Citation
A study of physical and chemical properties of the fats from the fat lobes of the moccasin (Agkistrodon piscivorus)(Lacepede)

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Title:
A study of physical and chemical properties of the fats from the fat lobes of the moccasin (Agkistrodon piscivorus)(Lacepede)
Creator:
McLaughlin, Joseph, 1915-
Publication Date:
Language:
English
Physical Description:
72 leaves. : ; 28 cm.

Subjects

Subjects / Keywords:
Absorption spectra ( jstor )
Distillation ( jstor )
Esters ( jstor )
Fats ( jstor )
Fatty acids ( jstor )
Iodine ( jstor )
Liquids ( jstor )
Nonesterified fatty acids ( jstor )
Snakes ( jstor )
Unsaturated fatty acids ( jstor )
Chemistry thesis Ph. D
Dissertations, Academic -- Chemistry -- UF
Fatty acids ( lcsh )
Oils and fats ( lcsh )
Snakes ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis -- University of Florida.
Bibliography:
Bibliography: leaves 66-71.
General Note:
Manuscript copy.
General Note:
Vita.

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University of Florida
Holding Location:
University of Florida
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This item is presumed in the public domain according to the terms of the Retrospective Dissertation Scanning (RDS) policy, which may be viewed at http://ufdc.ufl.edu/AA00007596/00001. The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator (ufdissertations@uflib.ufl.edu) with any additional information they can provide.
Resource Identifier:
022564559 ( ALEPH )
13856227 ( OCLC )

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A Study of Physical and Chemical Properties of the

Fats from the Fat Lobes of the Moccasin

(Agkistrodon Piscivorus) (Lacepede)










By
JOSEPH McLAUGHLIN, JR.









A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY









UNIVERSITY OF FLORIDA JULY, 1949












TAM C CNI NT


PZWAM

2VWmmcnJ x'K, Pzr OF ijTVI M&A ..... ... .............
Itatiemt of Resarch Probl 1

Prparation of Moccs51R Snake 01.

Methods of Sepaao of the Pattr Acids 2

opalitptivo Inmstigtio of Ca nn 1W~ MUO

Premparation of Mixed 1att AciAI from a rat

benval of Usapaniablle Matte fr the ixed

Yatty MAc 6

Ram~wal of the law N.1.culat Wel tIsg MA*UL6

Vou t Absorptt of Mixed Yatty Maif7

banzlvamt Cryta~liation

Slvat 0WIllis.Ua1 7

Sepwation of PooyetewA Acis by Lhvm Stts S

Sepazaton of Mixed Patty MAcis bw ftypalutia

frm Slvwts at Jaw Tmperatmr,



s Distllation of Hie Patty Amid UstOr7

at, low Pmemuse3

Sam Psatures, of the Oe2.1ation of the Oopusitleft of

lmudvina Natkw 7ractio 17

Details of the Method~ ot Omloulation ?bvolve is Iste-atius of the More Saturated or SoIl" Acis 20 4-








-.U-


Pap

lbatew .tions of the )Wwe Thsat=&tl or ,Lqu4'









Me IC Amorpton Spetra2

fte Idatiflatio of the Wtty MaW 29





Ta 1 of l sb1 32

StaI=6ul Polybwosmi&. from Mkocain 1st 33 VSUtUation of Xat)~1 Etter bttose from 3*asis ht 34 Tabe U 36 Ublb 111 Table IT 36



Table VII4 Table VW U 40r.o 1 ;m Ldo sta *a& its Uvaluit FPWmtt of the AlksUm #1?o.1 S.t~s Ii Table Ix


11041 50 Usuess of 1gw. 1 51 BRte~m13at1= of lim1e1,s Limente, ma Aaehlhanio 52

MIUe in Mocasin SW*k Oil by Spetwophotmetrl.,
Amalyvi#









-lit.


PWq

62


Tau*e II de nab:e :11 6 %ble Iy 61






S...**.......**o~e~e*eteee .ee .#9e 6

SOGSI1M ~ 7
















PWA


The i .jeati oa.asW in this teserta-tio is pre.eted it f aoawt similar to that nod f Lo the artici. Inee ftis va fou to be thae most sash or f pw 1iStmlatit of the soveiel part. of the ftvetgt. The manrof listing mfrufmoes ts the os~qj one for teohnical wok ini orpsid eemuitr of eouepm *lo logh an seVe* Joural abbu'v'iai 09 t ofici ofe

Us conformity with proet u&s In rosear*L pabim~ti all laeu" am an the Canters %maue
























AD

3 �lf r TN3













SNoks .11 as used in this dIss. Ue *** Ve.s to the g3yeet# btab A flo the fat IO-s ot the maseI. f!is vwmat project inv.1v's a *tW of the ahal "d physifal Poperties of sm il POboi I oe.psig the fat lbes f issa.o. l (A1e.t aow Puarsohn (espoa). f. *f vtivo to a qwu3.IvtI =Al Oiti-. itve ammIIS of the fatty asid preet Is moo t msml oUol.

Pow a #807 of the work one ex ma *t oils ~ to this to soe data 1e" oou w~In, Sa P.IaA a tat of U asall ge





ft orftial IavOSUtiOlo s this Ub.awtowy, whish wwo* p41 foVMus V~o poIO sales of ,ew~fl -. 11AIII 110, 1&emt* t"a host~WOLSIC PPwOOMOS sam ObAms In the fs"W eelow lially Proo".
0.14-WOesiagof the fat l*o of on - vA-~ spGies 4vM" as =s imit Aterua for investigation. On the bulS of the rwicum rwseasb

IW~~s. P~VU1,VS amd POl. old.pwssed macmoin smab oil I"e --e - to be M" e sir'able forwuo In tbls Igssttwgt1.a of fatty


The fat lobes of tba s"at"e A~stw. pisaivoa (i.op&) 00O4*Y '' asu the dtmmutih Nb..141. ve Suplie 1w ** bus All0II of the Ross Allen Reptleo 2Ina or 811',. 4pwtgs, flwid.s.


-a.


STATXMMT 07 "SFIAMM PROX=












WIPa reavw3l of the lob.. from the *a* the 'boo sa dals

off, axM the fet IOU. were dried on filter paper. fhe lobes (1a 25pe quatte) vare vwppol vith ievaoea In filter cloth to that the prwveu ua. vm44 not fox". 1awa aumts of soids into the oil. Vw 1U1d Pressed mut ont4 stae ol, hem2yso %l.04, blod oells, ut~w, and som. un e tat sell.

A oetrw oqvippA vi1h two 250 *o. ctwt~ ftbos e* ope.stel at 14QO rwpm for tva on"w. Rash ra pvot l abot 350 amof puzqfiel snake 01l.


07 O SR1**ION O7 SM 7*!! ACIDS

Am 4q kNOW140 Of the fatty "I"to the l~i~o

OW 200 of Oaty aid mwwah. So sagle fator has contri5ttI W"~ tOWSm4 VhiS bw d te the eWW twestigatie. vhich hav bss

0140 A*W eqamftea of the fatty* esid. It is softe to oae

th" MWO aftesat 11 O this ft"I vill be intimtely assoclatst with

t~ wslamt M" off *$fet.* itbds, for tas seertim of tbess


0* mm preetsss vhiah have been Iwsetigat tor fatty aelt

.Pmatn eh S O"'Oyatai$on of the "eids or their~ deivivesw. distilUtioa of the eAd&$ ow their eaters, s"& other meW em 10 YONAM7 LUVIM& into two SweuVa. ft& fist of these LIudes these P"002000s whish afe vz~ftl for analytieal or identiftestion pwos only and Ad4*#. %ooms. the are time-ossoudng or costly, hwe *017











little or no eo ig application. Ths .o& g .mtsto of those proesss *oh are dptal. to the a . pewati of the a.is

I rW $glatia of the method for fatty sei& a

to wt psible, rosy of the c proces are also e4. lIMOUn t *3 pa s xxB*ftx*rsa mW oou,,t -m of tbe mWy ustetr a ial mh in t omo ially feasitle pof.em.

Amn theh predu, whih ha ges td fo the se of e fatt ais aredistiling, ystali A c uging, too. @~h with hoxab& ons of thee mthods. Usixgle proewe to ti13 sat aer.ty fo Obtaea pr* faty a oif. A omineation of two erUw pocues better remtl . this Is partiularly true In the laboratoqjaeile spparation of fatty .iadd sixtu~, since a m bet of erytailstions a" often combined with seerl fftaetjo*l &1tIlU. late s. @evull the me.l esters of the sais are oedlos I& , U dist1llattea proeba , The choles of the metbn& of sepasil.. E~spe" w~f the stattla. at$.s wra.iab1e "~ vpo the objeetiv to be attala. Ld Un the fonlle, dIis.ien It appears legisi tro eonsider eom af the va~ou =it pwoaseas sepsateWy'. It should& be reslised, hoey , that tbey ca frequently be arata...uhly combined eM tht the vauis aethes difer gveatl with rega d to their -prtica, l~rt ,.
SIc this "s.erh is bes vp analytical szanatises ofa

ntal fat, it sems desirable to ImlvA6 a di sen of the ari. rantm methods hs s ftr foal &eW tme in the quantitativs Of m8W1i.Mtttl #tM* of the zatWal fates. A fairly Nil asosunt







Ii
I.
II!


'1
iiii

t
~Ii 11


'

jt
'I


I
I j I
ID
I
I


i Il
If'


ii











mi.mor c in the solubilities of the edA salts of saturate and wuat4. fatty acids ham* beenu tliue& for spstato. This separation is simplified if aid* of low 34leaaU1 wS~t ar s t. aotioal ar alslimstls from appIat* solv*s at low te1m t o also be emo for separ mim sa.

When fats contain apgeiabe ouats of acids of ow mlular weit then *emio fo(apxfy4), It is n to remove the more wolatile said of low molecula weight before pswe.d4i to separate the higher satuwated and umsatuwated aides. This only applie to "ial tnes of fats uich as Milk fats an Porpoise oils.


P2WARA1TI (W TEE 41MIE IPA= AO!D MM3 A PAT


The quantity of fatty "sids reqlx*4 for an aemwt wvi1sts

8aireMs VPa the amlaSity of tbo *Lxtmat oqxe acUde. Th. ant, rans f w 20 gr for a simple type o fat to 500 g fo a comlex

taeof fat muftan flok alie.

Complete by4"I"Is of the oriw a fat is essmtial. T saonif 100 PSO IV VWi of fat, &M4 30 13sxts by weight of potassim Wroxido ia sboat 5w pat. of saso (9500 Pew 840t). Vet for "e o s# . and tbea wesve most of tba lcoho by Uistillatis.a te, saspe&e 41sstlv4dJ ater, ax m ap~fUl matter is mmve at We~ st, It U60 .s-. Th fre fatty side ere fomd by add$* war*,, d12.te hydrochloric acid. If Vissaturated "s, ae pwsmt In qmnit, ..ae meet be ta to pzwomt oxtUtioa. e the aals hew been lbewat4











thq axe extracted with etber and are dr&mao vm &t loof.
Woen higb3r unsaiuto& "Ids a" pwm*t In qusatity, it s W 6e4irablS to uIse too uch s1lcal otw to pweloug the saponf aties. The h1i1cuustta fatty side 008117 UJ S som J utuASSl4 IS the pressing of a ss alkali ma ).eag1O eAsting. Thrfs vith
suhadA. it to &.s*vsxe to ae very little alko Iis lam of VO MW auntbmA* retlodred.iw and to heat =dr wet~ awa as lef as Is recdl.




iM~ TI XL%= SAM AGTM

met# It mot all* of the usp~ib atvpomti

toodo to go" with the .tbw.4*l 1455s isalt "at t 0 e atmxtea OIVU 'l1S.1 fmls. If the tmil of =smfa3.MWes In the ftIs lee. the 0? peW em.t romvs is not nuse". ft tho mos oft snakeail*1 the uneqsfiabo wfsS e n-ot waswl Sim the

tm' .1 VMS .a7 0." Per *a&.


RMVA.L Of TIM UN MI WZRIO 1*YT AOIW

If apreabe attte of lower s'ena" weism fstty metu a"e present. they saul& be uimov51 before the h1l.W fatty ad". a"

~pewae&. ne ute Of WemIOWIa the lower umlleulaa' weS*It fttr sOles it Iq distillation In a Va'2t Sf stemo.


.4.











The amut of low mlealaa weight fatir aide n 1A Mea t "

*U is found to be vex7 sliht (0.13 per cant); the"f.-r, it is not n tessa t mwe the low mlecula weight fatly &G .q


MMMOGR O A M OFW == FAM ACfl


fa this pzwblm t use ft chrostswgae kpsar" to offr me aesats.e oe se5tan other proeees.



WOM-OLTMOTSALLIZAYOW

te ms-solvent pewatioa of the fatty acids dpSo vo th faet that tbhq -- o3 y miseleble ia the liUA tat,. After the solutions a" coled, the higher aeltiag acid crystallize, arM tby em be separated fwou the liquidI acid* IV suitable mamm rns, such as psin.
'N copete iwstttel ses-oolvent orystallisatio leSie hs slume fficient .l oil an eipsat for lowvq3t atuve wrk wr not waildale* It Is problem that nons-olveint crystallizatlam woul& pote a SNA se aration betwfa the nvtm amd poythaiA "ids present in snSO oil.


SOLVM OMY$TAUJZO3

fe of the boest prelimsls methods of separating the va us asid is IV t*e use of thUs differing solulities of the lead slts of the


-7t.










ft* sois la eth.,. Per a, oompete &widption of the toomiqu U9.8 Is the 104l salt-otbow mete No the pods~u etvm IW the A.O.A.e.P ON 60 Vo At moolwn ad Pouv.



WAUA3TO W OLMMO C ACMS Tr =in SAXt
Ooasideble voik hau hem rwopwto in tbe llteratoo m _. th soPawati et f pelthM aeltd tq the u. of lIth sat*. ad* solve t Is use is the aesoo-wato sOhti. IM a &A",I .swo at is Pwo~ see I1unt",bltn.




SWIWID 0M XU 4W 1AT ACIDS BrCITLIn


low a ~1ou of tSIheil11-1 - a q s ad dis~vantaoe of this mntb sos Xllitd1. Jditatift*s of material aMA .qaumt prelvdd vse, of tUs moth" la this stmf. sops"tim fat ty mot ,y lowm.W.msr. wilt w tmt solvents', is ait* satweatuy
*m IMP MASe of fa us iaIAble. Its WO whent the qusztity of fat to qodte sMMl is not a a=to.*o .




fth fr eab AistIl3atia ef fatty seol esters has em *Ao, sObR s"Ised - a nmea of sepmitiou of WMa at I . "a is. the fwae "iatie Of tlhe mZ l esters is fruently UNA for the laber, tOW preParatio of the IMdirifaml *e*W. fte ast iaptmt er]wl~w st4











eonornedwithi the fractlioaal distillation of fmtty acids and their*

eaters are those reports&IW rafft and co-orbers,, whow inweetiated the 'boiin poin* of the acids -nd their estAe under high va.

Caltwl and rtt e port the boiling points of laie, myristie, pasInti. stearle, anc olelo acids in a oathode W using a O*Ws VwV with a Croe tU between the receive arA r , ad obtai m vtalus materialy lver than those priousl reported. Thes mtbors, state that at a ver lov prevwr a liqid has bellUK peint; it solms or evworats. Just s ater des In air, at a rate a t dspe nt upon the naturs of the sbstme. e4xrell am brtlq e1so decribe a method for fmtionation of the fatty aci of Si ttov and eo.t oil.

Brna1 "ascribe asm paao for the distillation of the fatty Aide at low peass. Us separation of paMtia and stwoaiid va = eistillattUe was reported 317 esti fs 4 aM 1y tres pe afr.w,

TA 19 6 the eratiaa of the fatty acit of eeot 41 an& o aMo l4v", OUl V y uses of their uet~l ester was first reported. the fttyi addM Of latter fat were separate& %V the frattme 6JistillatIeS of their methlq' esters, and )sdan20 21, 22# 7a 241 2S# 26 ots the SePeatiVA of the fatty Adds of both .oowmt ad paa bWM $3 byr a 0siMla Method. The fractional disti11atie* of metb2l es M was also reported for the separation f fatty acids of cottonsa o.i2 ro, ntoa heru o , On *11k29, .110.

-4-











and~ pst an 4 UO "il"vs the fwatienel 8Istinattg of the mothyl este? of O~e ot oil f vty &Si, aA Amt ng, A3lma, s Mo.eeP*t the eVtrftUo Of *e fatly *eS& Of OCO rt oil ad pasl k"l an 1 the frationa Ostitlatua of thelt


I* , Bro and 2sal a od t e uspamwtieS of 41* mVW meters of the fatly aoS~s of usubsAw oil Vy Ym distillatim~ !h~y oal*=&~ that It is possible* to mk &. rov sepois of* 4idds accringto their olilaw v$*ts this motheL Tater

*MWs3 St .ted that the separatio of th et1 esters of the f-t .ids Of UsIA lpOW uW be maA 1 tractiomal distillation at pros vw7tI ftm 4t ?,us. %a sepaa of the h1ably VIm. stm fatty a5Aus of beef *&baI 1y fvrntional &stillattan Of their *Up%~ esters id not susosa. borw an&AuI. I s t that it to possible to ditsi Lw *tIool2,y the motbyl estoes of tbe Itbnlate" *W of eattle. US, sa Asep Wains.

fte labrato*y separatioa of fatty &"i~ (as Adds or as their
*Stw$) t the use of fmetiowl 4ittillatim tI eon. ared to gve iua lotm sepamits. 1)wm , ad Goldag* clatlat the U600ol Is of little 9vsafttt vala.. Sitm 19"O iwesStlti Of the �toel Aieti3.latit at ftty "Ul mixtw"s 1 use, of met*1 'tors has ben aH iife

2"pllbuiwa betw#" the Yv~r ad lIuM lihase is wot obtalel I* O va .b ems. As. the OClm ratio and the rate of ditIlleo.

-,0-oo















%ionapparats a" ulesowleby & A~bw of 1*t~e3,8"4.1 hi 1930 Iaanusa xA~ fTie*bO .lop& m 4.tLAvjmv heAted p~m 400 Uhidh V lag U84i an64 a w -W-e ja.u to Putmet it OSSamt towomtw fllot~m. ftl* apyasbu *iasmo In the sepatI of Use uAtWql ..es of Pa2*Itie an St..st &SIAS. mi the 4*viq Vi &1.0 aNVIOVA 1 thowe *th.,'s for tb. f ctloatsA of the h~igWeli slA of pat *11. 0 &pmx* vwa aemsoatir YmI~ft4 IV lat 1eft&v4, a Rmsl* ON& the ofw~* .upIAMwasl UNA 1 6b.sw


~uPK*U1 plultleam& ouic "ids. b~etOn f is~

*SeWvd In t0w sefmttIo of the hijww au"64 %ot that004 0Oj

kawm~ a 1swtbo41 Swat *owo.q a 1~

b...zapw 4s 13 pestutut e kt1 **fl a ma triti e~ dstillato .11..tlma. MWe .ob is V~t f& a vwil Ltwt mtor sbul to Ujawalu thewma flww&A$#*a

Attace& to the 00UM aW* & 4is~ittX3gk setW wA~ oft, M &mea. stsm~AS torw a. joints a"e vam.1v Ow3q4e, Th* t~aeratmv at then top of thoe*3AM to mamurek 1W & thwmter. "M tUs PrOOSuWO JA ta413.4 IV & uteww. lbs 41U.12II. fla* al the C.M wei Olatftel 3' heatol stA & ecstat tmpun~w Ie ua0tabOA 1W AGeetst. Mb. 81" M is .estwll*~ b*&W. In


-U.











order to piwea the dtotiast frox solidiMSr. Um pw..w. Is aintaiue4 by a Steadava vmi Po f W to a 1ap. tM& otesi drs *la ms. Wl preeome chapo duing the �Istihatma ar vmestrab1. M- laboatory colvo are. vuma1l th to ft fet


1819.3 Upm tho tip. of p. mt"44a 4M0t -rem wase *to.,

I~tril oos are um? of tho Wd*w t" to vsxIms Slates =wt a toabm^t$V wA tkm gift th. ffe# of mitlple Usitllati....# 34&4p~ In the appeatco Is too emt for iaamT. distillaton. It Is .adptubl. oar to 1arpeeeao, Contnmm W0k. P.*lmi 1ovm vsA*A give mtata12V los b.1d4W an am q ate satsatory In &Itl2-lrf mal Sm~. of matweAl. NA W to* yvetizations of me iiffoxt tpvw of pakif ustew bw to
p.~ei. JR1)M Whttini ad mA dow~iba * s~ pafI vlt laal rlp (5 2 5 "a.) am" of 5DO* eqmw sin; M a s yeo? Vilee. Parker, wAMLUgha' 4setibe a salv pmm with a
-LS usdlfiatica of the 11 O sod vire boliw vtd* UA U =. & *rl..ler. voo 1sr&~ a txie*tjo+j &= Iil* v" a modifle.*Joa of that dewrJelI Ooop M y&W4

aw aemttmulv mtitis ot OWSAV0*01). Unkasl to the 6"Olopmet of off lut, sinlLueml A1sietltioft Ogpwt, M MW Of his VOMalfe mW OSNIse In Mes U:ol t arile pv& 34*.l Ua 193. IA this axti4. be 40.wtlbe hise ..lm an M U.hs I somin4tolas for frtmsa Ais~$u41aiom


Az-












Mar" reported the epmwgtioa of the metJ71 esters of p.alitIe. toeaze, lei. ad elai acids 'b the use of the PodIelaisk RAftatus. GsPertitte stuio. of tiverelfidacking ete- iaI bon be bees I6 P ?.ssb. aan d q a These =tiaez state, that vire or &s hellees, oewal teet4 sA J.* abiaJe wee feval to be tbs %tt psbts.

