A study of the relationships between chromatographic behavior and structure of steroid hormones

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Material Information

Title:
A study of the relationships between chromatographic behavior and structure of steroid hormones
Physical Description:
ix, 155 l. : ill. ; 28 cm.
Language:
English
Creator:
Hartman, Iclal Sirel, 1930-
Publisher:
s.n.
Place of Publication:
Gainesville
Publication Date:

Subjects

Subjects / Keywords:
Androstane   ( lcsh )
Chromatographic analysis   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: l. 148-154.
Statement of Responsibility:
by Iclal Sirel Hartman.
General Note:
Manuscript copy.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000423960
notis - ACH2365
oclc - 11045751
sobekcm - AA00004950_00001
System ID:
AA00004950:00001

Full Text







A STUDY OF THE RELATIONSHIPS BETWEEN
CHROMATOGRAPHIC BEHAVIOR
AND STRUCTURE OF STEROID HORMONES







By
ICLAL SIREL HARTMAN


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







UNIVERSITY OF FLORIDA
April, 1963


I
























hfI dL~magstem is dedaredt to p ~ebad.





(p


___I_ ___

















fe sMlume ad emnnMMRmvat of a~ tmli eu, N. Ol amJ

Bat. Las W. n Iear wan ofe te t eo vltal to the athnr as a

n a e at M tof pt'Mttl I d Sppot ad m n meat gIraeul
anmmshemldg.


mtf]mtr hienk mn elms dau to fR. Awda L a egp sw hiA
adeus and tams9pxt te the padust. Et shift 4W the
aLmBftr.B


The



th apls


ad of Da. -Inm I. eInr.Jr. la the cattletaln s
of th as me ip deeply .appsesa .


asbM alm v wlhem rto e eleda the *="at i nm--AltNo
Japoyn at tL be i entdatiea of mso of the dataU a tla
of thi dimemrtatiem.


itI














TAB Or COTIr



ABWSUMe iLL


LST or TABUL vii

LIST OF nIFou ix

ItllRODUCTI 1

Statemat of Prpos 1

Chrtntography 2

Historical Bftkgroud 2

Gas hromteoraphe IMethods 4

Principles of uas Chromtography 8

Basie Qas Chtrotogsaphle Apparatus 16

Aadrostasme 1

Conafortiome of Aadrotame Ria System 18

Iseartim i Andtostames 24

Aldrostau t somers roosesstau Hotorm A tivity 25

Methods of Assay for Aadroesu and 17*Ketosttroid 2s

Chmieal sad PhysI aI Methods of charimtertioati
of Steroids 28

Noa e latutr 30

XXUMIBTIUIL 32

Iaturals and iMthods 32

Comp dl Studied 32













'ass
8yrapenti t oC arraitrs Ma
tista~ a tL r f Stered se ther er, atvw 34

iAster m StnDd 36.




anet .*7 lbes. 37

ft8olpga ftsm Cino 3







a s2 lbaroeb ne bm t sttees wYin M( 3.3) At
new cartoa Itn Ve sti"ms Ifte st
Xeb1ylaestylo sodatoe rPolyrstr (S30) 31
M.ee p s. 1tr A4t o rostr (8mX0) 51




IZepentyylg S Lebse0s Polyester (Ub5) 53
IlnraIcss |6trU nad S1150 jpa4 X-llO41 53
q9--1005 nlorieshyl Polyzsr (Q-1-0065) 56

UWsssesa oft pasiUtary swritmnts so
Coepartiave Suprtmnita 62
1emIase o Cof MGi tiqve srmnts 3
mC *tsiemi of los Itsnuy Pfhas mu Uese Urns. 1a7
of "posn


_ --?:;--




-T .


seprMtio et rwy 17uletoirettid 11t

EnMm Mxttres 113

Pqrepretto of Ure temple 118

Kteue esr of the Seperatti of Writtr
17ltetoestwroid 118

Study of theb Dpedimae of Relative atra oa Tims oa
TerpeIatue end on the i1turo of the aerewse Stsaard 121

Dtseutui of latow Affecting UsteatLoa Time dlaes 121



ustetiship of Cphvtovsraphie shelter to Chantal
Struetwo 125

attention Ptterns 123

Iffst of Itsustwu oan tention Dta 128

The.ootieal Aspects of 7Prtitilo ChraMtoeg hy:
as1tilmkip of Struste" to 6h,.inteoapis
Mobility 131

The Appliteitnt of tOssliquid Chawatorasphy to the
Isolttim and 0Ismemmmt of UatSnry 17* tos ids4 141

A athebd fow the U8mlsstoem SepeUst of ai O2
wmltCi317~aKtetwoide and Progte.rm

Smmry 146

mstaauws ulk


IORAPURCAL hImGH












IU=T TAXUBa


able
1 Sriwstrm f WWAMSM "tmv with o$ isa l Tinmaw
-md rtesbte a M i0 Mol f 43
seW adtm s 1see auN otm Va 4m
S a Igmn -m1 3SWS5 $*S 45
3 lW-fti Ta h* as X W* mol-i
4 aMesUf Isme "da R 88me so X 8640 Gob= 4e
4 8e0t-I- ls i d 4-prattc as1. e 1 U.n3 e Ql 47


s.tmpss el ~aes u em UesS war M)t ea ue*
S'a).me Stm sm so


8 UsmmSe mt Sem m* sa31 noey p a0
S oherm i

0 assematem rat et .1imanum seutd see Ms 11
bqsmltrastr AdspW emIta 5a


11 A Oeugaee! 1 h1e 3fahtto ae3i e 4a ft
u A---met alm d M fSeim vte @9 Tmfs *-tf


5 asleesed aAp nasmum nme
Nt yl)*see o- a, sos et 99m





1l So ol b Iaotw caseates Tamo 3,ss n mu S
IRlo t Tiemoid 74t m t
tr gwtttt- Xff7


vu










btr kog






18 aa f OSntewer Istaktt oft the law ur
o seeatems aId rk ase 92

-Memsm (lIa -Iam*) 94
t uer.isn **g-as- ros est 1M( / Ip) 1s
21 *r U- at If fttpal a7atm teU f ealree

22 so s o& the fw seS atandad as he dos att
l A -rr~-- sAf Mal-**tl* WeFtwth af g!*&* A- ftettI-


oafM out*" O8as f M OBfA eI
XIwso fctr 14


Tili












LIuT Oa r rEW


1neas
1 dattostat iotln u 4
t. A uthed far ualcratla ab of th lttesl plate 11
3 artum. of t arriw oa velasity an solftm ettsLMy 19
4 St rld males=


6 Aatl r l bds. B-Asdal bemad. 19
7 erklotal n2
8 de ma sdnaUtem n

9 OiPtRqud ham ta o a tseu f ko.t ate e d
trtithy1tly1 then M* t 031 **40 m 11$
1o musu qiti ebrmawssm eo a adutwe f aem smeet
attlhyailyl the oea r aBt f4f 81 o*mm 11
11 Oul*MU eid iremIugm of a a ue f tuem .*6td
at1akylstlyl oehes a 1X UMb NMmI 117
Is s* ylaastd. otersm 2 ,of glastey nm
mtarthiylAtlyl s1thes 3X no4w eolim it















It-atm t of Purpose


this study had two mia purposes


The iusstistit o t~ t the a liquid eemsul-to aph bOeaver

of a wrs ef Leemmr amdreamate with the adt of deasrlMtiU ehw

adt stwasture to hr-atwographs ability.


thwrsh the homledge and 1asl~ht 1t0ed La it e aWe of tae
ahowy satdy to afply the ps-Uqdd eumte ap m hs ushod as m

aslytsal tachPque in the tiolatift and m msuranmt of 01 stsods
of aportae e In n, to. r ultuls objestiw as the actual maesmw

Mat of thsse stoaoids la biological fluids


ta the ftx part of the study thwnty-e isemat anmdgwes
wee WtilIsed. bhee UMwe etbes as e bsi s t thetl rtielr

saotrsto al tIa shps. The steetuwes wneS tr f thm t msbt ntted
mstamt uaews to the disbstitated stmd. he Smubttment oas

aa mlngE f setia In all cases


ft ta sopretits stody of biolo isaly sgpaifitmel odvMskCm

tam proasm Le mm we insladed v*Lth *e r tosgeet with dt Ue
steroids la b1*0l8imal flids.


l* I-

















Tasom A smasrly ivos sdt e the am eesmieI of t e
sahm 1f whIe-iWnfm alM ho to Ss0 aq "nn a 4- p r ,dS
1. 1. W bes d 4 obsie a Smetsat W MwsA M k MA be
SitM o Mpnpe ra meamsemph (i1). I l is mni () spm*
-agd the pmSlm a ael. leea tas. mm """I mmins an
oSe hats eolm *o sausm *se=.eae. Sme a" sPaess by,
INleti as TYI- l m SM fteniIt of nmm.r t hanu as he
tlom, nst -m so d poeess lamsrmphky. t pse sem han
dmsese toM a s.iy epst oo SeMl tee mt a1, Se tshe se M
hi waosmoosy Is a asu. I


te, peepIt ae' f Ik eesenrLy tM d ahMe ea fmt. 9 imsetl
eaUae Ms *AS. Nou .aPastem dqla i qand diesf r eltw
beamtt biSe *m t pew.a. le dChllLse erd a fa m r bL e Uq
W eitd ar She gwe phise is 0iLeo an my be lSAd mg aS. So
0 -t wasbe ooze"s" a as tedamesd ats mtd befstmes beatse
*ns HUulg tm s asfo dlUt ed alm.e Mf dot S I s debn a naWu.

ala. he peasnimly tott o deatwes meos o eeinssvd La she
PliYfleta ro thedo distswrts proeine. Mhets toPte e =- is
amel dasL eby ma e*ltiti mset 0s eE ele set fhe we s
stn wI me 40hel Mef do f vury fbsmy asadt ased phase. s e


- 2 -


Il~arnb~irll kl~filllll~L~









pe e s *I mI-t auId mns iatte e notes eamle t le dis att
butie o*9 the solute betwe the *I-al of the am prhses sad toe
atmei -- of evi alnm In a vwy sm1 1 rl of time.


as ler bsel type opf etermMtegepy ae atdnepa n ad
pfratie md are daistiag end by de assure of th promes f dA tse
botism f bte asolte a btw the phaes. (Seae nt r 14) 2 ed*

erspetes qtms, the deUttuib m is dapr- d e ht so- do-
ef te ad*Mase that Is albel d*atrl set. At my m

tah asd epL aiothm. rWeeMMMitt as tne tte the Mltty
f "bte Is, N phset- ad hke total miusa tate spey is A Owrle.

S* eaeWpstie Istar 9* toff +reted by the psamee si otahe arseias-
Is the sast IW s i eemrbly due s toh Usndted rfaw es af

e solud lpha. ese"Weft to the dmsptlor Seeths, tiM tCiteam

aos to pearitims ystym (lSquida or liqIU4e d qnd) e Uwasw mer
a elde "nr o ee -- ostei oef trbe olWe as wll as ftena, uidi
Me-s-tm I-m O et a bsaesI i. the sem. Atis 1a e uaew
ditgeiAda" fIt s of i ratit.t sytese adi asseos so these urw
mim son*" (3).


lAth er Bmaepto at the wa vrt of 2A0a ealy phases .of e
dins|ms > eof emsespahy WMere Uitd so senbm eesrae py.
br th eWtnd fiAl d beet adAepe m esat prttI reltheld fSamd wide
inlt 41 ma


S3 *









































ML



FIGURE 1

Distribution isotherms:

A Adsorption isotherm.

B Partition isotherm.


-4-


- I --p~- I~_ ~n --_--~---~--k------ ~-- -


ML2









Two typ of adsorptieo methods wer poyed by the early
iavestl pteor. The firt type irvold the use of lars soloma for

lars smple d were primarily preparativew i purpose (4.). athe

rand type wU for sepuattim and tetimaten of Sma qumatities of

maplt em mUl olums, This m thod femad appl" atita is the

IpeartaiLm d e ti tiat of urlnsr 17*ketoeteroide (6*7) mod of

ed4n l sterldso ad muaboletes (8). Alumina, memlass sad tilIa

gel w r used mt fequmRtly fe esolum peakta.


