

layer that surrounds the droplets and the factors that influence it. The electrical double layer

refers to the volume around the emulsion droplets that is influenced by the charge on the

droplet, if any. This volume can be broken down into two distinct regions, and this is illustrated

in Figure 1-10.

The electrical double layer is composed of two layers; the first is known as the Stern plane

and is characterized by a linear drop in electrical potential, and the second is referred to as the

shear plane and it is characterized by an exponential drop in potential. The presence of counter

ions on the surface effectively neutralizes some of the surface charge on the droplet. These

counter ions may be introduced in the system by the presence of an electrolyte.

The electrical double layer defines the region of influence of a droplet caused by the

surface charge. If the double layer thickness, also known as the Debye length, is large for a pair

of similarly charged droplets, the droplets will be electrostatically repelled. If an electrolyte is

added to the system to decrease the double layer thickness, the repulsion will decrease and may

be low enough that the droplets can get close enough to be attracted to one another. This

phenomenon would result in destabilization of the emulsion. Therefore, in an emulsion in which









the droplets are charged, any additive or parameter change which influences the electrical double

layer thickness will influence the stability of the emulsion.

1.2.4.3 Phase inversion in emulsions

Phase inversion can occur in emulsions due to a number of factors. For a given emulsifier

concentration, the viscosity of an emulsion gradually increases as the phase volume of the

dispersed phase is increased. However, at a certain critical volume fraction Oc, there is a sudden

decrease in viscosity, which corresponds to the point at which the emulsion inverts. Oc was found

to increase with increasing emulsifier concentration.56 The sudden decrease in viscosity is due to

the sudden reduction in dispersed phase volume fraction. Often Oc is in the range of 0.74, so that

upon inversion, the dispersed phase volume fraction reduces from 0.74 to 0.26, thus reducing the

viscosity significantly. Oc should theoretically be in the range of 0.74 for spheres of equal radii to

be at the maximum packing," but #,=0.99 was found for paraffin oil/aqueous surfactant

solutions"" and #,=0.25 was found for olive oil/water emulsions.59 The phase inversion of

emulsions can be brought about by several parameter changes, listed in Table 1-5.

1.2.4.4 Emulsion creaming

As described previously, "creaming" is a special case of emulsion instability that occurs

when there is no change in droplet size or size distribution, but buildup of an equilibrium

droplet concentration within the emulsion occurs. Creaming is not so much a breaking of an

emulsion as it is a separation into two emulsions, one of which is richer in the dispersed

phase, the other poorer, than the original emulsion.44 The more concentrated emulsion is

referred to as the "cream." The creaming phenomenon can result from an external force field,

such as gravitational, centrifugal, or electrostatic. In most cases creaming is undesirable, as in

pharmaceutical products and agricultural sprays where the product must first be shaken in order










to uniformly disperse the droplets again. In some cases, however, creaming is desirable,

such as in the separation of isoprene droplets from water in the rubber latex industry. Regardless, it

is important to understand the physical parameters that affect the creaming, or sedimentation

rate.

1.2.5 Demulsification

As discussed previously in this section, it is not always desirable to have a stable emulsion.

Often an emulsion is present in a system in which it is undesirable. One example is the presence

of aqueous emulsion droplets dispersed in crude oil. Crude oil is always associated with water or

brine in oil reservoirs and also contains natural emulsifying agents, such as resins and asphaltenes.

These emulsifying agents form a thick, viscous interfacial film around the water droplets,

resulting in a very stable emulsion. Therefore, demulsification is very important in the crude oil

industry. Many physical methods have been developed for demulsification, depending on the

industrial application. These methods are described in Table 1-6.

A wide variety of chemical additives for demulsification have been developed in recent

years. These additives are all relatively high molecular weight polymers capable of being

adsorbed at the O/W interface and displacing the film. The primary advantage of these additives

is that they can be added to the system even before emulsion formation, so that they act as

inhibitors.

In the petroleum industry, demulsifiers have been considered for breaking the common

fuel oil emulsions. In this area, chemical demulsifiers that have been investigated include ultra-

high molecular weight polyoxiraneS60 and micellar solutions containing petroleum sulfonates,

electrolytes, and cosurfactants.61









It is evident that there are many methods for demulsification. The nature of the emulsion to

be separated is the key factor in determining which methods) is best for each particular

demulsification problem.

1.2.6 Surfactant Selection for Emulsification

Often the selection of surfactants in the preparation of either O/W or W/O emulsions is

made on an empirical basis. However, in 1949, Griffin62 introduced a semi-empirical scale

for selecting an appropriate surfactant or blend of surfactants. This scale, termed the hydrophile-

lipophile balance (HLB), is based on the relative percentage of hydrophilic to hydrophobic

groups in the surfactant molecules and ranges from 1 to 40. An HLB of 1 represents a surfactant

that is highly oil-soluble and an HLB of 40 represents a highly water-soluble surfactant.

Surfactants with a low HLB number normally form W/O emulsions, whereas those with a high

HLB number often form O/W emulsions.63 A summary of the HLB range required for various

purposes is given in Table 1-7.

The calculation of the HLB number for a given surfactant, as developed by Griffin,62 is quite

laborious and requires a number of trial and error procedures. Simplification methods were later

developed by Griffin62 that applied to certain surfactants. Davies63 developed a method for calculating

the HLB values of surfactants directly from their chemical formulas, using empirically

determined numbers. The HLB number can also be determined experimental through several

correlations that have been developed. These correlations relate the HLB number to such

parameters as the cloud point,64 water titration value for polyhydric alcohol esters,65 and the heat of

hydration of ethoxylated surfactants.66

Another method that may be used to select a surfactant suitable for forming an emulsion is by

using the phase inversion temperature (PIT) method. The phase inversion temperature (PIT) is the









temperature at which an emulsion experiences phase inversion, as described in a previous section. The

PIT of non-ionic emulsifiers has been shown to be influenced by the surfactant HLB number, so the PIT

can be used similarly to the HLB number in selecting an emulsifier.59 The primary distinction is that the

PIT is a characteristic property of the emulsion, not of the emulsifying agent.59 Due to this, the PIT

includes the effect of additives on the solvent, the effect of mixed emulsifiers or mixed oils, etc. In other

words, the HLB number is actually a function of all of these properties, but only the PIT completely

analyzes a given emulsion system. The PIT method is useful because the PIT is a measurable property

which is related to the HLB number. A summary of effects of PIT and droplet stability from different

investigations is given below:59

The size of emulsion droplets depends on the temperature and the HLB of emulsifiers
The droplets are less stable toward coalescence close to the PIT
Relatively stable O/W emulsions are obtained when the PIT of the system is some 20 to
650C higher than the storage temperature
A stable emulsion is obtained by rapid cooling after formation at the PIT
The optimum stability of an emulsion is relatively insensitive to changes of HLB value or
PIT of the emulsifier, but instability is very sensitive to the PIT of the system
The stability against coalescence increases markedly as the molar mass of the lipophilic or
hydrophilic groups increased
When the distribution of hydrophilic chains is broad, the cloud point is lower and PIT is
higher than when there is a narrow size distribution.
The PIT can be measured by the following methods: 1) direct visual assessment,67 2) conductivity

measurement,34,68, 69 3) Differential Thermal Analysis (DTA) or Differential Scanning

Calorimetry (DSC),70 and 4) viscosity measurement.71, 72 Both the HUB and PIT methods for

selecting an emulsifier in a system have been widely used and adapted to meet industry needs.

1.2.7 Applications of Emulsions

Emulsions are desirable for many different applications because they provide a system

having a large interfacial area. Historically, cosmetic emulsions are the oldest class of

manufactured emulsions.44 Emulsions are desirable for cosmetic applications because: 1) they

increase the rate and extent of penetration into the skin, 2) they open up the possibility of










applying both water- and oil-soluble ingredients simultaneously (e.g. deodorants), and 3) they

provide for efficient cleansing.

Emulsions are also widely used for pharmaceutical applications in the form of creams or

ointments and as drug delivery vehicles. They are also ideal for use as polishes (e.g. furniture

polishes, floor waxes, etc.) paints, and agricultural sprays. Many foods are manufactured in the

form of emulsions including mayonnaise, salad dressings, milk, and margarine. Another industry

where emulsion technology is important is the asphalt industry when the principal requirement is

the production of water-repellent surfaces. Emulsions are also used as polymerization vehicles to

aid in the production of high polymeric materials such as plastics, synthetic fibers, and synthetic

rubbers. These are just a few of the many applications of emulsions.

1.3 Microemulsions

A microemulsion is a thermodynamically stable, isotropic dispersion of oil and water

containing domains of nanometer dimensions stabilized by an interfacial film of surface-active

agent(s).73 The term "microemulsion" originated from Jack H. Schulman and coworkers in

1959,74 although Hoar and Schulman originally described water-in-oil microemulsions, which

they referred to as transparent water-in-oil dispersions, in 1943.7 As implied above,

microemulsions may be of the oil-in-water (O/W) (see Figure 1-1 1) or the water-in-oil (W/O)

type depending on conditions of the system and system components.

According to Bancroft,49, 50, 76 phase volume ratios are less important in the determination

of the microemulsion type that will be formed (i.e., W/O or O/W) than the surfactant

characteristics (e.g. HLB). As previously mentioned, the Bancroft rule states that whichever

phase the surfactant has a greater affinity for will typically be the continuous phase.









The creation of a microemulsion entails the generation of a huge interfacial area, which,

according to the following equation," requires a significant lowering of the interfacial tension

(usually << 1 mN/m):7

W = y *A (1-7)

where W is the work performed, y is the surface or interfacial tension at the air/water or oil/water

interface and AA is the change in surface or interfacial area. This ultra-low interfacial tension in

spontaneously formed microemulsions is achieved by the incorporation of surfactant(s) (typically

a surfactant + a cosurfactant, especially when ionic surfactants are used).l Figure 1-12 shows the

thermodynamic explanation for the behavior of macro- and microemulsions. As can be

concluded from the graph, there is an optimum radius for microemulsion systems where the free

energy of dispersion becomes negative, thereby making the microemulsion stable and its

formation energetically favorable.79

Schulman and others first noticed microemulsion systems in 1943 when they observed that

the addition of a medium chain-length alcohol made a coarse macroemulsion that was stabilized

by an ionic surfactant become transparent.75 Even then, Hoar and Schulman recognized the

important role of a very low interfacial tension in causing spontaneous emulsification of the

added water in oil.75 They concluded that the role of the alcohol is as a stabilizer against the

repulsive electrostatic forces that the ionic surfactant head groups would experience.

Schulman and others used a variety of experimental techniques (e.g. X-ray diffraction,74

ultra-centrifugation, so light scattering,74 VISCOSimetry,s and nuclear magnetic resonance

(NMR)82, 83) to elucidate some of the characteristics of these microemulsion systems following

the groundbreaking work of Schulman and Hoar. These studies were instrumental in providing

them with information about the structure, size, and interfacial film behavior of microemulsions.










They were able to determine the size of the droplets and they found that the presence of the

alcohol within the system led to greater interfacial fluidity.