The uhaps and die of~e the Paehlng utoWt oelxpv

atesnmtIv . fters the U offielW of frctioatis a e eL 1. NOIWOV, the 2 rt&*M Of SIR& ftm as the contest **t* Old the pwvpe w aab .amt teo - so&. eiwd, *UZI gsA 7sw4*1 sowrii a& effi~am fwaftmtt elv P WLih amta glass, hoMes. Paeftag uateulal OOM'eseA of viie bout.. Is Ou"UNA to be .xtywey off5elant" - 4 5 A cilu p~i of Olome-fitting 'III. holloes vat l no* and UittembergS56 fer te tiomAl"tIO of mtkW POaldta" fa'. ~ etaswmfe.

?he use of fatty 44iA ester di. tilit mthods of tat S2627vs


Us eonlea.l type of padcb vas Aoewihs I Stdow -"* A dotail desription of this type of packi*S sad results of wasus of .fficimy tests Vere, gives by3wa

I* weest yeve the, vse of the nolesula)' still has bees of gret iqpots. In fractiontbW vwW )ighbobeIin asbete. gaeiaw AGIsttUaie of the WSW2. vnsataated adLds of fish *o vs rpwte IW ftner ad Va Ass jewraftI.


OM33,-











IRWIMzAL DIT LL*?OF 0 HIM FAT AM~ 3U1M Al L)W PMSBU2S

As already state, thoce potion of the mui ffttr asids whieh a" to be futh? reelve 1 emetioe we cout . *4 into metb es tem bY beili n with abft fm timmo thIr w0.* of vmetl4.*l In the proe of appoIe3l - per sent 7W wilt of s.itoA valftric &014-o ut*. m sterifed asid IS ee3Id 1W wahing the ote soluti of Us osters with di1l.t* petasob c Ste soatIM, Lte e el e into mets *ste U *wwaly 92 PUT ent OffOCUtO-, If Pw.~tiGR1 It thiAeMA flatt the wer' fWs &e*Us ob10 bew*A mevre a1 -o.stewifuel.

lb. metbyl 40tam s use& prlasrily became of thelir 414bt1 lower %oil-ing pIate as 0.qm.vd with those of etbw1 eetei.. with the operating Pressurs wnbg fma 0.1 to 0.2 me now isemly obtainable with the orinary. votary oil ~* ttis faster to lees 040"&Oleat than form127. Itovevr, It is eouvmimul to eoaMMU Using the Mthyl etrn SImS. so MWe dataL eat4 b nesrooe fer mihyl estrn.

Th. reesrdied beilirg Vointo #A th. heel of the tftmtl& Colu a"t not af aclentifie vt 1!fi.1 In this %Mp of distill. tica. Samoew. the meb head ts~Woft" Should 4 e ~tmtel2y Mmr~ed; uime., in monjumtioa with thors af the heating beth. tbqr

afoda reliable LVAMUCoMi Of the =06thtbas of operation, and thevfe of the effl1iemq of the frastionatim.











At 0#2 M. prmes the eo heM& termem~turss tor atbr




V1alss B laW~ frscti=z Of knw ra98 in being celleate& tor rew fractiona~tioun. the vei*t of OW* ests-frao.tlen =U3 got greatly 40 .b~t ten gr- em =7 beeas little as twoo tbM pm. SM I#" it to reamonaabl cartaft that a mixtur of costat copoj tion is distilling In lan quantity. It Is better not to exceed tU ainmt stated. Mrws In the analytical detxwaion of sepeutiesicae .qudwasate or Ioine Val1ues ez minizise If cariM out s emend smfl tfstieas rather than on e v-ey 1a&%W cut. Ifhe miutum witf of a fration is determied uim3W ty the aut reqred for 6cats, detrimtIon of its analytical ahaot&Wvist*S.

"% sheifa3 or no-att~r wtter CenmwUr rengto In the reiua, nndisti3XM eetwr fwetio~# The emount of spifkb, r~isi mq be deeide remnvSg it twos the plkalim 802ation obtained after dete~matten of the &pp~'st ejuivemt of the meidVA1

*stew. ftis i. fOUovbt b7 reewvey of the fatty aside weM" a4etrintion of their eqmtvalsnU, te, go of esters is It vs ofa residuti1 estot is then:





fte offet of beat Vuz n satuwated fatty AV temss W th ftnetim atisn is reported ) 7 0Ioi'I SL, Thar wepewt thowt **to"s of aside











with thueort r f Usm siftble bm# mS wm mUbtinta117 vz affet.. It to their opldale that wr gma al I pe24rettds. (*.C. fink 011) wid e e ls little altered althouah &li*t ro

arr~umntto .ajwm**de 1"miid, folimie 1W nown diumsa1tta

aw tae~ plaos to a vmw e mal der.. Aw materiel so st.Ie aper An the residual. vadistillel e*tewa, The" mtbo oncluded that tbo

5*du 1fe." in the eoV2 of a fairly prlonge &lutlUAtla -WI tUsigtmt.~ Tbq state that thea action of raagots (aihell) go SOl'Sate ftrig ~4"VO jo trmatuent of the fete prior to fmtls.m

*Us to * No Supotmt poetiale. oof at t,*.4later~. Atigat theaf MW GOM = -11 O vs.um aduifs twoatuul diutllatift at 2Av VSInSM. It Am" Is pointed gu that eaters of oemJftao h r "VtW asde .adfw Oqlisetif A the twk* VSMtmt at *l~h tkeW dill JA ated, fteetioaatiAt ..1 of the

deeibd tp mdthat s.h estOWO (#4C. gntqi ,lag"stswftt) esWOt to distilled Ia a eo.u at too big% a tmpoati.

Noris m a OZA7 m the piels 'boiling point data for the folloving eters at wo ur. prammt metb2i Wyitats. nAo; ~nt


rnt~l11210020"at. 1A.$. Altss and TrIsbUO els Mrd *M dat on metbyl olemte, mebr la.41at., an Ml .1ilate. !he1r #1.13 that the tollig point of the tbn*, adid ~ atexns 11% wMOM e2'd fraU that 9t~ 1W VWrri and Tery. All ap that the IxillIz points of all thz'ee of the O moMid a*0 otwea etramar 44.55











SOW FAM O T" OLf?&"! 0? "MCM0Mf 07 INDYWAL 9&.V&

The pturpose of the estez-fractio~atian wocs~ is the pouto

of srls f -str-fttinsfor use in the anali of a xiboo fa*ty eas. Nach of these.. e-ra m Al contain not xoeto

*w unatviate ester. ft wpoi of so& ftwaw~ am be dir..t* 2r calmlated fro their~ saponifS..t$a eqraivlea arnd Z.41. Values Me1 U. tru of most fat* in *Lich the onl unsatuxtedL .somns N Ift to USe On series at .i5s Weis,4 1tio , and lilnes.

Whsas is uwaall the oss., be* olee and Ui4.tei acids a ra the awa Iodine Yalw of the C wwatarted estes ca be eoand Zmet for a neg1igie erro. It aan be asg(e that the distillatiow of these enters Inpoprioal onastant tho~w*the opratics.

The he1lx point of zetgr 12almest is very coe to tha~t at maInW oleato. If Unolwmici toL 13 s ex~ S w7 p them.14d acid

preen, complete dteruaation of suok uqemfacin a be ao

fte doterminatina W be mae 17 use of Iodin Value and sapon~ostiou eqivalnt In coosjvaon with the spetrohtomtri dtemwteno the UllAl acid.

In *afatt. Osteeiafll those fwnsuatat Soum* tbahe l

at =stw atd acis. Partina4y in the 02 and *is. Us v

**Wlex. Yhe emstiati*A of meW of the poW34them" BSIP to still ue..rtaift, 83A It Is nna,,' toa~ a mre Llfftta at mvluatum thes etrtieti














As 1w.ed etstol, th*eaWi aap~ttae a ette-fratesa *1**
contain* "ster. of ax4 tnm isatuWet aei wth the-- atiasideU 41 of the *am AU contt amb to~ dot e ft" e* uivuefmt sal


It a fma~ttz of wiot v. swivelent 1w, "~ ldift Tab*. X

otau a VOW esters (a ths am 0m6m n ,et. 04g 4i) with S4im Value h m squ~vsawt l~



,, ~~~~ a v,, .(- IV , 'RI7

sal a"^ u e .iyaust, 1of ate *9 * .Aw te Oo(v it) Preent









f .1wetgte te Le . I
I t I e w lg o tsatumt eten m aesl" - a M

mW mixtmr of the bovol1.m (..g. CIA ad ) belve the a~ly4wUt of *t* h is .

~AI eetev4bwaotIeaa h iaalud Le I'M Id 4iv

at aegds of wf svv~ Ia the ka.).e OAWle of Ue palustl M.

-Ou ties their eqite ~sIuv~e. ts WWI wa*&"- tho oe. tatiom ).e am1rst wo #wqlt#Ato ela the vot*. of am thus tus" IS, is .inintsMere lv.


VBTAnS (V TEM MEMD Of CALMLATION IWMTW











If X, Y, and, S a" the i.spetiV ei4 9 O a the OW fatrstel NA~ toe tw V~asatat a~*m ina fm0.tion of Vw V, ad 360 ',. 36, 16 * the coMsqedis equvalwmt eM 11 ?ss ZV, we- th mm Padua to"" yalese then

(1~) x+ s r S=V
(2) 4* # N 11 = WN



(U ftU (2).I the GfkM~iiaiO T&WVIrTo Va eu



TM values of r &AM S. the Unseatiated cmoms zfm 0
tleM Ail*, f*1 4bat at X* the bis* mu1tu of satt* *sw


It aq Ue pointed mt thai Is us. the *qU* (1), (2), ad

(3) ab .s* i~uifild bW a~3.qSn In Oqti (2) thp

v~a~v1 f the equivo1ents time* 10
i.pmsei asthod.a ovuslvatift of the oste...ttt daft I*
lo given -0" 40%48b 1OM the paitleta *I Infets. ft the A&. loir of saaZmseu if the mo 4"fwist #qq* at frookotiUu es4 hks Uem used, It is St m..es0q to0 isolate an 4t.1mi the *Mft 14at of Uhesas &M4ecrn~ of ans et-4wetief.














les 1A "s t totwo. ana VA.4 Sm Wie are fror *, ,t1Ie in otbW. aim* te vmSU 1te hinas JA fraetIow of wvam 'below 2% MW be sMtibatll soIA21 *=j 4*be tU Nalest I

otflgb-famt Thi not" hae bern usel Vbf *.eIVA *%M bof %Mr Alti~sd wift s, AMUl distLll*&rn Spoat

Umr the, slurtwica.l heatel ..bf is OVa" it1 .is "Us tOat trase of *wee aois we dt*Ua* In the aj am 0% *OA fm*t14W. Ret svub O&W it wy Ue asom that the aatrs M =94tated powtla of the frmtion hban tho so VvU&

Ue LastUSe %a utzSed the satw*Wte 5*tm of a freetift e Ums 'Solatel a Useir *~n the awint evanow gives in the pwea~f ow Is Vi Aith xa the vw* of 00*0001 .s~r* Of tewsbs&d Oo1Arnt, U.y~Lte"nAae& equi leat Is sw ta" beo '02,0 to a~ate x SO a Mmlr mItr of sau.






Is fteotions with eqv1vaimts belo these or maat4 01

.stmo tbm w e *a* pa~mitates " go& lewgo askato se g~itstes.

Tim saturated "tore peraft La these fzactums m be bslat.& ad the O=~ t *aloulatad br the Cmml .41t1.a &tO0 Zf 4^ .oicien ft~intj O~b VM UrNi. Clatlo. of the atem f>stiae Of equivaos belm ZW 04 the as.wtba tbat th*


(W THR 9M SATURNM M











setamtoe AV#t* Darts bin th. "M eavumt t d






of Palattt*, bmxamis, =&4 unutamtet CI *tom Wh 0


.a in the nlquIA' addsMW Wfe tAew.d to Ue of 31MS INO6 ta-pe

Wtt~mo eftwetbw AW17 of unsatura~ted. ewt ami te evaluted vith %Ike folowLxg fats in xjt~

$0 IMS am the =1auO Oe 0*04*8tici do1*so XW of olnte. ith not m tn 10 to 20 per eet of 11ne1t*, the dateaimo ~te14almtScrrva .1e with thoe, OatA ~ Ole"* aMA lIUInemt I"M Tal".

hA tb&*trfmUwcnan2eg rprieo ilae

the 4tisdae eqIVAlets at Uaawo Ole Owhertim to& to Ue appre.Ia2 low thm m hose owa 1W) the idmUw vou. The ma not bern peom Ut it mw be ft to the pres at tu of autO8selu estow *toh lar MNW 4M ir r~l~f with 60MwlI ptaIn bmis It shvU be 1km -in AU& that the 4et~wmin *md tmU Of the *Pr uatwatte C 9~~ tWW We WM&UZ 021I** low than the taw vau,


*.21.











Ii otrP-taun 1s41atea7 pr~od1m thoe imah 00aat
*.I)ly of vaatr4 figtO~ the" a" vej' =gall gwts O s"to "to". Atus 4604aa~a of the mes *et4vae1.t of the40 mitewrfr..ft"Sn prv Aimmlt, eA reults an not t"e r.ab.e It b* UeM fourd that It Is bottew to a.w04 th~w to pa1mUiU thma to %a* their *oMN *qdidmsto
%41 -L1 limleatoeM at. *MM pnrnWt, the vpwi of ee*d felloW AUPU 9*0 to eim~n vaUM.
Nuatinu with vpu~taleae abw thoseof t--&-u a11e1 be*1dQto4 to biu XdxWmW of A" %e amd 21

-toi eiw ea V
*A1 apt f the amirm ai & savorn tie **teI in the PmgOPift pawrV, it line bee fmz that zellable zlyeg aq bo obtalmd fte21wa*~r of ee*wraetisu. Mob ase M"a be ca"M* WM It* Aftft S1~ d a S the UlaltOS Sbmld ta Wae oceaust the wVi& eutero pwoiamt Ia the xmItu. Th* Mape
ofth m r Imm* fw414RbAL 1 eSOOWW qucy " ar

*WUl be takes to &VOIft 02IU6116 With pwapW pweemtM, it is possible to Obtala41600 e�A 4&Ua of te ar&W of kwtal seted b*v

XWW tS1RA"the vpaoxbte eelson of t*8 f l.u It

inP&U4 a ofwtit s~o vtter to ottuin the eeOitinAt of the *"eMO suM 'liuU*A Mt... i this it to ponwible1, to #4allate th ,I ,0 'Ot eae fatir S"' Pteest ft the oDWlga4 1000-1, In the -.1A Ot











awlxfate the operation re ext1ud Imme arA teift=As OldA a fiml acamm of not uore tha. 22 =Its me to ewbm in this


Uma with the somit S~be bm~enm at the s f or L sm at the mrh4;b2g umaWate i sompos of f is wA &121d fat$* It he. UmL olid tbiat tho oav is. compaeb with that for mW othoel ustuisi petbts of a eoupwble com1.etr.,

Us. MOW1 featga" .014AS IbalSa a e*PO~ SdI4 sma1s
AbR1& te O.umtd at nto 4 "t", &14 thmiw twaetioma datLStU* ties ..INVI46 tbw I* Without 6"mq.

0400. ft ir.-mtim or dbeeme, &4* at this mm wAtmxwateC. 147W a'n 1laU to vdWpo2lpmr or ether' ob~ &j the t00rfwtiets. Ibis h bar UtShowt to be *Vooially libmg to 0" de1ua 0030 atl~o the mlual said 17 wom of the e alsobo. Iu musk lastange It to mesaig to (&) "P34w the minia .r0nteaa of sulfwiU a.A t oge ~ j .ex0te ttIU"%ditift


isUtillato, bat to pow te nreao ulztwm Sate .e)4 vatr MA presed~ at eas to extret the eetrs f*M the diluteal ve ith othe.w fte lattw pw..LS~on is rn3 1VWtt.

te ft.liM sow is~l carried outa. asul ea

.outa the *4elo of ar otems or hismor satutated &W Prmt, mxtrl 611 the lulti. .44, a exsl1"walle proportioat mW wITIStOOA VMelL ond~t oller1~ prevw CC MWg lower aturteI











*a5*. "Wi Will also )51uo of va t* m&4S Awr A P of .1.1. ad&. *us-tol aciL of Ow Ot or~ I*A mtserie,




?A addition to moo all the olele aoA pe tb.Us 1I~cVw

w~tm iwlA* msot at U wrt msau e acids of th. 00 9 Mh v seres the *WLmA i of the %A lwev *or'**

-'h of tba .astasud %, 610, 01, x~r awate of sr~t~Ad trae at tbo Palate.




OwbW to tbo presec of the daule badc In the owo~ gm, the satwmta fatty set how & tpptoe 4mOrtem spevj Th e~leale ..4 mt W4 .xtibit sa ahserp*1s dcto th*ea*mw 400 Ut 4.0 show ws.i stle &bsa*Uqavai ?#2oe#en~t v the, mer and the z'.atatv s i*tla of the dmble UrA.

Sim*e the ult,,wiOet *orwptSa propeties of tba oatde faty . Ami M* 4www the pr#** of the&"pb4 abV. gro, it is evUest tOat the a&sarptbearaeb c of the satuated meid shuld be qw3.ttv eilja. UWaRmw M As.ociateod iwestigat.I Ahe Ultar..valet al.erpte proeries of tomb,. aceste.btrs arl.cpie, la, #1pb,,1t , sw~iItt*, Vaa e and ate&A~e swAoi between the wfe leng of Moo o

2.55$ AW*. OWa st4.e th at the abiewptiMs ..offb41mt St











the hld~eT mo~but of ewotlt O$uil. Mues .uths come1 that $two the lexgth of the chsU nQs ot matori$l d4 MW sbheoipne efficiemut, metbrlax metlme goups emt 1e 4i0#.

*tiaS'~. as, choe ros Ia. this reion of as espetrm mie1ft ad OWtL4 a bd stut a iaoer, of sAtuv*ad* ina x the -an 224 Ut 2W0 Astft oarth.? mad WA oam~a that anm avw A the 1ath of USe h~drseswt eh"l wma &tt..de& I a *MIaJ^ the aheoiptiou Uv twa the red av mas, J S% uemi the =W. tWW o atl Wbsorptlea. UOMW, LW and AreuWW67 stted tbat this

.htsdo"a toS t QPU to the hib saUM*A SMS. 10

XmnrdA ONOmd the va1w. Of A for eseti. andp tI ar~ for vawm" Value of the MOU"I" "tbntift "Mdttimnt CA.

StRUSS, of the absorpt~a esoffi&ents of the satumtd el Ms

heW* 14A U tMe POSsW1t thet I&th 11W liUAtate the *WS *AsA Sw
* 4Seo at Is a aws.teeat f- P* " t tn*

4ese not matal off**t the Ohsiwt" gt the, abU**Mttt haa bUea ebwa tbat the valV..e of the vaieum asters MIfte OM shLt14 frm those at the pareat aeiA*", bet AMrtA 10 veaIeM1~




06 itoneSIty am the potte of the &aett basm USe obV

*AM be*aA OW $.0m3 the SS~ 90t ale.0 to te.~et bet*- O=JW~tl** is attmd4 b a MrW shift In the abeotpttoa Is" towL the teA, VhiS lys~~ ~s has beeA the .SbJNet











SAM-""a has bees PzMRse4 for ws in 41ti~o~f m


at the .m.wmtief spoti Ot UmatuatdA asw Taf am*dt


tive potlea of the *UWq1mU Us&*. UXVU ad S~ xpAteL thes pmmes Of a maft s 2700 Avpst'a a~b-00O mdi. Ma Isolate meUd totmum easmbt a bes pows s Olo

***pwpt ,7 "W UpOO ms, Utehef sma ft u-7 stei tSA f~m t i~vtlosi comlomto, aselati&, beads In theou

-ea ot ww o*Q loaw thma 2M Amswm t mbw*~% latls at--- lixmlia~ or lbmaest. ae$4. 3w sp mime rsmW oxteado& the &Uaopftl studies of the wsatwtu awie to 2)00 Aotm as not" that the aside on w ofts~

M*that is a ofbia~sa the start asiA Ww*Tptlmenv

v a abe."Ties 40.1 t to the eftuesh am. Th o bweadt

*lgi. scU ooe ot a lawr wr lug O t W abserWewvor 3. hpt.ow, an& soels of the iborptles #Soota of "e4. 1 lae WuA litalaf MA4 vb that the a~dtt of ehrd Uws pro

ds*a furhe shift of tWbA tousal the 2*New

watewite lotgs"*.m i.t3

establishing the steta of is"Mwi 1 1 % -1 -94. 77.* 76 OL SWe AMw a stawg ienr$&e absowtle. bat this alxerwt JS A""S~ is a tm. fow.


~.2.