The flst uaseseful patitesat ometh were developed by

ktt. linret mad wmor s (9,10) who utilised polite esoums, ShO tly

after the pbllsatti of then mthods, Iateasive raseerh w dit

rested teard partitia enhrmtoerapy tebatqwae. fhe preble Ot

the lt amtlitty of storods to systems based ea wter as toh

statieeMy phase s venom by the development of .eemt sytes

ud a whabe me fi iU of prttittiom c.h irteraphy of steroide beam

avutlabl, AmOg the pImemr tfa this field ere Mortis ad i klgrS p

(9,10). Z fftwrel md his assesiate (11), etamm(l12), ksd (1314)
md S eard (15) to ume a fi.


The system of pe"rtites hreoatesnrpy seestatd -f thwee

CeemaMee a majwr empmat of the ambdl Ipha whish au telattivly

Me-polaw, r a mpeas of the stattie ry phose mre or lees poet Li

eharste to ehome the solabtlitty of the steed sad a third ePn-

peams of ma a11 Smt of Wter. The Mwter s laser ematted fie
ystemS usmlag alprg ted paper ad fr eartala eobma methods. E


* 5


_ ~~____ __ ___









weme* ** perti stte ahemsre- yphj synmt, hit mbtal e ase

WOe platr the dte a IMy in seaS a-d ts pprst W- sPcy*obhets.


the gPtnPaest twe5 UdlUquid sad Sfhltid 'prtitLes shllit
toNIql y aw detikbed by arwti ad $'mge is u341 (u) bthe sesaept
*f a pr sse mwM phase me t ~ ppled mtil 1Mw u J-s asd

terdta pUlidted thestLr am lasttl papr (17). Mssa at ee1 0,
anansa 9me1=46 gas t b e a tp I Ibeeo mete4d ws lJi lao

esel i thtoqgin the wortd se meay types pof rems. Sh Pest

OtaWe ta Vmteie "em of o -a01t0e1eeghy sto deto a14 wide
Snpg of ppUesatmi ap.nd of aSaloyj aid the aseMititty of de.

tettes i oee stieetu.ae of sep. .







Seo eohtnai rphy to the p esaM by IMtaha Iftues It sIpeY
8a4 "t its isnt G-iar- by a gsasesou mbtl phase. If
the satr7y Iha"e to liqMid, the ithod Is sailed paeI aid

ob t0mp1s)pSyh ad the *faptioa s dIue so te diamratAtl M liobUity
ft da miatrtal betssee tahe Usald aid Vas. f the satssay phase

to a swU, he mathed ito sled gasoeiud Jhbemetoepi sy sad the

pYeMties to de to the ad frpeat of the 4ple se*ra on the
mUd mvbe.


ITh twu type of gaM shem"t Mgr hia bthebods ae fSort di
tiae ed *by the na- to bateh the s1mpe mveM thrwsgh tse eo-.


___I __









IIrtde, d4UI mnMt id rbatal Malyse" awe these tabam e
whh isy be usd lI pus-*old ehbramet ogp. hbe *latia
tehMque s used almet easluastly Into gas-llUqAU eha Ea y.


omtaul eaalyst s the pemsae of a eiantieU flor of
srita gs smatntata dhe sample mixture th a a eohma of
adeeatt. se idew of usa a atUmwans strem of tbe ample

Itself a a dtqlmp j a eash soluae attale its em I *eem tte
ademprese e1quallbiwm wam the so trltbm of itStelti (18). Sepr,*

etias ft shieved ly for fabst eapoame u h mnees stiaf fme
the solue., oMnquatly fre-al easlyais to of UIted ao as a
Maskn d eof harper t.


Da lasm- esstl ia, Asia we elaborated by classes (1s),
it the thoed of diepUltaa thr uampe arady a tbe eolve by Msee
of pastuc a eatfmous flew of a eaeriw gas wls eastesa the
ope of a sabstese esalld the dieplasa. ib d4splaswe is as
esem thaf it is ae atstlely adored ateo te eela tN Mesy
ememmt of ate simple admIx the epqlr diereU d by the
disp r Iet a Vrg dtisplaes aethat emoape t of the simple that to
less srsa ly aderted. shlr the Csaonens of a ample ov al
the colom at the sm rate that t displaear iL satursta toh
eolam bekad the temple.


Xalteiar ersMenp" y taminero the ofatteMr passage of aa
umdmbed ia or UaiUqd the ktl the c.romhtmgUSe systan. A
wlati*sed emple is Lasrodwmd lato this sCtea o ofs *ad i eatWred


S-7









dam the eelm. oImpes *f the sl mp diArtheft theme
btomn a be mitha @e.ms phase sad ta stiea.y phese. 206

erw- eal nllUy mr~ I ten the deoi tm af the pas se o

*a dmariete Wa of earrie gau. Soe miniqe Ibsne eof
*Iattes mrin* eaqc ed to froatl ad displf a1e lys #e
I bso aema~s of die oelue am a peak. Ste rvtims tlm tal

deslftd the laslm of tel bete the tameoduestla eI the mq sle

it the lhermmutraplht s*yeta and d othe mus of bts pek a*d-
a Lo a maMW of qiuUteotfiW id^Cttneitem ao Me aMpead. 19M
pea a em ma mllIy be elated disLrtly to de m tm AmL met im







A dlAt4gst s eet s gs t hemsteomea is tmhe iUttUefsm ad

boo p-l-- t ho Is4w In moads the t u Ow to do vowu stak.
the e i e sei dmqm oef 11l4 ue amm Wes the ofrptIs,,,tr emt
1% S4 111-I-sow y eoo -te a eide a so -l maspseN. t1e Steb

a e 4I mo*llled *...u -_sp to O1ls to Ua ht,4uq- "etitam
a"d t p*oIaiqid ebem Is onelsMai to a dtlettasmm **lms.

inm,* thw to rs mjeW dUSfam be"em the ta syles, %hah
to *f rest peasIteal temrtiM. ft system of 1edUd4 IrMaMi*
t lpegby he to is tWeaeme w itah mauetio. dee tao ss- w.
At the tala ok a g -Uiqsd e4hama-aphy -SIlm dtwe at L ee mr
- **l|*atmi bstsesm th ee m-mse slhe a m-le bmt w thr w *

aglrt et the brbm, the lat eel --f thes -p with tht


. f


_ ___









u14 hte so fur aotl awy mu1 L ofesef (). we eas
s Mto *selti "kh aj trih tadIem datly ef My Oeabw




A thesd Ul plate teostem is app ~ bl e to beotkh U d


a aintssa t ohe as s to lsAkh t6h w, eer a owa s 0h sesstea e
dhe sao stia that Ve1d be *a *qumlibutm with 0e nwm so *s.
easastots e tatid etisft- twia that steuisa. eas w seo .


FshrttsB to piei s o hanIs ela itilLrtuiiLa siemes li shef

t e .tatlMd iftor eask htafLM mrs af thf at ebi phle.


the **cttows pet plate (slaate.U faste) md the set
&Wit oft plam ) s0 attia to a eolm d4teseas tLhe install
esalMia ehirwsd. ow empeses eof the "it the atseme
a* *e omtthT7 pyhas *f the 4*1-"m to be -eXed Ita desusw
the spme Irt Stasr. hibs fbter is 4wtt. d as the mati* et otf
nSesti tim of tue *,m ess to ash ether. pmetim can be
*Wigpi by sdoe uas a lUqiqd phse with spedi&s* Asusmsrettes,
oab ow seotti e eteostlim .of mes ImUfat-- imbi beafs a




Us tmph of lnm ft uhst quilthUim w9a oeet m is dotmed
- as she betat. af eo satts thea-mest plate (It). thas 1mr of
thseswttwst plats Oemobitd a* o l forM a Osaiftl liqid phase,


* -









solts and tep~gsta ean be ealsulated La a m-ew of eps aI of*

whtlh Wsare a uUat of the dgree of peak spread reltie to tes

resldeaMe to the l1eoM. The lngth and diSte of el the e.lum

the ditfftiste seme ant of the solume in tih two phase, the mmrt

of stUttUsary phMa, mioStee. ty of solom pekiag, the gete e* flwr,

tempietwre ad the ustre of the eaerire as 11 sl mwrialls

afftetIN tShe vale f N w. 0 getral, N is lasgef "d by an t suee

at length *f eolim dd a dressa tI its *da**Lt.o


Tke equatitm seei ly udd for the saltlatiMea f i toa




*fen ts t h r w ett t tie m mswMred ftri the tasndsttoi of tihe

esMple and At is the width of peak bea. (See tunij 2.) the' nits

my be tim et lm8 th provided both ta ad A t en ta thea s4 t siu.

this eqastio fore i a smi that the st its m et bein stated is ia

equili drl vwth the eoiwum. I lo lated ta this imsr is stitfi-

semt aaly fto amnestrsal poh sta ie ste abk equ stam toii bi ad4

p~eability theuy1 l).


the rhsst equtialiat to a theretteal plate is givM by the

equation L /

wher L the lenrt ot the celm, usually tin leliMtU e. he

vale of is aw en- l liraw er fe msr outtati g as hih dffiatIsmy

and demiseM with tastnmuMas reusimton tei. She plate tLheue

peevUes a entful Mss of we ltstitn eolmn eff~fOte y. The


- 10 -


__








































0

t
I
I \



Time










FIGURE 2

A method for calculating number of theoretical plates.


- 11 -


I




-.-- ..,, .-q


nUv tlaMip of wrtms tnm whulh detana the Vlse of 1 we
Stwa is the van Deamter equatia (22).



i 2,3 + + .amA U.U. 4L
S-( + k')2

uhm X to a qummuty ehnsrarlrti of the pmsU L thW

argr pmrtteUl dim& ar, y ti a erresmie tesr or t the
aeurteIty f the pOath f e gr oa ithe lat eperiie l pae,
S thw daiftMia esfat mst of the solute I tohe sswm- bam ,
ua A the Ulase gas veleaty, k' i the etie of the fnetle of

mwp1 t e tai qtd phaws to the fietlt i ttL wOpM phase d
s e tume as et the Utliqd 11, sad tuq i the diftusia
effistet ao the slate la trhe tiqd phase.


In the simprlfLed wr a-emer *uatla

fi A + 3/u + Cu
A rpneams the addy ditffl tem, r o ttam, al e lt tple pth
afteet, 3/u ten i t he oUelsular diuttat O St tha IS S tm at

deewrbe the weenie to am tumafer. Se *My dit fIaest m
the result of the ixnulE Ipath vhash the ma follws th a th
p eted **mIa. Ite iweeartle to this peth eptrlbsee t a betoad
Aa of the Jpa tspfphtle bued. A edetie o ta pttIelSe si (4d),
=Mif pmkit sed mi"tm pitil ae tee ted to deseMse tbe seetrl-

beatei to ot this tom. Ie wseed fate to Lthe affti.a e the
*ample Mboesula t the emeler gs, weh riualu ta bad b irdeag.
bI lowT the WIleerl wartot of the warim *a, the pretm it this


- 12 *









fiuta. Diffusiote asrses with tIsrese ton tmpetUat .

tesmste Ia pgose- on the eoer had, by rassessu the desity
of the Pa toids to deesse the diffalrui sr this rtsea,
hlSh tmperatwe s as euplced toethe with hih pemses of the
esrelr gas.


hse tem Cu, vesiter se to uass trames rtheldew sevwal
pimraten hbe valm of k' is depedent as the parttiteb ea-
fatest as wll as the walattUv ralas of the liquid and the passe

bLuse is the elem.r k' is a-saily greater thian m beasius the
eeletl3ty of the solts I the hiuid phase is uMsally treeer them

to the wgsee phe. t, 2a% my tasese Ia, the solability of the
samp i the i quid chase will deease I by a se1 ll mnet he d4
ten deflad as te e*tetiv thihass of 'the Uquid tlt, IS eited

to the uaesod eeMr it the v Dtemstet equttesi, Its etattibotl ,

will thelfre be qgisYeable. A tedtinetta df a mlts tl ir*a ased

sasm o9faituwsy, w suth my be coueatrse d by a smilmltmean dicases
ta k' if the iamot of liquid pha is t deded. eM liquid diffsusi

se.ft.lesi (Uq) to dependent a the wisosity of the lquid yhae.