Later, in 1967, Prince74 prOposed a theory that the formation of microemulsions was due to

the negative interfacial tension that results from high surface pressure of the fi1m. Prince

explained this negative interfacial tension based on the depression of the interfacial tension

between the oil and water phase that occurs when surfactant is added. The principle behind this

theory is described by the series of equations that follow. The surface pressure of the film at the

air/water interface, naw, is defined as:17, 74


awIM = 70 T (1-8)

where yo is the surface tension of the pure surface (without surfactant) and ys is the surface

tension of the surface with surfactant. In the case where oil is the second phase (oil/water

system), the surface pressure of the surfactant film at the oil/water interface, now, can be defined

as:


ow Yow o f/w s(1-9)

where (yo/w)o is the interfacial tension of the "pure" oil/water interface (i.e., in the absence of

surfactant) and (yotw)s is the interfacial tension of the oil/water interface in the presence of

surfactant film. Rearrangement of Equation (1-9) gives:

70w) =7/wo ow (1-10)

Based on Equation (1-9), for a surfactant film that can generate a very high surface

pressure (now), the interfacial tension of the surfactant film at the oil/water interface (yo/w)s

becomes negative. This is only a transient phenomenon because generation of a negative









interfacial tension leads to a negative free energy of formation of the emulsion, which is an

unstable situation. This is illustrated by the following equation:

AGronn = yAA TASconfig (_1

where AA is the increase in interfacial area, ASconfig iS the configurational entropy of the droplets

of the liquid that are formed and T is the absolute temperature.84 The negative interfacial tension

accounts for the spontaneous increase in interfacial area that occurs in the formation of

microemulsions. When a transient (unstable) negative interfacial tension is experienced, the

system will seek to stabilize by spontaneously generating new interfacial area, thereby raising the

interfacial tension back to acceptable, stable limits. As previously mentioned, in order to form

microemulsions, it is required that the concentration of surfactants be greater than that required

to reduce the oil/water interfacial tension to zero and to cover the total interfacial area of all

dispersed droplets. The transient negative interfacial tension that is generated facilitates the

spontaneous break-up of droplets.

The nature of the thermodynamic stability of microemulsions has long been studied. The

stability can be attributed to the fact that the interfacial tension is low enough that the increase in

interfacial energy accompanying dispersion of one phase in the other is outweighed by the free

energy decrease that is associated with the entropy of dispersion.79 Furthermore, the free energy

decrease that accompanies adsorption of surfactant molecules from a bulk phase favors the

existence of a large interfacial area and hence plays a maj or role in stabilizing microemulsions.79

One must also understand the role of cosurfactants in microemulsion formulations. The

addition of a short-chain alcohol to a surfactant solution to enhance microemulsion formation has

long been practiced." This cosurfactant (short-chain alcohol) serves to 1) fluidize the interface,

2) decrease interfacial viscosity, 3) destroy the lamellar liquid crystalline structures, 4) provide









additional interfacial area, 5) reduce electrical repulsion between droplets and also between polar

head groups of surfactants by acting as charge screeners and decreasing surface charge density,

and 6) induce the appropriate curvature changes."

In 1972, Gerbacia and Rosano86 inVCStigated the formation and stabilization of

microemulsions, with emphasis on the role of the cosurfactant (in this case, pentanol). They

suggested that a critical aspect of the mechanism of formation of microemulsions involves the

diffusion of the pentanol through the interface. This process has been found to be a necessary,

but not sufficient condition for microemulsification. In essence, the alcohol transiently lowers the

interfacial tension to zero as it diffuses through the interface, thereby inciting the spontaneous

dispersion of one phase into the other. The surfactant then acts to stabilize the system against

coagulation and coalescence. NMR data and calculation of free energies of adsorption of the

pentanol into the interface have proven that a strong association between the surfactant and

cosurfactant is not necessary for microemulsion formation.86

The stability of the phases of surfactant in microemulsions results from the competition

between entropic and elastic contributions to the free energy." The two intrinsic parameters that

ultimately affect the structure of the aggregates existing in solution are the mean bending

modulus, ic, and the Gaussian bending modulus, K The bending energy, Fb, is directly related to

the mean bending modulus and the Gaussian bending modulus, as can be seen below:


Fb, 1 -~T C2 2CO,)2 1 2C
2 (1-12)

where C1 and C2 are the local principal curvatures of the surfactant layer and Co is the

spontaneous curvature. Bellocqs states that the contribution of the bending energy to the total

free energy is a crucial determining factor in the type and characteristic size of the structure. In

Equation (1-12) above, the first term represents the amount of energy needed to bend a unit area









of interface by a unit curvature amount, and the second term is important to the change of the

membrane topology and the phase transition.88 The spontaneous curvature, Co, is determined by

the nature of the interactions between the surface-active molecules; i.e., the competition in

packing of the polar heads and the hydrocarbon tails of the surfactant. If the dominant

interactions are between the polar heads, then the surfactant orientation will be concave to water

and a water-in-oil microemulsion will be formed, whereas, if the dominant interactions are

between the hydrocarbon tails, the surfactant orientation will be convex to water and an oil-in-

water microemulsion will be formed.

The addition of cosurfactant (short-chain alcohol) can have a profound effect on the

curvature. Bellocq reported that there is a very efficient lowering of the bending constant, K, of

the surfactant film with the dilution of the system with short-chain alcohols.88 This lowering of K

was attributed to thinning of the interface, and this attribution was confirmed with deuterium

solid-state NMR.

The chain length of the cosurfactant (alcohol) was found to be critical in microemulsion

formation. The chain length determines what types of phases (bicontinuous, lamellar, sponge,

vesicle, etc.) will be of importance. These phases play an important role in the type, structure and

size of the microemulsion that will be formed.89 In Simplistic terms, a short-chain cosurfactant

acts to prevent the formation of or destroy lamellar liquid crystals. Since there is a significant

difference between the surfactant chain length and the short-chain alcohol, there is a tail wagging

effect due to the fact that the excess hydrocarbon tails have more freedom to disrupt molecular

packing through conformational disorder, increased tail motion, and penetration and/or buckling

of the chain into the monolayer.90 This motion of the hydrocarbon tail prevents or disrupts










ordering of the molecules and therein prevents formation of or destroys lamellar liquid crystals

and enhances microemulsion formation.

It must be noted that cosurfactant addition to microemulsion-forming systems is typically

only applicable for ionic surfactant systems. Microemulsions that incorporate non-ionic

surfactants may be formed without the need for cosurfactant addition; especially in the case of

non-ionic surfactants of the polyethylene oxide adducts (POE). This is because these surfactants

are composed of a homologous series of varying composition and molecular weight," which

serves the same purpose of enhancing the interfacial film fluidity. The important factor in the

formation of these types of microemulsions is temperature, because this class of materials is

solubilized in water by means of hydrogen bonding between the water molecules and the POE

chain. Hydrogen bonding is a temperature-sensitive phenomenon, which decreases with

increasing temperature. Therefore, the temperature conditions under which a microemulsion is

formed are important to the type of microemulsion that is formed. Above a characteristic

temperature, which is commonly known as the phase inversion temperature (PIT),91 the non-

ionic surfactant changes its affinity from the water phase to the oil phase. Below the PIT, O/W

microemulsions will be formed and above the PIT, W/O microemulsions will be formed.

1.3.1 Formation of Microemulsions

Now that the some of the basic principles of microemulsions have been discussed, it is

easier to understand what conditions must be met for microemulsion formation and why they are

required. There are three maj or factors that must be considered in order to form

microemu sions.5

First, given the importance of achieving an ultra-low surface tension at the oil/water

interface, surfactants must be carefully chosen so that this may be accomplished. Secondly, there

must be a large enough concentration of surfactants to stabilize the newly formed interface such









that phase separation does not occur. It must be mentioned that the type, structure and

characteristics (e.g. Hydrophilic-Lipophilic Balance (HLB), degree of ionization, etc.) of the

surfactants may potentially play a maj or role in determining just how high the surfactant

concentration needs to be to stabilize the interface. The third required condition for forming

microemulsions is that the interface must be fluid (flexible) enough to facilitate the spontaneous

formation of micro-droplets with a small radius of curvature (50 500 A+). That is where

cosurfactant structure can become very important.

1.3.2 Applications of Microemulsions

Microemulsions have a range of industrial applications. They are useful in technologies

such as enhanced oil recovery,92 pharmaceuticals,93 COSmetics,94, 95 food sciences,96 and

detergency.97 98 Microemulsions have been extensively studied in regards to their use as drug

delivery vehicles, and are now gaining attention as possible mediums for use in detoxification of

blood to remove free drug from the blood stream of overdose patients.99

Drugs that have significant hydrophobic functionality have been shown to partition into the

corona (area at the interface) and/or interior core of O/W microemulsions.99 It is generally

accepted that this is largely due to hydrophobic interactions. Recall that the formation of

microemulsions leads to the generation of a large interfacial area. It is believed that this large

interfacial area facilitates the uptake of relatively large amounts of drug into the microemulsion

in a time efficient manner, thereby significantly decreasing the bulk drug concentration.

1.3.3 Nano-emulsions

As early as 1943, Dr. T. P. Hoar and J. H. Schulman75 discovered that he could prepare

transparent water-in-oil dispersions of nanometer size, which displayed stable thermodynamic

characteristics (i.e., the water droplets remained stably dispersed indefinitely if left unperturbed,

with respect to temperature, pressure, or compositional conditions). Anomalous systems could









also be prepared in which oil droplets are dispersed in water. Later, other scientists, including Dr.

Stig Friberg,89 found that they could develop transparent nanometer size dispersions of one

medium within another continuous medium, which were not thermodynamically stable, but were

kinetically stable (i.e., given time, there will be a breakdown in the stability of the dispersion that

will lead to phase separation. Such systems have come to be referred to as nano-emulsions

(thermodynamically unstable systems). Thus, both Schulman's microemulsions and nano-

emulsions start in the nanometer range, but the microemulsions are thermodynamically stable

and maintain the same size whereas the nano-emulsions coalescence and display an increase in

size with time, which ultimately causes phase separation.

In order to sufficiently comprehend the difference between microemulsions and nano-

emulsions, there must be clarification of the nomenclature of the two. As previously mentioned

microemulsions is a misleading title considering that their average diameter ranges from 10 -

100 nm. Light scattering and X-ray analysis have indicated that microemulsions are, in fact,

coarse mixtures as opposed to molecular dispersions.7 Nano-emulsions lie within the same size

range, but may have diameters that considerably exceed the size of a microemulsion droplet (20-

500 nm). The emphasis must be placed on the fact that nano-emulsions are in fact emulsions of

nano-size, meaning that they are kinetically stable, (thermodynamically unstable) heterogeneous

systems in which one immiscible liquid is dispersed as droplets in another liquid (as emulsions

are defined).

Although they are on similar size scales, microemulsions and nano-emulsions have

markedly different characteristics (see Table 1-8). Nano-emulsions are formally defined as

thermodynamically unstable, generally opaque, sub-micron-sized (20 500 nm) systems that are









stable against sedimentation and creaming.100 They may have the appearance of microemulsions,

but they do not necessarily require as much surfactant concentration in their preparation.1oo

As early as 1981, Rosano et al8 6 found that certain microemulsion systems, which they

termed "unstable microemulsions", displayed significantly different characteristics from

traditional microemulsions. These systems were dependent upon the order of addition of

components (i.e., the mixing protocol) and their formation was contingent upon having a large

enough concentration gradient to allow diffusion of amphipathic materials across the oil/water

interface.

El-Aasser et at. lot performed studies of a miniemulsification process, which produced O/W

miniemulsions (another term for what we refer to as nano-emulsions in this paper) having an

average droplet diameter in the size range of 100 400 nm. The miniemulsions that they

produced were generated by means of a mixed emulsifier protocol, which was comprised of a

mixture of ionic surfactant and long-chain fatty acid in concentrations of 1-3 % by weight in the

oil phase. They stated four "distinct and significant" differentiating aspects between the

necessary conditions of preparation for miniemulsification systems and traditional

microemulsion systems, which may also be produced by mixed emulsifier systems:

1) Concentration of the mixed emulsifier system: only 1-3 wt% (with respect to the oil

phase) is sufficient for the formation and stabilization of miniemulsions, whereas

microemulsions typically require 15-30 wt %.