Btaiso of the llttalt uAbops epoetra of the saame fxttp seids and of olo, Ua.2*L.a. linolmd, andat bon teft extended to 2100 Agstion W 3k an& a#8ce@9. Sia am of the uns-tmted aa.U* rose. a pek at LQAor o theee stu~ies wr later etended to the etrums ltimwlItj aa mW $.twttg o.sowtts vosmAW A aoqarwsez of the abnoptem

spe geo xw5istic &aid dissolved 5i ethanol, It4ectanel and hepta Ahewa that Use po.2.t Min.t, em, skiftg the absorpt maim to a loverwo U ngth sod 4>v* love aboolte valmefo xtite

*.gfiients. AU to i.Oat" m"4 ber a bmed, U" with the ~ao at a)mt 2M vp0 s and a Intns ab.ortlat Uta 2I850 ma 1rO Angtroms hatawftti in the fatty seU prefts. abontib sIMlav to that at the lmsatuwt st~uiw Maial4~. sattlin of *le madi sh~ta Ohe absorption toward the visibl. em& of the apeotmi. %a available spearotbtometwi data a the fats, fatty' .44. 3*A theti etaws )law iemt bo aiel


uethods, u~fe foannt primfti to the d4.W4~ a& ..ti~mmat ofw pw'ptinm of .OW490" 3*ssinm poRM3* Is fats, V11., amd Aes te usthM is 83M# Vp~ab?* to ftW mWtewras h IgWhh ppofesof thoe~ copMs

*4ifieatlea incld taret o for emoptiea W uera

at. , we o alk1b & me s #a Iemnsutie **I Ia ase smati.. posa* a m.mesi of shewoi "at ex th0












1uomaid produc. fte abserptim U &a oagtiint MMs sbto Om pine for the bsoiptia IW oonjWgtOR SinautS which werf iS the mateWl beore the Lomstitm.

Amite on som now spatepamZiaas~jm of faW *sift


la 194 114m sottd tht theis w" vae mapsteuat mtmle haape In f.*s am~grwditain
lmwlber vw* he bi done on the paphtnr svlmo

tb* o44Maioa of fats tbvo& the .a absorptio a thra4 o p~tto in fatty~ esters. faweo86 Imaipa th oal ship of 0101 U abeipie bead 4areippat in the "etm at this Sa=U Wl l to** Sams, The W& "Pt =at bo

*vmU"4e .aaeftlj bOmm *W(njso #VW

Boone" pasatl p*1abhiel a vwl for the ns. of vltmviolet spcrphtmr In the OxA of cb.sp ft the &MlobmI grows of ft MCA. lie pwooedt" to clalus to be of Speei4 bolp U a" e.*e~ bs* qajp~ aid~ g 1.R1 g $ 1 1


RU~it&e $Rd OXIivetRIMS VOdr thet the AWORIntioa 49 lMUMOl so$A % tbe *ptmphotmwtw* a~~ p" a M~w valf Ums the aft Of lIawum~c aclL Pemat should Oin. U wam, tim m" offn" fe tails *eus @Wvo t "it"~l,
0#00=0m Obtaiind zeMIxto in the datmmiatls at the aipla ad bet. loommw of slese$ eld in tang.11 b~y the use at a











. Yb wmt Obtaia f"S wpmt. towett #A


to I" Ne~l~ jj gj- discuss the application of %u3traV102lot sotbeevyise 4r$t oil 7mWd In their artiele tb" tismm the dwepat of tsts for the Astandwtia of fatt acm* IV g, -- --01mgmtri 2 0? t YW




ft. msthe( V*04foe' the t~iflatioR of u Ovgm 5
ol d for th c owo of tue woMan-,llyfprli


to all aIo1sts, ftfula constant M&* asth a* 4tb pain"a, ow tive iaMis 4e epeA .Wi t. -- ,y r .ONAtVtim to the identifuiation of ftme amPW. The poattur of a .hesmt4tsb &*O"4.t1v fwer oq.t3 t fb" pwed ef the identity an j4ty of the x In ttOam.

fbtfowit42, WX7 few .optaDu 4au'iw of the f fatty acidAe. b o eeA . * of 1 stmwig.s h* do. "vkoa 44r 'eath" Of th e"tme 6 vhmeh hb* b e to be vw us" t i the 14mftfim&Ue of tem ompws Yb. alas* Stmvr.1. t .lw of the VA o ** fa AM possible the use of a avow of amalytifl psw ~A

-r"O to be it -e vubw.
ftuh .ostmos as the utva1a5m t the gm berU4, amOVI valuo. m" th.e reom thoeraat. =uh" m


..V












uU.~ tom A1lhav bwo Q~pUio to the tat "~ fat .aS fm, mw Year** Vitut attewtins to dotract from tho vusas ot m maytSIM2 elbtat, it AmA to pebmAt "t. tbA thw me ftt ja i WfO~ ot th it~t7 or paUifqof a in1w~na aoiC Poi U2a an oma loouws ml~u" of pg taM =d $W* a"" vni pesem tha atn1Lata~ equivaIi of wau14t *d*m a Z&Vkwo 4e Ima*tea aAs w poseeee. th tiiw ele lu

oof s*34ds emL


-30-



































1 P DTIA AND DIS=7SIOI












DATA ABm DISUSSION




Data on Oi1 fm~ 0oldA'lssed Yt L&I of 16ocesiu

CAietroda Piscvemu) fJas.ee)


Nee 8p n V.oatil Natter.a a*a.

spocifte 1.vti WON,) . .. W t 4. Z8 of (25). .a � � � ... �. � 5apeificaa ,.w (.., ts.. , I o, �






10sAW. .*0ls . a . . . . . . . . . . . .




SafVrato AdA.* . . . . . . . . . . . .



v"&I ft2 '1A"A a a * e a * a * * v * * 0 # * * ft" atty.~eJ A"".. .** a, * a a 0 * * * * * w a





Spm e.2.l. .. . . . .. . . . . . . . . . . .

fPq5abur VtmR a . . . . . a. . . . ..


o .. .1., f



1 aa92

* . .. 2$2.b109.1

,. . .1


a a . , a 0.07 %


, . � a .o 22#

# 72.7% a * . a . a,1

* a aw 0.6 % .o * 0.00 %

0 a & 04P00 % ... .a,- 01

a a a.a


-YP.-


U


- !p ! .! .. . -.. -! -! 1 -[ I-- --I- W.. 'N 0 i M � " - ,_ - , -ii~ - , ~ O R . o












TO S tt"MOf TAI


Vwo. the data is TaO I, it to sa that the at of soluble etis s 0,l3 per ot. the solable vl&tils ads or& 0.07 W at,# sad the isoluble volatile aSe ar 0.0 pew sout. This information shov that the ewnt of low sa alar wight f atty ads In m.saf tmko oil to Very law. Low Mlea-lar val fatty aci se tho .1e of lower mlomlar Weight tha l ii ote teA.

"Is Vasapeaf1&Vo reside s imptezit *zme the sreat of hig zoleaaraw eight alodbals present is shows ty the quatity of the wesiw 60. hta Indisats, that the at of h~ mleular wight ales bole (of the cholesterol type) ppessat In moesasin snake oil is 0.4 per cent. $"*=Il color tests "a on the ="~jjbomtter show tbat cholstoltpe aleohols are present.

An seotyl value, of 4~.1 -" tomu. ftis value is aetuialiy the %Warweat ssetyl VOlWa ratbar tha the true aetyl vaaO At*ws SOubtmtims the acat72tiou due to the free fatty addq the acetyla' tiesdue to the free hyrmW S~pe of the Slpearl (prodaue d the te fatty edit Voe foiead), and taking Into nout the eiwt metal erro in the detereaation of aet$ val%s, it us fowA that the Otme aoetYl value' Is very small. It to.evident that the esout of 1qydrWo &*Us present In miocosix snake oil is very sligt. fte aestyl value is used for the pu~pose of daeowaii the a8mt of k$odrw ad present In messi ank sal ol.











It shbo'i e pointed out here that the dlffwso )etvim the Win Valu en the tiocyan value of a unaturated acidmi tw to a measue o poy-natrse *O pri 9.$in tbe vae of the moea.* snake oil is 77.2 &Aa the Iodne Value of the 2oeosela snake oil is 4., itk apmst that te -w of po1yA=at. teo acids preset should be abamt 26. per et. analysis of the fatty w.d proeet in aooc"ga snaka .1. vabtate


It nt be poine ou that the toin*e mmer of a * is not alvays a t asure of its unsatumtio.o An examiatio of the theor behi the usa o iodiu n mbe in the uaturation of 4fferent mpd bmived soveslIm portant factors. ports hay* ste that ojugated, dblo bUd fail to gvo the eret1 toEdm Mbw sw. that the aistown of the double %ad from the cat% beql sAl the zethovl v'op &a = a44t affeoto the iodim adtlm.


116010 MMIMIM O NO@OSN1 The poxents of othe-woslitb2.po.yroidl4, Aseasa a 4**ibA b7 t4wltd . v a ProliziavyN asttmte of toaum of usatated 0 0m fatty selAs. Pom 63 gi v at OiquI&O saids, (obtane frm the lead salt-ther sethad of separator) the"~ wre -prove a total of 25 of etbwr-Inboble, poyrmds ?rIMP this uSiXtOZO of Poqasud me bteas(m a fraction ial be. Sea to blaolia at 200%C., =d a& further' heatisg %Us fratiou Loepe%"











on fwrthew hmt1sw. A4Wzdi to Jv4sthiS IS tYPtosJ. Of a debAenie asoi Wh L&vitd& ma* this Otst=t it was thmuth that the p-irteal ftmla of .1sa&c Wa aud that of 1A*4i. V" eas 2. At the present tim the epirieal fomvaa of eo )eU*66S14 soi appears to U. 02eM2 r that ot

.m#nesc1L is still susidee to beOW322

OA the basis of the amut of oths-n4_ue ble p.)$aies .k taee, it was esolated that te original snk all Ctain at l.0.t 7 per seat of the Mody unatnuw 02 aA on ratty "I&*.

It asl be posted ont that the etheiasWb3' )b1Aro do ataWe' Siv the aetusi gaantitr of th* ighly iunutal 00 anda fatty aeliM prseeu%.




FMW MAS P5O AR A


SUIy gp ! of IiqUiL fat*y ads, otusned1y the uso of the,

leLO-e4at-the neth&, 'are eail to 200 Cr~ of usthamel Vam 3 Pam of ooma ate& -ulfl.e siL The =IA=* van bilel for smt -fif te= minutes. an the tho mzes wthaoiml 419lttle off until ab ut 80 per .st of the mothl was nuwil. te niitane w then pem iWe 1 liter of U.stillot water, and 2M0 crw of ether were aIet. the ether laye vas tMen washeL with a dilute solution of potamoiw obonate After the w~aoted fatty acids he& been removed fro the et 1W washing rpetedl with petas*iu aremate solaitiOmm










ad Us otbhr vawsisti3*& off, l$ieT1* Us I o A 92. pr Goat larau t S the meotl. *ter restated.

91* dat a ~ WMU1 wwe obtained wlie 453 jiw. of ut~r
ectew vere distilled at about 1 n. pesse i a L"4 aM Zve11 9 "p 0 2* mtk esetm vere obtaimd fiw t e *liquiA &CiU sepaaatd tq' use of the load-aIt..ths pw.obm. 2he distium latiest fraction wee eszladm. for refwt*s iM~ess. sapmilogtiea equivlets, a n dlum Valus. With tMe-i foMtij it W& possible to US the Uldtch 110m09 of m1ouatio for the emposition of the imlnySb eetrSAIse6 to the po*slure as ijomsud In the "eView of the 1iatu~rs.















Data. Otaln f~m Ditillation at 1 inm. of 145.3 Gw


of -etql bter

*,lql4' Acids Pro4dze


of 1iqul4.' Aei"8.


fewM


21071-21 0,2. 1.4"256?
n7.) o.a 1m3 0.0 50.g.

12ax110 1.44"" 230.0 3.
1.7 1.4w 26.5 70.3 S 2.4483 2 0. 0 . . V1S440 LI, 1.45M 2".2 0.



U1-S65. 1.2 1.1459 29%.0 M

10 0.9 1.4 ....0 120.3 3* 14@ 1 ., 1.4 6M 2" .0 12 7-2 4.2 1.4 32. 21.


UD- il














7"M th .apoxilficattes sowvAlats, 0 wiative, Latta" ad Iodlne embers, the brea.H4m of the warlomi fratios are glv In Table III



lbtew Compsition of lmtons 0btalne& ftom Distill.-tla at I mw.

of 4, eDZS of Ketb1 Ito" at n1vUM A15. *iquid*

oUe P. oued 17 o.a t-Ether Ut"


at? mn ...o 1,-a
t n ate ate do li
Jln g.) otdnt

1. 0.2 0.08 0. 2 0.7 0.32 01

1.0 0.22 0.13 0.6 1.7 0.11 0." 1.2



S 3.'?0.lI4 1. 1


S ~.23.36 @.M.
9 1.2 1.2 10 0.9 .9 11 1.9. ..1

12

0 N
KA" a .32 a;


Us 4.2g . of 2001af


po"esent I, 0.3 g. of Uwapoadf&Lbo ompose& of bj" uaats& aw


residue and


3.9 6. at











Ybie #Uqui~fatty sl traction obtained by the lead #A$

other mo Is. 71.0 poer st of the total sm , oil. 0* cvwet I*& the valves for the varlaes mtb' aeto, s aova la able III the folloving pervestasas of vAeds ewe obtirAm4


Peromt.co @oqaetta of NLqufL' ?ttj Aoid 7wat

(Ce6"a*t. fre Mata, li ! II 11 and I)




lh.ma N o f~A, 6a
Samai at mi,




6.43





Rrmehi__ _a d !OM n.00


-A-,,












fh. (at.a TAU T us obtaluM I frattioatta( the m.*1.
*Onow fr.. the 'soUL soM.

TAX& T
W&t Obtesu. tim 3stllatift st 1 nu. o 20.0 laum

of OAVI Uston of NUUAjo LAU.,


0~a 610 i. R~~.) y ~ h


('0,)

-_ _id2.5'






6 116-450 1.2 1.43 277.0 6.5 7 1 .0 sou 2.9 U.? 6 1q 3.9 S" 299.5$ 9 Imlk 0.? l 292.5 9.,











Y. the Sponifiation eqvalmts, ,efretlv* 5t . and Iodine lues a na In Table V the bwmak.-dw of the vwa.* Ova. tiew tve a table VX has beem oaluate.


Uter Compodtiou

of 20.3 I
A"&4


of Yvwtcfms Mbaim& fres Distillation at 1 3m, at bt*1 Rstoes of "U11I' A.ift. W8yI1L'


(in a.) wri. bom e... I it. *lst Owi i...


1 0.2 0.194 0.0m
2 o.3 O."Se 0.0o1

3 4.1 0.0" 4.027

.S 3.& 0.855
3.3 2.0" 1.221 1.2 0.f9Z 0.091 0.2m
7 1.0 o.5.5 o.o 0 .3 8 3.9 0.390 3.510


10,a, 19.2 0.92 0.081 U.438 0.0 6.5"












fte %lI$A fatty .ide fraction obtaied~ IW the lea&4-altstho? metho4 to 24A per coat of the total sake oll, On convetIng the values for the va-Ious methyl t- e, as sho in Table, VTIo, into the perentag at the owiglwa snake oil the folowing owult are obtaleat


TAM T
?.WPOSStm of 7atty Aelds in the

(Calculated twos TWOl


*Sid*fw


oi of AC.7


Ib~isU.0.62




Olole 0-~77 6t3'1m


Whoa the .olA pereentatg frout the Oliqul' and *solid" fatty asod fra~tie Ver saw the data given on the: follovig Pg In, Table VIII were ottalwai.


.41.














of au u 011.
9Pmotoor booftei Ol Oil.
lsmatios ObtalA 1 the Uatt-bethd

INat AfMU





5t4u1, 8.37 oN-.em �43 0Oli. 35.25

L~melel,16.00 Arahadmic a-A o 10.97 ..l i9I.82


ble II to UAsed upon the distillation data howm


in Table. U t VII,


31''r'IIUMAL DNA .. 20*YLR& ACM

fts data obt8aie frfm the fractional distillation of the *04144

fra-tion of the mooasix snam o11 In iv in the ection on dittillation.

After separation of the '.11.4' fatty acids fwo the '14U1V

fatty acids 1 use of the lead-ualt-octhbe proos , It vas found that the hve70 Molecula Voiat of the *sol.d" fatty act fraction is 2A.l, Aile that of the 'liUqu fatty ae]i freativa Is 290.











wree exubg the Asta oebtalaa. Us , lea&salto-tb

Spa tion. the saMPle was t6etd for pOSSIete eZiAt4ie of the vasWtatsted saids. .th fofloving data vwe oUalnA
lodme Ime (Ranns v Qj aX n S OI ..1.

Ioe_5 *=e ( ), ,eld. 7 , ~,....... -7

Ioi Vabr0m ), *Liuid.* a. .. ...

ft* eas( ledie Talbes at the *liqu x" m Os2JAi fatty agid ftatiso .ewt for 99.2 per emt of the unnaturstioa of the origial snake Oin.

Vor with the fsoUl' sal fretion showd that it e.nta d a high ,wmtap of pmmitie and stearic uSae. hyore, It ws 4mmrU* to b a fractioa which eontabal only palule am t Is asde. Thi purpose was ashiste I* use of a soluti of at saad nd water of a spmf1e raviq o 6.,11 at 0. The solution was abm-tel. with palmitie and te.L aci, and to this setwated solutu ms adWs 0.9132 C. of the ffsolid' aside from uossin snako oft. Mtw the mixture bad wemael oversight In a 7rigidair., the preelpitats e a i*.adt ea a tota at 0."~ g. af fatty a"l~ vas obtaie. VMer the conditions of the e~erimut, only a *iteur of palatio ad sta$.o acids heold have, preipitatd.
e fretn point of the preipitate v dte wi.mA to be . Aeasrdt -to Esstoa9", the f yeaslag poWi of a biMs7 mixtez of palitie and ste ,i acids in 5h the mole fiction of pabaitie io

0.7 ad the mole fraction of stearie is 0.3, is %Ae. The Wey-.e












nwaean' wW of nalstnts rniz=to vas 265.6, vhnle the sotAl awm~ muelawr w1~gt of the preipltte wo 265-2.


S ~ ~ UA!MO A AND I"~ IrAwArnON

fte sposmep1hotmt5? w4l vas a. Sso n hbeal M vith �L 1. 2301 Ultrwvlet Acossqr Set atteeheG. Vb. ultsatlot weewy set providea the eeey eqlet for M menae in the m length regon at 350 to 220 zMWeins.

?be abe apparatus was tst*& and s were sf t standards and do it for off i.eI of Seemtiam Yer the stanard m ethods at apeswtin the reaae It roferred to the varus Blec * let

The prooedw*e used in this phase of the i'eseerdhiIs on ih~e as devsed IW Mitchell, Krqbll, and Zsd~alee* and valater .e).uomteg Vr ISdi. and lraq(-bi11. They report that it Is possible to eterm1 the - Of Islele, llolonlmS, ad arach14en1e

SCANI lfteftwl the double, baninto oosJtA* p..itiMS in e% Galbum solatlon of ethrla 61764.

fte metIUA VMS& we a fellmmw

SOVOW 0.1 and 0.2 g. at mocasIn snake oIlv a el

VOI*hL and placed in the tne of small vial used for lodinew oe detexuluaticmn. In a 15 x 2.5 on. (6 x I in.) test ULbU wer played 10 9l. of alkaline glel rf-sget. The test tdwba weavered loosely with a &U" a% a va kapt izmmrs* at a *etaut depth in an on %Ot at 180O. When the temperature of the agentt k the test soft












hA rsaa1 280%. the vial o3utz8Ain the noesin snace oil '8 oppel into the tctte. . test tub as m 3i'rl three t s at 030-Mute sItervals to mix the fat with the 917)00 1oalttiOX.

After 25 intes the tunb was r V& ffti the bath antL VUS

.la r-apidly uer the tp. %a. esitmto of the tube we" trWused to wa shat the tte, and the voluss wa s lute with 99 per eat lethal.

The sales steo In a retf4wlefto overA1. The mterils rmwel frm the tue In the hot alkali solution W pree1pitateC Us. solution in the volvumtrie flask wms briuht to rem tempr~are# nAi a p rtio of it vas ftlter*&. Proper dlaut* for absorptio. MssmIata w" A" with 99 per emt othaml.

It vas aseesary to ewn7 a blank solution of alkullus C3y"Il d the itlep woedw., i13n01xg dllut1i for ww in the solvent e6l2


PxU%&WI or ViR AZKAI OLT=OT Sa0 ow

Potaeiw1 hydrxid (745 S. assaing 85 per oaSt) was .44. to 2M0 21. at st*1... glel resulting In a 1.3 XLPotaesim b~drxi. Solutioft. While preparing the recent, the lyool solution wa b*" IS SA bl40MMq flask until the temperature weahel 1wo At of the ut*r vs rueuaM 1 boiling. o that it va possible to maintaft a tweeratare of 1800. while the saple ws bebg heat*&.


Jb0.











The formla wed to tzula eplfic aiph Ist


SP601igt AlpbM6 1*3
*I

Alf oabs.orption ..officien

to w Wswi1ty of rgAation trastt 17 the solmt

I = at sity of r.diatim Uvandtt4 IW the e.Iante

a g cowomtratla of so3ato In n per 1000 il.