As tuAtese teaer e alght be aepested to redase the vlisesit
but *ta xiwveas teipeLatue f est k' and bg adverO ly, tlh ase
we ult is dffitsult to asses.


oe last ter la tfhe w e Deetr ePsIttle is the raft of flew
of the earlier as. Theeotteally eddy difftuest is 1 a epe mt *of
the at of flw. The eomtrlbatien to I of lesular diffusita is i
twme seti to the nate of flow. aessttme to mas tt-asfor,

13S









hoemaV, Vwies distly with the vels ity of the Wp. If alt
othe vw*rabiles re Ipt seortst, there will ast ea eptimm wte
of flo for the met fflSlmat opoatle of a soalm a tadisted
tois FPre 3. At flw v lUu th1 s the opetim vlue the seartti
bttiom of molelart diffudAoe to willi be apprMsable. Above optimw
flew rate reetatee to msu tuemfer basemes taperte. Bmwasr
this pet of the ewsr is mlatiwly flat sad e-settams bwoe opteim
velsety will m t hae too d letewimr a m efft in 8. The Uleesul
diffuash tear is larSe fe vier ases of high ditffwbiltty.
ebrlstems to m 8 ttuafw ill be deMlem t for itquidS of hioh
viesioty Rd fer htih *semMtatie of stetae y phase qualitative
Md qmUetite e predtiete of tshe effa t of I of *eheg is the

prMaese eo the i r eeW equBater at e su meised by IPetea (23),
Bmos- aId Peellu (24) and tall (25).


te awr peed solMaen the oests a presswe drop betwr
the aslet a ud te eRtsut ends of the esem. A la utal tA the
aboUflte 9esue at either end of the eolm or the use of a taper
olma with a wideriag dimste t etrds the eatlet at er peusble
elIatieu to rraUiSt the pussut drtep. A eauetlt ftstett for
the peiasse e dep aUess the eolm haU bee dweried by Jmw e ad
Martial (17,24). Tb tinatlem veoum defined a


whoae is the v.lro tri f1w rate of 8, oan he greeted l to give
the Ilditiag ratemtiea velme whh is tideIpeent of pMaur (27).


e 14 a

































Moleculat-at Resistance to mass
diffusion transfer



Eddy diffusion


Gas velocity, cm/sec.


FIGURE 3


Influence of carrier gas velocity on column efficiency.

N


- 15 -


'









t/Va %/


e 4 vt is the 1usttas emstnm Vwema ats *S "mws drop
ssrw the olum, R La she aet neat Tvel pO ia the Selsm

llWe the a elt eat d ea d tLs the d weos e wa lm peasusno.


VzRluu sepeelms hbme berM dwvisd to est the pplaUa*
bUiity of the v Da etma theory d will be maeLdr ed the
itEslnUee eatio. 0Is *heMy has been ueLftl In attatlta

bih solum, estataftu.



L elm .. eLLaemar le. Ammam

220 qraS OMes omay gOW e fle-magggt p0smtterm mn0a1ts8
ef a .erst gr m supply, a tiu veplster, a pe PMuseN ue~, a
ieewdewl, amMle pt aMd a ptpet Lee tatrodt1g tshe meia, a
eo~0 a deAsteW sad be*Btlg wtite La the lfes I he ite ms.


bmwasr as Io supplied fus a eepassed somes ftd it
f.r vea thrasgh h sole L a Malated by a press vavle. 2he
gS Psusem thene a she detefttr ed pMrs eds so the tampl part. "h
sample oL LatrOduOd Ltee the 8ampl pert Whekr t it heated to fst
veoslis ettm. bs ematnlo ga emawies the tsaple Wepr at. the
eo*ms whome theasiesrt p tabes plase. .he spemrste
fem.peatt ft the farls te atAwr ga so ter the doesesaft M
piw sa to stespe or to a eollestioa ppeafts Ow taift stady.
ntimenV esatwet Oet the appaens. emp st tis as umpenft eea
*ideasite itn Lae MaaenteVPubhy. theb atrl m dteteig t A.tmi

U *










dumld be that of the 0*li to p~sr t e**^*dmt of welts

ibteh wemld .mwult iS nha e i eeo temtlU, IThe Mapl pes

trempesw should be adjustable to pesaut the ds

t***>*i s re tela tly hAib Ir the wqptd wvrlsstl of

seomte.


* 17 *













AmIdrsmom


lAdrotams belem to the broad Viap of biologisally

0t p mst asot o-gm amto l4 the netuteS wUthih peses the

<2hU *al n B Mln~i- -(~1~ Olavl oof L401"0 444100l*< lei-

(tis mastatWe laseluds imbote prevteesty7 's ene as 1t0s1
sh.lam5s) the mloss sbtaola*s 17 ahbms asimes whith ams up

the *-ook" yotsm *e ewar tifa (opem 4). the tis Wte

dets s atd as As I, 6 sad 9 #ad the arbem alte as| mibsd as

laietSed is the AbWse stlr states the Imo SsWar asthyl vgeO)

(desip eSd asu rases aste t 18 d 19) project to Ser tAet Of the

stfreod mlessle. tit projeatime i desesbutd as po *Meed Ia

is epemed by a seoUd libe. Slailaarly, sOy jtOSs o tO hei

testf tah tia soyst is desitgled as paMrrtemmd ad i co iMdgrsited

by a dotted UMe.


the twmo SOMbeMtMU doeteibee the diffesut spatial easage
moi ofe the stem in a siblt eolastuil swuatwe at ae-Wt tOgtem*

tsfEmenmttes ftr lo mge-mat to amotLhe take place byp the

simple rsattie of a *iagle broad. The best tesm asmmMlm we the

chair sad bet eesw of the syclhws -medel (nlam 5). the dtfftr

ease to mao y whisk eas pririly des to itfOtmrmse Il the


. s -W


An..Ir- _














18


FIGURE 4

Steroid nucleus.


A
4 1 4


4Y3 2


FIGURE 5

Conformations of cyclohexane:






A


B



3 2


A chair,


2fF


FIGURE 6


A- Equatorial bonds.


B Axial bonds.


-19-


B boat.


I:-" -~~I`~-~-----~-- -.~--- ---~-


--.-.'--


ii
:r
I
-;i
I









Uapwtbl at-es u1sotly appgsrrmbLag stemo am- wnrtsn rf.
**te destee the stability of the pmrtesUlr eanfamsti. ft


iMs -stable a fs,. the emsltted mEUesuie sl t is thle sel *.

east. *of XIa6t MSay ad thwbgfts of highet stablUity (S),.


2th beads of the ebiab ratm in a eyetlI amlu ml*st s
of t typms (ngre 6):

1. tw0 usr er low in the ples of the rwle an
suaed equat.sL.
2. Stnw plpdiaU lat to the pleas f the rta8 aM
ealld s al. thee were early 46itePM
as polWr bemds.


te stability alatlm-hip sea be lateed by eaNtUdN0ast 9 f
m*'beied Bit tmate stas ( V a de WMils erwtl ). ~SaeM these
me tetau eeitrlWe to a tt ttatity to a eate t& th is es

with dNatails itmseina diteoas, t h tfs4et csW t seed ts
*tM th d vlatit siabltittas of dufftee k#amEBlss (i), b

the belt to"d the 2,3 ed 56 wreep, awe clseW to sbek hetr thf la
the Shalt 9s0 Theslmfs, the .hltt i the emse tables *eOae616
Zn addi4ite w 4der W"ts revrlp elersue deasity a Ge am atSal
bakds has boe sY rtsd ft be 4 beaotr i* stawtis iatmtem (), h w
lateteeie disease baeem ray pea) of xia l grnps a -elara tha
the~ bht equtl raps, Oehbeses, OV" dal ggei mlbbit W -es*
Wt VWslds tesmrd eauk ths. tuIs, a OaquInd sit.y, eartl


- 20t









msftitumet to leo stable thae the eeevpenmmag eam und with

eqmatmerl substitumeat, whe.e muan4aed rpllsems are at a




rTe ga meriMsate l that sm be ard flr oem i*d tug

Oytl- u4e up of Sta aerrad rtae mush as the etaseaid is that
the stable strsters we chair seotaisties with as my a

possible of the erubstmn la te* equatoral peltiwee. As a

eewsemtstiw saftemtu oLf th steod lemro, th ehlastmOl
alMslb (Opge 7) ay be awnlad. in t us lele s rlgs
and c an Isied ti the shalt eamnwstie b ty .~afubionm stagr

A sad D. hag A is far to aseum the be te or haits te bot
the tastabitty assestated with the best tam. of the eyelshaina

olmaulte it auoinestd by the Stsetetes between the methyl pemep

ea 0*10 ad trh hydrwesl tiOp as C0-3. SilaUrly tio sleshtm

ttrhe 3hydireg. sad the 0-10 methyl stomp op she boet fto (29).


It the androstaame olame (tLgA 8S) the ria system of
sholetamt is present but the side *halt at 0-17 is laskita. Ite

adrestame soi ulel it deseibed as bhair three siezrm ied chair

rslgs and me fIlw -mbrd risa. Ihe anal hydroga at ptoiitmt 1,

3 7, 9 and 14 e it a lime puarlll to am amotaer. he ertbee
pa tl 2-3, 5-10 ad 7-8 U li n am plan a d earbes trs 1, 9, 11,

13 sad 14 Ai la a eseoad pltu parallel to the ftlrt (29). SIe-
iatia .lt &is predusea a slightly psherd tag due to the beaSifg
at the beds. the hy ogms attlhe to 0*15 and @0Sf alo selie


* 21.






















a a
a a


FIGURE 7


Cholestanol.


FIGURE 8


5a-Androstane skeleton: a-bonds ---,
The equatorial bonds have been omitted


p-bonds_ .
for clarity.


- 22 -









aod the eqaswial and axial soanept it not anpliable, the bet
at 0615 asr G-17 have ual-exiau l (a) Mad t~-st.eqmttal (e')
welatsIehtp wtth Ipest to tiag C


VeMftl Sgmeu s l ties vgdiag stability and wasetivity
of quatwetal ad menal kma titlMts has bean dWled (1.34)
* fw of the pratfiar t e0m my be b arMLiad as BdeliU

1. A mIubstt.emt is gmMally wer stable it the

Meqmulal them it the extl otesatu re. Tihs,
it *ub tituet es be oqid lbested is dtflemt
wteamtesi the equaotral will pedolnmst la
*equiltt waixtea.
2. As oqeatetal hydrsxl roup to am*e aMseelble

tbhe the aextl smd th~bwe s mets am vapidly
then the daxtl M9p Io eaterifteaste or hydetplyt
rge NUtlems.


tortalX aebeol I msme otrWsly adoebed them its
axial ten.*
4. The equaeterl a sUtal rtetaUette substttef tlt

is oftie flMsted ta its lafmasd s partLa *spstm.


- 23 -













Oratesttta s o "btita s at spesifte pesiateu

*>mgia ted below (35)


5a~csownt.

~~ ~3na

a 6

a

a

* a

aCAD)~

*, a(5)


* a

a S

* *





* *(A)a ()


amd SA.semal


SZi



ar


. 24 *


2

3

4

5
i


.


*1


XI~rr~ la.ldmtus


i- T i -rrr ow









The spatial orietati~e of a su ttamb1t i lrO p t isat
ated with nSfwr to the s gal r methyl gormp bteh s

p*r-isted. thu a prmp prejestimg lo btet of the islMs is
retired to as beftg j to the methyl proop d a peup paejesat

tag the sr a is to them.


IhM toe major *lass e tf isors adstams re d-et4
ated as So- ad p radvresea e sad ia ftr i the spatial rmfisu

WtOt. of then hyeass an 04. Sa the Sacft e ltroti the ruw s

A med lut to ash th sa d la the S seutesn they noe ul

sitaialy, the 3Soby rtprm is to the Mgslar methyl feompg

sod the Spiyk g4me is fag to thbe. IubseIttatti m of ay winl

hydrEnes forthea resltso t tihe preodutire of st -oeer M ts pIai.

Thes SOiedwstamn ol and Joa esdVost p-ol differ oely In the

ems1tat1e of the hydremyl rap on 3.