2) Droplet size: their miniemulsion droplets range from 100-400 nm in diameter, as

opposed to microemulsions, which range from 10-100 nm in diameter.









3) Chain length of the cosurfactant (fatty alcohols or acids in this case): miniemulsions

require at least a 12-carbon chain length, whereas microemulsions can be prepared

with alcohols of shorter chain lengths.

4) Order of mixing of the ingredients (mixing protocol): successful production of

miniemulsions requires that the ionic surfactant and the fatty alcohol be initially

mixed in the water phase for 30 minutes to 1 hour at a temperature above the melting

point of the fatty alcohol, prior to the addition of the oil phase, whereas order of

mixing does not affect microemulsion formation.

El-Aasser et at. lot performed an array of experiments to help them to better understand the

miniemulsification process. They found that the process was a spontaneous phenomenon based

on results yielded by both dynamic and static spinning drop experiments. The oddity in this

discovery lay in the fact that the measured interfacial tensions were unexpectedly high, ranging

from 5 to 15 dynes/cm. They attributed this finding to the formation of emulsion droplets by

diffusion of the oil (in this case, styrene) from drops into the adj acent liquid crystal structure of

the mixed emulsifier system. They also stated that the presence of mixed emulsifier liquid

crystals, which was confirmed by birefringence observations, significantly improved the

emulsification process and led to greater emulsion stability. They concluded that the mechanism

of formation of miniemulsions was by swelling of the mixed emulsifier liquid crystals by oil (in

this case, toluene). This swelling of the liquid crystalline structure led to its break-up or sub-

division to form small emulsion droplets, which were stabilized by the adsorption of the mixed

emulsifier complex at the oil-water interface.10

Although emulsion stability is generally known to increase with droplet surface charge, the

miniemulsions that El-Aasser prepared displayed contradictory behavior; their stability increase









corresponded to a reduction in surface charge. These results suggested that the steric component

of stabilization was the dominating factor.'01

The mechanism that has been attributed to nano-emulsion breakdown in a ternary system

of brine, oil, and non-ionic surfactant is a 3-stage proceSS.102 The first and last stages of the

droplet growth process were found to be due to Ostwald ripening, whereas the droplet size

distribution of the second stage became too broad compared to the expected theoretical

distribution (as predicted by the Lifshitz- Slezov-Wagner theory) to be due to Ostwald ripening.

Katsumoto et al 102 made this assessment after plotting the cube of the z-average radius, rz, as a

function of time, and obtaining a linear relationship. In addition, their group found that the

volume of bound water on miniemulsion droplets plays an important role in obtaining a

homogeneous miniemulsion.32 The storage temperature of the miniemulsion solution and the

molecular weight of the surfactant were determined to significantly affect the system's stability:

as storage temperature is decreased, the rate of coalescence increases, and as the molecular

weight of the surfactant is increased, the rate of coalescence decreases. The former Einding is due

to an increase in surface tension because non-ionic surfactants become more water-soluble as

temperature is decreased. The latter finding is due to steric effects that become predominant as

the surfactant size is increased.

Nano-emulsions may be formed by a few different experimental methods: condensation,103

low-energy emulsifieation methods involving phase inversion,100, 104 Or by high energy input

during emulsifieation.los Forgiarini et at. too reinforced the concept that was proposed by El-

Aasseriol concerning the importance of the mixing protocol in the formation of nano-emulsions.

They found that they only obtained dispersions of nanometer droplet size when the nano-









emulsion was formed via stepwise addition of water to a solution of the surfactant in oil. If other

methods of addition were used, the droplet size ranged from 6 10 Clm.

Izquierdo et at. 104 analyzed the formation and stability of nano-emulsions that were

prepared by the phase inversion temperature (PIT) method, in which emulsions were formed at a

temperature near the PIT and then rapidly cooled to room temperature (250C) by immersion in

an ice bath. They proposed that a change in the interfacial curvature is critical to nano-emulsion

formation, and that the presence of lamellar liquid crystals was probably a necessary, but

insufficient requirement for preparation of nano-emulsions.100 They concluded that the key factor

for nano-emulsion formation should be credited to the kinetics of the emulsification process.

Nano-emulsions have possible applications as drug delivery vehicleS,105, 106 in drug

targeting, as reaction media for polymerization, in personal care and cosmetics, and in

agrochemicals. It is also plausible that nanoemulsions may be used in most industries where

extraction will ultimately be required. This idea is based on the premise that the nanoemulsions

may be designed so that their stability characteristics will coincide with the requirements of the

application.










Hydrophilic
head






Hydrophobic
head
Surfactant .Reverse
11icelle
m olec ul e ...........................................m icelle


Figure 1-1. Schematic diagram of a surfactant molecule, micelle, and reverse micelle.




Osmotic Pressure

Turbdi~ySolubilization
I~\ / /Magnetic
I Resonance

I Su rface Tensiocn
Equivalent
Conductivity
/ // Se11-Dltfusion




Concentration of Surfactant


Figure 1-2. Properties of surfactant solutions showing abrupt change at the solution critical
micelle concentration (cmc).















Adsorbed
film


Mi cell e


/ I Monomer
Water
Figure 1-3. Schematic design of micellar solution showing the three maj or species that are in
dynamic equilibrium: 1) monomers, 2) micelles, 3) adsorbed film.








A B


C D
Figure 1-4. Schematic diagram of the four major micellar structures: A) spherical micelle, B)
cylindrical, rod-like micelles, C) unilamellar vesicle, D) lamellar micelle.

















Fast relaxation time, microseconds


o~-~b
P P
a,


II
II
I I
\\
\\
\\
\\
I I
,I
'9 ,I
..


~---~p


~ 's
z
2


Slow relaxation time, milliseconds to minutes
Figure 1-5. Mechanisms for the two characteristic relaxation times for a micelle in a surfactant
solution, zl and z2, above cmc.


C (An)


Aggregation number, n M


Figure 1-6. Typical size distribution curve of aggregates in a micellar solution according to the
Aniansson-Wall model of stepwise micellar association. Region (I) corresponds to
monomers and oligomers; Region (III) to abundant micelles with a Gaussian
distribution around the mean aggregation number; and Region (II) to the connecting
"wire" (heat transfer analogy) or "tube" (mass transfer analogy) between Regions (I)
and (III).













N Na N
Na -N
Na _Na


Na+ Na' Na
Figure 1-7. Schematic of sodium counter-ion "cloud" around SDS spherical micelle.







dlroplet ft
mrnonoriers Frais~ele




Figure 1-8. Schematic diagram of the adsorption of surfactant monomers from the bulk to the
oil/water interface during emulsion formation


Figure 1-9. The emulsion droplet size in the hexadecane/SDS solution system after 30 s
emulsification at: (A) 50 mM, (B) 100 mM, (C) 200 mM, (D) 300 mM, and (E)
400 mM



















Shear plane


C'r?- Ion


~T~L~I11~1' Ioll

O o


ol o

O
~> o ~ O
Oi
O ~
oi Q


di stance


Charged
surface


Stern plane
Figure 1-10. Schematic depiction of the Stern-Graham model of the electrical double layer.


WATER


Figure 1-11. Schematic diagram of an oil-in-water (O/W) microemulsion













+ I I \Macroemnulsions (IFT = 1 mnN/m)
AG of
dispersion

0 Droplet radius


Mi croemul si ons
(R* = 10-100 nm, IFT 10-3 mN/m)

Figure 1-12. Thermodynamic explanation for behavior of macroemulsions and microemulsions










Table 1-1: Summary of methods used to produce emulsions
Metlul Related to Drops mainly EnergX Mode of Restrictions"
Metlul disrupted by" density Operatione

1. Shaking 4a CD L B N

2. Pipe Flow
a. Laminar 5 V L-M C V
b. Turbulent 4a T L-M C N
3. Injection l0a -L C
4. Stirrmng
a. Simple stirrer 1,2b T,V L B,C
b. Rotor-stator (5) T.V M-H B,C
c. Scraper 5 V L-M B,C V
d. Vibrator 8a ? L BC N
5. Colloid Mill 2a,4c,6 V M-H C V
6. Ball and roller mills 5 V M B(C) V
1. High-press. (2b) T,C,V H C N
Homogenizer
8. Ultrasonic
a. Vibrating knife 4d C,T M-H C W
b. Magneto-striction C M-H B,C W

9. Electrical l0b Elec. Charge M B(C) Several
10. Aerosol to liquid
a. Mechanical 3 -L-M B.C
b. Electrical 9 -M BC Several
11. Foaming or boiling Spreading L-M (W)
12. Condensation Several
a V-viscous forces in laminar flow, T-turbulence, C=cavitation
b L=low, M-moderate, H=high
a B=batch and C-continuous
d The continuous phase should be V-viscous, N-not too viscous, W=aqueous









Table 1-2: Common tests for determining, emulsion tvoe (W/O or O/W144


Name


Description


Without shaking, a drop of oil is placed in the
emulsion. If W/O type, the added oil dissolves rapidly
in the emulsion and disappears. If O/W type, the added
oil floats on top of the water continuous emulsion.

Electrical conductivity is measured. If W/O,
conductivity is low (like oil). If O/W, conductivity is
high (like water).

Viscosity is measured while a few drops of water are
added to the emulsion. If W/O type, added water
increases viscosity by adding more droplets to the
dispersed phase. If O/W type, added water decreases
viscosity (or shows little change) by slightly diluting the
continuous phase.


A parallel beam is shined through an emulsion of low
turbidity. If the beam converges, an O/W type emulsion
is present. If it diverges, it is W/O type. This is due to
the relative refractive indices of oil and water.107

Without shaking, a few drops of water soluble dye are
added. If O/W type emulsion, the dye dissolves rapidly
in the continuous phase, changing color. If W/O type,
the dye dissolves very slowly in the dispersed phase,
and most will sink through the emulsion.
Filter paper is dipped into the emulsion. If O/W type,
paper turns pink immediately from being wetted. If
W/O type, paper stays dry (blue) for a long time since
the continuous phase does not wet it.


Dilution Test




Electrical Conductivity Test




Viscosity Test





Refractive Index Test





Dye Solubilization Test



Filter Paper Test










Table 1-3: Types of breakdown processes occurring in emulsions
Breakdown Type Description

Buildup of an equilibrium droplet concentration gradient
within the emulsion. This phenomenon results from
No change in droplet size external force fields, usually gravitation, centrifugal, or
(or size distribution) electrostatic, acting on the system. "Creaming" is a
special case in which the droplets collect in a
concentrated layer at the top of an emulsion.

No change in basic droplet size,
but ithbuidupof ggrgats o This process is called flocculationn" and results from
the existence of attractive forces between the droplets.
droplets in the emulsion

This process also occurs when creaming or sedimentation
Flocculated droplets in an results in a close-packed array of droplets and these
aggregate coalesce to form larger droplets coalesce. The limiting state is the complete
droplets separation of the emulsion into two immiscible bulk
liquids.

This process does not involve actual coalescence of
droplets, but rather the transfer of dispersed phase across
continuous phase after solubization occurs. If the
Average droplet size increases --
emulsion is polydisperse, larger droplets will form at the
due to the two liquids forming the
emlso expense of smaller droplets due to the difference in
ermulscion bin ot ttal chemical potential for different size droplets (Ostwald
ripening). In principle, the system will tend to an
equilibrium state in which all the droplets have
combined and are one large droplet, or a separated phase.