I wwaf 1inh n coetiptam of solt~a tbwou* vhich tho

wa4iatift paM-e


Table SW.m th stan~ai value reportel7 -,be az

Xrqb- f tbo ..t.i.nat.. of fatty &old Um.a li4*4 lIAlUw$, wAI arail~ai acis#u



WRIfMW Valus for U.. In Ipetropbotomtric Auaois,




1"Pri~aT&NW vd -s-e~eAsoptonCoeflgn

9"M*a 2Y4.0 2W31










In ord*4 to evaluate the 9V"etb%*.wfte data fr the 15mrLag uwas* enelm oil it was eessay to ru a samplo of the o*ut~l 1,. After Imctigatis it was f y=.1 that d4stbl .tbe is a sol2t. In spctn-t.t* vm* It is mmems7 to hav a sent vbich does not abeotA vltrmlolt 14;tt so tint all absorptIon abawa win be that of the mi lto to ix. tog. the ortgOmal . .i ouatO te A 10 em of Mms on to wI* we S44.6 i

4.atl'l athov to ake a total volow of 100 3d. This gives a 10 per aout solution of moomasa onmb oil.


pectiophotomer at on Paro No e oe..ia es O.l Vwe loneil Sq Sp.if
LaAlIpha, U Oam-e. Extntion


3500 0.0020 0. 1 0.8 m. - 2.?7? 34100 o.002UT o.V7 0.8 m. 0 - 2.260 3300 0.00326 0,32 0.81-. a -246 3m. 0.005 o.534 0.8 M. 0 - 2.M33 31 0.005M 0.5% 0.8 m. a -Z.2321 3150 0.0&4 0.&14 0.8 U= 0 2. 218 3230 o.0@Qa* 0.610 0.8 M06 0 .2. 2147 3120 0.00605 0.45 0.8in0. a 2.21M17


Oftthe O We toatiu sM. 0 1a41ates AL~tbhr1 Othe solution.


10 p.r cent uecasis sa , oil-..


,4-










um x (oftum.,)


spetropIhostric 3Dta *a Pwo, OoA.powmd Urnasin Snakes Oil

Ia Alp4ha Log 0 a.ti

....6y 0,617 0.6 w . - 2.2m 3050 0,00782 o.28 08 0- 2.140 S 0.01050 o.262 0.

2950 0,01328 0.332 0. m /4i-192
2900 0.02005 04M0 0.8 m. 0/ i. .6M 5o0o38. o..,917 0.8 K. P4/ - 1.4328 2800 0.0512 0.321. 0.8 X0. 0116 1.2907 20. o.342 0.8in,. o/.. - .262o 27, 0.O5 0.42? 0.8 -. 0/1 -.
-o 0.0m o.M o.8 i,. 016 - 1.113 2650 0.0665 0.104 O X0.n 1.1772 260 o .0742 o.11? .8 l - 1.13 2550 O1.u 0.221 0.8 w. oI0- 0.85 2500 8#7 0,167 0.6 X0. 0/4_ -0.40
210 .36 0447 ova I. ./%-0.05 *001.270 0 ,191 08 ma. 0/256 +.0.10 2350 L. 13 0.92 O.8 mn. 0/2%, +.0.18 2300 1.567 0.612 1.8 4n 01256 40. 20 2250 1.7 0.5"5 1.5 CAM 0/5+..0.17'


-we













In order to g the daa givft in f ble X it is noe"lla to s the 1g SpOific e tintiox Cofficeat. T hls m It possible to pl00s al Of tZhe 9mqh a Ow M . The Log Specific Exti

0.6ff iieat is o14ted t tal*g the Log to ben 10 of the Alpa value In 2a L For thi-es&~ sas 7 r I..


-49-

















i E -1, 1 A ,tA _Ln,
j
5 C 1"RA F W. CAS N S N A i'--La



































Ll
(\J 0. cli WAVE- ALENCTH IN ANGS)"i0i'&













DISCUSSION OF PlM I


Yhe absoptin at 2Y40 Agstrow to .msd 'by the 4loo eo~wvgaties. MW alpha aV4 is 1.525. It 100 per cmt conjugated l*1014 ..14 ivos an alpha of 86.0, if 100 per cet en.fated liolmnle aci gies,. a alpha of 60.9, .a It 100 per cent ongated ara**donie "id ge s alha of 0.3, thean it to posible to 0 the s t of oujuaMtim preet fom the results feuMnd. It aU the aboorptioa at 2W Anetwo as=e" to be lineolele, thean thew i proseat In the original Meamia snake ol I.?? per e M of a conjugated limleic add. * the s prosed it was found that the perosta0 of eoaJute trims in the original moeoo.4s saks oil 0.15, an the pereatag of onjugated te.twasn e in the vel sommas euaf oll was 0.04. The trieon alworytion shov at the 2680 Aagstvom v longft. The tetrm absorption shawe at the 3010 Angstm and the 3160 Augstron wwv lone*.












=w XuTMu Of L2IGJMK0 LIMLUP AM ROM)AO

VW H)OASI SNAU OIL BT PM HOOI CA SS

ft .a oil v<.&n Ia this d.t.,*atis was isa"As" In an 3k&lizw gyoe so..ti in aeoar o with the mthod of Miell. K" 1, and sei1 which roooer a &eaaple of tin 0.1 to 0.2 grsx A savle of U0oftsin sn)a~ oil, 0.135 grm w's saft v to a volo of 10 ml. with 9 per .t ethal, eeoz ProoItre *1*h vas Gz*4io In &*WlI in the Pree.4v section. li,. solution of 0.1355 Vvs af ssla ol per 100 id. Is r-pwemt. at in the table' 0 In the ttion ea.

A ftal== Speetweowter was vzo, ad readigs versee,.ow at Istele of 10 Ansty frm u the rema frea 3500 to 2270 APUIM*a the esalts awe reported In Table X1. Sine the log &IpU is the asm as ths log petac . ltintio @oefficit. the log a for each ways length ws not tatulatod.



UtOURIaat oU of Unletlo"* !4e e Arahitoni A IX


2. Slit canan. pe ~50 0.80 010 0.8 in* 0/io .00

0.84 042U4 0.8 Sao 0/10 0.05












Ia a mt(1


I I i tI as I_ __ _ __


-wo





"@30 ~30 350







-3M7


0.80



0.75 040 0.41 0.61 0.61
,I 0.648 0.g4



0.68




1.02 1.04 1.6 1U10 1.21


0-.117 0,167 0.108 0,102




0.083


0.082 0.083


0.0" 0.0" 0.107
o.ud

0.1L24 0.138

0.14 0.147 o.149
oL1 0


U


0.8m.

0.8 M.



0.8 mm. 0.8 M. 0.820. 0.8 aw.


0.8 i. 0.S.6


0.8 0.8



0.8 am. 0.8 n 0.8 SO.

0.8 A


0.8 VAR


Clio C/10 o/10 0/10 @110 fo/x 6/10 @/10






0/10

@110 0/10 6/10 0/10




10o 6/10




eho


-0.05

- 0,0755




-21





.0.106



- 0tmw




- 0.102h .0.0653

O.


.a"g




*


I










'TAM xZ (Ouni1~e)


3260 1.12 04157 Ol8 an. Cli@ ,O-aw



.V0 1.34 0.182 '0.8 NOL 0/10 o 01*7 2.5 0 o."3 8 m. 0/10 0 .,7a =0 1.70 0,232 0.8 . 0/10 0 .* 32M 1.92 o,26o 0.8 . 0/be . 0.28" S 2.38 0. 0.8 m. Clio 0."


0 2.53 0.3 0.8 m" 0/10 , 3260 2061 0,3*0.8 0/bc
3 21,60 04353 0.8 -. */o 3o2 oI o.2.n, o/,1o !!4/1 3130 2.3 0*323 0.8 -a. 0/l .
;2.60 0.302 0.8- 0/10 + .m
3110 0. 02w 0.8 0.@/i3 21M3
3104L 04269 0118 U0. 0/10 04 3090 1410 0,20 0. 8 c. /10 814 0,20 0.8 m. 0/10 * o.2 3070 246 0*2k 0CA . +/10 . t 3066 2.23 0.302 0.8 MR. C/10 40M,
















Alpbs


16*


T V -


3050

3040 30 3020 3010

3000 2990




2960 2950

2"0 2950

2w2 2910 ON0 2890


2m7 2m4


2.74



t.6 2.90

2.8



2.3
2.60




2.48




3.36

2.74





4.15 4.39 4.00


slit VIMt


twatift


- - - was..


MW apsmZ2.0
Ittinetaft


m . L . IL _ - -e


0.32 0.388

0.3n 0.3% 0*370 0.35 0.322 0.318


0.31i7 0. y6
OAS 0.5w



0.522 0.'77


0.8


0.8 0.8 -.

0.8 -n






0.8 0.8 m.




0.8 m. 08 mM. 0.8 Mu. 0.8 nn.




0.8 mm. 0.8 in. 0.8 3. 0.8 1. 0.8 mm. 0.8 -m.


0.8 s-.


I


0/10
0/10


0/10




0/10
Clo


Clio








0/10 0/10 0/10


0/10 0/10 o/10o




0/10
eflo


e/",o

Clio a/,,o


U


____________ -- p -0.7wI 9
loop"55-


,Var


S0.4M .0.4,0




. 0,,%6


. 0.T


+ 0. 5
* O





*0.4082 . 0.4425 p0.4857 0 0.5263

* 0.5709

*0. 610


+ 0.661


TA xx (amumm")










TA30 Xt f@.taflus)


2840 2020 2790


2m~

2m5

2mW 2740 2720


2m 2690
2680 2670 2650

2640 2630


5.90
6.32 6.61


6.03 7.03

7.00


6.89 696


1.06
7.36


7.88



8.31 8s.18 IL17 ?.55
7.28 6.92


0.800 0.856

0.896

0.940 0.952 0.952
0.9w


0.948


0.9"3 0.943 0.6
0.9"1 1.037 2.068 1.130 1.126


1,10



0.9%

O."s


0.8 m. 0.8 -4




0.8.

0.8 0.8 -.

0.8 m. 0.8 0.8 n.





0.8 us. 0.8 rm. 0.6 -.u






0.8 0.8 m. 0.8 -o


0.8


H/10 toio








0/10



1#110 0/10)


ol"


*/io


@/o C/io 0/10 0/10 60/10 0/10 0/10 0/10 010


A0


00-8R26 +0."


9"3. .912 .0.""






+.88M


Uc spew,*
AUU Uwlr* VidUL tratim Ittizwtim M*l comea-


JL










TAmLE XX (0.ntim.wi)


Slit ona.. S

2620 6.6 o .895 0.8 u. @/10 + 0.8195 2610 6.61 Q.8W 0.8 u. 0/10 *0. 820 2600 6.64 0.900 0.8 s 0/10 ..0.8222 2590 6.60 0.89 0.8 u 0/10 0.95 2580 6.57 0.890 0.8 u. 0/10 , 0.8176 2570 6.6 0.87 0.8 u. o/o 0.8102 2560 6.37 o.863 0.8 In. 0/10 ,0.801 2550 6.31 0.855 0.8 us. 0/10 . 0.8W00 2b 6.35 0.861 0.8 i. /"0 * .8o 2530 6.57 0.892 0.8 . /o , 0.8a 2520 7.02 0.950 0.8 u. 0/10 * o.8 43 2510 7.6 1.030 .8 u. 0/10 * 0.81 2500 8.28 1.116 0.8 m. o/*0 0." 24" 9.7 0.132 0.8 af. 0/100 + 0.9m 2w 10.63 0.144 0.8 M. 0/100 * 1.0265 2 .66 .158 0.8 s. 0/100 * 1.068 2460 2.62 0.171 0.8 M. 0/100 * 1.101 2450 13.43 o.8 0.8 . v/o . 1.121 241.26 o.193 0.8 u. 0/100 * 1.15 5 2430 3A .9 0.2" 0. 8 us. 0/1oo * 1.175


-m-II












waZI 119 (Oontts





za*oo 15.& 0.222 C . 0/100 1.94 2310 WM c.222 0.8 m. /100 1.Z4 240 17S12 0-230 0.8 -. 01100 ' 1.20

MyO I IS.OI 0.5 0.8 =. 0/100 ,1. m 2M6 ig$o% o.258 OA =u oftoo * 1.* 2M 2350 190 02& .8 Va. of/100 * 1.29M3 2350 20.008 . 0.20 ow 0/100 # .26 2,30 20.1 oo .2 0. 8 . 01100 + 1.30103 2320 20.22 0-274 0. 8 0/100 * I."OS '2310 20.15 0,27 1. 0 . @100 * 134 2300 19.76 o.268 1,0 -. /100 + I.2M63 2m9 19471 0.267 1.0 m. 0/100 1 * 24 22*0 19,01 0.258 1.0 . /ioo
2m18.60 0.252 1.0 Ul. /100 *1.2695 220 16.30 0.2m 1.5 an* 0/100 * 1.2625 2250 17.46 0.237 1,05 +.010 2240 16.83 0.2*8 1.5 M. 0/100 2230 3A&(2 0.217 X0 . @1100 + 1.2"4


-AS.











TA= ZI (Coat iaMe)

In lb " Width rtat rfw 2m2 15.50 0,210 1.5 -. /100 + 1.1903 =0 IL.9 o.Mo 1.5 mm. Oftoo +1.175


20 14.61 0.198 2,0 =1. /100 *.164 2190 1395 0.89 2.0 ua. 0/100 . m 2180 233" OQ4& 2. 0 ma. 0/100 *1,1I5 21" 12.77 0. 2.0 ... /1oo 4 1.1o2


S of the dat f240 %US I1 Ii pIolenlId gI q b .al3 .


Use SPOific Abeption 0oosffjcts fox from T&al XIU a


It 23NO *A4t~t, *b* vndm etal 1" 20. 00, At 26M Apiam. tbs va* obtds V- 8.18.

At 3010 A. the va&e obtainA u 2.90.
Mt 320 *WW the vaus obtsin e 241.
Ina *b3 ?X it was found that the abor Tausws ve *qvaleA to the f.oVW pft sen a t "a#
At 2340 A~atim# 15.4 PM ..t of lin.1$.. aid.
At 2680 *A~xow, 3.9 Pew cmt of Iaolai aid,
At 301 Autrw 11.24 Per ctat of arohdncwid,, At 3160 Agtmoin 11-57 per amt at .wschihose &cit.


-596-











A


i"Xi"I".", A"





































/*























C, C)
LrIl
cl, N C, cu W\
ONE-LENGTH !N










Aa exzatiox of the data Indicates that the follovwtalu" a" more pro1We7 cwrott
L4ioleie a"* o. * ...... 0. ... * ....... &. 5.4 per t
A.akiwd aA ec~ acd.. 11.4 perat

ftomet. t at. fo the rm fro ZO to MW Amsts"M it apweIro that the sbeovpttl m.w hame een eazoel 1w a vieUla or I vitmix sabotmness. It is pibabe that thie to the re n tat IUtch an& SWaivsAmmS foud to hi&h a ve3m for linolento .td ty the speetropbotommUtw e tha. Yita3a D, Vbifh Contains three .dobe bUndo in conjagaWo position, wu2& %W *30,0"O to AhW Ve In this Y&W on the ispeetwopbotams*ta'?


.0621.
















snake oil a"e Used~ a go**** YV~r In. tie tievstl~w



Posemibq of MAWt A"" AA Nocas Sof Oil

Wso at AaU.ta









01510 35.25



TWA1.1 1540




?w0 partial tests of the reliabilt? et thi~s analysis mie

(1) The Ioin Vain*~ of the 801 of faftt aids foud as pft

to the orliasml vncosa sake oil, a&

(2) ?h. saponlfiation equivalut of the am of the fafl ad*
fiuM as compared to the origna moosa snake oil.

TAX"e flI and XT Siv, the resultsebti&


.4b.













00" 0 aniL Iodine UIue. at 74Vr Ad"

of )bomcas Sak ftl







+~~Nme of 11. oA
f ITOW I"

1.77 04,000

15.930.000

8.3701000
bas46.53 0*0633 35-25 0017

?4ol 5.7 0, 283 Pu
�b4 and salupaf 11.4~ 0.3%Z grm



t total toda. YTale of the o.g.ral sna oil was
1,071 9fm, whc is 0,,023 go g'ee t ea the to for the s d. Th. totmienatea vw bse.( uo a on su saupi. of the or?11a MOOOaSIA snake oil.


-61 N-












TA=~ UT

Parsmae 8mpovtlexandz Saosfiatiou Rqluivaen

oftheYatty Acis of Mocsn nke Oil


Xm* at AeU o~~f Total Spnfcts Paluitie, 3549 steer" 6.55 01.56 35-25 70.1 Awaai~14nU andI M1uaw4.od 11.4 21. TOAL. 19 2.


The totl eSlflitIOR Oquivaleut of Mats oil m 192.6, v5ek to 0.1 p los The. aleatioas wre umd a s one-am uoessin snob otl.


the origimal womitis than that of the al4. sal of the ori4iial


-A.


















3sd upo the data sho w n this disseUtiet , the flowin C ue inuctds


(1) X unaturm t f�atty oisa" not easily distlle.

won at pwrea.es as low as 0.1 s. to 0.2 M.

(2) Is wpotrphotMtric detozinatift of fattY ..I& e~rf

ew met be takma to esalat* the data obtaiM.

(3) U aq detemimstie of fatty smis in moccaXIt

oil, w *ax* motbet is ab.1ntoly reliao..














1. XaL=*&ja. :., and Pollard. C. B., Unpubished Data (H.S. ~Tss).

2. Youg. V. 001 Jr.* aw iPol1ard, C. B.. *wSbisbed a


3. Xaw4taah I.,, 'Mwmica1 Technology and Analysis of Ols,

Tate, an Wa5,1 Uddiian an& Co., Lizte4, Tole. 1, 2, 3

(1915), hfth Miatioa.
4. 0=n, A,. Arl-s da Yet 2B ja Vol. I (Der.in, 192).

5.30to, R. R., 'ffts and Patty~ Foods* (London. 1IM). 4. *a. 4. .. O'ible Oils and Ta.te (Lo ado, 1926). . D"s K. ., 'Vtilisift Of Pate" (London, 1938).

8. ilAitct . P., Lhe Misicl Constitutio of atu al PatWp ,;obn Wiley & Sam. Inc., *It SItIon.

9. Official and. ntetivo Nethods of Anal ris of ths Ass"Itoa of AMrieult''al Chlustw, Sixth RMittta (194,5) # pmublised~ IV~ the
Association of Officilal Agrioultaral 0h..sts, P. 0, Doz 3

awaula 7rawd in Station, yaanu ,4 D. 0.
10. iltitoh, T. P., he Chicsl 0o.titetio of Utu .-. at ,

hMo Wi14V & Sons, Ins,, SoooaCi Mitift (1947).
11. tlsst=. A. V., 'Ptty Aside mod Their Dwtvativ"r John Viley

a S", ins.. (1we).

12. M .lt . S.. Notv Y or*, 1 . te(-So Pulshers, Um.,


.66..


BITOG:











816 (1889); tt a&, Dye, M, Z 2583 (W): MrM (oa V7. Ca4. 1. M 1316 (196 ..




18. c .Z eM a t1 L 1. , 2, 8-3 (190). 15 oln la a15 hbq.1 , , 1 (1918).






SK, - 5 ( 19 3).

17. Xms a ,af jr1 (191) 19 , I bLa4 Z 35M (106Ltg ~ 31(9






21. &Mt " M r (,gj. 3 ( (.). 26, mla. G , P5 , *(r.3e3). szd 78.t'a).













32. 3.



36.

38. 3N.

40. 41. 42. 43.


I44. 45. '4.
41k,
4.
49. 50. 51. 52. 53.


Ametn . A13m, and ore, 3 .. , 1431 (1925).


Zftvn an& 31 , 12 (193).

Clarbs aM his. ~ 763 (9. ,3. 12)
z aM bit, _. .m.. 2b 16 (190). WW. L. . _Cgt L- I&4! (19).






aia m fths, . .. a (2., , 2 (93).



vu, Co-d, Lv s, , Arc..lL aM lU', S ). Ana,, l.al.- 941 & z, (1930). ,). Nt r an 38.)",. & . -. AMl , 3 (6(2) ). 82wblau W& ""4" 1.- EnM USA 3, Uo (193). 3*m2*# PamihLoosr, Arc~ibal , an yooow * i (m


A~l, 14- 2p 30 (1930 ).



Ikd.. a .iiak , ].I&. , n . a. a. 11 4 (93 . lIa#e bmgb.rs aA Lonb1, g~m g. &, rM 39(193).


XeIsg, tt aMZ 616 I (1938). 2 (93) Yemw", t"&5, an& 1,3731 (1934).











34. !ougsw& 3 .ov*U, and Ysk., W. -%. ~0 Z 957 (1937). 55. 7 ea. LavrmM a , 2 f g , M (1).




36, Sco ae, and Ri,, b, L. I "o1, . MM., j 155 (1937) 58. .dma, U. S. Patent 2.014,4441936); Om. Patent 361.01,3(1936).





39. StemA Cv.A. . ;. R2MAI



59. $tde,g j. la. 38 (1). 60. Bro W,- *m. Am .Rd.. 1. M (1939). 61. am = &M r eu_ e ..a , ;_- . .. ,g (.8).



63. rriie P. A., and K. , 3. 5., l am, Sg Z. &~. 3Idslawl s&awl, AM., f, 1304 (1913 . 65. ea.mXmx.adOvf ,2W XS.
13% (193D).
66.3Ik aml Iwis bb Akf 13W4 (1913). 67. UradAm#L W 2-U 7 13) do. hw ad Miller, Q m. A=, Zb 221 (1923). 6Y. ImtMEsgh . 521 12)


71. halq4 ad R1.hawaezv I. bL Qa. 963 (1940). 72. latcohi a Kabll. _CW w. Anal- WI.. Z 765 (1931);
Si~,m 21 i-98Ss 1940.
73. Mitabell. tmqbill, ad ZubMi*,. bL OM Anal. I.s Up
1 (1943).