The taelattea mad etwetuwal eld~sdatie of asdulm s w ti

SEbltbt hbem a activity pud a p roble to the olrwy ts wstitlee.

the smmeZ a of stieea beumoes hiabi ea be isolated ftum tse s or

adromel serea is very mall, honver, Imaue ameits of the sterfid
herames or tir tMUstabeUets are really foend it srita. rawmtien

halnstefristg the testwelar bhomen instlde derltapMnt f s eedar

sel aebaestalsties. Amg the early usthods of bleussay of hbiAl

elttvity, the mu t setik stery mM was the uthod based a the abllty


* .25









of the hwbofs to pr tee see, Fwh t Ps iaew (M*37). ksesdt

(38,39) ustas rwie an thke es of baml, w base the Eflt to
Isolate ad tdieafy a of the prtsipl *i.ethe f the mlI
hIres. the Isolated evsteau eb s tted baleSteal activity.

ha-eil htrnLermtsMd ee tiadiested it to be a *tmel-Utbe betes

Mad IStemedt amg" the be e 4samdredeam, the s atesnts etf
Bareeame ra f thbe elsuideed by partial synmaet ftm
hbelesm.l by rbtesa nd his asselates (40-41). the sjaiMlt

eepeMd me Idemfea t totimt Iselated ftbee aM by BItsmmat,
bekh smtiamaly and phya'ologsl lly, sad tu strweamr u wae to
be 3ahytr--y*. --m-sewatr-ams. To Oeare bItoegu amy astiw

semIs of dw arses e nythewis sd "g dteiatin dahtly twwus
afte*. IBeeM we ~hyd Sp-ey apgspa-lT7ems sa *dfnr y
S0e0a etmsa-17-em (40-43), both of whieh bhave st been tIslated
fem b maMurste (44,45).


JMme the e(ig 1 a drsteaem the fist to be Isolated we

17p"dthiydgpW m .5n eOestm 17-mea, whhish uwse elleid by the lUlatim
of 3 sohyd "i-s em5adtr -e1.17-dios, 306hyd0may75p rhwetaae
11,17-dimAw d 3, 1l3pdihydrtm-5yll -edesa -17-e (4468).

hmgIenis aetlwity ws dimtretd I fr 3,s,137pMthyde pmy.Sen.estm-
17-e (49).


NMm urt.e also e ttas sterldes hlrti a dewtl bead be.
twieemn eS es 5 sad 6. 3-tedat aod his eo-eobe (50,51)
seelated and Idietled pt- .yd.r.mdrSr-ea*17-em.


* 26 *









Uebaem S d hi* scr p C() replete the iotIattie of 5c
Mdrmeste -* 17-4ie *atd S3-mdnetae-3,17-die ftem am1 ma
amd fuese Mrims.


DBrtag the iet desede tho ee sales cpheatf the seuei d
useless ad of abmelt all of the naturally ewianusg t m1dsi hbar

born a eer rted (S)S6).



motm C o Uf a Ale rMMMEt fMd 17ttis nwida M t

sIgseems itsseaId brm w ge a t t" s""s wee fat eadtaied

(38s,3) by bitreel y method to detunmte btols eal aittity. ay
defattie ealy these awMtem iwtmer ew tbittag bliaelgeal
eettvity a sesued by 00b Iropth of caese em be ealIed sodseaM.
brta has been tahe mjr se ef s ei ster eid o the w tset m tr
and sime met of the ustawy steroids wiaek en bit dmljwgsmt

atiWtty &l" esatsta 17ketem Proup, the development of ehteb al

mntbods hba bees diestd tunard spesifie restitm of thits tap.

STh psedure meet widely used eor the detamiMtl of 17testeaed

is the *eolutMtr uesvureiwme baud as the a amamness srtestai (7).
Tseurrs of a 17besen with jrdatlwbesJuaw la the preesm f

eaUiU Stw1 taumsiem e rnstie piod t of volte 6oler but f mu-
baom etruntamt he oolered *epmdbU haU abeTep tte mamI m at
S20 i ad is mrffi~etetly stable to peamit wngeeslblk redhp.

lhewea, the botme prs p -s ewba 3 em luterwf with tL w Utes.
l utedat *f Sthe abov premdrvw hve bees verimee by ZmL --.(5m ).


. 27 *











the shardan properties of a steoid lI these of any other
r cia omprl shald be eaidmred a the wr4asto of the
hostlrnal pr of the stearod maolwal. It m be asume- at
s isolated emtismul pep um ad eest ls wth v t Unti r -W

tearoo" m enthe gro s. Is cases f wer tw o pouiqas e
ftfleetly *,ose to talmusese seb otbrs pe lIperte, thbet
vsetaie m-t be asmaidend together.


s elslatie nvemt tes eof dial &i t nrtaral easbteU.Mn
ha aelmady beem emtl e, ed geMral, the hyde 0p l tor a the

otlod U m el will ldmne the e mem reastifmte es bol thi

sreu, *.e., ertetfl0.tie, ether formtiem poiade 1a-tiema,
waplmein by a hlos cat. Sese esmaties ban beesn utiliail

ia th isolatieB a meaalysls of staeoids. ft as mas Mr Imt Oest
tieom t autNh maeosd emploredl were, tiftoaties fa she pyrastiss
of asety s trflruwesestyl d"wittw u d Simr w f"if a t
raesftoan X traiethyuilyl sthmo eof the *e&e*ds.


Ihyteal mthods employed ia the e aseeenltsastoe tO stoWis
lmlu4 te attwiele and tasted beorpeti e epwtlAl etaty di.prt L

t6ay reeallrlphy ad charmteraphta elyst.


WlitwMelt abmripta was Latredstd Late the sterm d F U
I the ealy 1m's ad besom mtsrmly popea as a tol the

deateft md dAte"anut e toe mMunts e" of sampsemd with


- 8 -









buhuiAi relapew. With the Oewulatli of data, *oumstloa o
stnetwa and U ytelt abseptel feUd.d a po neful O WB hed

rw tbe lesidaetim of the stmraet" of a Iwr vciaty of stereMB ,
es fLtld has been s mwoed by rtem (59). hrs rmeautly Joe
sad his *smwhr s (0) tSeat ~bi s the 14a"m abeeItsm shel-
terlstles of stee r, the SOutdttlea of aMial and equatoetal
artataetl lat In ee duse n tafrarl speatal rulysts. oM wiv
atlra of optiel aetltt with the wve length of the light s kha
and usd by sely lt"watates uatil 1840 whbs 3sR developed hi
odim laiM sad a mhn,_mt_ Ight seae nre bse-m awtlabile, he
alpUatiMs el wuatey dispewsit ma hods to tbe stearid field u
pteiammd by Djwral sad his asueesoit (61,6). 2he abseteptI
bdts l roestery dispenim mneur nts r easitiv to eatfr*

e tinl huaes sad ham been sful In autabliUrtb the wlldity
of oweia strtust l wrlatimehips. x-esy dlft istm pattern of
steunis bse born usefl it eupplirMntia itati d awslysrts idt m-

a pelte detelastiom (63).


With the advest oCt hveestrahie teeique a v lblo tool
we added to sterod awlysis. Ade rpties hrmae y us aN p lod
to the pviftleatls sad snematieo of stevLds (4). Seolvaa ysteaw
of diffLest potarittie w de igned fer ueasewstl sepe tties by
partstieo ehrnumt sphy ,(9.11, ). amoed (15,63) lavemtfal ed the
behaviorf a waido ne of ato asad CI6 keb t4t s sad saeeabUied
the appIltsabity of the li rse-peropyleo glyeol yate to the
soeltttie f e the eteeatw ld. Cue taa sorrelatim beiwa
*heinmteaph i mobility and structure beg to emeas. he MabiLUty
S29 -









of the steroid olule was observed to doemr, witth tlasesla
*mker of mag ftettieos. The les them empe ted stnrdatias

la the ability of a steroid with a 8*21 khydroyl slbstitmit 1we .

tatsugp ed as an iAdiatiO.a .* tatllesemr hydsgeO barit". "e
grmal pattern of webilities observed by Sawd w eeMs tt with
the ematp developed by afteo that steroids pese.tg equMtetl

iydPWra~ yl r s m uhibit lower tI tti-s tL'm thL, saene eal ..
iteam with as atal hydwtmyl pgrs (31,"). resthImNra the
earrelaiem betmmns ubilityl umber ad amu of e ep ttMl ,
poities sad eremeate: of hydimyl rmepsi tgetba wlth the.l lsets*m
ship et wmbtiLtte between SO* sad 04*tisaum prowded gmeteiUsatle
whih we is 48lreeum with the obetrvatsi"e of. TUsli-n sa h:i

prap (52,47),. M the hteltieslhip of shro t Stoe.tr eltiste
ttu to ateetu~, eartlaa slaes t the nature of ehmm eaiunMds

moe obtasied (). rt tepraphy bera" sad after seslattes

embled the tbwetipe to "ass the lbUr* of, setyble bldrinyl
aroup (0,70).






Stweld isem latue is based Ms the aule deblad at the 1950
tib W~eodatLom Cotam s mme Io Lad (71,72). ~te m ule whe been

pwelrl~ mally apprwov by the late ratimal abtein o Ire al d Applied
Ch-istry. 8lm the Sameltature system is of reent orlta ad

stime rwy tavestiater still ealey eamo- MMs, a brlef summary
of the approved semmslate e will be gitrs.


m 30.









Aoetdlg to the a r Syta the Me f the *tuoed tr
band as its hydOesrbs melaess to whibh prefixt s mad smfftmi

ao added to Late ast the astue the subetits ua tL pSiCts

of thw embastitmeae -e ladieatod by the mobr 16 the eatra

at= to whdsh they awe ateshted. the poJmtiona *f the sdoeti*

tomst to the fret d to the wenr of the bJy*eeorbu m t e tl sWe

dedtga d as p ad a. vreepetively.




*s, ad with Meak -e, the O.antt tisn tat C is ladisetod -by

the pmefax a Sp. eaatuestle to Iadiseated by the stfftw ** ,

eg. S.ras. the rues tfr the subatituats state that ady ea

ktadi f umbetitme nla soah eampoa d ti ladsatd by a suftlx, th

tiats sur otitu e awe ladlfeted by petimes. ta substitute

to bh dlsl esrl by a mftCs L h eshm aoseod a to the ItosIeUsl

a6rm ofa piorL tys eambsolle aid r dw uItatrive itudlasl ertm

*eaibrl, alohoel, eam, Clhar, halogls.


*31 -


















slewltuls ad nhm*de








tleeds tinetited in tlahe ae teto s*dy m
sheen 5e ttCr pi etlar aset mat nlteanI d btlilgaelt

i ampet. fAl m ese tm w wone meret n samo*, Ae









smeiaAems, g.1pa. mtinntr m-, jai .fal p*dt.. bwhyd


Slef*a nteS r LkI ... pI. a.m t UpAnhyI

3. 17mdtasne1**, 1)eud tkdsmc w 1n*se b*nrSC'
l", iMYees Im 3LJo dt.Bhydg-*1a sedeesal7**-, 17p *m-d ep*


ererue vil,tar7< .


t* a a


I eI I I IM










.ni, Aft 3IS^n-to ef *11 tas SiBamso USaW

bsv iwn studieiu *ul-omsly with the Sbe*meeinld. 1s

ammtes is to msIly the sia as stet stsi N m t t

detlosaUits 1ll be A -stitad tor 5 to assh saori e.







t ny Wms,* I assnesm ,uaerw p a waston ma
hpIsbdqeq--gs4. -Sm17*em, whish is italuied beam.. @t t a












1lle 1 eoaoWne *ill be ande to sn* u1e 'a
aB aso "ftft of t" nWk iON&lA"^ thft **-t *fWe a

T'LtM ci ww -w tuvAUtY go" lla IMI i taftemo --it It t

em sIsi are still 1dely used It the em e, mp.siadlly it

the rs *f ianistl awwoom iAasna. she .eSA ain sh atesei

names at use --as ase su misei belas.


-as-L~rr



itieshe~dlmlsu


1hC~Evh-euainl
If~-~ -wn rd~~a-


"-"eWMns

Ptl- "*


*.kimy.Sa~ardwsee* 7mSUtse



b, Ul3p*Mhhpese.b'uml..m4l7.Y

sp yna- lgsse*flte*atel
S*.9we *.nebmiaeme

bm.7.Ipwm.*smsrr-% -


* S33


I- -- -1----


I









te the ttel smrtm g the vsesmlte balm ta ts
apady, an atbbelvtaW f of *-asa 1L oa4 Ma l st ms Y-peIr

ot thIs itnetiStU wa tihe mrMta of Ia/S Iem-me plt ,in
tet eatsta"eI a t s to written as the dpo Lax to UMh ahbretsntd

e. lor eas.e, ~ t 40 qflh s17.Is to aeVtaed
au jao. mollt17mae nd pIhr;y3mip, .4iurr 17emts o ab MnsWvl
eM. 1 Sp*IA.p*1,17*.m.