This is a questionable "breakdown" process since
essentially another emulsion is formed. The inversion
Emulsion type inverts from W/O
to O/W process can be brought about by numerous parameter
9 changes, which will bue discussed in detail later in this
section.





Table 1-4. Factors influencing the stability of emulsions


Description of Effect


Factor


For mechanical stability, a surfactant film with strong lateral
intermolecular forces and high film elasticity is desired. A mixture
of two or more surfactants is preferred over a simple surfactant
(i.e., lauryl alcohol + sodium lauryl sulfate).
Significant only in O/W type emulsions, because of conductivity in
continuous phase. In the case of nonionic emulsifying agents, charge
may arise due to adsorption of ions from the aqueous phase. The
repulsion or attraction can be influenced by changing the thickness
of the double layer, which is described below.
A higher viscosity reduces the diffusion coefficient of the
dispersed droplets, resulting in reduced frequency of collision and
lesser coalescence. Viscosity can be increased by adding natural or
synthetic thickening agents. Viscosity also increases as the number
of droplets increases; so many emulsions are more stable in
concentrated form than when diluted.
Uniform size distribution is more stable than an emulsion with the
same average droplet size but having a wider size distribution.

As volume of dispersed phase increases, stability decreases
Phase inversion can occur if dispersed phase volume is
increased enough.

Usually, as temperature increases, emulsion stability decreases
because of increased frequency of collision.

Addition of polymer that adsorbs at interface can influence stability.
Polymer chains can prevent coalescence due to bulkiness, but
they can also enhance flocculation and decrease stability.


Physical nature of
the interfacial film



Electrical barrier




Viscosity of the
continuous phase or
of emulsions


Size distribution
of dispersed droplets


Phase volume ratio


Temperature


Steric barrier










Table 1-5. Parameters that affect phase inversion in emulsion and the effect they have.


Effect on phase inversion
W + O + emulsifier & W/O
O + W + emulsifier & O/W

Bancroft's Rule Making the emulsifier more oil soluble tends
to produce a W/O emulsion and vice- versa

Oil/Water Ratio increased in an O/W emulsion +W/O emulsion and
vice-versa, as described above in the text

If surfactant can be dissolved at least partially in either water or oil
Bancroft's Rule If surfactant is dissolved in water+ O/W emulsion

Depends on the surfactant and its temperature dependence.
If emulsion is O/W type with polyoxyethylenated nonionic
surfactant, phase inverts to W/O with increase in temperature due
to increased hydrophobicity of the surfactant.

Strong electrolytes polyvalentt Ca) added to O/W (stabilized by
ionic surfactant) + inversion to a W/O type
Because of decrease in double layer thickness around oil
droplets, droplets coalesce and become the continuous phase.


Parameter


Order of phase addition


Nature of emulsifier


Phase volume ratio

Phase in which
emulsifying agent is
dissolved


Temperature




Addition of electrolytes










Table 1-6. Commonly used physical methods of demulsification


Method


Desription
Basic centrifugation techniques are applied to separate emulsions of either O/W or
W/O type.
Centrifugal methods are advantageous when:
1. Viscosity of continuous phase is not too high
2. Droplets are above a certain minimum size dependent on the viscosity
3. Density difference between continuous and dispersed phase is low

Inexpensive, simple process in which time is the only parameter Only useful for larger
size droplets, and usually used only after one of the other methods has formed large
droplets

Filter with large surface area is used to collect droplets until enough droplets combine to
form a large droplet, which breaks and moves downstream Wetting of the fibers by the
coalescing dispersed phase is desirable Fiber bed thickness is not a factor, as coalescence
occurs on the front face of the fibers only
More efficient than gravitational settling, but not very efficient for small drops


Low intensity of vibration is necessary for coalescence of droplets High
intensity would cause loosely held floccules to separate

Emulsion passes through a granular bed which coalesces droplets May be either
gravity or pressure driven process Common filters include anthracite coal, sand, and
gravel May have a combination of beds with different filter material at each stage

Widely used on large industrial scale due to low cost of electricity Works on both O/W
and W/O type emulsions For O/W, droplets move towards electrode of opposite charge
and coalesce For W/O, electric field induces a charge on the droplets which causes
them to collide and eventually coalesce at the oppositely charged electrode Since O/W
has a high conductivity continuous phase, charge dissipates rapidly but droplets attract
due to rapid travel of charge through the medium For W/O with a low conductivity
continuous phase, droplets hold charge for a long period of time which allows time for
droplets to travel to the electrode In general, W/O type emulsion coalescence is faster
than O/W coalescence

Heating above 70oC will rapidly break most emulsions Coalescence is increased at
higher temperatures due to diffusion of droplets

Freezing of water droplets in a W/O emulsion will cause ice to form, which expands
and breaks the film + oil envelope. Requires repeated freezing and thawing due to
elasticity of oil envelope Generally uneconomical due to apparatus for repeated
freezing and thawing


Centrifugal separation'c







Gravitational settling



Filter coalescence,
Ultrafiltration



Gently shaking or
stirring
Ultrasonic vibrations


Granular bed floatation



Electrically
induced









Heating


Freezing


Table 1-7. A summary of HLB ranges and their application
HLB Range Application
3 to 6 W/O emulsifier
7 to 9 Wetting agent
8 to 18 O/W emulsifier
13 to 15 Detergent
15 to 18 Solubilizer












Nano-emul sions


Stability Thermodynamically Thermodynamically
stable unstable
Droplet size 10 100 nm 20 500 nm

Surfactant Concentration Usually require 10 30 wt% Can be formed with
surfactant 4 8 wt % surfactant
Formation Independent of mixing Depends on mixing
protocol protocol


Table 1-8. Microemulsions vs. Nano-emulsions.
Characteristic Mi croemul si ons









CHAPTER 2
A NOVEL METHOD TO ELUCIDATE THE PRESENCE OF SUB-MICELLAR
AGGREGATES INT SURFACTANT SOLUTIONS

2.1 Introduction

It is generally believed that surfactant molecules in micellar solutions exist in equilibrium

in three states: 1) as surfactant molecules that are adsorbed at the interface, 2) as monomers that

are dispersed in the aqueous phase, and 3) as micellar aggregates.l Little, if any, attention has

been given to sub-micellar aggregates, which may in fact be a significant fourth state of

existence, particularly in micellar solutions with short relaxation times (< 100 msec).

Given that micellar solutions are used in various technological and biological processes,

one must consider the potential impact of the often overlooked sub-micellar aggregates. It has

been shown that the monomeric form of surfactants displays significantly different properties in

solution when compared to micelles, especially when considering the disruption of biomembrane

structures and when using micelles for solubilization of proteinS.109, 110

Midura and co-workersll illustrated how one might manipulate the relative concentrations

of monomeric versus micellar forms of various surfactants. They have shown that the monomeric

concentration can be determined by filtering the surfactant solution through ultracentrifuge tubes

having a nanoporous membrane with a nominal molecular weight cutoff that is smaller than the

combined molecular weight of the surfactants in the micellar aggregate (see schematic diagram

in Figure 2-1). They have also shown that the critical micelle concentration (cmc) of a surfactant

can be determined by means of ultrafiltration. "l However, they did not acknowledge the

contribution of sub-micellar aggregates, which will conceivably play a role in such processes as

biomembrane disruption hemolysiss), solubilization of hydrophobic molecules in aqueous

solutions and in drug delivery via micelles.









When one considers the dynamic nature of micelles, it is conceptually obvious that at any

given time, there must be sub-micellar aggregates present in solution in significantly large

quantities, particularly if micelles break and form rapidly (i.e. relaxation times less than 100

msec). If micelles were infinitely stable, then one would only see the existence of monomers and

micelles in the solution. But if the micelles become more and more unstable, the concentration of

sub-micellar aggregates must increase in the solution. In order to prove this hypothesis, we have

performed ultrafiltration experiments of sodium dodecyl sulfate (SDS) surfactant solutions

followed by analysis of the filtrate by the dye complexation method to determine the

concentration of SDS. We have taken ultracentrifugation tubes having a 10,000 molecular weight

cutoff (i.e. molecules or aggregates that exceed this molecular weight, aggregates greater than 34

molecules, will not pass through the filter), and measured the filtrate concentration for SDS

concentrations ranging from 1 mM to 100 mM. Given that SDS has a molecular weight of

288.34 grams/mole and an aggregation number of~-65 molecules/micelle, micelles definitely

will not pass through the ultracentrifugation membrane filter. If the notion that micelles exist in

only three equilibrium states is true, then one would expect the concentration of SDS in the

filtrate to increase as a function of total SDS concentration up to the critical micelle

concentration (cmc), and then for the SDS concentration in the filtrate to remain constant with

respect to increasing total SDS concentration beyond the cmc. If sub-micellar aggregates are a

significant entity (i.e. a fourth equilibrium state) in the micellar phenomena, one might expect

that as the total concentration of SDS exceeds the cmc, the concentration of SDS in the filtrate

will increase, albeit at a different slope (lesser) than the pre-cmc slope due to the presence of

sub-micellar aggregates that are made up of 34 molecules or less (which may pass through the

10,000 MWCO ultracentrifugation filter membranes).









Here, we present the results of our findings, which support the existence of sub-micellar

aggregates and allude to their significance in technological and biological processes. To take our

hypothesis one step further, we examine the effect of micellar dynamics on sub-micellar

aggregate concentration by stabilizing SDS micelles with dodecanol (C120H) and

dodecyltrimethylammonium bromide (C12TAB), (as shown by their relaxation timeS2), and by

changing the counter-ion of the dodecyl sulfate.

2.2 Experimental Procedure

2.2.1 Materials

Ultrapure sodium dodecyl sulfate (SDS) from MP Biomedicals, Inc. (Solon, OH) was used

as received. The following chemicals were also used without further purification: n-

dodecyltrimethylammonium bromide (C12TAB) from Tokyo Kasei Kogyo Co. (Tokyo, Japan)

and 1-dodecanol (C120H) from Acros Organics (New Jersey). Double distilled, deionized

Millipure water was used for all solutions.

2.2.2 Ultracentrifugation

Centricon YM-3 and YM-10 ultracentrifugation filter tubes, having a 3,000 and a 10,000

molecular weight cutoff (MWCO), respectively, were purchased from Fisher Scientific. Two

milliliters of SDS solutions of various concentrations (ranging from 1 mM to 50 mM) were

placed into the top portion of the ultracentrifugation tubes and subsequently centrifuged at

~2900g (10,000 MWCO) or ~4500g (3,000 MWCO) for approximately 10 minutes so that less

than 10% of the total solution volume was collected as filtrate. All samples were centrifuged in a

bench top IEC Clinical Centrifuge (Damon/IEC Division, Needham Hts, Mass). The filtrate was

collected in the bottom attachment and diluted to the micromolar concentration regime for

analysis by a slightly modified dye complexation method112 and compared to a previously

prepared calibration curve.