_7?0


75. Dlev11 an Mwmas. I ago. 899 (29 4). 78, D.1a., ,r , andiam. L EaI-, 1 12 (192)�






81, Rusaft. 3.1mm. adww ZZ,.omt O90 (i4 7).





89. OarmOL, . I .l D 0., an Poloa La, I1%. 0.. 2lo s
S37 ().
84. 'OeRa, 1. f., ves mi . d ., O-s sh, ., ( 945, 85. f 12ma. a B). M M (1?, , 82, Iirlas, T A ., faMn N Szlta 3. 1f, 3. 7vat W. (0.47). 83. 01 onrs T, Y. * ftin".1 . D.0., az3wa, . ., a gpd



1Z. ,45 ). . s ba , 2 (1).





90. bdwsaow. R. , 3. P.. D. C.,um ,., J. * a U.















5.. 9(1948).






92, 'i, I, A.,mo. 3 &,. a pa, 1 (S-)
9.b13..~Ae *I,,S L 3., (o. 1* Anal* A 94. Mitchellg. ty. ., Jr. aMW~ It.i~o R.. an t"I YomPna




Iter-Beien Publishers, Ina,, 1vw York, 1. 7. (1942).


















fto Sthe eogo his si1e? appresia1u to D. 0. I.


Us ireted this wrearh. %ao ome of the unea~ to dolgely to Dr. Polards ~ndums and coustictiv* eriticsm.

Bler appriati is he.r e o s for the oopemsti of Mr. be. AIU&a of Siver Sr gws, Yria. Wr. Allen kindly tonstsi ths oils aid matrisIs vsM for this s*.


-?U ,


















:00"a Malmd zin, Jw. was +omu a .OtoibeT 115 . t
Marietta, OhS. ie p m hs t stude at the MAversity of lorida, ,iere he veeve& his hdM r of Ree, Deozree Inl 1939.
Ve author'x fWate vozk at the iversity of lorida we vitapt ee b fortp-ttro =ats soi@. in the AM. !vinty mnths of this Avs serise Sp spent as hvist In the, )ioloa1 warfoxe IPJect at Camp DOWWA4 Isq~l. So. received his Xweter at Se * fee fm the %1veeity of Florida in 1947. M2 atsuixg the Grauate S*Ael of the bJ~ven.tV of 13nilds he hIA tie POOition of ,lXabator Assistant for to yea and a baato @mmil ]eUevshlp ftr tbree yea..

Re is a m of the An eleaw Obmj*4 Swsety aM ot h1.$4


ill l .


310m I I s















Ibis isertationat m rpm v ulw Use dirctio of the Chalmof of the caf atf %pew4..wy @omitt.. aild baa tow approved 1 all uner of the Coitte.. It was rSabtt&d to the Graduat Ousl and was .pproved as patl fumiflU t of t .. m..remt. tw the .... of 2L E









Nut -ff+A-1 � . .wl+




Full Text

PAGE 1

A Study of Physical and Chemical Properties of the Fats from the Fat Lobes of the Moccasin (Agkistrodon Piscivorus) (Lacepede) By Joseph McLaughlin, jr. A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA JULY, 1949

PAGE 2

TABLE GP CONTHJTS Page muii INTRODUCTION XTD REVIEW OP L1TERASUEE * 1 Statement of Research Problem 1 Preparation of Moccasin Snake Oil 1 Methods of Separation of the Patty Acids 2 Qnalitptive Investigation of Component Patty Acids 4 Preparation of Mixed Patty Acids froa a Pat 5 Removal of TJnsaponif iable Matter from the 1'i.xed Patty Acids 6 Removal of the Low Molecular Weight Patty Acids 6 Croaatograph Absorption of Mixed Fatty Acids 7 Non-Solvent Crystallization 7 Solvent Crystallisation 7 Separation of Polyethenoid Acids "by Lithium Salts 8 Separation of Mixed Patty Acids by Crystallization from Solvents at Low Temperatures 8 Distillation 8 Fractional Distillation of Higher Patty Acid Esters at Low Pressures lh Some Features of the Calculation of the Composition of Individual Ester Fractions 17 Details of the Method of Calculation Involved 18 Ester-Fractions of the More Saturated or "Solid" Acids 20

PAGE 3

-iiPag* Eater-Fractions of the More Unsaturated or "Liquid" Adda 20 Absorption Spectra ^ She Modification of the fatty Acids *9 E3CPKRIMBSTAL DATA AND DISCUSSION.. • •• 3* Table I 3* The Significance of Table X 32 Ether-Insoluble Polyhromides fro» Moccasin fat 33 Distillation of Methyl Eater fractions from Moccasin fat Jk fable II 3* Table III 37 Table IT 38 Table T 39 Table 71 Table Til 41 Table Tin kZ Spectrophotoxnetric Data and its Evaluation Preparation of the Alkaline Glycol Solution ^5 Table IX ** Table X *7 figure 1 50 Discussion of figure 1 51 Determination of Linoleic, Linolenie. and Arachidonic 52 Acids in Moccasin Snake Oil by Speetrophotometric Analysis

PAGE 4

fable XX 52 60 Figure 2 SUMIART Table XXI Table XXXX Table XIV COSCIUSIOHS BIBLIOGRAPHY ACKBOWI^BMRBT. . . biographical items COMMIT THE REPORT. . 62 62 63 64 65 66 72 73 74

PAGE 5

PREFACE The material contained in this dissertation is presented in a format similar to that used for the articles in Chemical Reviews . Y • • This was found to he the most satisfactory for proper interrelation of the several parts of the investigation. The manner of listing references is the customary one for technical works in organic chemistry of comparable length and scope. Journal abbreviations are the official ones of Chemical Abstracts. In conformity with present usage in research publications , all temperatures are on the Centigrade Scale.

PAGE 6

ISTROEUCTIOH AHD KBVISW or THE LITERATURE

PAGE 7

STATE? IKHY OP RESEARCH PROBLEM Sna fee oil, as used in this dissertation, refers to the glycerides obtained froa the fat lobes of the moccasin. This research project involves a study of the chemical and physical properties of snake oil produced by cold-pressing the fat lobes froa moccasin (AgldLstrodon piscivorus) (Lacepede) . The chief objective is a qualitative and quantitative analysis of the fatty acids present in moccasin snake oil. for a summary of the work done on snake oils up to this time, see data by McLaughlin and Pollard 1 and that of Young and Pollard 2 . PREPARATION 07 MOCCASIN SNAKE OIL The original investigations in this laboratory, which vere performed upon pooled samples of heat-rendered oils, indicate that heatrendering produces sane changes in the fatty acids originally present. Cold-pressing of the fat lobes of an individual species gives an excellent material for investigation. On the basis of the previous research by McLaughlin, Young, and Pollard, cold-pressed moccasin snake oil was determined to be more desirable for use in this investigation of fatty acids. The fat lobes of the species AgkAstrodon piscivorus (Laeepede) , commonly known as the Oottonmouth Moccasin, vere supplied by Mr. Ross Allen of the Ross Allen Reptile Institute of Silver Springs, florida.

PAGE 8

Upon removal of the lobes fro* the snakes, the blood was drained off, and the fat lobes were dried on filter paper, the lobes (in 25grern quantities) were wrapped with extreme care in filter cloth so that the pressures used would not force large amounts of solids into the oil. The liquid pressed out contained snake oil, hemolysed blood, blood cells, water, and some unbroken fat cells. A centrifuge equipped with two 250 e.c. oentrifuge tubes was operated at IhOO r.p.m. for two hours. Bach run produced about 350 grams of purified snake oil. METHODS Of SEPAEATION Of THE fATTT ACIDS Present-day knowledge of the fatty acids is the culmination of many years of study and research. 50 single factor has contributed more toward this knowledge than the many Investigations which have been made Upon the separation of the fatty acids. It is safe to conclude that future advancement in this field will be intimately associated with the development of more effective methods for the separation of those acids. The many processes which have been investigated for fatty acid separation, such as crystallisation of the acids or their derivatives, distillation of the acids or their esters, and other methods can be roughly divided into two groups. The first of these includes those processes which are useful for analytical or identification purposes only and which, because they are time-consuming or costly, have very

PAGE 9

little or no commercial application. The second group consists of those processes which are adaptable to the large-scale preparation of the acids. A rigid classification of the methods for fatty acid separation is not possible, since many of the commercial processes are also excellent laboratory procedures. Future research me y consort some of the now strictly experimental methods into commercially feasible processes. Among the procedures which hare been suggested for the separation of the fatty acids are distilling, crystallizing, and centrifuging, together with combinations of these methods. So single procedure is entirely satisfactory for obtaining pure fatty acids. A combination of two or more procedures gives better results. This is particularly true < | in the laboratory-scale separation of fatty acid mixtures, since a number of crystallizations are often combined with several fractional distillations » Generally the methyl esters of the acids are employed in the distillation procedures. The choice of Hie method of separation depends upon the starting material available and upon the objective to be attained. In the following discussion it appears logical to consider some of ... * • * ;*. f •»•**' the various unit processes separately. It should be realized, however, that they frequently be advantageously combined and that the various methods differ greatly with regard to their practical importance. Since this research is based upon analytical examinations of a natural fat, it seems desirable to include a discussion of the experimental methods which have to far found acceptance in the quantitative or semi-quantitative study of the natural fats. A fairly full account ‘ 3 -

PAGE 10

di«m

PAGE 11

Differences in the solubilities of the lead salts of saturated and unsaturated fatty acids hare "been utilized for separation. This separation is simplified if acids of low molecular weight are absent. Fractional crystallization from appropriate solvents at low temperatures may also "be employed for separating aired acids. \$hen fats contain appreciable amounts of acids of lower molecular weight than octanoic (caprylic), it is necessary to remove the more volatile acids of low molecular weight before proceeding to separate the higher saturated and unsaturated acids. This only applies to special types of fats such as milk fats and porpoise oils. PREPARATION OP THE HIKED FATTY ACIDS FROM A FAT The quantity of fatty acids required for an accurate analysis depends upon the complexity of the mixture of component acids. The amount runs from 20 grams for a simple type of fat to 500 grams for a complex type of fat such as fish oils. Complete hydrolysis of the original fat is essential. To saponify 100 parts by weight of fat, add 30 parts by weight of potassium hydroxide in about 500 parts of alcohol (95-100 per cent), reflux for three hours, and then remove most of the alcohol by distillation. The soaps are dissolved in water, and unsaponifiable matter is removed at this stage, if necessary. The free fatty acids are formed by adding warm, dilute hydrochloric acid. If unsaturated acids are present in quantity, care must be taken to prevent oxidation. When the acids have been liberated

PAGE 12

they axe extracted with ether and are dried under vacuus at 100°C . When highly unsaturated acids are present in quantity, it is not desirable to use too much alkali or to prolong the saponification. The hi^xly unsaturated fatty acids easily undergo isomerisation in the presence of excess alkali and lengthy heating. Therefore, with such acids it is desirable to use very little alkali in excess of the amount theoretically required and to heat under reflux only as long as is required. removal or tmsAFOR ifiablb matter FROM THE MIXED FATTY ACIDS Most, if not all, of the unsaponlf table matter present in a fat tends to pass with the other-soluble lead salts into the unsaturated or •'liquid* fraction. If the amount of unsaponlf iable matter in the fat is less than 0.7 per cent, removal is not necessary. In the moccasin snake oil, the unsaponlf table residue was not removed since the amount was only 0.46 per oent. REMOVAL OF fHB LOW KOLEXJULAR WEIGHT FATTY ACIDS If appreciable quantities of lower molecular weight fatty acids are present, they Should be removed before the higher fatty adds are separated. One method of removing the lover molecular weight fatty adds is by distillation in a current of steam.

PAGE 13

She amount of low molecular weight fatty aeids in moccasin snake oil Is found to he very slight (0.13 per cent)? therefore, It is not necessary to remove the low molecular weight fatty acids. mmi^soGRijmc adsobptioh or mixsd pasty acids In this problem the use of chromatography appeared to offer no advantages over certain other procedures. 1I0H-S0LVMT CRYSTALLIZAS10H The non-solvent separation of the fatty acids depends upon the fact that they ere completely miscible in the liquid state. After the solutions are cooled, the higher melting acids crystalline, and they can be separated from the liquid acids by suitable mechanical i»ans, such as pressing. Bo complete investigation of non-solvent crystallisation is given here since sufficient snake oil and equipment for low-temperature work were not available. It is probable that non-solvent crystallisation would produce a good separation between the monoethenoid and polyethenoid acids present in snake oil. SOLVENT CRYSTALLIZATIOB One of the best preliminary methods of separating the various acids is by the use of the differing solubilities of the lead salts of the 7 -

PAGE 14

fatty acids in ether. For a complete description of the technique used in the lead salt-ether method see the procedure given by the A.O.A.C.^ and the work of McLaughlin and Pollard 1 . SEPARATION OF POLTSTHEHOIC ACIDS BY LITHIUM SALTS Considerable work has been reported in the literature concerning the separation of polyethenoid acids by the use of lithium salts. The main solvent in use is the acetone-water solution. For a detailed summary of this procedure see Hilditch 10 , Ralston 11 , and Markley 12 . SEPARATION OF THE MIXED FATTT ACIDS BY CRYSTALLIZATION FROM SOLVENTS AT ION TEMPERATURES For a discussion of the advantages and disadvantages of this method see Hilditch 1 ®. Limitations of material »nd equipment precluded use of the method in this study. Separation of fatty adds by low-temperature, with or without solvents, is quite satisfactory ehen large amounts of fat are available. Its use when the quantity of fat is quite small is not advantageous. DISTILLATION The fractional distillation of fatty acid esters has been extensively employed as a means of separation of natural fats for analysis. The fractionation of the methyl esters is frequently used for the laboratoiy preparation of the individual adds. The most important earlier studies

PAGE 15

concerned with the fraction?! diet illation of fatty acid* and their esters are those reported by Krafft and coworkers^ , who investigated the boiling points of the acids and their esters under high vacuum. Caldwell and Hartley^* report the boiling points of 1 auric, myrlstlc, palmitic, stearic, and oleic acids in a cathode raccoon, using a Gaede pas?) with a Crookes tub# between the receiver and pump, and obtained values materially lower than those previously reported. These authors state that at & very low pressure a liquid has no boiling point; it sublines or evaporates, Just as water does In air, at a rate and temperature dependent upon the nature of the substance. Caldwell and Hartley also describe a method for fractionation of the fatty acids of butter and coconut oil. Brown 1 ^ describee an apparatus for the distillation of the fatty adds at low pressures. The separation of palmitic end stearic acids by vacuum distillation was reported simultaneously by Krafft 1 ^ and by Krels end Hafner 1 ?. In 1906 the separation of the fatty adds of coconut oil 1 ** end of 19 cod liver oil 7 by use of their methyl esters was first reported. The fatty acids of batter fat were separated by the fractional distillation of their methyl esters, and Elsdoa^ 0 * 21 * ^ 2 * reports the separation of the fatty acids of both coconut and palm kernel oil by a similar method. The fractional distillation of methyl esters was also reported for the separation of fatty adds of cottonseed oil 2 ^, hydrogenated herring oil**®, human dUc^, chanlmoogra oil^°.

PAGE 16

and peanut oil 31 . Stokoe^ described the fractional distillation of the ec thy 1 esters of coco neat oil fatty acids, and Armstrong, Allan, Moore^ reported the separation of tie fatty acids of coconut oil and p al« kernel oil hy the fractional distillation of their methyl esters. In 1923, Brown and Beal 3 ** announced the separation of the methyl esters of the fatty acids of menhaden oil hy vacuum distillation. They concluded that it ie possible to make a rough separation of fee acids according to their molecular weights hy this method. Later Brown^ 3 stated that the separation of the methyl esters of the fatty acids of "brain lipoids may he made hy fractional distillation at pressures varying from 4 to 7 sm. The separation of the highly onsaturated fatty acids of beef brains hy fractional distillation of their methyl esters did not succeed. Brown and Ault 36 stated that it is possible to distil fractionally the methyl esters of the debrominated acids of cattle, hog, and sheep brains. ©ie laboratory separation of fatty acids (as adds or as their •;7S esters) by the use of fractional distillation is considered to give incomplete separations. Channon, Drummond, and Golding^** claim that the method is of little quantitative value. Since 1930 investigation of the fractional distillation of fatty acid mixtures hy use of methyl esters has been intensified. Equilibrium between the vapor and liquid phase is not obtained in the unpacked columns. Also, the reflux ratio and the rate of distilla-

PAGE 17

tion are not under control. Separations performed in such equipment are consequently highly unsatisfactory. Improvements In laboratory distillation apparatus ere described “by a Somber of investigators 37 ’ 38 * 3 ** 40 * *1 . In 1930 J ant sen and Tiedch**^ developed an electrically heated, packed column which was enclosed in an evacuated Jacket to protect it against temperature fluctuation. This apparatus was used in the separation of the methyl esters of palmitic and stearic acids, and the device was also employed by these authors for the fractionation of the hi^i-melting acids of peanut oil. The apparatus was subsequently modified by later investigators** 3 * , and the Improved version was used by Lepkavsky, Peskov, and Evans** 3 for the separation of caprylic, capric, lauric, myristlc, palmitic, and erucic acids. Indications of association are observed in the separation of the higher acids, but they could be completely separated as their methyl esters. Modern laboratory distillation apparatus employs an electrically heated, packed column which permits a high reflux ratio and an adjustable distillate collection. The column is packed in a well insulated 'W outer shell to minimize thermal fluctuation. Attached to the column are a distilling flask, receiver, and condenser. Standard taper, ground-glass Joints are usually employed. The temperature at the top of the column is measured, by a thermometer, and the pressure is indicated by a manometer. The distilling flask and the column are electrical ly heated, and a constant temperature is maintained by rheostats. The side arm is electrically heated in 11 -

PAGE 18

order to prerent the distillate from solidifying. Low pressure is maintained by a standard Tacoma yajap attached to a large, evacuated steel droa since rapid pressure changes daring the distillation are undesirable, the laboratory columns are usually three to four feet la length and are permanently mounted. Their efficiency depends largely upon the type of packing material ©alloyed, reflux ratio , etc . Industrial columns are usually of the bubble-cap type. The rarious plates exert a scrubbing action and thus give the effect of multiple distillations. Hold-up in the apparatus is too great for laboratory 1 distillations. It is adaptable only to large-soalo, continuous work. Packed columns usually give materially less hold-up and are quite satisfactory in distilling small amounts of material. numerous investigations of many different types of packing material here been reported. In 1932 Whitmore and Lux^ described a column packed with Lessing rings (5 x 5 mm.) made of 50-mesh copper screen; and the next year Wilson, Parker, and Laoghlin^ 7 described a eoluate packed with a glass modification of the singleand double-tarn wire helices which ho _ had been used earlier. Weston reported a fractionating column which wee a modification of that described by Cooper and Poseg^*. The extensive investigations of Podbieleniak^ contributed much to the development of efficient, small-scale distillation equipment; and many of his results were iummariaed in hie excellent article published in 1933. In this article he described his column and gave hie recommendations for fractional distillation. 12 -

PAGE 19

reported toe separation of the methyl esters of palmitic, stearic, oleic, and elaidic acids by the use of toe Podbielnlak apparatus . Comparative studies of divers if ied p ackin g material have "been made “by Fenske , Tongberg, and Ojaiggle^, These authors state that wire or glass helices, carding teeth, and Jack chains were found to he the hast packings. The shape and distribution of the packing material are important determinative factors in the efficiency of fractionating columns. However, the importance of such factors as the contact surface and the proper drain-hack cannot he over-emphasized. Tongherg, Qpiggl* and Fenske 52 describe an efficient fractionating column packed with one-turn glass helices. Packing material composed of wire helices is claimed to he extremely efficient 53, 54 * 55 . A column packing of close-fitting wire helices was used by Sehoenheimer and Ritteriberg 5 ^ for toe fractionation of methyl paladtate from methyl stearate. The use of fatty acid ester distillation methods of fat analysis was reviewed by Longeneeher 5 ^ in 1940. The oonical type of packing was described by Stedman 5 ®* 5 ^* A detailed description of this type of packing and results of numbers £fk of efficiency tests were given by Bragg . In recent years toe use of toe molecular still has been of great importance in fractionating very high-boiling substances. Molecular distillation of toe highly unsaturated acids of fish oils was reported by Farmer and Fan den Heuvel^. 13 -

PAGE 20

FRAOTIOHAI DISTILLATIOB OF HIGHER FATTY ACID ESTERS AS LOW PRESSURES As already stated, those portions of the mixed f tty acids which are to he further resolved hy ester-fractionation are converted into methyl esters hy boiling with about four times their weight of methyl alcohol in the presence of approximately one per cent by weight of concentrated sulfuric acid. She unesterified acid is removed hy washing the ether solution of the esters with dilute potassium carbonate solution. She conversion into methyl esters is usually 92-98 per cent effective. If production is below this figure, the unesterified acids should he recovered and re-esterified. She methyl esters are used primarily because of their slightly lower boiling points as compared with those of ethyl esters. With the operating pressures ranging from 0.1 to 0.2 mm. now readily obtainable with the ordinary rotary oil pumps, this factor is less significant than formerly. However, it is convenient to continue using the methyl esters sines so much data already have been recorded for methyl esters. The recorded boiling points at the head of the fractionating column are not of scientific significance in this type of distillation. However, the column head temperature should be systematically recorded; since, in conjunction with those of the heating bath, they afford a reliable indication of the smoothness of operation, therefore of the efficiency of the fractionation. 14 -