So m.Se.i wee obastad ium oemaM-etl s e4 aMsd weo
of 3sestfty t pw oSe dioset asn. f s h Wu d~sem

sdmse -amouse I et suptat who meiu by pa shwSassesset.
thr .1 she 17-gtastotegStda< (bY ea3 -I*efa desn17.eme kstE
hwslSydir Ip fartM*17*ies 3pl" a esommrbyta*4*7*)
Ibms s*lymed by Mewr hlotsp4 hi ImkAlisi as tMlAtU





ift dnrtWtw ofa the *tWOuse wn powsems by the foUlanI




Amt as. a ses td (2-5 S) wa eUlmtn to s rts Wtah
*aste mbytdt (0.5 aI) ad prIdtatL (0.8 f1) at 67 ta a day be*h
W s heows. th .lv wn qepts ew a 4wesoO aMd
bthe s d se, seate us dIW setolwd is astems.


* 34 -









azLtemf fta UI1. as smwtd (21s m) w
*llmid to .jst wAth aenttsesetle ainf tddt (0.5 al) and
petda (8.2 al)) tMhe ~UetlI as "em af to a taw states
a e. t empq1g (73). mthe s vwoes eman ap ate as





,2m1sbto t sthes aL So se elevd (1-0 M)
Wse at2ed t. one wv t aI a th lsssans (0.5 01) sad a
lw d4pe ot rlte, mthestmst as ectatrle owr 4 hom at
ram mep su (74). lhe restsLa bLutem vs erthug a
the miontmst slst3s vww pevesed maee utm c IMh
tYwi* w trtse sd ith bomas d II stt s4. tL uq
wrStm wM4 .Su aSpllted md Wttcr nogm sad the IhmthSletlyl
et ase5 s edt4 wm dMarulved ia sn .Y..a. (s7).




,Sdams a mhe som 3amwd sa she"a mltgmtv

ths trbee etmtdy. atesate etv Wriiufseetes wis ehine
mqassghed bra emotes sstKn, tHim t-a l ustLrttrt sl ne wogm
ebre .nprash.eS a sem f 0h.2s1as soluti,. AfL* lutt"s. -l
awe It st. lbams of 0.2 11 tI l s we Ud %WO IaJest Se. AtU
~swemts wroe ae "la aLpivat.


* 3 *









Q: emKple of eash derilvtive wu slysed by laft ed
sptresey to ae rtala the mbstituttes of the destred gmqp

ea she stertU mules, Saeo of the drvts ttves hsued sall

emmta of the 6rs etvolrd whish vere asly Idestifted by theyt

*haseseteitU pesoI Ca the shremtoe iNs.



MAdama
aelestme was used a the slreasf emedard for daily
-m smemt A stuMetd eol.tee of .ehsleat iu -seaM ee wU
tjested iato the **Ium g sh 4ay befase y sa es of mosw wee
earned met mtd "u sepgeaed wey to or tI'm hues. Who IM letim

tiaw *f the mples ere thtn related to the vtea mA tat eof

Shoes tese d we emesed a relative mts tues.






Speatal grade aeste eitherr lsatmtie ge.) and t*r t

-re MalesltkKebv OWNSasm or"aa"l he"tleIs), "ih0w0f900
psids, heaMs dad aette ashydride (all trea fise kSetastftle
go.) ve" aqmloyd. pyyAinsA a"d wmast sahydide were ftAwbly
dstilled beftae use. m1t`hyiit amM, tamethylhlsooeeteei

ead etflteeOessDtese ShydtVid we obtaesad K + K abst Ieen e asd
we see vu tuhat ftathear 9psiet tion.


- 4-


~











G mefnrllty obtead asid-lwshed ( ad l th eoa of .1he
ls t ro, stlistsed) dB4itsWems em0wt eMse ne d "as 01m
suggests. VWOO wvers

rhemis. V (AnUit ae i La *besesges,5s esetU",nft.)
e o n (rn,11 Gorp., PrttOt*M fA.)
Aas Ass (salab., Eafmas, nette*U )
Maatam er (r + asieeta Cosp., AbMiselef,.)

afflmest amk sts sopl ee o*w tadgod under ta4*Idesl














n5323 usthyl phimyl *iae iteuyll
**Ima prmqelet, es,



AssMlMr.ilase
th o1,tfa tcmetally obtott pnos werVe ,itaeB










xslUO rttLi Uhestl pLyme:
2*30 uyl r east p eothyl)r
ZIAM llUat r *
xF.1150 tmarlle enteow peo1or
(St MIA Per t ~yeaMsakyl)
XAlw-S otrtle atlaume polhrw
(5 rsol per soeess evemathyl)

the aban players were obsAted burs srl Sasty
e pa as esort a M W v ft .


* 37 *












od 50MX ee8hylmahayl 1 "lWrin. huis Is .bsimed











sDew oraf h4lt vcIm wtlises pise f ( rltlwme)
(Ityl etate mast) ore abwe et eam obetaied

ftr. ew Sstag Coeepr, MltdlJM, iflgom.




sahylemegI. ishaltate (05)
nhyt ngLwostl .msetaste (6)

pem1tyl*lyl edigte (IMd)
mepatylslet l Msbueaoe (M)
Item ws eoktaled hse AMaslts, irt, 8MtlssW.

tlf1t 81 Dlr0stky0el0hs6yl0e"us

Iks s abtstined ftm AptLed satemse 4b4CItorss,
se"ae Cellos, tIm yiilvs.


. 38 -











( geo)4a 20
SiAL s-m-r m w t oenid Lm sas uma epand





INmgdism Iat Ift u

'T dhe tyi mOMmt Et tlUVd lbas- was wls d ea4d dUisseo
to a sell emma f *k eldo (fo ilAUo ptyesm) ) rfcm
(ior all *athir hes). The toutate wms trmaKlsee4, with Isttl,
ts a b ee O4seMMlSb the wejsd Msqpsrt epmIMas ta the Ws

l"set a "e tar 4i8semlba the lirid polymyw. the ulae w. s
he"td tly with eesmat stitrtag vatil the rw hG t had empe.

vated aqpletaly sad a Itmr flowiat utifemdy seated aUppof was




Wh matched of depetitle of the lquid phaIee ase a s0up10t
wms Mliable ed mep dwtble a d ws prfefsble to he hfilaotim
abo4n (706). I the latte t whod, a s1owry o9 lf id |ie#e, s% hemt
and smemo r wi s ltamd sad thea dvid vtth at welft, te mB sa
e.gteft hio e hiAld hiase deitted fas he up t w s the do.

pa dest o tMe nsh *Ie of the support as wil as e the vw, *of
wsoht Used


* 39 *


__









The seaed support w packed, with gentle tapps, late
a copper or staialss steel eohma ome and of which was plunged

with lass wool. The open and of the colum ws flared for ese

of peekift. A vibrater as as aid in peaokag we foend to be o-

dstrble as it resulted in ftraejtatton tf te suppren d *Is

pyeducd tightly peeked eolms. The epe O ad of the eolm us

plagged witth glss wol and the e*lum was bent Into a shape,
taking *ra not to eeastr4te t h diameter of the tube at the wMr.

Ibe eolmm was the suspended i the a ebemItqIeah ead eaoed foe

24-48 hoers t the desired t eMrtwo namdar 1 pts of presmue of

carrier pU Darti g the 4m itag press. tih outlet sad of the

olam was sot iemnneted in ordo to prwet the possible seomW
latie of voUtile sb stetmes i the deteter. After tihe 4hm

au eowd, eamtma s So teo dfetest. ore mad and t'he m ole ws

saturatd with a 1-3 pg quantity of the rvtereas stdad bletatese.

he so called 'prnaiag' or satuttia press was nesseary as hoese

eolmes wtee a certain a m t of seaple bss, attribWtable to iwe-

versible admeptiee ws observed. Ats phemmas weted fts

*olum to som and was depadent on the mature of the sample. Pta

emmple as te X*.40 sloe, sthe was me obserable tlass otf msad as

while eatrogens wers ade sed itrreeribly.





Mesm w nts Wre m des on two g a ohrea1mteaphqbi imtsaui
Meodl 600 itea beseereh Spealties coi man, IUtsroad, Califaents, em

equipped wtth a potimisatioa dotestor (77) sad the other with a fla


* 40









tit satia detester (78). Slightly higbr smeitlvtltUL wae

ob rwed with the flu ioaa tioses iat. It has bn reported

(79) that atiple hydr ayl prups ted to depress the wesittv-

ty of the p-lotiation detector.


Both tastrmmnte wre equipped with a taiajMttioa syt
desired for Tismy and lanrl type alsredippfer ajeat (80).

a tbhe amther' eqperewe this type of itajeter us rae savemsn it

to use sad Smv amu ahproduibale result ttha syrae I type taIjatres


The solim tmperture wM esetrolld by a proportial
smtrollsr. A teo aside the heatita eablaet wa ealoyd to

tioue atfiaf distributioe of haet thrwua me the alimr.


The errier seas used ver arten for the p-lansiatieo

deteste itr tand nitreges fer the flame mat. Ia the latter suit,

aueased aIr sd hydmif e ver ewloyed for the flm.


* 41 -













p"Umiumary Kgxpfiaft


Ra1ults of p0rlieuary x will be vw ftaed bwiftly

and oely fer a rfew irumWtatti aues. All 'msu-m-nts we

ade aM to hreoaitar h equipped with bth p-iAriss r detester

atesw flatested othwiato. Cohalms wro pasked sad swed as do-

sribrae. Itbiatit tims (tx) a e reported i wlaue. All itajet*

tlas vre ude with a 2 pi aUquots of 1 pg / pi sawttaw., he

p* iisatt i dt ster s Opeaited at 1500 volt. Dettet imp'.

ate e 2453450. 'aepriser timpenrt 290.310o.




a3*3 Ith JB.arl i... ftlWst at*1
ithes elues diffrttl g la smetrataeet of Sn,30. umb tel

etf ~sfoper m the lgth at dimnto of the seoluwm e eplo4ed.


it A olam of 6 8C-30 coeted as 30-60 ush Ch-'mebet V

to a foot 3/16" (o*d*) eoppw tube was peipaed

Vta Ateue of reteatita tis with t.rsan of the

e**a amd peUssue e~ the datlewr s ane give ita

table 1.

2. A e**ln of 31 81-30 coated oa 30*60 uesh Cbe.he.o b

la a 5-feet 3/16" (od.) copper tube ws prepared.


* 42 *


___


L; --






















'-


o -"
L *
r.% f


0

a Ln i o
I i i *

1-1

e4


I-I
0
U
0


bo

0



0
0








p.1

44










4.1
0)








a,



U



.I



E
4J









1-1
0
g






njl
1-


co co C14 ON t
.





.0 0 0O Cao < o




0C 0 t C14 0 00
.
oo 0 0 0; oo oo 0;
r-4 -4 r-4


m

0
1-a

0
4J























C,,























o
O
0
.1l




u


- 43 -


1.

0
0

I


I I I *


fl


So mr o P4 o o o 2
i N






0




* CM O O O O -
S- I N NI C
E-l


"' --- --- ------ I--r


-









Separattio factors for adrostfa ismar. wader

diffwrt eoadittema of temperature d pressure

were masured (able 2). Aetate derlwctiv of

the steeids were prepared with the ai of Ltprow

lang speSatite fstersO CoqMnCar e st etiotn

tam wvlMas *f free steroids sad sterold retates

art gives a table 3. Colmin sodittls:

T M1~5 P 1s ps.


3; I A -l*m of 3% 5.30 seated an 5040 mesk Kcnt UC

in a 5.5-foot 1/4" (e.d.) copper tube w p~npeed

IRtesstta v le and sepuation tfatenr of thle

174*testred and sa the reepfstiv Setates Mw

givm i Table 4. Colnum eoditise: T 200,

P 20 pet


Iltdlidual empsamm eaoald iet be rselsed ken a mlatus
eetal4mig the tree parimlpl wurtiay 17)e tee dsr"e w hxe. t*e

grphed. Ametylatte of the steroids did eot vesrsl ta i fapsed

aewa tim. (osf sestimms i mad 3 bshev.)





1. 8X30 sad DW-tlieee

A s*Im eoated with 31 85-30 sad ItX s*eIllsem was f owd

unsetsfteotory. Results nre sot reprcedibla* h esema

we dissaded.