2.2.3 Two-Phase Dye Transfer (Methylene Blue Complexation) and UV-Vis Analysis

Methylene blue dye, chloroform, sodium phosphate monobasic, and sulfuric acid were

purchased from Fisher Scientific. Methylene blue reagent was prepared using these materials

according to standard preparation procedure.113 Two milliliters of methylene blue reagent were

added to two milliliters of the diluted filtrate from the ultrafiltration experiments. Two milliliters

of chloroform were added and the solution was shaken on a Vortex mixer for approximately

thirty seconds. Any SDS that was in the fi1trate completed through electrostatic interaction with

the positively charged methylene blue, became oil soluble, and thereby partitioned into the

chloroform organic phase. The solution was allowed to phase separate and the organic phase was

removed and placed into a separate test tube. This process was repeated two more times and the

organic phase was then analyzed by UV-Visible spectrometry at 652 nm and the SDS

concentration was determined upon comparison with a calibration curve. A Hewlett Packard HP

8453 UV-vis spectrometer was used for all UV-Vis analysis.

SDS solutions were also prepared with 1-dodecanol (C120H) or n-dodecyltrimethy-

lammonium bromide (C12TAB) at various SDS:C12X (X= OH or TAB) ratios. These solutions

also underwent ultrafiltration, dye complexation, and subsequent UV-Vis analysis to determine

the SDS concentration in the fi1trate. It must be noted that the dye complexation method was

specific to SDS; therefore the presence of any C12TAB or C120H in the filtrate did not interfere

with the detection of SDS to any significant degree.

2.2.4 Foamability

Ten milliliter samples of 25 mM SDS, 25 mM SDS + 6.25 mM C120H, and 50 mM SDS +

6.25 mM C12TAB were placed into 100-mL graduated cylinders and capped. Each cylinder was

vigorously shaken 10 times by hand and the volume of the foam is recorded immediately after

shaking. Each solution is tested at least five times and the reproducibility is better than & 2 ml.









2.2.5 Fabric Wetting

A commercially gained 50:50 cotton:polyester blend fabric of I in.2 was placed on the

surface of pure water (control) or surfactant solution at 250C. The surfactant solutions that were

used were 50 mM SDS and 50 mM SDS + 12.5 mM C12TAB. The water or surfactant solution

displaces air in the cotton surface by a wetting process and when sufficient air has been

displaced, the fabric starts sinking. The residence time of fabric on the surface of the solution

before it was completely immersed was measured as wetting time in this study. This wetting time

in each solution was measured at least 5 times.

2.2.6 Dynamic Surface Tension

Dynamic surface tension was measured using the maximum bubble pressure technique.

The pressure required to form a new bubble in solution is measured by a pressure transducer, and

the reading is transmitted to an oscilloscope. For these experiments, the dynamic surface tension

was measured for micellar solutions consisting of 50 mM SDS and 50 mM SDS + 12.5 mM

C12TAB. All dynamic surface tension measurements were taken using a 22 gauge needle tip with

a gas flow rate of 3 cm3/min (Which corresponds to 6 to 13 bubbles per second or approximately

77 to 167 msec per bubble residence time at the needle tip). This flow rate was chosen because at

higher low rates the nitrogen gas forms a continuous j et in the surfactant solution at the needle

tip. At lower flow rates, the results are similar to equilibrium surface tension results.

2.3 Results and Discussion

2.3.1 SDS Surfactant Solutions

During the 1970s, Aniansson and coworkers discovered the existence of two (fast and

slow) relaxation processes and developed a model of the kinetic process of micelle formation and

disintegration.21 The first maj or assumption of this model was that the free surfactant monomers

are assumed to be completely dissociated and the size distribution of the aggregates in a









surfactant solution is assumed to follow the behavior that is shown by the solid line, A, in Figure

2, where monomers and micelles are the predominant species. The curve shown by the dashed

line, B, in Figure 2-2 represents a micellar solution where a substantial fraction of surfactant

exists as sub-micellar aggregates in the solution. This representation suggests that the sub-

micellar region (Region II) is larger and that the actual micellar region (Region III) is broader as

compared to the generally accepted model for micellar solutions.

Aniansson's model suggests that the only important contributing species in micellar

solutions are the micelles themselves and monomeric forms of the surfactant as shown

schematically by Figure 2-3A. Over the years many researchers have based their thermodynamic

models of micelles on such a perspectivell4 (i.e. monomers and micelles as two main species in

solution).

We propose here that sub-micellar aggregates are another maj or component of micellar

solutions as shown by Figure 2-3B. If one were to filter either solution from Figure 2-3A or 2-3B

through a nanoporous filter with pore size smaller than the size of micelles, then in the case of

Figure 2-3A, there would only be monomers in the filtrate as reflected by Figure 2-3C; whereas,

in the case of Figure 2-3B, there would be both monomers and sub-micellar aggregates in the

filtrate as reflected by Figure 2-3D. If the surfactant concentration in the filtrate were plotted as

a function of the total surfactant concentration, in the first case, one would expect for the

surfactant concentration in the filtrate to increase proportionally to the total surfactant

concentration up to the cmc, and then the surfactant concentration in the filtrate would remain

the same irrespective of the total surfactant concentration as shown by Figure 2-3E. If the

second case were true, one would expect the same initial behavior (i.e. increase of surfactant

concentration in the filtrate proportional to total surfactant concentration up to the cmc), but









beyond the cmc, the filtrate concentration should continue to increase with a different slope due

to the fact that sub-micellar aggregates (but not the micelles) can pass through the pores (see

Figures 2-3D and 2-3F). In Figure 2-3F, the plot suggests that the monomeric contribution to the

filtrate surfactant concentration is represented by the region from point Q to point P, whereas the

sub-micellar aggregate contribution is represented by the region from point R to point Q.

The concept of sub-micellar aggregates contributing to micelle formation has been

considered previously by Zana39 and by Kahlweit,22, 42 but only for very high (~ 25 times the

cmc) concentrations. It has been well established that micelles have two characteristic relaxation

times associated with them: a fast relaxation time (referred to as zl), which represents the time

that it takes for one surfactant monomer to diffuse into or out of a micelle, and a slow relaxation

time (referred to aS z2), which represents the time that it takes for a single micelle to fully break

down or to fully form. According to Aniansson's model, the slow relaxation time, z2, Should

increase with increasing surfactant concentration.21 However, it has been reported that for some

ionic surfactants, such as SDS, z2 fifSt increases, passes through a maximum, and then decreases

again.38, 39, 41 This behavior in the slow relaxation process of ionic micelles is not predicted in the

Aniansson-Wall model. Kahlweit and coworkers, using their own T -jump and p-jump results,22,

42, 43 COncluded that in ionic surfactant systems at high concentration, the reaction path for the

formation of micelles must be different than that at low concentration. Based on this conclusion,

they came up with a new model for micelle formation (Figure 2-4).

This model is based upon the principle that ionic micelles, including sub-micellar

aggregates, can be considered as charged particles. When ionic surfactant molecules such as SDS

are added to water, the surfactant molecules dissociate into negatively charged dodecyl sulfate

molecules and their positively charged counter-ions. These counter-ions are present in solution









as a cloud surrounding the negatively charged micelle. At low surfactant counter-ion

concentration, the micelles are stable with respect to coagulation due to repulsive electrostatic

forces. Consequently they can grow only by stepwise incorporation of monomers according to

Aniansson's model. As more and more surfactant is added into the system, the counter-ion

concentration also increases, which compresses the electrical double layer around the micelles

and reduces charge repulsion, allowing the micelles to come closer to each other so that

attractive dispersion forces (i.e., Van der Waals forces) lead to a reversible fusion-fission

coagulation according to

Ak Al 7 Al k I = I (2-1)

where k and I are classes of sub-micellar aggregates. Kahlweit42 then represented the micelle

formation reaction path by two parallel resistors, R1 and Rz (Figure 4b), and compared the

formation of micelles to the discharge of a capacitor through two parallel resistors," so that the

change in the monomer concentration with time was given by

dlnA, 1 1
-+ (2-2)
dt r,, r,,

where t21 TeferS to the reaction pathway corresponding to the stepwise formation of micelles by

addition of one monomer at a time, and z22 TeferS to the reaction pathway which corresponds to

the merging of sub-micellar aggregates to form micelles.

At low surfactant concentration, and hence at correspondingly low counter-ion

concentration, Rzis very high due to electrostatic repulsion between sub-micellar aggregates, so

stepwise aggregation dominates and RI determines the rate of micelle formation. As the

surfactant concentration is increased, the counter-ion concentration also increases, and hence, R1

increases as Rz decreases. The concentration where R1 equals Rz is the point where 1 = z2 paSSeS

through a minimum and z2 is highest (for the SDS micelle, this occurs at 200 mM).116 If the









counter-ion concentration is still further increased, R1 becomes so high that R2 determines the

rate of micelle formation according to the reaction mechanism in Equation 2-1). Kahlweit' s

model suggests that the concentration of sub-micellar aggregates in micellar solutions only

becomes significantly large at very high (25 X cmc or above 200 mM) surfactant (and counter-

ion) concentrations. Here, we propose that sub-micellar aggregates are present in relatively large

concentrations even at lower concentrations (3-4 times the cmc). For example, in a 25 mM SDS

solution, the sub-micellar aggregates account for ~ 11-12 mM SDS.

In order to determine if sub-micellar aggregates are indeed a significant component in

micellar equilibrium, SDS solutions were prepared at concentrations below and above the

reported cmc value."' Upon ultrafiltration and analysis of SDS in the fi1trate, we have found that

the SDS concentration in the fi1trate increases nearly proportionally to the total SDS

concentration up to the cmc value. However, beyond the cmc, the SDS concentration does not

remain constant but continues to increase with a decrease in the slope of the curve (Figure 2-5).

Since the SDS concentration in the filtrate does not remain constant beyond the cmc (as

would have been expected if there were no sub-micellar aggregates in the system), we have

concluded that the increase in SDS concentration in the filtrate must be due to the presence of

sub-micellar aggregates that are made up of fewer than 35 SDS molecules. This graph is striking,

as it suggests that in a 50 mM SDS system, given a cmc of ~ 8 mM, more than one-third of the

surfactant molecules (~ 17-18 mM) are in the form of sub-micellar aggregates of less than 35

molecules. This tells us that sub-micellar aggregates represent a significant portion of the

micellar solutions. This is the first conclusive evidence that sub-micellar aggregates represent a

significant portion of micellar solutions and thereby, cannot be ignored. Such a finding has

potential significance in applications related to flux of oil soluble drugs with respect to









controlled drug delivery (sub-micellar aggregates would carry a large percentage of the drug to

the target organs), hemolysis of red blood cells (a system with a large sub-micellar aggregate

population would cause much more hemolysis than a system that has few to no sub-micellar

aggregates), and even possibly in predicting the anti-microbial efficiency of a given solution that

consists of surface active bactericidal agents.

In order to further prove our hypothesis, we decided to make the SDS micelles more stable

by adding C120H or C12TAB in various mole fractions. The Shah research group has previously

shown that one may tailor micellar stability by the addition of long chain alcohols or oppositely

charged surfactants2, 28 (Such as alkyltrimethylalmmonium bromides, commonly referred to as

CnTABs) as shown in Figure 2-42 and that the stability is especially enhanced when the additives

have the same chain length as the SDS.30 The long chain alcohols enhance micellar stability

through charge shielding and the long chain TABs enhance micellar stability through

electrostatic interactions between their positively charged head group and the negatively charged

head group of SDS. We have noticed that the addition of C12TAB tends to better stabilize the

micelles when compared with C120H.

Given this previous knowledge, we began by adding C12TAB in increasing mole fractions

to the SDS system to see if the concentration of SDS in the fi1trate decreases. As shown in Figure

2-7, the filtrate concentration does indeed decrease for C12TAB mole fractions up to 20 mole%

and subsequently levels off. It is important to note that by adding up to 20 mole % C12TAB, the

concentration of SDS in the fi1trate was reduced from 26 mM to ~3 mM. This is an amazing

Ending which suggests that the addition of the C12TAB made the micelles so stable that only 3

mM SDS was free, presumably as SDS monomers, to pass through the fi1ter (i.e. the

SDS+C12TAB micelles are behaving somewhat like rigid spheres).