PAGE 21

At 0*2 ran. pressure, the column head temperature* for methyl laurate, methyl palmitate, end methyl oleate (or stearate) are approximately 75-80°. 110-15°. and 130-135°. respectively. Unless a large fraction of known range is being collected for refractionation, the wol*$it of any one ester-fraction should not greatly exceed about ten grams and may be as little as two or three grams. Even when it is reasonably certain that a mixture of constant composition is distilling in large quantity, it ie better not to exceed the amount stated. Errors in the analytical determination of saponification equivalents or Iodine Values are minimized if oarried out on several small fractions rather than on one very large cut. The minimum weight of a fraction is determined simply by the amount required for accurate determination of its analytical characteristics. • •• V ** The unsaponif lable or non-fatty matter generally remains in the residual, undistilled ester fraction. The amount of unsaponif iable residue may be determined hy removing it from the alkaline solution obtained after determination of the apparent equivalent of the residual ester. This is followed by recovery of the fatty acids and redetermination of their equivalent*. The amount of esters in R grams of a residual ester is them R x ( e< ? u l va l ea ^t of recovered of fatty acid 4Ifr) apparent equivalent of residual esters The effect of heat upon unsaturated fatty esters daring the fractionation is reported by Harris et_ a if 2 They report that esters of acids 15 -

PAGE 22

with three or fewer unconjugated double bonds are substantially unaffected. It is their opinion that sore un saturated polyethenoids (e.g. fish oil) acids are also little altered, although slight rearrangement to conjugated i some rides, followed by some dimerization, may taks place to a very small degree. Any material so altered appears in the residual, undlstilled esters. These authors concluded that the changes induced in the course of a fairly prolonged distillation were insignificant. They state that the action of reagents (alkali) and solvents daring hydrolysis and treatment of the fats prior to fractionation is a much store important potential source of structural alteration than any condition encountered daring fractional distillation at low pressures. It should be pointed out that esters of conjugated higher fatty acids undergo polymerization and/or cyclization at the temperature at which they distill in heated fractionation columns of the described type and that such esters (s.g. methyl elaeostearates) cannot be distilled in a column at too hi^x a temperature. Korris and Terry^ gave the precise boiling point data for the following esters at one xsa. pressures methyl myri state, 114°; methyl palxdtate , 136®; methyl stearate, 155*5°; nethyi oleate, 152.5°; and methyl llneoleate, 149.5°. Althouse and Triebeld^ also reported some data on methyl oleate, methyl llneoleate, and methyl linolenate. They claim that the boiling point of the three acid methyl esters are in reverse order from that given by Korris and Terry. All agree that the boiling points of all three of the acid methyl esters are extremely dose. 16 -

PAGE 23

SO MS FEASUBSS OF SHE CAIOOLAfflOU OF THE COMPOSITIOIT OF IHDIVTDUAL BSTHR-F8AOTIOBS The purpose of the ester-fractionation procedure is the production of a series of ester-fractions for use in the analysis of a mixture of fatty acids. Each of these ester-fractions should contain not more than two unsaturated esters. The composition of such fractions can be directly calculated from their saponification equivalents and Iodine Values. This is true of most fats in which the only uns; turated components belong to the series of acids (oleic, linoleic, and linoleaic). When, as is usually the case, both oleic and linoleic acids are present, the mean Iodine Value of the C-^g unsaturated esters can be determined. Except for a negligible error, it can be assumed that the distillation of these esters is proportionally constant throughout the operation. The boiling point of methyl llnoleate is very close to that of methyl oleate. If linolenic acid is $he only poly-ethenoid acid present, a complete determination of such esterwfraetions may be made. The determination may be made by use of Iodine Value and saponification equivalent in conjunction with the spectropho to metric determination of the linoleic acid. In some fats, especially those from aquatic sources, the mixture of unsaturated acids, psrticulorly in the G 2 q and C ?2 series, is very complex. Em constitution of many of the poly-ethenoid acids is still uncertain, and it is necessary to adopt a different method of evaluating the ester-fractions. 1 ?

PAGE 24

DETAILS OP THE METHOD Of CALCULATION INVOLVED As already stated, the composition of an ester-fraction which contains esters of only tw saturated acids with the unsaturated acids ell of the sane carbon content can he deduced from its equivalent and Iodine Value "by comparatively simple calculations as follows} If a fraction of weight w, equivalent E*, and Iodine Value I v contains a weight u of unsaturated esters (of the same carbon content, e.g. C^g) with Iodine Value % end equivalent and the mean equivalent, E a , of the saturated esters (v— u) present follows from the equation! from £ f the weights of saturated esters are calculated as a binary mixture of the hoaologucs (e.g. and C^g) between the equivalents of which Eg lies. Again, ester-fractions which include only unsaturated derivatives of acids of two groups in the homologous series eaa be evaluated directly from their saponification equivalent. In certain cases the computations become somewhat more complicated, since the weights of more then three independent components are involved. u a v ( ) w— u 18 -

PAGE 25

If x, y, and z axe the respective weights of the one saturated and the two unsaturated esters in a fraction of weight w* and 3 § x » ®y> S s , are the corresponding equivalents and I y , I 2 , I v a*® the corresponding Iodine Talues , then (1) x 4 y 4-s tt w (2) x/lx 7/»7 */*, * w /*w (3) y-iy *** * v.i* (In equation ( 2 ) the saponification values Y x , Vy, V 2 , T v can he used if desired, the equation becoming: x.Y x *.T X 7.f x x w.T v ) The values of 7 and e, the unsalurated components , are thus determined while, from that of x, the binary mixture of saturated esters is evaluated directly from its equivalent . It may "be pointed out that, in use, the equations ( 1 ), ( 2 ), and (3) can he conveniently simplified by employing in equation (2) the reciprocal of the equivalents times 10^. She proper method of evaluation of the ester-fraction data in any given case depends upon the particular circumstances. In the majority of analyses, if the more efficient type of fractionating colunm has been used, it is not necessary to isolate and determine the equivalent of the saturated components of an ester-fraction. 19 -

PAGE 26

ESSER-mCTIOBS OF TBS MOTE SiffiUBAEED CB "SOLID" ACIDS The lead, salts of tetraand hexa-decenoic acids are freely soluble la ether. Since the small Iodine Values la fractions of equivalent "below 296 nay he attributed solely to methyl oleate, the calculation is straight-forward. This method has been need when "solid" esters hare ; * I been distilled with a simple distillation apparatus. When the electrloally heated column Is employed, it Is evident • 1 that traces of these acids are detectable in the and eaterfractions. In such cases it may be assumed that the saturated and unsaturated portions of the fraction have the earns equivalent. In instances in which the saturated esters of a fraction have been isolated and their equivalent determined, the general equations given in the preceding paragraphs may be used with x as the weight of saturated esters of determined equivalents, !j,. The determined equivalent Ej. may then be used to calculate x ae a binary mixture of saturated esters. RSTBR-FRAOTIOHS OF THE MOKE TJSSJOTAEED OB "HOTTED" ACIDS In fractions with equivalents below those of unsaturated Cjg esters there are sane palsltates and much larger amounts of znyristates. The saturated esters present in these fractions may be isolated and the components calculated by the general equations given. If an efficient fractionating column was used, calculations of the esterfractions of equivalent below 270 on the assumption that thdbr 20 -

PAGE 27

saturated and unsaturated parts have the seme equivalents is sufficient. Ester-fractions with equivalents between 270 and 290 are calculated from the general equations. They are calculated as mixtures of palmitic, hexadeesnoic, and unsaturated C 18 esters. When lead salt-ether separation has been employed, the presence of stearic acid in the "liquid" acids may he considered to he of little importance. Tractions consisting wholly of unsaturated esters should he evaluated with the following facts in minds .... • • . .. . '-i,. • . .v • • So long as the unsaturated C^q ester-fraction consists mainly of oleate , with not more than 10 to 20 per cent of linoleate, the determined equivalents correspond closely with those deduced from oleate and linoleate Iodine Values. When the ester-fractions contain large proportions of linoleate, the determined equivalents of unsaturated O^g ester-fractions tend to he appreciably lower than those observed by the iodine values, fhe cause has not been proven, but it may be due to the presence of traces of sntodxidiaed esters which have undergone scission during refluxing with alcoholic potassium hydroxide. It should be home in mind that the determined equivalents of the more unsaturated C^g ester fractions are usually slightly lover than the true values. 21 -

PAGE 28

In ester-fractions immediately preceding those which consist wholly of unsaturated C 18 esters, there are very snail anounts of satorated ester*. Actual calculations of the mean equivalents of these ester-fraotions prove difficult, and result* are not too reliable. It ha* been found that it 1* better to credit then as pala&tate than to use their deduced equivalent*. When only linoleate and oleate are present, the proportion of each follows directly from the iodine values. Fractions with equivalents above those of unsaturated C^g ®ust be calculated to binary mixtures of (^g and (fo, or C 20 and 0g 2 , according to their observed equivalent*. In spite of the numerous variant* and assumption* suggested in the preceding paragraphs, it ha* been found that reliable analyse* may be obtained througi calculation of ester-fractions . Each ease must be considered upon it* merits, and the calculations should take into account the methyl eater* present in the mixture. She analyses of the more unsaturated fractions rfvould be completed quickly, and care should be taken to avoid oxidation. With proper precautions , it is possible to obtain component acid data of the order of accuracy indicated below. Easing determined the approximate components of each fraction, it is a comparatively simple matter te obtain the composition of the "solid" and "liquid* esters. From this it le possible to calculate the amount of each fatty aeld present in the original sample. In the case of 22 -

PAGE 29

complex fata the operations are extremely lengthy and tedious, and a final accuracy of not more than 1 2 units may he reached in this calculation. Bren with the somewhat greater inaccuracy of the figures for some of the more highly unsaturated components of fish and allied fats, it has "bean claimed that the accuracy is comparable with that for many other natural products of a comparable complexity. The "liquid* fatty acids obtained in a component acid analysis «) ynO ri be converted into methyl esters, and their fractional distillation carried throng without delay. Unless das precautions are observed, adds of the more unsaturated types are liable to under©) polymeric or other changes during the esterification. This has been shown to be especially likely to occur during concentration of the mineral acid by removal of the methyl alcohol. In such instances it ie necessary to (a) employ the mlnlsam concentration of sulfuric acid to secure nearly complete esterification (probably as little as 0.5 per cent), ad (b) remove no unchanged methyl alcohol by distillation, but to pour the reaction mixture into cold water and proceed at once to extract the esters from the dilute alcohol with ether. The latter precaution is very iiqportaat. The "solid* acids obtained in separations carried out as above contain the whole of any stearic or higher saturated acids present, nearly all the palmitic acid, a considerable proportion of any mjrristic acid present, and smaller proportions of any lower saturated 23 -

PAGE 30

acids. They will also Include, of unsaturated acids, minor percentages of oleic acid. Mono-ethenoid acids of the or lover series, and poly-ethenold acids of any carbon content will, on the other hand, pens almost entirely into the "liquid* acid group. In addition to nearly all the oleic acid present, the "liquid" acids nay include nost of the ©ore unsaturated acids of the Gig or series; the mono-ethenoid adds of the Cjg or lower series; mch of the saturated Og, C^, 0^. minor amounts of myristic; and traces of the palmitic. ABSORPTIOH SPECTRA Owing to the presence of the double "bond in the crbonyl group, the saturated fatty add* show a typical absorption spectrum. The ethyl enic acids not only exhibit an absorption due to the carbonyl gxwg> but also show characteristic absorption values dependent upon the number and the relative positions of the double bonds. Since the ultraviolet absorption properties of the saturated fatty acids are dependant upon the presence of the chronophorlc carbonyl group. It is evident that the absorption characteristics of the saturated adds should be qualitatively similar. Ranart-I/ueas and associates^ investigated the ultrar-violet absorption properties of formic, acetic, butyric, caprole, eeprylic, lauric, myristic, palmitic, and stearic acids between the wave lengths of 2000 and 2 555 Angstroms. They stated that the absorption coefficients of

PAGE 31

the higier members are essentially similar. These authors concluded that since the length of the chain does not materially affect the absorption coefficient, methyl and methylene groups cannot be considered as chromophores in this region of the spectrum. Bielecki and Henri® had studied a number of saturated acids in the range 2144 to 2600 Angstroms earlier and had contended that an increase in the length of the hydrocarbon chain was attended by a shift in the absorption band toward the red and also by an increase in the magnitude of total absorption. Severer, Ley and Arends^ stated that thie generalization does not apply to the higher saturated acids. Birr and Miller® compared the ralnes of A for acetic and palmitic acids for rarious values of the molecular extinction coefficient 6 . Studies of the absorption coefficients of the saturated acids hare led to the postulate that in the liquid state the acids exist in a dissociated and in a noa-dissociated fora^* Bster formation does not materially affect the character of the absorption curve. It has been shown that the v allies of the rarious esters differ only ‘ slightly from those of the parent acids*^, but absorption is radically changed by salt formation^. Unsaturation in the hydrocarbon chain produces a change in both thb Intensity and the position of the absorption bands, the absorption being due not only to the carbonyl group .%pt also to the ethylenie bands. Conjugation is attended by a marked shift in the absorption band toward the red. This phenomenon recently has been the subject of 25 -

PAGE 32

extensive study and ha* been proposed for two in distinguishing conjugated from unconjugated adds or esters^ 1, ^ 2 * After a study of the absorption spectra of unsaturated acids Tan dor Hoist' reported characteristic maxima which depended upon the number end relative positions of the ethylenic bonds. Dingus 11 and Thompson^ reported the presence of a Maxima at 2700 Angstroms f*r alpha-el e o s t earic acid. An isolated double bond between carbon atoms produces an absorption at 1800 Angstroms. Mitchell and Krayblll? 2 stated that from theoretical considerations, absorption bends in the spectral region of wave lengths longer than 2200 Angstroms are not characteristic of non-conjugated linoleic or linolenie acid. Burr end miler* 8 recently extended the absorption studies of the unsaturated acids to 2100 Angstroms and noted that the unsaturated acids gave an absorption curve that is a combination of the saturated acid absorption curve plus an absorption curve due to the ethylenic group. The new band for oleic acid occurs at a longer wave length than that observed for 3~ heptene, and a comparison of the absorption spectra of oleic, linoleic, and linolenie adds shove that the addition of ethylenic bonds produces a further shift of this characteristic band toward the longer wave lengths. » Determination of the infrared spectra are frequently useful in establishing the structure of isomeric unsaturated compounds . 77 . 78 . 01s acids show a strong infrared absorption, but this absorption im absent in the trans form.

PAGE 33

Studies of the ultraviolet absorption spectra of the saturated fatty acids and of oleic, linoleic, linolenic, and araehidonic acids have been extended to 2100 Angstrom by Burr and associates^. Sines none of the uns turated acids readies a peak at 2100 Angstroms, these studies were later extended to the extreme ultraviolet; and many interesting observations were made® 0 . A comparison of the absorption spectra of nyristic acid dissolved in ethanol, iso-octane, and heptane shows that the polar solvent, ethanol, shifts the absorption maximal to a lower wave length and gives lower absolute values for extinction coefficients. All the saturated acids have a broad band with the maximum at about 2050 Angstroms and an intense absorption between 1850 and 1730 Angstroms. Tins aturat ion in the fatty aeids produces absorption similar to that of the unsaturated hydrocarbons. Elaidinization of oleic acid shifts the absorption curve toward the visible end of the spectrum. The available spec tropho tone trie data on the fats, fatty acids, and their esters have recently been compiled®*. 82 Brice continued his research on existing spectropho tome trie methods, modified for application primarily to the detection and estimation of lew proportions of conjugated and nonconjugated components in fats, oils, and soaps. The method is also applicable to fatty materials having hi g h proportions of those components. Modifications include corrections for absorption by interfering substances, use of alkaline glycol as an isomerization medium in the analytical procedure, and correction of absorption data on the

PAGE 34

isomerized product. The abeoiptlon data correction io needed to compensate for the absorption by conjugated consonants which were in the material before the isomerization. Result! on some new speetropho to astric analyses of fatty acids were reported “by 0* Connor®^ In 1946 tajAberg 8 ^ stated that there were various speetropho tometric changes in fats during rancidification. Further work has been done on the speetropho to metric studies of the oxidation of fats through the oxygen absorption and ehromophore production in fatty esters. Holman®^ * ®*> investigated the relation^ ship of eacygen absorption to absorption band development in the esters of the common unsaturated fatty acids. The data reported mst be evaluated carefully before any conclusions are reached . Beadle®? recently published a method for the use of ultraviolet spectrophotometry in the study of change# in the double-bold system of fatty acids. His procedure is claimed to be of special help in cases where both conjugated and non-conjugated coss>onenta occur. Hilditch and Shrivasteva®® reported that the determination of linolenic acid by the speetropho tome trio method gave a higher value than the amount of linolenic acid present should give. Ho explanation was offered for this excess over the true values. 0*Coimor®9 obtained results in the determination of the alpha and beta isomers of elaeostearle acid in tung oil by the use of a 28 -

PAGE 35

spectrophotometer. The remate obtained from spectrophotometric sad chemical analyses were not In complete agreement. la 1948 Hendrickson et. a£. 9 ° discussed the application of ultraviolet spectrophotometry in drying oil research. In their article they traced the development of teste for the determination of fatty acids by epeetropho tome trie methods. SHE IDEHTIFIGATIOB OF SHE FATTY ACIDS Ijhe methods used for the identification of pore organic compounds and for ascertaining the components of mixtures axe generally familiar to all chemists. Physical constants such as the melting points, refractive indices, and specific gravities are universally recognized as contributing to the identification of organic compounds. The preparation of a characterizing derivative frequently offers further proof of the identity and purity of the compound in question. Unfortunately, very few acceptable derivatives of the rmsaturated fatty acids have been described. A number of investigators here developed derivatives of the saturated acids which have proved to be very useful in the identification of these compounds. The close structural similarity of the various series of fatty acids has made possible the use of a number of analytical procedures which have proved to be of great value. Such constants as the neutralization e^sivalent s , the Iodine Humber, the acetyl value, and the more recent thiocyanate number are 29 -

PAGE 36

well-known, tanas which hare "been applied, to the fats and fatty acids for many years. Without attempting to detract from the usefulness of such analytical constants, it should he pointed oat that they axe not, per se, proof of the identity or purity of an individual acid. For instance, an equimolecular mixture of palmitic and stearic acids will possess the neutralization equivalent of margaric acid* and a mixture of unsaturated adds nay possess the theoretical iodine number of oleic acid. 30 -

PAGE 37

EXPSRIMEHTAIi DATA AKD DISCLTSSI05

PAGE 38

EXPKRIHEBm DATA A3® DISCUS SI OH TABLE I Data on Oil from Cold-Pressed Pat Lobes of Moccasin (Agklstrodon Piacivorua) (Lacepede) Moisture and Tolatile Matter. • less than 0.1 $ Specific Gravity (25®/4°> .................. 0.9268 Index of Refraction (25°) 1.4690 Saponification Ruaber (Koettstorfer Humber) 192.6 Iodine Roaber (Hams) .................... 104.4109.1 Soluble Acids ........... 0.13 i Insoluble Acids .94.85 % Reichert-Meisal Talus (Soluble Tolatile Acids). ....... 0.07 i> Polensko Talus (Insoluble Tolatile Acids) 0.04 $ Saturated Acids .22.7 ‘p Unsaturated Acids ............ .72.7 £ Tree Patty Acids. .. 0.31 £ Acetyl Talue. .... ........... 4.1 Unssponifiable Residue. 0.46 £ Ritrogen. . 0.00 f Phosphorus. ......... ....... 0.00 $ Specific Rotation —0.12° Thiocyanogen Talue. 77*2

PAGE 39

THB SIGUmCAHCE OF TAHJ8 Z From the data in Table I, it ie seen that the amount of soluble acids is 0.13 per cent, the soluble volatile acids are 0.07 per cent, and the insoluble volatile acids axe 0.04 per cent. This information she vs that the armunt of low molecular weight fatty acids in moccasin snake oil is very low. Low molecular weight fatty acids means those acids of lover molecular weight then 1 ccuric acid. The unsaponifiable residue is Important since the amount of higher molecular weight alcohols present is shown by the quantity of the residue. Data indicate that the amount of higher molecular weight alcohols (of the cholesterol type) present in moccasin snake oil is 0.46 per cent. Several color tests run on the unsaponifiable matter show that cholesterol-type alcohols are present. An acetyl value of 4.1 was found. This value is actually the •hpparent acetyl value 1 * rather than the true acetyl valued. After subtracting the acetylation due to the free fatty acid, the acetylation due to the free hydroxy groups of the glycerol (produced when the free fatty acids were formed), and taking into account the expertmental error in the determination of acetyl values, it was found that the "true acetyl value* is very small. It is evident that the amount of hydroxy acids present in moccasin snake oil is very slight. The acetyl value is used for the purpose of determining the amount of hydroxy acids present in moecasin snake oil. • 32 -

PAGE 40

It siiould be pointed out here that the difference between the Iodine Value and the thiocyanogen Tslue of an unsaturated acid mixture is a measure of the poly-unaaturated acids present^ . Since the tiiiocyanogen value of the moccasin snake oil is 77.2 and the Iodine Value of the moccasin snake oil is 104,4, it is apparent that the amount of po lywuns a turated acids present should be about 26.8 per cent. The analysis of the fatty acids present in moccasin snake oil substantiates this finding. It must be pointed out that the iodine number of a compound is not always a true measure of its uns: turatioa. An examination of the theory behind the use of iodine numbers in determining the unsaturation -v °f different compounds shoved several important factors. Reports have stated that conjugated double bonds fail to give the theoretical iodine number and that the distance of the double bond from the carboxyl and the methyl group in any acid affects the iodine addition. ETHER-INSOLUBLE POLYBBOMIDES FROM MOCCASIN FAT The percentage of ether-insoluble polybromides , determined as described by Icvkovitsch? , gives a preliminary estimate of the amount of unsaturated C 2Q and fatty acids. From 63 grams of "liquid" acids (obtained from the lead salt-ether method of separation) there were produced a total of 15 grams of ether-insoluble polybromides . From this mixture of polybromides was obtained a fraction which began to blacken at 200°C., and on further heating the fraction deeooposed

PAGE 41

on farther heating. According to Lewkowitflch, this is typical of arachidonic acid. Wien Levkowitsch made this statement it was thought that the empirical formula of clupanodonic acid was G^^O,, and that of arachidonic acid was C^qH^oO,. At the present time the empirical formula of clupanodonic acid appears to "be and that of arachidonic acid is still considered to he ©a the basis of the amount of ether-insoluble poiybromides obtained. it was calculated that the original snake oil contains at least 7 per cent of the highly tmsaturated 0^ and C 22 fatty acids. It should he pointed out that the ether-insoluble poiybromides do not always give the actual quantity of the highly unsaturated C^q and C 22 fatty acids present^. DISTIELmOH OF ESTER FRACTIONS FROM MOCCASIH FAT Fifty grams of "liquid** fatty acids, obtained by the use of the lead-salt-ether method, were added to 200 grams of methanol and 3 grams of concentrated sulfuric acid. The mixture was boiled for about fifteen minutes, and then the excess methanol distilled off until about 80 per cent of the methanol was removed. The mixture was then poured into 1 liter of distilled water, and 200 grams of ether were added* The ether layer was then washed with a dilute solution of potassium carbonate. After the unreacted fatty acids had been removed from the esters, by washing repeatedly with potassium carbonate solution.