* 44 *












TABLE 2


Variation of Separation Factor with Temperature
and Pressure on 3% SE-30 Column


Steroids


5p-A-3a-ol,17-one

5a-A-3c-ol,17-one

5a-A-3p-o, 17-one

5a-A-3-one,17p-ol

5a-A-3,17-dione


2240, 15 psi

1.00

1.08

1.11

1.11

1.18


Conditions

2100, 14 psi

1.00

1.06

1.14

1.09

1.16


1920 11 psi

1.00

1.04

1.16

1.11

1.16


- 45 -












TABLE 3

a
Retention Times on 3% SE-30 Column








Steroids Free Acetate

5p-A-3a-ol,17-one 8.9

5a-A-3a-ol,17-one 9.2 12.5

5a-A-3-one,17p-ol 9.8 15.9

5p-A-3-one,17p-ol 10.2 14.0

5a-A-3p-ol,17-one 10.1 -

5a-A-3,17-dione 10.4

5-A-3a,17p-diol 10.6 17.5



aIn minutes.


- 46 -












TABLE 4
a
Retention Times and Separation Factors on 3% SE-30 Column


Steroids


5p-A-3a-ol,17-one

5a-A-3a-ol,17-one

A-5-en-3B-ol,17-one


tR

6.3

6.5

7.1


Free

Sep.fac.

1.00

1.03

1.14


Acetate

tR Sep.fac.

20.5 1.00

20.3 0.99

21.2 1.03


a
In minutes.


- 47 -










2. -. a nth1 ulmlysBtl XItehalate ("aI)

hees *Olimn er proevd

a. 1.1 8U* + 80.5 g?

b. 0.s%1 w

e. 1.5I 6C3-

siiPaes sesMMlet obtaIlA a the tohs seoniM

me givw tla tbio 5, whiwe the tMsdidsil euet.

bt ehus 99 iek h se Ist tsble, e.g., to g. tb.



3. Sad nd l ts*tfc ae l)ea) r uMsd uts*

Sevs ..iie em enpred,

A.* i Il. tfMbKspphaSy1)*.Rber (Abbeedesed
-s phuV a fabl s*.)



,* X 1is* ueieqehimb )sth+ x we"3 (1 2t e o)

*o asBee-ew 8 n*30 (dot ae t a)


Cee seesditatnt e 1u* 4, in 30 pet.







StnrI Nhtb (3 ud 36 M"os)* hp Im. e*sMI ut be
eaied d-e-e -, on eek sawis ga senL iin t *n .1 a se -

Oftis" 60 Uf p lrc am -w d ofM rtlas *f Ck y-to

Aftti a rbmt wte4 aW mot, tealUW bisast puttksiM tale

tols d so -rwspaets tw= s we aUwbed as bis asmos ms

*NaOe waef wes a o**meAie d.


. .


:








-

'''
i -
i







.'

I







TABLE 5


Comparison of Separation Factors on SE-30 and
Ethyleneglycol Isophtalate (EGIP) Columns


FREE
5p-A-3c-ol,17-one

5a-A-3a-ol, 17-onea

5p-A-3-one,17p-ol

5a-A-3 -one, 17p-ol

5a-A-3p-ol,17-one

A-5-en-3p-ol,17-one

5a-A-3,17-dione.

5a-A-3a,17p-diol



ACETATES

5p-A-3a-ol,17-one

5a-A-3a-ol, 17-one

5p-A-3-one,17p-ol

5a-A-3-one,17p-ol

A-5-en-3p-ol,17-one

5a-A-3C, 17p-diol


1.57 SE-30 +
0.57% EGIP
1.03

i.00

1.33

1.18

1.17

1.08

1.36

1.10


1.06

1.00

1.34

1.57

1.43

1.20


0.57 EGIP
1.00

1.00

1.12

1.12

1.03



1.32

1.10


1.17

1.00

1.75

2.00



0.96


1.5% SE-30
0.70

1.00

1.30

0.87

0.83

0.87

0.80

0.87


1.25

1.00

1.25

1.05

1.40

1.20


The values of these compounds were used as the point of reference
for the calculation of separation factors of the free steroids and
the acetates, respectively.


- 49 -


- -












TABLE 6


a
Comparison of Retention Times on SE-30 and
Bis-(m-phenoxyphenyl)-ether Columns


Steroid Acetates 3% Phenoxy 3% SE-30 and 3% Phenoxy

(1:1 ratio) (2:1 ratio)
R Sep.fac. tR Sep.fac. tR Sep.fac.

5a-A-3a-ol,17-one 17.6 4.4 21.5 1.36 12.5 0.54

5p-A-3a-ol,17-one 12.0 3.0 23.0 1.45 15.8 0.69

A-5-en-3p-ol,17-one 8.0 2.0 22.5 1.43 14.0 0.61



Cholestane 4.0 1.0 15.8 1.00 23.0 1.00



a
In minutes.

%


- 50 -









*5 LMethsrl asml tiamen hlro CB0,5-2)


A eolum of 1 Sl-52 coated on 1OO110 al h Ansahw ABS is &
4-foot 3/16" (o.d.) copper tube wa prepared. Reteitim tie- values

we given t Table 7. Colm csnditiou ss X 1, t 2W5 ps
and flaw ate 2 l/ulln.


bhL w. s a obsrable adyntage to be gaied by the mws of
*-52 polym as eepm.m d to W-30 phase. The tfae M&ak ppesr t
emsed a oeauider ble desrese in flow rate ad it wM dreid4d to pe

fem s bequ mrens on earsew thba 100 tesh supports



Ba. 9mha rak Veman aluoam eans

A e**tu of 305X Cs1iUeoma coated ea 4050 mesh AsaeMam AS
ia a 5sfoot 3/16" (o.d.) eopper tube wU prared. Colm wau toe

retentive MM aftew it was out dowa to a hingth of I feet





A lum of 12 M goated on 30O0 ase h hmal wom tb W 25-
foot 3/16" (*,d.) eow pp eelmaR M poepawoed Cdim me sV ety
fetemtive; so pek. Uwe obaeved.




A aolma of 3X MIAd coated on 30-60 .ah thaQemaseb W tl a
4-foot, 3/16" (o.d.) opper tube was pripaei The- celmm required


* 52












TABLE 7


a
Retention Times on 3% SE-52 Column


Steroid tR Rel. i


Cholestane

5p-A-17-one

5p-A-3-one,17p-ol

5p-A-3a, 17p-diol

5a-A-3a, 17p-diol

5a-A-3p,17p-diol

5p-A-3,17-dione


14.6

2.9

6.7

6.2

6.5

6.7

6.7


1.00

0.20

0.46

0.42

0.44

0.46

0.46


In minutes.


- 52 -


.i










toi g*mle so .mmmeeMr T VAtn e 3 pet, ee Mo *
Up iddw.,



as ws d s Ua mi ssm an 4.fbes MU oem ums weI,
as. u0es0-mm a uoh em mem ente4 d t .Sa maW emueMb l
B ha .e 31WP ( .$.) pepper t6 e m pWamiA. Ias pss dm
-na 1N.sw e sea e m Owpi-aie to b s watmI
nmiSlltml. lla aa lpa asI,, aml ameis awS4iML.








A -- b ad I U *G&W- goe -0-- EhWAI 0 1
esse 8 obu o thstem was aubjestdl o bl@ ****ugme..... ew

b a teassm em h ,sq emee ma. R em dmesss.





A weln et 31U.WS. -0ee 34 a 5,41 sna Uma t aa
$fat*s W" (o.d.) eqp r s uboe g111s9 a s mme
eaalsl ame lnd Mote g m s IS dIeea, go em Vas
doamle1d.


*S,*










TABLE 8


a
Isomeric Retention Times on 3% Neopentylglycol Adipate Column





Steroid Rel.tr

Sp-A-3,17-dione 5.8 3.06

5a-A-3,17-dione 6.5 3.32

( 5a-A-3, 17p-diol 7.7 4.05

5a-A-3a,17p-diol 6.7 3.53

S5c-A-3a, 17p-diol 6.7 3.53

5p-A-3a,17p-diol 6.7 3.53

a-A-3a-ol,17-one 5.4 2.84

*5p-A-3a-ol,17-one 5.5 2.89

5a0c-A-3a-ol,17-one 5.4 2.84

5a-A-3p-ol,17-one 6.1 3.21

(A-5-en-3p-ol,17-one 6.4 3.37

t5a-A-3p-ol,17-one 6.1 3.21

5p-A-3,17-dione 5.8 3.06

5p-A-3-one,17p-ol 6.7 3.53

5p-A-3-one,17p-ol 6.7 3.53

5p-A-3ac-ol,17p-diol 6.7 3.53

(5a-A-17-ol 1.0 0.53

tCholestane 1.9 1.00
a In minutes.


- 54 -













TABLE 9


a
Retention Times of 17-Ketosteroid Acetates on
3% Neopentylglycol Adipate Column


Steroid Acetates tP Rel.tR


5a-A-3a-ol,17-one 23.5 1.6

5p-A-3a-o1,17-one 25.0 1.7

A-5-en-3p-ol,17-one 27.5 1.8



Cholestane 15.0 1.0




a
In minutes.


- 55 -









A colm of 3% xp-1105 heated on 3500 ass Kr mat A it a

6-foot 3/16" (o.d.) copper tube was prepared. Rteation data sad

separatla factors awe given in Table 10. Colur eeodition:

T w 250, P a 35 psi, flow rate 20 al/min. The flow rete wa




Eve tkouh the separation factors ldisated thorweteial

separation, actual weolntioas of the compo-Rts of the. ixtre wer

not accoplishad. aThe ide peak area, presase of seam sketrang ad

the high retentivity of the eolbm in spite of the high taperatmN

of operation were major drawbacks.



WrlbQ065 tFhmreahlw NImatr (atO40065)


A colum of 31 QF1M10065 coated on 70-80 oash Audaem fi a
6*feot 1/8" (o.d.) stainless steel tube wa psaparend. MeaurmIM t

were usda on the chtu tograph equipped with the flam lasisatiMM

detestcri Collm eoaditionst T a 2380, P a 14 pei. It se

hoped that seletive retantioa of this esom a de katonaes cold be

used to advantage tIa ffeating separatitoas Less of phase aad

irregulat ehrwmtgraphie patterns were obserued sad ln spite of

m-ear effort, a reliable data could be eobtaitad. Qn ataGrpe

tati; o of the data was that the he had roasted with he glass mel

plug


- 56 *










TABLE 10


a
Retention Times and Separation Factors on 3% XF-1105 Column


Steroid tR Rel. tR Sep.fac.


5p-A-3a-ol,17-one

5a-A-3a-ol,17-one

A-5-en-3p-ol,17-one

5P-A-3a-ol,1i,17-dione

5a-A-3, llp-diol,17-one


Cholestane


13.8

16.8

24.0

25.0

37.8


22.0


0.63

0.76

1.09

1.14

1.72


1.00

1.22

1.74

1.81

0.74


1.00


1.59


a
In minutes.


- 57 -


mm I I












Ile elfse d ote proper eeor t fm thbe stafisay p hee
dth fltSet seeasd4est fo this tmdy. ae weefsied ras syt 1m et

t s*meltd Melse prowde *$ fairly wide me *f hydris t.m
deliase for Ittewatius with solvaet m1mle 0la. eosaWe of bhe

rlativ rlidity of the sklesos and the hers arg Ue fftsew s

of the rea daw eIs tl es eoa f atumtla beirass the Solute ad the

emEnst il.Uiu, thu* intiasteetu .see setiv to starts fteets

(3). TIr tie-ttias bawes the palr of th rse"s ia the

e*lwst nelam ls *guse with latenasioaw bua s the s lmwEt mil
eulu themes aselM *see of a platr srbts+ se ta p lOt

selvem my tbwhete be seA stde ed ep the b ility of tbh

pOls gaps eof th seluat to attnest purs e th e solsnt uelms
with a fltos eeaItabl f to Sstam tbh that mwe the nsolmet

emlesls thewselw. A rawepltr Olut, wMhih erase Mrt seh a

fer e* atteettem to ouepenm eot of the pe.t oslvet Oad the awml
sattxstive fe aWw Ia t he pure t Wee v-eetabliasbd. 8e6mptlr

slate as eth etdr hbad, xhimit few se a sebat e ateUsstw
essee thia polar ivUN l O. Ike eeL61e *f aWI-~ laM fs*it s
te attested to e- mother by relattily week dtispu ioa enees

ad a smpela slste may ex hbif the swe weak at eti as o
the solvent molsale themelves. A polls eolute tatwee ie fate a
s*pt solvM t is upmated to have vry little ofr as Iatoasrte

with She eWeet.


o* So


atmMr7e 1a 7es7l~aa 7Imeirm7s-









It hial*y mtvwaSr d uoleales, *Smsr irs tl my
-awr d te do ttmlimsft of eastras and ealit So an uease

la the 4dtpum ia fusnse (8i). Ite Metrstit basis f muse tlow-
eteu s s til l mleSt*, but umplrtallty i to lawm that gm
s-t **a tMusuous tsmd o ttesAt f s oete Br wes o*etly ham
they atsst elaSud u1sumile. fUt"ledy, vsaewd gero

atwast thd o olas of polar sueesgeom M" mae surely lab
do Mstuwed g*Eps* e -t-*ed mem my dnm be asswed to be




a6 tfhe Prema sWseeft t tah U sd fMoI 1t -e-em s0s62404
Ats addas te se *eio ital t asmpety s*oideii inowt*t ahbse
bad to east as m of the isllraAg equirmte af psatMbi at dth
seMpte of usat ettsm" lw T asltlltyr, s w l slbttMsyl, 1
rviwSeetty eau *h touPoss. 1v easpoas an Im I, as

loved tLa Mhis sty, she ckoe *f lbhses owa 1tf.. as Wsiesy


A ses o Itr avoistL ty


tes s aqB satifsted usoitads of *eatiwly hi Letity
wee* to be studied stintse-m ly ith thei dsewt t b ow ar
loe platty rtw bota typwe f tISOd gseisee pn aodSted. A
pelar *easiMMy phase st e "hae he l*a.tesia e5 se p6tdr wsse
of the aterti rlel with h phase and a smoer pham wsle U be
aeted proewsmaitaUy to attree t he mS-pelar s.tIoms.