If this is the case, then if we reproduce Figure 2-5, but with the addition of 20 mole %

C12TAB and C120H for all SDS concentrations, then one would expect that the curve beyond the

cmc should shift towards zero slope, with SDS having the steepest slope, C12TAB having the

near zero slope, and C120H falling in between the two curves. As can be seen by Figure 2-8, the

addition of 20 mole % of C120H and C12TAB did indeed decrease the slope beyond the cmc,

with C12TAB leading to a virtually zero slope line!

This confirms our hypothesis, but in order to completely eliminate any possible doubt, we

decided to perform one more critical experiment. If there are indeed no sub-micellar aggregates

in the 80:20 SDS:C12TAB system, then if these solutions were filtered through a filter that has an

even smaller molecular weight cut-off (MWCO) than 10,000, one would expect that the SDS

concentrations in the filtrate for a given SDS/C12TAB system should be the same irrespective of

the MWCO. We ran the samples in 3,000 MWCO where aggregates with less than 11 SDS

molecules can pass through the pores of the membrane tubes at ~ 4500g for 10 minutes and

plotted the results together with the 10,000 MWCO results and as seen in Figure 9, the SDS

filtrate concentrations in the two different filter sizes were approximately the same in all cases

where C12TAB was added. In the case of 50 mM SDS (with no added C12TAB), as expected,

there is a significant difference between the filtrate concentration from the 3,000 MWCO tubes

versus the 10,000 MWCO tubes. This suggests that in mixed systems of SDS and C12TAB, there

are no aggregates larger than 11 molecules, whereas in pure SDS micellar solutions, there are

aggregates at least up to 34 molecules and possibly having even higher aggregation numbers.

Various other physical properties of SDS systems, including osmotic pressurell6 and

conductivity,120 have been shown to increase proportionally to the total SDS concentration up to

the cmc and subsequently display a change in slope just as seen in our ultrafiltration studies. We









believe that the presence of sub-micellar aggregates contributes significantly to the deviation

from "ideality" that is observed in the osmotic pressure of SDS as reflected in the work of Amos

and coworkers.116 We are also convinced that the rise in conductivity beyond the cmc is partially

due to the increasing concentration of sub-micellar aggregates in addition to the number of

micelles formed.

Micelles have also been used throughout the years as a vehicle for carrying out various

reactionsls and it has been shown that the reaction rates are dependent upon the micellar

stability.ll We believe that the underlying factor here is the contribution of sub-micellar

aggregates, which may very well solubilize some of the reactants and thereby significantly

influence the reaction kinetics. Therefore, this is yet another application of micelles where the

presence of sub-micellar aggregates must be considered when utilizing various surfactant

sy stem s.

Sodium dodecyl sulfate was used as the chosen surfactant for these studies because it is an

extensively studied surfactant.46, 119-121 However, Midurall illustrated that this phenomenon of

increasing fi1trate concentration beyond the cmc holds true for at least two other surfactant

systems: Triton X-100 and Chaps ((3-[(3 -cholamidopropyl)-dimethylammonio]- 1-

propanesulfate). This shows that the presence of sub-micellar aggregates is not limited only to

ionic surfactant, but that they are present in non-ionic surfactant systems as well. Based on these

Endings, care must be taken when modeling surfactant micellar solutions and when designing

micellar solutions for usage in controlled drug delivery, anti-microbial solutions, for

solubilization and denaturing of proteins," and when considering the hemolytic activity of a

given surfactant system. These Eindings also provide great insight into the actual mechanism of









micelle relaxation in surfactant solutions and allows for further correlation to various

technological processes, such as foaming, detergency, fabric wetting, and emulsification.

2.3.2 Effect of Counter-lons on Sub-Micellar Aggregate Concentration

Another interesting aspect of micellar solutions is the effect of counter-ions on the micellar

stability. Pandey et. al.125 have shown how counter-ions affect surface and foaming properties of

dodecyl sulfates. They have illustrated how changing the counter-ion in dodecyl sulfates from

sodium to lithium, cesium or magnesium leads to distinct differences in the corresponding

dynamic surface tension of the solutions. Dynamic surface tension is a measure of the actual

surface tension of the interface at a specific point in time where new liquid/liquid or gas/liquid

interfaces are rapidly being generated in a surfactant solution. Dynamic surface tension directly

reflects the surfactant concentration at the interface at that point in time, and hence the

availability of monomers and sub-micellar aggregates to diffuse to and stabilize the newly

created interfaces such as those created in the generation of foams and emulsions. As such, it is

heavily dependent upon micellar stability in that a more unstable micelle will provide more

monomers and sub-micellar aggregates to diffuse to the interface. Dynamic surface tension can

be measured by the maximum bubble pressure methodl26 and can be represented by using the 6

parameter which normalizes the dynamic surface activity with respect to the equilibrium surface

activity as follows:

6 = yd Teq w eq), (2-3)

where yd is the dynamic surface tension, yeq is the equilibrium surface tension measured by the

Wilhelmy plate method, and 7, is the surface tension of pure water at 250C. The value of 8 = 0

(or yd = eq) indicates that the surfactant adsorption under dynamic condition is the same as that

under equilibrium conditions and the micelles are labile and the monomers are diffusing fast,









whereas 8 = 1 (7d = 7,) indicates no surfactant is present at the interface under the dynamic

conditions existing during the bubbling process implying either the presence of relatively stable

micelles or monomers with high characteristic diffusion time.

Pandey et. al 122 meaSured the dynamic surface tension of four dodecyl sulfate solutions

(having lithium, sodium, cesium, and magnesium as counter-ions) and reported the 6 parameter

as shown in Table 2-1 below. The 6 parameter values are lower and similar for lithium dodecyl

sulfate (LiDS) and sodium dodecyl sulfate (referred to here as NaDS to distinguish the counter-

ion), while they are higher for cesium dodecyl sulfate (CsDS) and magnesium dodecyl sulfate

(Mg(DS)2), Suggesting a higher dynamic surface activity of LiDS and NaDS. This finding

suggests that the LiDS and NaDS have a lower micellar stability, while the CsDS and Mg(DS)2

have a higher micellar stability. Readers may refer to reference 33 for a more detailed

explanation of the factors responsible for the counter-ion effects on micellar stability. Pandey et.

al found that the trends in dynamic surface tension correlated well to the foamability behavior in

these systems as well.

The Shah research group has shown that more stable micelles tend to have low

foamability, but high foam stability.2 The stability of foam depends on how quickly liquid is

drained from the foam lamellae.90 Nikolov and Wasanl23 extensively studied the micellar

structure inside the thin liquid film of the foam lamellae and they showed that the drainage of the

liquid film can be explained by a layer-by-layer thinning of ordered structures of micelles inside

the film. This structured phenomenon is a reflection of the micellar effective volume fraction,

stability, interaction, and polydispersity.

Based on these studies of counter-ion effects on micellar stability, we decided to determine

the relative concentrations of the sub-micellar aggregates in LiDS, NaDS, CsDS, and Mg(DS)2.









We prepared 25 mM solutions of the LiDS, NaDS, and CsDS and a 12.5 mM solution of the

Mg(DS)2. The Mg(DS)2 WAS prepared at half the concentration of the other solutions because it

has two dodecyl sulfate chains in every molecule. After centrifugation in the 10,000 MWCO

filter tubes, we measured the filtrate surfactant concentration by the dye complexation method.

We were pleased to discover that the trends in filtrate concentrations relative to counter-ion

correlated well with the dynamic surface tension values reported by Pandey et al.122 (See Figure

2-10).

Figure 2-10 clearly shows that the filtrate surfactant concentrations are significantly higher

for LiDS and NaDS than they are for CsDS and Mg(DS)2. This behavior gives further credence

to our speculation that the presence of sub-micellar aggregates is directly linked to the micellar

stability of a given surfactant system.

2.3.3 Importance of Sub-Micellar Aggregates in Technological Processes

The Shah research group has shown significant evidence correlating micellar stability to

various technological processes including foamability,124, foam stability,2 emulSion droplet

size,46 fabric wetting,125 and detergency.126 The effect of micellar stability on these processes was

explained on the basis of the micelles ability to break and supply monomers to the bulk that can

adsorb at the interfaces that are created in each application. For example, in the case of

foamability, a less stable (more labile) micelle will break rapidly, giving up its monomers to

stabilize the foam against instantaneous breakdown. However, this explanation has been met

with a bit of skepticism over the years because the timescale of micellar breakdown

(milliseconds) is so much shorter than the timescale of these processes. This argument does have

merit and until now, there was no sufficient explanation for how micellar stability extended to

effect technological applications. We have shown here the intimate relationship between micellar

stability and sub-micellar aggregates. It follows that the operating molecular mechanism in the









dependence of the aforementioned technological processes on micellar stability is directly related

to the flux of not only monomers, but also to a larger extent, to the flux of sub-micellar

aggregates to the newly created interfaces in each of the processes. The more labile the micelle,

the higher is the concentration of sub-micellar aggregates and higher is the flux of the monomers

to the interface.

2.3.3.1 Foaming

A foam is a coarse dispersion of a gas in a liquid with the gas making up most of the phase

volume and with the liquid in thin sheets, lamellaee" between the gas bubbles.127 Foamability,

the degree to which a surfactant solution is able to generate foam, is a relevant property for many

industrial applications, including detergency, food processing, and mineral floatation. One of the

maj or factors that affect foamability is the ability of the stabilizing agent (surfactant in this case)

to adsorb at the newly created air/water interface to prevent immediate breakdown of the foam.63

Therefore, foamability is highly dependent upon the concentration of monomors and sub-

micellar aggregates which can readily provide the monomers to the interface. Figure 2-11 shows

a schematic diagram of the lamellae of a foam.

In 1991 Oh and Shahl24 Showed that foamability is influenced by the average lifetime of a

micelle. We have shown that the sub-micellar aggregate concentration is very high in solutions

of SDS alone, lower in solutions of SDS + C120H, and almost non-existent in solutions of SDS +

C12TAB. After measuring the foamability of each of these systems (25 mM SDS, 25 mM SDS +

6.25 mM C120H, and 25 mM SDS + 6.25 mM C12TAB) we found that the SDS system, which

has the highest concentration of sub-micellar aggregates, also displayed the greatest foamability

as shown in Figure 2-12. The foamability decreases with increasing micellar stability, with the

SDS + C120H micellar solution generating less foam than the SDS solution, and the SDS +

C12TAB micellar solution generating the least amount of foam. Since the SDS + C12TAB









solution had few, if any, sub-micellar aggregates, only free monomers were available to adsorb

at the air/water interface of the foam. The free monomer concentration is very low in this

solution, and as such, the foamability is low.

2.3.3.2 Fabric wetting

Fabric, or textile, wetting is another process where the presence of sub-micellar aggregates

is important. Due to the large surface area of fabrics, equilibrium conditions are rarely attained in

the time allowed for wetting in practical processes.3 As such, the kinetics of surfactant adsorption

at the solid/liquid interface of the fabric is a controlling parameter in fabric wetting. If there is a

high concentration of surfactant that is available to adsorb at the interface, making the fabric

more water-wettable, the wetting time will be short. The hydrophobic tails of the surfactant

molecules adsorb onto the fabric and lower the interfacial tension so that water can penetrate into

the interstitial spaces of the fabric weave. Therefore, one would expect a solution of SDS (50

mM), which has a high sub-micellar aggregate concentration, to have a faster wetting rate than a

solution of 50 mM SDS + 12.5 mM C12TAB. Wetting experiments were performed on these

systems, and as expected the fastest wetting time was found for the SDS solution, as can be seen

in Figure 2-13. The fabric wetting time was also measured in pure water as a control.