PAGE 42

and the ether vae distilled off, leaving the methyl esters, A 92.4 per cent conversion into the methyl esters resulted. The data in Table XI were obtained when 45. 3 grams of Be thy 1 esters were distilled at about 1 can. pressure in a Leefey and Ewell93 type column. The methyl esters were obtained from the "liquid" acids separate* by use of the lead-sal t-ether procedure. The distillation fractions were examined for refractive indices, saponification equivalents , end Iodine Values. With this information it was possible to use the Hilditch method of calculation for the composition of the individual ester-fractions® according to the procedure as discussed in the review of the literature. • 35 -

PAGE 43

TABIE II Data Obtained from Distillation at 1 nan. of 45.3 Oraas of Methyl Esters of "liquid* Acids. "Liquid" Acids Produced by Leed-Salt-Sther Method Fraction Temperature (°o.) Weight oi Fraction (in g.) Saponification Iodine Value l...™ 107-317 .. 0*2, 1.4452 .. «• 56.3 2 317-128 0.7 1.4445 230.0 ‘ T* : , r"' l 50.8 • y 128-132 ! 1,0 1.7 1.4449 250.0 63.6 70.3 80.1 * 133-138 1.4460 263.5 5 138-148 3,7 1.4483 280.0 6 149-153 13.2 1.4520 298.2 102.9 104.9 f 153-157 8.1 1.4532 302.1 ft 158-162 4.2 1.4542 293.5 104.9 * 162-165.5 1.2 0.9 1.4598 296.8 87.4 10 1.4679 290.0 120.3 11 168-169 1.9 1.4679 276.0 109.1 12 170-200 *.3 1.4805 333.3 212.0 , 13 (Residue) » | 4.2 4 J 1 I *: f 36 -

PAGE 44

From the saponification equivalents, refractive indices and Iodine Numbers, the brerJ&-Aovn of the various fractions are given in Table XII TABLE XXX Ester Composition of Tractions Obtained from Distillation at 1 an. of 45.3 grams of Methyl Esters of "Liquid 1 * Acids. "Liquid" * Acids Produced by Lead-Sal t-Ether Method Sample ! Weigit of Traction (in g.) Amount of Esters (in g.) Methyl myristate Methyl palmit ate Me thy hexadecen oate . Me thy oleat > Methyl i linolate Methyl arachidonate Methyl clupano donate 1 2 3 4 5 6 7 8 9 10 11 12 11. 0.2 0.7 1.0 1.7 3.7 13.2 8.1 4.2 1.2 0.9 1.9 *.3 0.08 0.32 0.22 0.11 0.11 0.33 0.44 0.12 0.38 0.67 1.26 1.67 1.59 10.56 6.48 3.36 2.64 1.62 0.84 1.2 8.9 1.9 0.86 3.44 TOTAL 535 —ML 0.88 4.10 —21*22 —J,k : — a The 4,2 g. present is 0.3 g, of unsaponifiable residue and 3.9 g. of of residue composed of highly unsaturated acids

PAGE 45

The "liquid* fatty acid fraction obtained by the lead "altether method is 71*0 per cent of the total snafce oil. On converting the values for the various methyl esters, as shown in Table III, the following percentages of acids are obtained* TABLE XT Percentage Congjosition of "Liquid" Tatty Acid Traction ^ & (Calculated from Data in Tables XX and XXX) Heme of Acid Percentage Myristic 1.15 Palmitic 1.39 Hexadeoenoic 6.4 3 Oleio 34.48 Linolele 16.00 Arachidonic and Clupanodonic 10.98 0.S7 TOTAL 71.00 j 38 -

PAGE 46

The data in Table was obtained by fractionating the methyl esters from the "solid" aeids, TABLE V Data Obtained from Distillation at 1 am. of 20.0 Grans of Methyl Esters of "Solid" Acids. "Solid" Acids Produced by Lead-Salt-Ether Method Fraction Temperature (%) Weight of Fraction (in g.) _25 D Saponification EquiTalen t Iodine Value (Harms) 1 121-124 0.2 1.4358 3.2 2 125-128 0.3 1.4365 m 0.7 3 129-139 4.1 Solid 270.9 1.7 4 140-143 4.5 Solid 275.3 0.3 5 144 3.3 Solid 280.2 0.0 6 145-150 1.2 1.4398 277.0 6.5 7 150-154 1.0 Solid 281.9 H.7 8 154-154 3-9 Solid 299.5 8.4 9 Residue 0.7 Solid 292.5 9.3

PAGE 47

From the saponification equivalents, refractive indices, and Iodine Values shown in Table V the break-down of the various fractions given in table 71 has been calculated. TABLE 71 Ester Composition of Fractions Obtained from Distillation at 1 an. of 20.3 gross of Methyl Esters of "Solid" Acids. "Solid" Acids Produced by Lead^Salt-Ether Method Sanple Weight of fraction (in g.) • Methyl nyristate Methyl hexadeeenoate Methyl paUnitate Methyl oleate Methyl stearate 1 0.2 0.194 0.006 ‘ .j 2 0.3 0.298 0.002 3 4.1 0.073 4.027 • * 4 4.3 3*645 0.855 55 3*3 2.079 1.221 6 1.2 0.972 0.091 v 0.137 7 1.0 0.575 0.063 0.36? 8 3.9 0.390 3.510 9 0,7 0.140 0.061 0.499 TOTALS 19.2 0.492 0.081 11.438 0.605 6.589 * 40 -

PAGE 48

The •feolid" fatty acids fraction obtained by the lead-saltether method is 24.4 per cent of the total snake oil. On converti ing the alues for the various methyl esters, as shown in Table TI, into the percentages of the original snake oil the following results are obtained t TABLE VII Percentages of Batty Acids in the "Solid" 7r act ion (Calculated from Table VI) Bane of Acid Percentage Hsrrietie 0.62 Hexadecenoic 0.10 Palmitic 14.34 Oleic 0.77 Stearic 8.37 TOTAL 24.40 When the acid percentages from the "liquid" and "solid" fatty acids fractions were added the data given on the following page Table VIII were obtained. 41 -

PAGE 49

TABLE Till Percentage of Patty Adds in Combined "Liquid" and "Solid” Fractions of Moccasin Snake Oil. Fractions Obtained "by the Lead-S al t-E ther Method Same of Add Percentage Juristic 1.77 Palmitic 15.93 Stearic 8.37 Hexadecenoic 6.53 Oleic 35-25 Lino laic 16.00 Araehidonic and Clupanodonlc 10.97 TOTAL 94.82 Table III is based upon the distillation data shown in Tables II to YIX, inclusive. BXPBRIMBHTA1 DATA — THE SATURATED PATTI ACIDS The data obtained from the fractional distillation of the "solid" fraction of the moccasin snake oil is given in the section on distillation. After separation of the "solid* fatty acids from the "liquid" fatty acids by use of the lead-salt-ether procedure, it was found that the Average Molecular Weight of the "solid" fatty add fraction is 264.1, while that of the "liquid" fatty acid fraction is 290. — 42 —

PAGE 50

Before myrnmining the data obtained from the leadU-salt-ether separation, the sample was tested for possible oxidation of the unsaturated acids. The following data were obtained* Iodine Bomber (Hamm) , Original Moccasin Snake Oil... 107.1 Iodine Humber (Hanna), "Solid" Traction 9*7 Iodine Humber (Hazms), "Liquid" Fraction... 145.6 The combined Iodine Values of the "liquid" and "solid" fatty acid fractions account for 99.2 per cent of the unsaturation of the original snake oil. Work with the "solid" acid fraction showed that it contained a high percentage of palmitic and stearic acids. Therefore, it was desirable to hare a fraction which contained only palmitic and stearic acids. This purpose was achieved by use of a solution of methanol and water of a specific gravity of 9*911 at 0°C. The solution was saturated with palmitic and stearic acid, and to this saturated solution was added 0.9132 g. of the "solid" acids from moccasin snake oil. After the mixture had remained overnight in a Trigidaire, the precipitate was weigied; and a total of 0.8234 g. of fatty acids was obtained. Under the conditions of the experiment, only a mixture of palmitic and stearic acids should have precipitated. The freezing point of the precipitate was determined to be 54°C. According to Balston^, the freezing point of a binary mixture of palmitic and stearic acids in which the mole fraction of palmitic is 0.7 and the mole fraction of stearic is 0.3, is 54°C. The average

PAGE 51

m locular weight of Balaton’s mixture was 2 65.6, while the actual average molecular weight of the precipitate was 265 • 2. SPKCTROPHOTOK-ffRI C DATA ABB ITS E7AHJATI0S The spectrophotometer used was a Beckman Model TJJ with a Bo . 2501 Ultraviolet Accessory Set attached. The ultraviolet acceeory set provided the necessary equipment for making measurement a in the wave length region of 350 to 220 millimicrons. The above apparatus was tested and runs were made to standardise and check it for efficiency of operation. Bor the standard methods of operation the reader is referred to the various Beckman Bulletins. The procedure used in tills phase of the research is one which was devised by Mitchell, Krcybill, and Zscheile^ and was later elaborated upon by Beadle and Xraybill^. They report that it is possible to determine the percentage of 1 1 nolei c, linolenie, and arachidonic acids by iso me rising the double bonds into conjugated positions In an alkaline solution of ethylene glycol. The method used was as follows: Between 0.1 and 0.2 g. of moccasin snake oil was accurately weighed and placed in the type of small vial used for Iodine Bomber determination. In a 15 x 2.5 cm. (6 x 1 in.) teet tube were placed 10 ml. of alkaline glycol reagent. The test tube was covered loosely with a glass cap and was kept immersed at a constant depth in an oil bath at 180°C. When the temperature of the reagent in the test tube

PAGE 52

had reached 180 0 C. the vial containing the roecasin snake oil was dropped into the tube. The teat tube was swirled three tinea at one-minute intervals to mix the fat with the glycol solution. After 25 minutes the tube was removed from the bath end was cooled rapidly under the tap. The contents of the tube were transferred quantitatively to & 250-ml. volumetric flask. Ethanol was used to wash out the tube, and the volume was diluted with 99 per cent ethanol. The samples stood in a refrigerator overnight. The materials removed from the tube in the hot alkali solution had precipitated. The solution in the volumetric flask was brought to room temperature, and a portion of It was filtered. Proper dilution for absorption measurements were made with 99 por cent ethanol. It was neoessary to carry a blank solution of alkaline glycol throughout the whole procedure, including dilution, for use in the solvent cell. PREPARATION OP THE ALKALIES GLYCOL SOLOTIC® Potassium hydroxide ( 7.5 g. assaying 85 per cent) was added to 100 ml. of ethylene glycol, resulting in a 1.3 H potassium hydroxide solution. While preparing the reagent, the glycol solution was boiled in an Brlenmeyer flask until the temperature reached 190°C. Host of the water was removed by boiling, so that it was possible to maintain a temperature of 180°C. while the sample was being heated. -* 5 -

PAGE 53

The fornula used to determine specific alpha 1st V* Specific alpha as c ^ Alpha * absorption coefficient I c sc Intensity of radiation transmitted by the solvmt I = intensity of radiation transmitted by the solution c < concentration of solute in grams per 1000 ol. 1 length in centimeters of solution through which the radiation passes. Table XX gives the standard values reported by Beadle and Xraybill95 for the determination of fatty acid mixtures containing linoleie, lino Ionic, and arachidonic adds) TABLE XX Reference Values for Use in Spectropho to metric Analysis I some rl zed fatty acid Specific Absorption Coefficient soap 2340 X 2680 X 3010 1 3160 A Arachidonic 59.3 53.4 25.8 22.6 Linolenic 60.9 53.2 Linoleie 86.0 S. i u * •*> | I* . 46 -

PAGE 54

In order to evaluate the spectrophotoaetric data from the is one rized moccasin snske oil it was necessary to run a sax^le of the original oil. After investigation it was found that diethyl ether is a good solvent . In spectrophoteaetric work it is necessary to have a solvent which does not absorb ultraviolet light so that all absorption shown will be that of the coE^otcad to be examined. The original concentration was 10 grans of moccasin snake oil to which was added enough diethyl ether to make a total volume of 100 ml. This gives a 10 per cent solution of moccasin snake oil. TABLE X Spectrophotoaetric Bata on Pure, Cold-pressed Moccasin Snake Oil Wave Length in Angstroms Alpha Log — 0 X Slit Width Concentration Log Specific Extinction coefficient 3500 0.00167 0.167 0.8 am. «• 2.7773 3400 0.0022? 0.227 0.6 mm. C 2.6640 3300 0.00326 0.326 0.8 mm. C 2.4864 3200 0.00534 0.534 0.8 am. c 2.2733 3180 0.00586 0.586 0.8 am. 0 2.2321 3150 0.00614 0.614 0.8 am. 0 2.2118 3130 0.00610 0.610 # 1 00 . 0 c 2.2147 3120 0.00605 0.605 0.8 am. c 2.2147 the concentration column, C indicates 10 per cent moccasin snake oildiethyl ether solution.

PAGE 55

TABLE X (Continued) Spectrophotooetric Data on Pare, Cold-pressed Moccasin Snake Oil Wave Length in Angstroms Alpha Log *o I. .. Slit Width Concentration Log Specific Extinction coefficient 3100 0.0061? 0.617 1 co 0 C 2.2097 3050 0.00782 0.208 0,8 mm. m 2.1040 3000 0.01050 0.262 0.8 mm. 0*4 1.9798 2950 0.01328 0.332 0.8 mm. C/4 1.9721 2900 0.020Q 5 0.501 0.8 mm. 0/4 1.6990 2850 0.03668 0.917 0.8 mm. 0/4 1.4318 2800 0.0512 0.321 0.8 mm. 0/16 1.2907 2750 0.054? 0.342 8 CO a 0 0/16 1.2620 2700 0.0685 0.42? 0.8 mm. 0/16 1.1643 2680 0.0774 0.121 0.8 ». 0/16 1.1113 2650 0.0665 0.104 0,8 mm. 0/64 1.1772 2600 0.0742 0.11? 0.8 mm. 0/64 1.13 2550 0.1414 0.221 0.8 mm. C/64 0.85 2500 0.414? 0,16? 0.8 mm. 0/64 0.40 2450 0.888 0.347 0,8 mm. 6/256 0.05 2400 1.270 0.494 0.8 mm. 0/256 + 0.10 2350 1.515 0.592 0.8 mm. C/256 + 0.18 2300 1.56? 0.612 1.0 mm. 0/256 + 0.20 2250 1.472 0.575 1.5 mm. 0/256 -•• 0 , 1 ? 2200 1-go —Q.-53Q , , 2.0 mm. 91256 -..^LOfiy? , 48 -

PAGE 56

In order to graph the data given in Table X it la necessary to nse the Log Specific Extinction Coefficient, ©lie makes it possible to place all of the graph on one curve. The Log Specific Extinction Coefficient is obtained by taking the Log to base 10 of the Alpha value in Table X. Tor this graph see Figure 1.

PAGE 57

LOG SPECIFIC EXTINCTION COEFFICIENT 35001

PAGE 58

DISCUSSIOH OS’ KOOKS 1 She absorption at 2340 Angstroms is caused by the diene conjugation. The alpha value is 1.52 5 I* 100 per cent conjugated linoleic acid gives an alpha of 86.0, if 100 per cent conjugated linolenic acid gives an alpha of 60 . 9 . and if 100 per cent conjugated arachidonic acid gives an alpha of 59*3* then it is possible to calculate the amount of conjugation present from the results found. If all the absorption at 2340 Angstroms is assumed to he lincoleic, then there is present in the original moccasin snake oil 1.77 per cent of a conjugated linoleic acid. 3fcr the same procedure it was found that the percentage of conjugated trlenes in the original moccasin snake oil was 0.15* and the percentage of conjugated tetraene present in the original moccasin snake oil was 0.04. The triene absorption shows at the 2680 Angstroms wave length. The tetraene absorption shows at the 3010 Angstroms and the 3160 Angstroms wave length. • 51 -

PAGE 59

BBEnWISATIOS OP LIHOtSIO, LISQLSIIO, A3® AKACHIDOSIC ACIDS II MOCCASIH SNAXE OIL BT SraOTROPHOTOKBTRIC AHAITSIS The anakB oil used in this determination was isomerlsed in an glycol solution in accordance with the method of Mitchell, Kraybill, and ZBcheile^, which re com ends a sample of from 0.1 to 0.2 gram. A sample of moccasin snake oil, 0.1355 grm, was made up to a volume of 100 ml. with 99 per cent ethanol, according to the procedure *hieh was described in detail in the preceding section. This solution of 0.1355 gram of snake oil per 100 ml, is represent« . ed in the table by C in the concentration column. ' A Bectanan Spectrophotometer was used, and readings were recorded at intervals of 10 Anstroms throughout the range from 3500 to 2170 < . \ 'i .'ft Angstroms. The results are reported in Table XI. Since the log alpha is the seme as the log Specific Extinction Coefficient, the log alpha for each wave length was not tabulated. ‘‘iV. TABLE XI Determination of Linolele, Lino lei c, and Ar&chidonic Acids in Moccasin Snake Oil by Speetrophotometric Analysis Wave Length in Angstroms Alpha *4 Slit Width Concentration Log Specific Extinction Coefficient 3500 0.80 0.108 0.8 ass. C/10 • 0.0969 3490 0.84 0.114 0.8 mm. C/10 0.0757 3460 0.86 0.117 0.8 mm. -c /10 °-0fcS5

PAGE 60

TABLE XI (Continued) Wave length in Angstroms Alpha r *0 Log -2 Slit Width Concentration Log Specific Extinction Coefficient 3470 0.86 0.117 0.8 m. 0/10 0.0 655 3460 0.84 0.114 0.8 ram. C/10 0.0755 3450 0.80 0.108 0.8 am. 0/10 0.0969 3440 0.75 0.102 0.8 ran. 0/10 0.1249 3430 0.70 0.094 0.8 mm. 0/10 0.1549 3420 0.64 0.087 0.8 mm. 0/10 0.1938 3410 0.61 0.083 0.8 mm. 0/10 0.2147 3400 0.61 0.082 0.8 am. 0/10 0.2147 3390 0.61 0.083 0.8 am. C/10 0.2147 3380 0.64 0.087 0.8 mm. 0/10 0.1938 3370 0.68 0.092 0.8 am. 0/10 0.1675 3360 0.73 0.099 0.8 am. 0/10 0.1367 3350 0.79 0.107 0.8 ram. 0/10 0.1024 3340 0.86 0.116 0.8 am. C/10 0.0655 3330 0.93 0.126 0.8 am. 0/10 0.0315 3320 1.02 0.138 0.8 m. e/io 0.0086 3310 1.04 0.141 0.8 am. 0/10 0.0170 3300 1.06 0.144 0.8 ML C/10 t 0.0253 3290 1.08 0.147 0.8 am. 0/10 v 0.0334 3280 1.10 0.149 0.8 ram. 0/10 0.0414 3270 1.11 0.150 0.8 m. -g/10 53 -

PAGE 61

TABLE XX (Continued) Were Length in Angetrons Alpha Slit Width Concentration Log Specific Extinct ion Coefficient 3260 1.12 0.152 018 ago. C/10 •r 0.0492 3250 1.16 0.157 0.8 sas. C/10 0.0645 3340 1.24 0.168 • 1 to • 0 C/10 t 0.0934 3330 1.34 0.182 0.8 £9R. 0/10 0.1271 3220 1.50 0.203 0.8 am. C/10 0.1761 * \ " i! 3210 1.70 0.232 0.8 an. 0/10 + 0.2304 3200 1.92 0.260 0 . 09 1 9 C/10 * 0.2833 3190 2.15 0.292 0.8 aan. 0/10 0.3324 3180 2.38 0.322 0.8 can. C/10 r 0.3766 3170 2.53 0.343 0.8 an. C/10 0.4031 3160 2.61 0.3J4 0.8 xnn. 0/10 0.4166 3150 2.60 0.353 0.8 an. 0/10 0.4150 3140 2.52 0.342 0.8 BBU c/10 + 0.4014 3330 2.39 0.323 0.8 san. c/10 0.3784 3320 " 2.23 0.302 ' * : 0.8 an. 0/10 0.3483 3110 2.08 0.282 0.8 an. 0/10 9 0.3381 3300 "i • 1*99 0.269 0.8 an. c/10 0.2959 3090 1.94 0.263 0.8 an. c/10 40.2878 3080 1*99 0.268 0.8 an. 0/10 O .2989 3070 2.08 0.282 0.8 an. 0/10 t 0.3181 3060 2.23 0.302 0.8 an. c/10 0.3483 54 -