* 59 *









UC-3 pllypmr ue feu to be atL.astfte munlsrlm
lhea. o, -.ph-i-r-,phey.l)..ar ws taeUstlag n teo adtu
nl .- paten of eo 174ttesetwe4d ***ne es ma $Us seastlmry
phase. ela 6). Namw shwe epeWod s at stdb e t a elmlai
*s*me s. rO pelyemter phases dehite th dest peltafty
but the etbs mas sresl to those uhtak wa thgumatables th
sqeutjes fastes the IdemItme t.mI sf 21fllS pbhaM Ws

pmitmlta, but betf le s pdasm seats the mlestll ptepatis
*of th0e stIeUS NatUet flutds d tshe ObsM1 stdility of sIUteam
pep~m sa bluien l


After prealuMawy lsasttU vgltw didffrwt Siuse
olstemas. sllUaes altr le peiames md wraeu pelywjer phases
bet* staly and ta utratsse, ftwr stwemay msem 4ah diell eff t
dree of polarity tad EUsativty wme esas Rl ushtyl subetemnsed
elsUms pm 0 s. Bo (smol.eate beht satUl). te silUems sftrtl
petlper M*4 (s Mtla e to silU mit ari fUle but mee table at
eltmeI d t-qsUeae) rn d aspmMtylglyel Msebso at m tt4 l
(eet stable, pelar) pwlater phases.


Whet ffet of ewyt th$e shm, l mlU f the *0 afstmimm Jy
Asa fSet the pma ofs a uide vmag of stesrAs had bee uaesgted
(U24*). L*pty S Ml mdnem (83) had epuotd t h temal sfabUty
md pele pepemtes af Musp~yllpeol sabaste. 3S.N stUieas
plpear had feemd wde e a M staUte y phamt samta the emly appli
saslus ON the s*U.liqd ftsr-me---ihi< maied to "soeei awame.




-: L":


X* 1 sad 1o.xff r me two mW peol os dvelolwe sp tS llUy st

hli t mI, re mu.


*61 -












6e"Iratiw DPImstaI~


All f the "smidties ad the Idn IaWt s utse" Uder
m *eals d Vaheds wee utilase to s pa*tlimd a) As stead. so
nmse-eratia of tkhe solutis wer 2 p/p1 mad 0. to 1 pdl aques

were talted. l dt estal mad i *asrter u u- eHe d ame
as %ban tge In 6 i.s. a p111idto. IMs OWN ehssepuA

quippt d with te plsaesmltim distetr (Oqrated a 150 etto) was

- wed eeles Iasdsated othebwte.


ell out telmaw wage Ned

ln A elim of 31 4*-0 eoted m 403-0 mek wsem AM
In Seie SA/ (o.4.) emale tube was pepbed*. Ae vm-epsa h

iwppel iitA the aime iMstmisi R detesear Iw plaeyp. esIm
ltt.rtes t 2. f 10 psi. letomw, ti Sui eheletamg *




g, A eol-m of 31 40 seI d eas *IW ask irlatepres
t a 0*ost 1/8" (e.4.) stale *s1eel tube veas Ine. e, s sheio

umetMh iemod wit the fltl me tosmiti detested rwa e-apId.
0*brm sdit es< t I a SOO*, P w 14 psti. a-sttm tie *f
rleletme4 "*9 viates.


* U *









OfflU.g A elum of 31 iest Bf 8s eated 40*SO ma
haem AsS la a 5s-ft S/31" (e.d.) ppw eo lu pIrt"ed.
Coetmdi at 238, 15 pe t flow vae 39 at.
rtmna stla fsre eblMtane 6-8 dimtes.


A eol of l 31*iftf 8 emmted an *IN0 se alrtaet I
n a inaft 1o/8 (0d..) s*teale. teal tae mes peme eIb.
emdi emua 2419, P 33 pi sad fLan ar e 24 /lAhas.

Atetfmas ts fer ehlesteme 5-7 dauaes.


MIlN. A oeIom of 3 UMb 1 an 3040 amh wemameb W tl a
6*fet 1/'8 (e.d.) statul.s stal tbe u w piepri,. lom
melamie< T 23t20. P 25 prSt ad flew rase 74 alXad.
beftiem tm foA r ysheleutm 8- *la u ftLr theb uri-em n
of the fto stteids md the start estatee and I11 d ites fee all
othae dempmd,


A kal m of X2 Mlb eo ated as 80-MW amH hlshm A is a
*e.at 1/8" (*.d.) stalda. *teel tobe ws pe spel ea olm ca-m et
T 231* PO 30 pe td la flw fLre .24 i/tA. atea i
thlessale 10 adtmas.


he val~tsiw reatim~ tLm i le val with repet to ehelaau
sueewd a- the asi eolsms under the eedistLie doeswrlbed ale em
a-mmio- lve M abl U1.


* 63 *








TABLE 11 SE-30 3%a
A Comparison of the Relative Retention Times of Substituted Androstanes

Steroids Free Acetate TMSi TFA
Sa-Androstane 0.21

5p-Androstane 0.20 -

5a-A-17p-ol 0.32 0.39 0.33

5p-A-17p-ol 0.41 0.52 0.49

5a-A-17-one 0.32 -

5p-A-17-one 0.42 -

5a-A-3p-ol 0.31 0.39 0.34 0.24

5p-A-3p-ol 0.29 0.35 0.31 0.22

5a-A-3-one 0.33 -

50-A-3-one 0.32 -

5a-A-3a,17p-diol 0.54 0.89 0.58 0.28

5p-A-3a,17p-diol 0.51 0.87 0.61 0.32

5c-A-3p,17p-diol 0.59 0.98 0.69 0.36

5p-A-3p,17p-diol 0.49 0.86 0.56 -

5a-A-3a-ol,17-one 0.51 0.58 0.47 -

5p-A-3ax-ol,17-one 0.47 0.56 0.46 0.28

5a-A-3p-ol,17-one 0.55 0.66 0.55 0.30

5p-A-3p-ol,17-one -

Sa-A-3-one,17p-ol 0.59 0.76 0.64 0.35

) 5-A-3-one,17p-ol 0.54 0.69 0.59 0.33

5a-A-3-one, 17a-ol 0.56 0.75 0.60

5p-A-3-one,17a-ol 0.53 0.61 0.47 0.33

50-A-3,17-dione 0.58 -

50-A-3,17-dione 0.50 -

A-5-en-3p-ol,17-one 0.61 0.64 0.55 0.33


-64







TABLE 11 XE-60 3%b
(cont.)


Steroids Free Acetate TMSi TFA
Sa-Androstane 0.27 -

5p-Androstane 0.25 -

50-A-17p-ol 0.60 0.71 0.38

5p-A-17p-ol 0.89 1.04 0.52

5a-A-17-one 0.65 -

5p-A-17-one 0.95 -

5a-A-3p-ol 0.65 0.70 0.39

5p-A-3p-ol 0.53 0.59 0.32

5a-A-3-one 0.77 -

5p-A-3-one 0.68 -

5a-A-3a,17p-diol 1.67 2.05 0.49

S5p-A-3a,17p-dio 1.68 1.95 0.50

5a-A-3p,17p-diol 1.89 2.21 0.66

5p-A-3p,17p-dio1 1.56 1.90 0.51 -

5a-A-3a-ol,17-one 2.00 2.14 0.87 -

5p-A-3a-ol,17-one 1.90 2.15 0.94 -

5a-A-3p-ol,17-one 2.10 2.44 1.16 -

5p-A-3p-ol,17-one 1.98 2.98 0.86 -

*5a-A-3-one,17p-ol 2.58 2.81 1.32 -

S5p-A-3-one,17p-ol 2.44 2.53 1.18

5a-A-3-one, 17a-ol 2.03 2.32 1.16 -

5p-A-3-one,17a-ol 2.14 2.14 0.91 -

5a-A-3, 17-dione 2.64 -

5p-A-3,17-dione 2.48 -

A-5-en-3P-ol,17-one 2.01 2.38 1.10


- 65 -








TABLE 11 Hi-Eff 3%c
(cont.)


Steroids


Free


Acetate TMSiL TFA


5a-Androstane 0.13 -

5p-Androstane 0.12 -

5ac-A-17poLi 0.71 0.58 .0.22 0.16

I 5p-A-17 oli. 1.48 1.27 -

5(-A-17-one 0.61 -

5p-A-17-one 1.15 -

5a-A-3p-ol 0.75 0.71 0.24 0.20

5p-A-3p-ol 0.61 0.57 0.15

5a-A-3-one 0.73 -

5p-A-3-one 0.67 -

5a0-A-3a,17p-diol 3.94 2.48 0.28 0.27

S5-A-3a,17p-diol 3.85 2.82 0.37 0.30

5a-A-3p,17p-diol 4.19 3.18 0.42 0.24

5p-A-3p,17p-diol 3.50 2.42 0.28 0.23

5a(-A-3a-ol,17-one 3.73 2.62 0.74 0.80

5p-A-3a-ol,17-one 3.58 2.86 1.03 0.61

5a-A-3p-ol,17-one 3.97 3.44 1.20 0.67

5p-A-3p-ol,17-one -

5a-A-3-one,17p-ol 4.61 3.88 1.27 1.24

5p-A-3-one,17p-ol 4.18 3.44 1.13 1.14

5a-A-3-one,17a!-ol 4.36 3.31 1.09 -

5p-A-3-one,17a-ol 3.97 2.91 0.81 0.78

5a-A-3,17-dione 4.00 -

5p-A-3,17-dione 3.64 -

A-5-en-3ep-ol,17-one 4.00 3.38 1.16 1.02


- 66 -







TABLE 11
(cont.)


- Hi-Eff 1%d


Steroids Free Acetate TMSi TFA

5a-Androstane 0.27 -

5p-Androstane 0.26 -

5a-A-17p-ol 0.74 0.65 0.29 0.16

5p-A-17p-ol 1.32 1.04 0.41

5a-A-17-one 0.63 -

5p-A-17-one 1.14 -

5-A-3p-ol 0.75 0.67 0.32 0.20

5p-A-3p-ol 0.63 0.57 0.25 0.15

5a-A-3-one 0.78 -

5p-A-3-one 0.70 -

5-A-3a, 17p-diol 3.23 3.27 0.88 0.27

5p-A-3ma,17p-diol 3.20 2.52 1.07 0.30

Sc-A-3p,17p-diol 3.60 2.86 1.18 0.24

5p-A-3p,17p-dio1 2.93 2.23 0.86 0.23

5a-A-3a-ol,17-one 2.88 2.43 0.78 0.80

5p-A-3ci-ol,17-one 2.92 2.61 0.99 0.61

5a-A-3p-ol,17-one 3.24 2.95 1.15 0.67

5p-A-3p-ol,17-one -

5a-A-3-one,17p-ol 3.84 3.28 1.24 1.24

5p-A-3-one,17p-ol 3.49 3.04 1.13 1.14

5a-A-3-one,17a-ol 3.18 2.92 1.11

5p-A-3-one,17a-ol 3.26 2.58 0.85 0.78

5a-A-3,17-dione 3.63 -

5p-A-3,17-dione 3.32 -

A-5-en-3p-ol,17-one 3.32 2.92 1.14 .1.02


67 -








TABLE 11 NGSeb
(cont.)