2.3.3.3 Dynamic surface tension

Dynamic surface tension is a measure of the ability of surfactant molecules to adsorb at

newly created interfaces under dynamic conditions. Dynamic surface tension can be measured by

determining the maximum bubble pressure when gas is bubbled through a surfactant solution at a

specific flow rate. When there is a significant concentration of monomers and sub-micellar

aggregates, the dynamic surface tension is low because the flux of monomers to the interface is

high. The dynamic surface tension was found to increase with increasing micellar stability so

that a solution of 50 mM SDS exhibited a lower dynamic surface tension than a mixed micellar










system of 50 mM SDS + 12.5 mM C12TAB as shown in Figure 2-14. This is an interesting

finding because the equilibrium surface tension of an SDS solution will always be lower than

that of an SDS/C12TAB mixed solution. Therefore, one must take care when choosing surfactant

solutions for various dynamic processes, because if the micelles are too stable, there will be a

low concentration of sub-micellar aggregates, and the solution will not be able to effectively

lower the surface or interfacial tension.

2.4 Conclusions

Here, we have presented the first conclusive evidence of sub-micellar aggregates as a

significant component in micellar solutions. The following conclusions can be drawn based

upon the results reported here:

1. The generally accepted notion that surfactant solutions consist of only three
compartments, namely, adsorbed film at air/water interface, monomers, and micelles, is
incorrect and we have shown that sub-micellar aggregates are a significant entity in
surfactant solutions making up as much as one-third of the surfactant concentration for
example, as in 50 mM SDS.

2. We have shown here that micellar dynamics are intimately linked to the presence of sub-
micellar aggregates (i.e. the more stable a micelle is, the less is the concentration of sub-
micellar aggregates in the system) and that by stabilizing SDS micelles through the
addition of dodecanol (C120H) or dodecyltrimethylammonium bromide (C12TAB), we
can effectively eliminate sub-micellar aggregates and reduce the monomeric surfactant
concentration to values as low as 3 mM.


3. It is known that counter-ions affect micellar stability. We have shown that counter-ions
also affect the concentration of sub-micellar aggregates in dodecyl sulfate systems (i.e.
counter-ions such as Mg2+ and Cs which enhance micellar stability, were shown to have
lower concentrations of surfactant in the filtrate, whereas, Li and Na which form
relatively unstable micellar systems, have higher concentrations of surfactant in the
filtrate) which correlates well with previously reported dynamic surface tension data.122

4. We have shown that sub-micellar aggregates, or micellar fragments, exist in the micellar
solution even at low concentrations, such as 25 100 mM.


5. We have determined that it is the presence of sub-micellar aggregates that provides the
missing link in our understanding of the effect of micellar stability on technological










processes. We have shown that when there is a high concentration of sub-micellar
aggregates, the foamability is the highest, the fabric wetting rate is the fastest, and the
dynamic surface tension is the lowest.













Sample solution
with large
aggregates


Filtrate large
aggregates are
excluded 4


Porous filter
membrane


Axis of
Rotation


Figure 2-1. Schematic diagram of the ultracentrifugation process. Sample is placed in top portion
of tube and the tube is centrifuged at ~2900g (for 10,000 MWCO ultracentrifuge
tubes) or ~4500g (for 3,000 MWCO ultracentrifuge tubes) for 10 minutes so that less
than 10% of the solution volume is collected as filtrate.


Force = 2900 g













i\\ B


II \
r' /' 20 \
IA \




Aggregation number, n M


Figure 2-2. Size distribution curves of aggregates in a micellar solution. The solid curve
represents a typical size distribution curve of aggregates in a micellar solution
according to the Aniansson-Wall model of stepwise micellar association. Region (I)
corresponds to monomers and oligomers; Region (III) to abundant micelles with a
Gaussian distribution around the mean aggregation number; and Region (II) to the
connecting "wire" (heat transfer analogy) or "tube" (mass transfer analogy) between
Regions (I) and (III). The dashed curve represents our hypothesized size distribution
for a micellar solution containing a significant concentration of sub-micellar
aggregates.










Figure 2-3. Schematic diagrams of surfactant solutions, filtration of solutions, and plot of filtrate
concentration as a function of total surfactant concentration. A) Schematic diagram of
surfactant solution above cmc consisting of three components; adsorbed film of
surfactant molecules, surfactant monomers, and micelles; B) Schematic diagram of
surfactant solution above cmc consisting of four components; adsorbed film of
surfactant molecules, surfactant monomers, sub-micellar aggregates and micelles; C)
Schematic illustration of filtration of solution from Figure 3A where only surfactant
monomers are present in the filtrate and micelles were not able to pass through the
filter; D) Schematic illustration of filtration of solution from Figure 3B where
surfactant monomers and smaller sub-micellar aggregates are present in the filtrate
and micelles and larger sub-micellar aggregates were not able to pass through the
filter; E) Expected results of a plot of surfactant concentration in the filtrate versus
total surfactant concentration for a system like the one shown in Figure 3A. F)
Expected results of a plot of surfactant concentration in the filtrate versus total
surfactant concentration for a system like the one shown in Figure 3B. The monomer
contribution to the filtrate is represented by the region from point Q to point P and the
sub-micellar aggregate contribution to the filtrate surfactant concentration is
represented by the region from point R to point Q.





































































I
I
I


Micelle


Sub -mi cellar
aggregate


Figure 2-3C Figure 2-3D


Surf. cone.
in filtrate


Surf. cone.
in filtrate


cmc


Total [Surf]


cmc


Figure 2-3F


Figure 2-3E





T


I
rr~rrm --


ii X,


Figure 2-4. Schematic representation of the two possible reaction paths for the formation of
micelles (a) and the corresponding resistances (b): (1) formation by incorporation of
monomers and (2) formation by reverse coagulation of sub-micellar aggregates.


Y, I






















Conc. SDS in
filtrate (mM)


0 10 20 30 40 50
total [SDS] (mM)


Figure 2-5. Filtration of SDS through 10,000 MWCO ultracentrifuge tubes for ~10 minutes at
2900*g. The point at which the slope changes is considered the critical micelle
concentration (cmc). The dotted line is the result that one would have expected if
there were no sub-micellar aggregates present in the system.


25 mM SDS, T, = 1 ms.



25 mnM SD)S + 1.15 mM CzOH, ty = 230 ms.



~IB 25 mM S)S + 10 mM3 C2TAB, r2 = 2000 ms


Figure 2-6. Tailoring of micellar stability by the addition of 1-dodecanol (C 120H) or n-
dodecyltrimethylammonium bromide (C12TAB).


















25


20





E 15



Total [SDS] = 50 mMI


0 5 10 15 20 25 30
mole % C12TAB in soln

Figure 2-7. Filtrate of SDS+C12TAB through 10,000 MWCO ultracentrifuge tubes for ~10
minutes at 2900g. The C12TAB mole fraction was increased from 5 mole% to 25
mole%. The total SDS concentration is fixed at 50 mM.
















35 '" "

g 30

B 25 (



S15

S10




0 20 40 60 80 100 120
total [SDS] (mM)
OSDS alone 480:20 SDS:C12TAB mixed micelles X 80:20 SDS:C120H

Figure 2-8. Filtration of SDS alone or SDS + C12X (X = OH or TAB) through 10,000 MWCO
ultracentrifuge tubes for ~10 minutes at 2900g. The C120H and C12TAB were added
at a molar ratio of 80:20 SDS:C12X (i.e. the concentration of C120H or C12TAB in
each system is 20 mole % of the total surfactant concentration (SDS+C12X)).














30


25


S20





15






7.5mMSDS/1.875mM 10mMSDS/2.5mM 25mMSDS/6.25 mM 50mMSDS/12.5mM 50mMSDS
C12TAB C12TAB C12TAB C12TAB

H3000 MWCO 10,000 MWCO

Figure 2-9. SDS concentration in the filtrate for 80:20 SDS:C12TAB systems after filtration
through 3,000 and 10,000 MWCO tubes, as compared to pure SDS solutions (50
mM). Samples in 3,000 MWCO ultracentrifuge tubes were centrifuged at ~ 4500g
and samples in 10,000 MWCO ultracentrifuge tubes were centrifuged at ~ 2900g for
10 minutes.












Total Concentration = 25 mM for all samples except
Mg(DS)2, which has a total concentration = 12.5 mM


18

16

S14

o 12

S10
o
-


L.


LiDS


NaDS


CsDS


Mg(DS)2


Figure 2-10. Filtrate surfactant concentrations for 25 mM lithium dodecyl sulfate (LiDS), sodium
dodecyl sulfate (NaDS), and cesium dodecyl sulfate (CsDS) and 12.5 mM
magnesium dodecyl sulfate (Mg(DS)2).























Figure 2-11. Schematic depiction of foam column generated by passing air through a surfactant
solution (left) and magnified view of foam lamella, thin sheet of surfactant solution
between adjacent air bubbles (right).


100
90
80
J 70
60
50
E 40

OA


solueion


0
25 mM SDS

High conc. sub-micellar aggs.
High foamability


25mM SDS/6.25mM
C120H


25mM SDS/6.25mM
C12TAB
Low conc. sub-micellar aggs.
Low foamability


Figure 2-12. Foamability of SDS micellar solution and SDS + C12X mixed micellar solutions (X
= OH or TAB)


Foam Lamella




Monomers and
Ssub-micellar
aggregate s











I


Low conc. sub-micellar aggs.
High Fabric wetting time



High conc. sub-micellar aggs.
Low Fabric wetting time


S60






L.
10


water


50 mM SDS

Solution


50 mM SDS/12.5
C12TAB


Figure 2-13. Wetting time of lin2 Strips of 50:50 cotton:polyester blend fabric in pure water, 50
mM SDS, and 50 mM SDS + 12.5 mM C12TAB.










50
22 gauge needle
45 _C6 13 bubbles/sec
Air flow rate = 3 cc/min
S40

35

S30

e 25

a 20



15




50 mMI SDS 50 mMI SDS + 12.5 mMI C12TAB
Surfactant Solution


Figure 2-14. Dynamic surface tension of solutions of 50 mM SDS and 50 mM SDS + 12.5 mM
C12TAB.












Table 2-1. Dimensionless dynamic surface tension (6) of different counter-ions of dodecyl
sulfates (50 mM) at a bubble lifetime of 50 msec (from ref 33).
Ion 6 parameter

Li' 0.138

Na' 0.131

Cs' 0.202

Mg" 0.353









CHAPTER 3
DETERMINATION OF DRUG AND FATTY ACID BINDING CAPACITY TO PLURONIC
F l27 INT MICROEMULSIONS FOR DETOXIFICATION

3.1 Introduction

Drug overdose incidences are a common and problematic occurrence both nationally and

globally. Many life-threatening drugs do not have specific pharmacological antidotes to reverse

the toxic effects that result when an overdose occurs.99 Attempts are currently underway to

develop procedures to detoxify blood in a timely and efficient manner.128-130 Therefore, the

development of an effective methodology for the removal of free drug from the blood of an

overdosed patient in a timely manner (less than 15 minutes) is critically important. In the past

few years, efforts have been underway to use nanoparticulate systems to accomplish this task.