PAGE 62

TABLE XI (Continued) Ware Length in Angetroms Alpha i*g-Y Slit Width Concentration Log Specific Extinction Coefficient 3050 2.41 0.326 0.8 mm. C /10 •* « r 0.3820 3040 2.59 0.352 0*8 ecu 0/10 0.4133 3030 2.74 0.372 0.8 mm. 6/10 * 0.4378 3020 2.86 0.388 0.8 rat. C/10 0.4564 3010 2.90 0.393 0.8 mm. C/10 0.4624 3000 2.85 0.386 0 . 00 1 • e/10 0.4548 2990 2.73 0.370 0.8 mm. c/10 0.4362 298O 2.60 0.353 0.8 rat. c/10 0.4150 2970 2.47 0.334 0.8 mm. c/10 0.3927 2960 2.38 0.322 0.8 mm. c/10 c 0.3766 2950 2.35 0.318 0.8 mm. »/io 0.37U 2940 2.42 0.328 0.8 mm. c/10 * 0.3838 2930 2.56 0.347 0.8 mm. 0/10 4. 0.4082 2920 2.77 0.376 0.8 am. c/10 0.4425 2910 3.06 0.415 0.8 mm. c/10 0.4857 2900 3.36 0.455 0.8 mm. e/10 0.5263 2890 3.74 0.507 8 CO . 0 c/10 f 0.5729 2880 4.1 5 0.563 • 8 CO • 0 c/10 4 . 0.6180 2870 4.59 0.622 0.8 mm. c/10 4. 0.6618 2860 4.00 0.677 0.8 mm. c/10 + 0.6990 2850 SM — 2 J 27 . • i 00 . 0 c/10 »°-re
PAGE 63

TABLE n (Continued)

PAGE 64

TABLE XI (Continued) Ware Length la Angstroms Alpha Log-Slit Width Concentration Log Specific Extinction Coefficient 2620 6.60 0.895 0.8 ran. C/10 0.8195 2610 6.6l 0.896 0.8 ran. C/10 t 0.8202 2600 6.6k 0.900 0.8 son. C/10 0.8222 2590 6.60 0.895 0.8 am. C/10 0JB195 2580 6.57 0.890 0.8 am. C/10 t* 0.8176 2570 6 . 1*6 0.875 0.8 mo. c/10 0.8102 2560 6. 37 0.863 0.8 mm. C/10 0.8041 2550 6.31 0.855 f 00 • 0 C/10 0.8000 2540 6.35 0.861 0.8 mB. C/10 0.8028 2530 6.57 0.892 0.8 am. C/10 0.8176 2520 7.02 0.950 0.8 BBS. C/10 0.8463 2510 7.64 1.030 f 1 00 • O C/10 0.8831 2500 8.18 1.116 0.8 mm. C/10 0.9128 2490 9.74 0.132 0.8 BBS * C/100 0.9886 2480 IO .63 0.144 0.8 ran. c/100 1.0265 2470 11.66 0.158 0.8 am. c/100 1.0668 24*0 12.62 0.171 0 . 09 1 4 C/100 + 1.1011 2450 13.43 0.182 0.8 ma. c/100 1.1281 2440 14.26 0.193 0.8 ran . c/100 1.1535 2430 14.91 0.202 0.8 ma . c/100 1.1735

PAGE 65

TABLE XI (Continued) Wave Length In Angftroae Alpha Lo/y,.JEiS Slit Width Concentration Log Specific Extinction Coefficient 2420 15.64 0.212 0.8 am. 0/100 1.1942 2410 16.38 0.222 0.8 ran. 8/100 t 1.2143 2400 17.12 0.232 0.8 m. 0/100 «• 1.2335 2390 17.86 0.242 0.8 ass. 0/100 1.2519 2380 18.45 O. 25 O 0.8 as. 0/100 «. 1.2660 2370 19.04 0.258 0.8 as. 0/100 1.2797 23^0 19.50 0.264 C.8 ass. C/100 1.2900 2350 19.78 0.268 0.8 ME. C/100 1.2963 2340 20.00 0.271 0.8 as. 0/100 «. 1.3010 2330 20.15 0.273 0.8 an. 0/100 1.3043 2320 20.22 0.274 0.6 zsn. 0/100 1.3058 2310 20.15 0.273 1.0 an. 0/100 1.3043 2300 19.78 0.268 1.0 on. 0/100 1.2963 2290 19*71 0.26? 1.0 non. 0/100 1.2947 2280 19.04 0.258 1.0 an. c/100 1.2797 2270 18.60 0.252 1.0 an. c/100 1.2695 2260 18.30 0.248 1.5 an. 0/100 1.2625 2250 17.48 0.237 1*5 an. 0/100 * 1.2425 22/(0 16.83 0.228 1.5 an. c/100 1.2261 . 2230 16.02 0.217 1.5 an. c/100 1.2046 • 58 -

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TAILS! 353 (Continued) ,.L^. x.„. • . V ... , -T-' 5. ' . . * \ i Wave Length in Angstroms Alpha i*s-4 Slit Width Concentration Log specific Extinction Coefficient 2220 15.50 0.210 1.5 mm. C/100 1.1903 2210 14.98 0.203 1.5 mm. c/100 1.1756 2200 1A.61 0.198 2.0 MU c/100 1.1647 2190 13.95 0.189 2.0 mm. c/100 1.1446 2180 13.35 0.181 2.0 mm. c/100 1.1255 2170 12.77 0.173 2.0 mm. c/100 A 1.1062 t Some of the data from Table 3d ie presented graphical 3y in Figure 2. The Specific Absorption Coefficients found from Table 3d were as follows^ \ > . J At 2340 Angstroms, the value obtained was 20.00, At 2680 Angstroms, the value obtained was 8.18, '* ' . " ’ * ' At 3010 Angstroms, the value obtained was 2.90, At 31^0 Angstroms, the value obtained was 2.61. From Table IX it was found fcliat the above values were equivalent to the following percentages of acids 1 At 2340 Angstroms, 15.4 per cant of linoleie acid. At 2680 Angstroms, 3.9 per cent of linolenic acid. At 3010 Angstroms, 11.24 per cent of arachidonic acid. At 3160 Angstroms, 11.57 per cent of arachidonic acid. 59 -

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LOG SPECIFIC EXTINCTION COEFFICIENT MVE-LENGTH IN ANGSTROMS

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An examination of the data indicates that the following values are more probably corrects Linoleic acid.. 15.4 per cent Arachidonic and clupanodoni c acids.. 11.4 per cent From spectrophotometric data for the range from 2400 to 2800 Angstroms it appeared that the absorption may have been caused by a vitamin or by vitamin-like eubstsnces. It is probable that this is the reason that Hilditch and Shrivastava 8 ® found too high a value for linolenic acid by the spectrophotometric method. Vitamin D, which contains be expected to three double bonds in conjugated position, would show up in this range on the spectrophotometer^. 61 -

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SUMMARY The following qualitative and quantitative analyses of moccasin . snake oil are based on research reported in this dissertation: TABLE XII Percentage of Patty Acids in Moccasin Snake Oil : ' * " > Same of Add Percentage % ' ' nfegf Juristic 1.77 Palmitic 15.93 Stearic 8.37 Hexsdseenoie 8.53 Oleic 35.25 Linoleic 15.70 Arachidonic and Clupanodonlc 11.18 TOTAL 9^73 Two partial tests of the reliability of this analysis are: (1) The Iodine Value of the sura of fatty acids found as compared to the original moccasin snake oil, and (2) The saponification equivalent of the sura of the fatty acids found as compared to the original moccasin snake oil. Tables XIII and XT? give the results obtained. —62*.

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TABLK XIII Percentage Composition and Iodine Values of Fatty Acids of Moccasin Snake Oil The total Iodine Value of the original moccasin snake oil was 1,071 grass, which is 0,023 gras greater than the total for the acids. These determinations were based upon a one-gram sample of the original moccasin snake oil. Same of Acid Percentage of Total Theoretical Iodine Value Myristic 1.77 0,000 Palmitic 15.93 0.000 Stearic 8.37 0.000 Herndecenoic 6.53 0,0653 gram Oleic 35.25 0,317 gram Linoleic 15.7 0,285 gran Ar&chidonic and Clupanodonic 11.4 0.381 gram TOTAL

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TABLE XIV Percentage Composition and Saponification Equivalents of the Patty Acids of Moccasin Snake Oil Same of Add Percentage of Total Theoretical Saponification Equivalent }§rriBtie 1.77 4.4 Palmitic 15.53 34.5 Stearic 8.37 16.5 Hexadecenoic 6.53 14.4 Oleic 35.25 70.1 Llnoleie 15.7 j 31.4 Araehidonic and Clupanodonic 11.4 21.0 TOTAL 192.7 The total saponification equivalent of the original moccasin snake oil was 192.6, which is 0.1 gram less than that of the adds. These calculations were based on a one-gram sample of the original noccasin snake oil. 64 -

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C0W3HTSI0HS Based upon the data shown in this dissertation, the following conclusions are indicated: (1) Highly uasaturated fatty acids are not easily distilled, ere n at pressures as low as 0.1 am. to 0.2 am. (2) In spectropho tone trie determination of fatty acids extreme care most he taken to evaluate the data obtained. (3) In any determination of fatty acids in moccasin snake oil, no single method is absolutely reliable.

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BiBnoc^.m 1. HcLesashlin, J., and Pollard, C. B. , Unpublished Bata (M.S. Thesis). 2. Yeung, B. C., Jr., and Pollard, C. B., ^published Bata Oh S, Thesis), 3* Levkowitsch, J., "Chemical Technology and Analysis of Oils, Fats, and Waxes,* McMillan and Co., Limited, Yolo. 1, 2, 3 (1915). Fifth Edition. 4. Gran, A. , Analyse der Fette und Wachse. Vol. I (Berlin, 1925) . 5. Bolton, E. £. , "Pats and Fatty Foods* (London, 1928). 6 . Elsdon, G. B., "Edible Oils and Fats* (London, I 926 ) . 7. Bean, H. F., "Utilisation of Fats* (London, 1938). 8. Hilditch, T. P., "The Chemical Constitution of Natural Fats," John Wiley & Sons, Inc., First Edition. 9. Official and Tentative Methods of Analysis of the Association of Agricultural Chemists, Sixth Edition (1945), published by the Association of Official Agricultural Chemists, P. 0. Box 540, Benjamin Franklin Station, Washington 4, B. C. 10. Hilditch, T. P, , "The Chemical Constitution of Natural Fats," John Wiley A Sons, Inc., Second Edition (194?). 11. Salston, A. W., "Fatty Acids and Their Derivatives" John Wiloy A Sons, Inc.. (1948). 12. Mark ley , F. s., "F a tty Acids," Inter-Science Publishers, Inc., 215 Fourth Avenue, New York 3 , N. Y. ( 1947 ).

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13. Xrafft, Ber., 1%, 1413 (1880); Xrafft and Hoerdlinger, Ber. , 22, 816 (1889); Xrafft and Byes, Ber. . 28, 2583 (1895): Xrafft and Welland t, Ber., 2£, 1316 (1896). 14. Caldwell and Hartley, £, Chora. Soc., 25> 853 (1909). i.M i *• „• % * J > 15. Brown, Proc.Ohen . Soc.. 26, 3*9 (1910). 16. Xrafft, Ber .. & 4339 (1903). 17. Xreis and Hafner, Ber. , 2766 (1903). 18. Sailer and Youssoufian, Count, rend., 142, 803 (1906). 19. Boll, Ber., 3570 (1906). 20. Holland, J. I^d. Cfom. . % 1?1 (1911). 21. Smedley, Bio chan . £., 6, 451 (1912). 22. Holland, Heed, and Bttckley, J. J£r. Research. 6, 101 (1916); Holland and Rickley, J. Agr. Research. 12, 719 (1918); Holland, Garvey, Pieres, Messer, Archibald, and Dunbar, J. act. Research , 2h, 365 (1923). 23. Crowther and Eynd, Blochs. £., H, 139 (1917). 24. Channon, Drasmond, and Golding, Analyst . 49. 311 (1924). 25. Bosvorth and Brown, J. Biol . Che®., lO^, 115 (1933). 26. ELsdon, Analyst. 2&> 8 (1913) J 22# 78 (1914). 27. Meyer, Chaa .-Ztg. . 31, 793 (1907). 28. Gmn, Chea . Una chan. 26 , 101 (1919). 29. Bosvorth, J. Biol. Chem. . 106. 235 (1934). 30. Hashiaoto, J. Au. Chon . Soc. . 4£, 2325 (1925). 31. Cohen, Terslag Atoad. Wetenachappen Ansterdaa . 34. 462 (1925) .

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32. Stotee, Analyst , 4& 577 (192'+). 33. Armstrong, Allan, and Moore, J. Soc . Chea . Ind., 44, 63* , 143T (1925). 34. Brown and Beal. £. Am. Cham. Soc . 4S. 1289 (1923). 35. Brown, £. Biol. Chtm. . §2* 763 (1929). 36. Brown and Anlt, J. Biol. Chem. . §£, 167 (1930). 37. Duftoa, £. Soc. Chea. Ind. . 2& 4JS (1919). 38. Clarke and Rahrs, |jgA. En£. Chaa .. 1£, 349 (1923). 39. Vidmer, Heir. Chlm . Acta. £, 59 (1924). 40. Peters and Baker, J^d. Er£. Cheia .. 1§, 69 (1926). 41. Cooper and Fasee, Ind. Chem. . 20. 420 (1928). 42. Jantzen and Tiedcke, J. piakt . Chea. (2), 1221, 277 (1930). 43. Brans, Cornish, Leptersky, Archibald, and Festev, Ind. Eng. Anal. 33d., %, 339 (1930), 44. Bash and Schwartz, Ind . Eng. Chen. . Anal. Ed. , 4, 142 ( 1932) . 45. Lepkorsky, Fester, and Brans. J. A&. Chen. Soc .. 978 (1936). 46. Whitmore and lax. J. An. Chen. Soc. . ^ 3448 (1932). 47. Wilson, Parker, and Langhlln, J. An. Chen. Soc. . 2795 (1933). '+8. Weston, I^d. Chen. , Anal. Ed.. 179 (1933). 49. Podhielnlak, Ind. Itag. Chen. . Anal. Bd., 119 (1933). 50. Xlem, Bature. 142. 6l6 (1938). 51. Fenste, Tongherg, and Qoisgle, Ind. En£. Chan .. 26, U69 (1934). 52. Tongherg, Qaiegle, and Fenste, Ind . Eng , foam. . 26 , 1213 (1934). 53. Fenste, V. S. Patent 2,037,317 (1936).

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54. Tongberg, Lavrovski, and Fenske, Ind. Eng. Chag|t . « 29, 957 (1937)* 55. Fenska, Lavrovski, and Tongberg, Ind. Eng. Chen. , 22., 2 97 (1938). 56. Schoenheimer and Rlttenberg, £, Biol. Chen. , 120. 155 (1937)* 57. Longenecker, 0i£ and Spaa. I?. 53 (1940) . 58. Stedraaa, TJ. S. Patent 2,047,444(1936); Can. Patent 361, 043(1936) . 59. Stedsan, Can. £. Research, 15B. 383 (1937). 60. Bragg, Ind. Eng. Chen. . Anal. Ed., £3^ 283 (1939). 61. Parmer and Tan dan Henvel, J. Soc. Chem. Ind. , 57 * 24T (1938). 62. Harris, I. A., Raeoff, I. I., Miller, E. S., and Barr, 0. 0., £. Biol. Chem., }&, *99 (1941). 63 .. Morris, F. A., and Terry, B. X. , Oil and Soap. 33 . 41 (1945). 64. Bieleekl and Henri, Ber .. 4& 1304 (1913). 65. Ramart-Lucas , Bicmard, and Grunfeldt, Conpt. rend. , 190. H 96 (1930). 66 . Bieleekl and Henri, Ber., 46, 1304 (1913). 67 . lay end Arende, Z. nhyslk. Chem. . 17B. 177 (1932). 68. Birr and Hiller, Chem. Rev. . 221 (1923). 69 . Eantzsch, Z. Elektrochem. . 29. 221 ( 1923 ), 70. Hartleh, Strahlentheraale. 39. 442 (1931). 71. Bradley and Richardson, Ind . Eng. Chem. . 32. 963 (1940). 72. Mitchell and Kraybill, Ind. Eng. Chen. . Anal. Ed., 1^, %5 (1931) { J. Am. Chen. Soc. . 64 . 988 (1942). 73. Mitchell, Kraybill, and Zsehelle, Ind. Eng. Chen. . Anal. Ed., 1£, 1 (1943).

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7074. Tan der Holst, Rec. t m. chlm., 54, 639. 644 (1935). 75. Dingwell and Thomas, £, An. Chen . Soc., 56, 899 (1934). 76. Bourgoel, Oredy, and Pisux, Conpt. rend .. 195. 129 (1932). 77. Gredy, fell, soc. chla. (5). g, 1029, 1951 (1935)} (5), ^ 1101 (1936); (5). ^ *15 (1937). 78. Delaby, Piaar, and Guilleaonat, Corvpt. read. . 205. 609 (1937). 79. Barnes. Rasoff, Hiller, and Barr. |nd. gag. Chen .. Anal. Ed.. 1 6, 385 (1944). 80. Bnsoff, Platt, Klevems, end Barr, J. ^n. Chem. Soc. . <$£, 673 (1945). 81. Basoff, Holman, and Burr, Oil and leap. 22, 290 (1945). 82. Brice, B. A., Swain, M. 1., Schaeffer, B. , Ault, V. C., Oil 2S* Soa£, 22, 219 (1945). 03 • 0* Connor, f . R., Heinzelman, D. C., and Dollear, 7. 0., Oil end 257 (1945). 84. 0* Connor, R. T., Heinselman, D. C., Carerella, M., and Bauer, Oil aM Soe£, 23,5 (1946). 85. LundBerg, V. 0. t Holman, R. T., and Barr, 0. 0., Oil end Soap. 10 (1946). * •» 86. Holman, R. T., Arch. Blochem. . l£, 519 (1946). 87. Beadle, D. V., Ojl and Soap, 22., 140 (1946), 88. Hilditch, T. P,, end Shrivaatwra, R. K. , Analyst . £2* 527 (194?). 89* 0*Connor, R. T., Heinzel»sa, D. C., McKinney, R. S., and Pack, T. C., £. Am. -Oil Chemists* Soc. . 24, 212 (1947). 90. Hendrickson, M.f tt Cox, R. P., and Konen, J.C., J. Am. Oil

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•71Chenlata* Soc. . 2£, 73 (1948). 91. Boss, J., 0. S. Patent 2,435.159. 92. White, H. F., Orient, B. M. , and Brown, J. B., J. Am. Oil Chemists* Soc. . 25. 73 (1948). 93. Lecfcy, H, S., and Swell, R. H., Ind. Eng. Che*. . Anal. Bd., l£ 544 (1940). 94. Mitchell, J. H., Jr., Iraybill, H. R., and Zseheile, F. P., I^L. g-nd Chen. . Anal. Bd., 1^ 1 (1943). 95. Beadle, B. V., and Iraybill, H. R., J. Am. Chen. Soc. . 6£, 1232 9 ^ t Shriner, Pol ton, and Barks, J. Chen. Soc. . * 1494 (1933). 97. Rosenberg, H. R. , "Chemistry and Physiology of the Vitamins," Inter-Science Publishers, Inc., Jtew York, H. T. (1942).

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Acoowmxmm Sh© author espressos his sincere appreciation to Dr. C. B« /'. ? * ' , \ « • * : % ^ ' rrt . . 1 Pollard, Chairman of the author* s Supervisory Committee, who has directed this research. The success of the undertaking is due largely to Dr. Pollard* s kindness and constructive criticism. Sincere appreciation is hereby expressed for the cooperation of Mr. Boss Allen of Silver Springs, Florida. Mr. Allen kindly donated the oils and raw materials used for this study. 72 -

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V BIOGRAPHICAL ITEMS Joseph McLau^ilin, Jr., was horn on October 7» 1915. at Marietta, Ohio. He pursued hie undergraduate studies at the University of Florida, where he received his Bachelor of Science £ Degree in 1939. The author* s Graduate work at the University of Florida was interrupted by forty-five months service in the Array. Twenty months of tills Army service was spent as chemist in the Biological ' : * v , • \ v ^ Warfare Project at Camp Detrick, Maryland. He received hie Master of Science Degree from the University of Florida in 194?. While attending the Graduate School of the University of Florida he held the position of Laboratory Assistant for two years and a Graduate Council Fellowship for three years. He is a member of the American Chemical Society and of Florida Blue Hey. 73 -

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This dissertation was prepared under the direction of the Chairmen of the candidate's Supervisory Committee and has "been approved by all members of the Committee. It was submitted to the Graduate Council end was approved as partial fulfillment of the requirements for the degree of Doctor of Philosophy.