Microemulsions are one of the systems that are currently under investigation. Upon injection of a

biocompatible, nontoxic microemulsion in the blood of an overdosed person, the microemulsion,

having extremely high interfacial area, can effectively adsorb and solubilize drug molecules, and

thereby quickly decrease the concentration of free drug molecules in the blood. However, in

order to fully grasp their function as toxicity reversal agents, one must understand the molecular

mechanism of drug uptake and be able to determine and manipulate the contributing interfacial

forces.

Preliminary results from pH studies have led us to believe that electrostatic forces can play

a significant role in adsorption of drug onto the microemulsion. Amitriptyline Hydrochloride,

shown in Figure 3-1, is an antidepressant and as of yet, there is no efficient method to reverse the

effects of an overdose in a patient; therefore it is the target drug for the experiments reported

here. Amitriptyline has a pKa of approximately 9.4 so that at physiological pH (~ 7.4), it will be

positively charged and can thereby interact through electrostatics with a negatively charged

microemulsion. These microemulsions are composed of Pluronic F l27, Ethyl Butyrate, and









Sodium Caprylate fatty acid (which gives the negative charge) and are prepared in Phosphate

Buffered Saline at pH 7.4. The obj ective of this study is to develop a better understanding of the

important interactions that occur between the microemulsion and the drug.

We have shown, through turbidity analysis experiments, that there is a linear relationship

between the Amitriptyline Hydrochloride solubilization capacity (i.e. the amount of

Amitriptyline that the microemulsion can accommodate before turbidity occurs) of the

microemulsions and Pluronic surfactant concentration up to a certain Pluronic Fl27

concentration. Above that critical Pluronic F l27 concentration, further titration with

Amitriptyline never yields turbidity. We have also seen that turbidity is not observed in systems

that do not have sodium caprylate present. Based on these findings we have concluded that at the

critical Pluronic concentration, there is no longer any free unassociatedd) sodium caprylate

molecules in the bulk phase, presumably due to binding of all fatty acid molecules with Pluronic

molecules. Therefore, we are able to determine how many molecules of sodium caprylate and

Amitriptyline are associated with each Pluronic molecule. Each Pluronic F l27 molecule can

associate with approximately eleven molecules of sodium caprylate and twelve molecules of

Amitriptyline at the critical concentration (i.e. there appears to be a nearly 1:1 association of

sodium caprylate to Amitriptyline). This yields further credence to ultrafiltration studies that we

have done as a function of pH which show that electrostatic interactions are important in

Amitriptyline binding to microemulsions produced by Pluronic F l27 and fatty acid soap. The

findings of this study will provide substantial information regarding the mechanism of reduction

of overdosed drugs and will allow us to approximate the uptake capacity of a particular

microemulsion system.









3.2 Experimental Procedure

3.2.1 Materials. Pluronic surfactants were obtained from BASF Inc. (Mount Olive, NJ).

Pluronic was used as a nonionic surfactant composed of a symmetric triblock copolymer of

propylene oxide (PO) and ethylene oxide (EO). The polypropylene oxide block was sandwiched

between the more hydrophilic poly(ethylene oxide) blocks. The block copolymer was denoted by

(EO)x(PO),(EO)x, where x and y are the number of units of EO and PO, respectively.

Amitriptyline Hydrochloride, sodium caprylate, sodium decanoate, and sodium dodecanoate

were purchased from the Sigma Chemical Co. (St. Louis, MO). Ethyl butyrate was purchased

from ACROS Organics (New Jersey). Sodium phosphate monobasic, sodium phosphate dibasic,

sodium chloride, and potassium chloride which were used to prepare the phosphate buffered

saline were purchased from Fisher Scientific Inc. (Suwanee, GA). Double distilled, deionized

Millipure water was used for all solutions.

3.2.2 Microemulsion Preparation. Oil-in-water microemulsions were prepared by first

solubilizing the appropriate concentration (3 9 mM) of Pluronic Fl27 surfactant in phosphate

buffered saline at pH 7.4 (physiological pH). Sodium caprylate (fatty acid surfactant) was then

added to this Pluronic solution in concentrations ranging from 25 100 mM. Lastly, ethyl

butyrate (oil) was added to the solution and the system was stirred until it became clear. The

ethyl butyrate concentration was fixed at 110 mM for all experiments in which microemulsions

were used. The microemulsions were subsequently allowed to equilibrate for at least one day

pnior to use.

3.2.3 Turbidity Analysis. Micelles, mixed micelles and microemulsions were prepared

with varying compositions of Pluronic Fl27, and/or Sodium Caprylate, and/or Ethyl butyrate.

The aqueous phase was phosphate buffered saline (PBS) with a pH ~ 7.4. Ten milliliters of the

micelle or microemulsion sample was placed into a vial. The solution was titrated with 0.2 M









Amitriptyline (prepared in PB S) until the onset of turbidity was observed visually. The systems

were sensitive enough that the transition from clear to turbid was very sharp (i.e. occurring over

a change in volume of 50 microliters or less). In some systems, prior to the system reaching

turbidity, upon each incremental addition of Amitriptyline, the solutions would exhibit a

momentary cloudiness, but gently swirling would lead to a return in clarity. During titration, if

the initial cloudiness was not observed upon the additions of Amitriptyline, copious amounts of

drug was added to that system; if turbidity was not observed, then the system was categorized as

one where turbidity would never occur.

3.2.4 Dynamic Surface Tension. Dynamic surface tension was measured using the

maximum bubble pressure technique. The pressure required to form a new bubble in solution is

measured by a pressure transducer, and the reading is transmitted to an oscilloscope. For these

experiments, the dynamic surface tension was measured for microemulsions consisting of fixed

sodium caprylate (100 mM) and ethyl butyrate (110 mM) concentrations and increasing

concentrations of Pluronic F l27. All dynamic surface tension measurements were taken using an

18 gauge needle tip with a gas flow rate of 5 cm3/min (Which corresponds to 3 to 10 bubbles per

second or approximately 100 to 333 msec per bubble residence time at the needle tip). We chose

this flow rate because at higher low rates the nitrogen gas forms a continuous jet in the surfactant

solution at the needle tip. At lower flow rates, the results are similar to equilibrium surface

tension results.

3.2.5 Foamability. Twenty milliliter samples of microemulsions consisting of fixed

sodium caprylate (100 mM) and ethyl butyrate (110 mM) concentrations and increasing

concentrations of Pluronic F l27 were placed into 100-mL graduated cylinders and capped. Each

cylinder was vigorously shaken 10 times by hand and the volume of the foam is recorded










immediately after shaking. Each solution is tested at least three times and the reproducibility is

better than + 2 ml.

3.2.6 Fabric Wetting. A commercially gained cotton fabric of 1 in.2 was placed on the

surface of microemulsion solution at 250C. The microemulsions used consisted of fixed sodium

caprylate (100 mM) and ethyl butyrate (110 mM) concentrations and increasing concentrations

of Pluronic F l27. The surfactant solution displaces air in the cotton surface by a wetting process

and when sufficient air has been displaced, the cotton starts sinking. The residence time of cotton

fabric on the surface of the solution before it was completely immersed was measured as wetting

time in this study. This wetting time in each microemulsion solution was measured at least 3

times.

3.2.7 Surface Tension. Surface tension measurements were carried out to determine the

critical micelle concentration (cmc) using the Wilhelmy plate method. In this method, the plate is

lowered into surfactant solutions of known concentrations and the corresponding output from a

gram-force sensor holding the plate is sent to a transducer and then to a voltage readout. The

system was calibrated using two known solutions (water at 72.5 mN/m and acetone at 23 mN/m).

The platinum plate was heated between each reading to clean off anything that may have

adsorbed onto the plate.

3.3 Results and Discussion

3.3.1 Effect of Sodium Caprylate Concentration on Drug and Fatty Acid Binding to
Microemulsions

As previously reported,99 we have taken a systematic approach to design a biocompatible

microemulsion system that would effectively reduce the free concentration of target drugs in the

blood. This microemulsion system is composed of Pluronic Fl27 as the surfactant, sodium

caprylate (SC) fatty acid as the co-surfactant, ethyl butyrate (EB) as the oil phase and is prepared









in a phosphate buffered saline solution at pH 7.4. Given that Pluronic Fl27 is a block copolymer

and sodium caprylate is a co-surfactant, if we can understand the nature of the polymer-

surfactant interactions in this microemulsion, then we can have a better understanding of the

structure of the microemulsion and the molecular mechanism of uptake of the drug. For many

years now, polymer-surfactant interactions have been studied extensively in relation to various

interfacial processes.84, 131-137 One of the methods of analyzing polymer-surfactant interactions is

through titration studies.54, 68 Here, we take various microemulsion compositions and titrate them

with concentrated Amitriptyline solutions to turbidity. We are using the results of these studies to

determine the pertinent stoichiometric ratios in our optimal microemulsion formulations.

For our initial titration studies we took the various microemulsion components and titrated

them individually. So our first titrations were of sodium caprylate (SC) solutions in phosphate

buffered saline (PBS) (pH 7.4). In this study, we found that at 100 mM SC, turbidity occurred

when 1 molecule of AMT was added for every 100 molecules of SC.

Next, we titrated systems containing only Pluronic Fl27 in PBS (pH 7.4) with 0.2 M

AMT. In these systems we found that turbidity was never obtained, irrespective of how much

AMT was added to the system. Then we added the ethyl butyrate oil to the Pluronic Fl27

systems and titrated these solutions with 0.2 M AMT. Once again, turbidity was never obtained.

Finally, we added our last component, the sodium caprylate fatty acid, to the system and found

that upon titration with 0.2 M AMT, turbidity was seen in these systems. For these systems, the

sodium caprylate concentration was held fixed at 100 mM, the ethyl butyrate concentration was

held fixed at 110 mM, and the Pluronic F l27 concentration was varied from 3 mM to 9 mM. One

of the interesting observations that we noticed in these titrations was that turbidity was achieved










for every Pluronic F l27 concentration up to 8 mM. Above 8 mM F l27, turbidity was never

achieved (see Figure 3-2).

Our next experiment involved titration of Pluronic F l27 and sodium caprylate mixed

micellar systems (i.e. no oil is present). In this case, the sodium caprylate concentration was held

fixed at 100 mM and the F l27 concentration was varied from 1 mM to 9 mM. We were

somewhat surprised to see that the lack of oil in these systems did not seem to affect the amount

of AMT needed to induce turbidity (i.e. the graph for the mixed micellar system is nearly the

same as that of the microemulsion system (see Figure 3-3).

These experiments provided us with two important insights. First, turbidity is only

observed in the systems where the sodium caprylate is present. Based on this finding, we can

conclude that the turbidity is arising from AMT forming a complex with the SC. Secondly, in the

systems where Pluronic Fl27 is present with SC, turbidity is observed up to some critical Fl27

concentration, above which turbidity is no longer observed. Based on this finding, we can

conclude that the critical F l27 concentration is the concentration at which no more SC exists as

free monomers in the bulk solution and that the turbidity is a result of the AMT completing with

the free SC in the bulk. Figure 3-4 provides a schematic illustration of this hypothesis.

In order to test our hypothesis we did the turbidity experiments for various sodium

caprylate concentrations. If our hypothesis is correct, we would expect for the critical Fl27

concentration to decrease proportionally to the decrease in SC concentration. As can be seen in

Figure 3-5, the decrease in the critical concentration of F 27 is indeed nearly proportional to the

decrease in the SC concentration. The critical Fl27 concentration is never reached in the system

where the SC concentration is 125 mM because above a F 27 concentration of 9 mM, the

solution becomes a gel.