Steroids


Free


Acetate TMSi TFA


5E-Androstane 0.09 -

5p-Androstane 0.08 -

5a-A-17p-ol 0.47 0.44 0.20 0.16

5p-A-17p-ol 0.84 0.80 0.32

5a-A-17-one 0.39 -

51-A-17-one 0.71 -

5a-A-3p-ol 0.47 0.47 0.21 0.18

5p-A-3p-ol 0.38 0.37 0.13

5a-A-3-one 0.49 -

5p-A-3-one 0.42 -

5a-A-3a,17p-diol 2.27 1.69 0.31 0.29

5p-A-3a, 17p-diol 2.08 1.89 0.35 0.34

5a-A-3p,17p-diol 2.44 2.20 0.44 0.41

5p-A-3p,17p-diol 1.93 1.66 0.28 0.28

5a-A-3a-ol,17-one 1.83 1.61 0.58 0.67

5p-A-3a-ol,17-one 1.80 1.76 0.72 0.73

5a-A-3p-o1,17-one 2.10 2.00 0.91 0.94

5p-A-3p-ol,17-one 1.88 -

5a-A-3-one,17p-ol 2.61 2.38 1.07 1.18

5p-A-3-one,17p-ol 2.20 1.98 0.91 0.97

5a-A-3-one,17a-ol 2.22 2.12 0.94

5p-A-3-one,17a-ol 2.05 1.76 0.62 0.50

5a-A-3,17-dione 2.27 -

5p-A-3,17-dione 1.98 -

A-5-en-3p-ol,17-one 2.09 1.99 0.90 0.84


- 68 -







TABLE 11
(cont.)


- NGSeb 17


Steroids

5a-Androstane

5p-Androstane

5a-A-17p-ol

S5p-A-17-ol

5a-A-17-one

5p-A-17-one

5a-A-3p-ol

5p-A-3p-ol

5a-A-3-one

5p-A-3-one

5cA-A-3a, 17-diol

5I-A-3c, 17p-diol

5Cx-A-3o, 17 -dlo n
5a-A-3p,17p-diol

5p-A-3pl,7p-diol

5a-A-3a-o1,17-one

5p-A-3p-ol,17-one

5a-A-3p-ol,17-one

5p-A-3p-ol,17-one

5a-A-3-one,17p-ol

50-A-3-one,17p-ol

5a-A-3-one,17a-ol
5p-A-3-one,17a-ol

5a-A-3,17-dione
5a-A-3,17-dione

MA-en-3p-ol, 17-one

Retention time of Cholestane:


1.82

2.00

1.78

1.86
b
12-14, 6-9.


Acetate TMSi TFA


Free

0.14

0.12

0.46

0.80

0.39

0.68

0.48

0.39

0.48

0.42

1.88

1.82

2.14

1.70

1.64

1.59

1.85

1.53

2.24

1.96


0.45

0.80





0.49

0.41





0.73

0.84

0.82

0.65

1.50

1.57

1.88

1.48

2.10

1.91



1.61





1.78
c e
6-8. 9-10.


3


0.24

0.39





0.26

0.40 -


-


0.36

0.40

0.50 -

0.35

0.60

0.73

0.88

0.74

0.96

0.87



0.63





0.84
f
10 minutes.


i 69 -













dtei M stton p* um Wi r-rm ta te Lh 1b .
g1sti ptte, 0 am t.mmm fpesm sto em OR al ".e sa



Th ealselosed growp ***o*et* teews foe as "ftnylo,
heesr mSertl am tr.mrmhylstlr l Ips a .re ste t uble 1). A

*6r1rtaem **s, mk med -sl fu erestemU| Umm ws oMed
e sMo o In k smmom m* snpe d*. ft Ublo 14.




esissle o meed rite n e *tqu*s.


( Irdw / wfree mor ) > 00 )' x

the Imasls e -mm irtel a table t 35.


Soe vem ~ eagse hto seemo t t ee ims pmmq to dtowas
emneta d the ofepi< d- O hn o ups O e at

ubetemR pN mpse am e-lm in table 1i.


table 17 im se w new of the IT tor se oaWom amssedat
S she setteeaspe*sh





A eeerqltea e tshe bime aeleast Iwe peepsitee of aes b
Sbom hkem tor sive to tIable 18.


S70 .








TABLE 12 SE-30 3%

Comparison of Retention Data of
Isomeric Pairs: Substituent Effects



Functional Group 5 a 5



No Substitution 0.21 0.20

171-o l 0.32 0.41

3p-ol 0.31 0.29

3, 17p-diol 0.54 0.51

3p,17p-dio1 0.59 0.49

17-one 0.32 0.42

3-one 0.33 0.32

3,17-dione 0.58 0.50

3a-ol, 17-one 0.51 0.47

3p-ol, 17-one 0.55

3-one,17p-ol 0.59 0.54

3-one, 17a-ol 0.56 0.53


- 71 -


-- .---~~---rp_.---- -- _~ZP --- ---


~` '' c~ '








TABLE 12 XE-60 37
( cont. )


Functional Group 5a x5p



No Substitution 0.27 0.25

17p-ol 0.60 0.89

3p-ol 0.65 0.53

3a, 17-diol 1.67 1.68

3p,17p-dio1 1.89 1.56

17-one 0.65 0.95

3-one 0.77 0.68

3,17-dione 2.64 2.48

3a-ol,17-one 2.00 1.90

3p-ol,17-one 2.10 1.98

3-one,17p-ol 2.58 2.44

3-one,17a-ol 2.09 2.14




















72 -




. N


TABLE 12 Hi-Eff 3%
( cont. )


Functional Group 50! 50



No Substitution 0.13 0.12

17p-ol 0.71 1.48

3 -ol 0.75 0.61

3c,17p-diol 3.94 3.85

3, 17p-diol 4.19 3.18

17-one 0.61 1.15

3-one 0.73 0.67

3,17-dione 4.00 3.64

3a-ol,17-one 3.73 3.58

3p-o, 17-one .3.97

3-one,17p-ol 4.61 4.18

3-one 17a-ol- 4.36 3.97


- 73 -









TABLE 12 Hi-Eff 1%
( .cont.)





Functional Group 5a 50



No Substitution 0.27 0.26

17p-ol 0.74 1.32

3-ol 0./5 0.63

3a,17p-diol 3.23 3.20

3p,17p-diol 3.60 2.93

17-one 0.63 1.14

3-one 0.78 0.70

3,17-dione 3.63 3.32

3a-ol,17-one 2.88 2.92

3p-ol,17-one 3.24

3-one, 17p-ol 3.84 3.49"

3-one,17a-ol 3.18 3.26





















74 -


; ** ; ~ _










TABLE 12 NGSeb 3%
( cont. )




Functional Group 5a 5p



No Substitution 0.09 0.08

17p-ol 0.47 0.84

3p-ol 1 0.47 0.38

3a,17p-diol 2.27 2.08

3p,171-diol 2.44 1.93

17-one 0.39 0.71

3-one 0.49 0.42

3,17-dione 2.27 1.98

3a-ol, 17-one 1.83 1.80

3p-ol,17-one 2.10 1.88

3-one,17p-ol 2.61 2.20

3-one,17a-ol 2.22 2.05


- 75 -


_ 1








TABLE 12 -NGSeb 17
( cont. )


Functional Group 50 5p



No Substitution 0.13 0.12

17p-ol 0.46 0.80

3p-ol 0.48 0.39

3a,17p-diol 1.88 1.82

3p,17p-diol 2.14 1.70

17-one 0.39 0.68

3-one 0.48 0.42

3,17-dione 2.00 1.78

3a-ol,17-one 1.64 1.59

3p-ol,17-one 1.85 1.53

3-one,17p-ol 2.25 1.96

3-one 17a-ol 1.82


- 76 -


";






TABLE 13 SE-30 3% -


Calculated Group Retention Factors
(log r = log rn + log ka + log kb)


Functional
Group


Frrue Free* Acet TMS


Free Acet T M5SI


178-ol
S 17-ol / Androstane
S3B,17-diol / 3p-ol
3-one,17p-ol / 3-one

17a-ol
3-one,17a-ol / 3-one

35-ol
3@-ol / Androstane
3p,17p-diol / 17p-ol
3P-ol,17-one / 17-one

3a-ol
3a,17p-diol / 17p-ol
3a-ol,17-one / 17-one

17-one
17-one / Androstane
3p-ol,17-one /.3p-ol
3,17-dione / 3-one

3-one
3-one / Androstane
3-one,17p-ol-/ 17p-ol
3,17-dione / 17-one


0.19
0.27
0.25


0.28
0.40
0.36


0.20
0.31
0.29


0.31
0.23
0.23


0.23 0.36 0.26


0.18
0.26
0.24


0.28
0.40
0.31


0.41
0.39
0.34


0.22 0.28


0.22
0.32
0.23


0.22 0.35 0.25
0.21 0.26 0.17


0.19
0.25
0.24


0.20
0.26
0.26


0.22




0.29


0.20




0.29


0.16
0.08


0.25
0.22
-


0.10 0.23
0.05 0.13


0.32

0.20


0.20
0.12
0.08


0.32





0.13
-


- 77--


~


Free Acet TMSi








TABLE 13 XE-60 3%
(.cont.


Functional 5
Grouo Free Acet TMSi Free Acet' TMSi


178-ol
17p-ol / Androstane
3p,17p-diol / 3p-ol
3-one,17p-ol / 3-one

17a-ol
3-one,17a-ol / 3-one

36-ol
3p-ol / Androstane
3p,17p-diol / 178-ol
3p-ol,17-one / 17-one

3a-ol
3a,17p-diol / 17p-ol
3c-ol,17-one / 17-one

17-one
17-one / Androstane
3p-o1,17-one / 3p-ol
3,17-dione / 3-one

3-one
3-one / Androstane
3-one,17p-ol / 17p-ol
3,17-dione / 17-one


0.35
0.41
0.51


0.42
0.50
0.63


0.14
0.23
0.23


0.42 0.48 0.18


0.38
0.45
0.51


0.41
0.49
0.57


0.16
0.25
0.25


0.44 0.46 0.12
0.49 0.47 0.07


0.38
0.51
0.53


0.45
0.62
0.61


0.54




0.60


0.47




0.55


0.55
0.447
0.55


0.62
0.51
0.57


0.32
0.20
0.24


0.50 0.50 0.13


0.33
0.24
0.32


0.37
0.26
0.50


0.11
-0.01
-0.04


0.28 0.27 -0.02
0.30 0.35 0.00


0.58
0.57
0.56


0.43
0.44
0.42


0.70




0.38


-
0.43




0.36
-


- 78 -


_ ____







TABLE 13 Hi-Eff 37
( cont. )


5a 5p
Functional
Group Free Acet TMSi Free Acet TMSi-..


178-ol
17p-ol / Androstane
3p,17p-diol / 3p-ol
3-one,17p-ol / 3-one

17a-ol
3-one,17a-ol / 3-one

3s-ol
3p-ol / Androstane
3p,17p-diol / 17p-ol
3p-ol,17-one / 17-one

30-ol
3a,17p-diol / 17-ol01
3a-ol,17-one / 17-one

17-one
17-one / Androstane
3p-ol,17-one / 3p-ol
3,17-dione / 3-one

3-one
3-one / Androstane
3-one,17p-ol / 17p-ol
3,17-dione / 17-one


0.74
0.75
0.80


0.66
0.65
0.73


0.23
0.26
0.24


1.09
0.76
0.79


0.78 0.66 0.18


0.77
0.77
0.81


0.74
0.74
0.75


1.02
0.63
0.71


0.77 0.64 0.08


0.27
0.28
-0.70


0.74 0.63 0.10
0.79 0.63 0.08


0.68
0.72
0.74


0.76
0.81
0.82


0.68




0.82


-0.30




0.76
-


0.70
0.37
-


0.67
0.28
*r


0.41 0.35
0.49 0.40


0.98
i
0.73


0.74 .
0.45
0.50


0.43
-


- 79 -


0.23
-
0.23


-0.05
-0.05







**f;. TIWV'


TABLE 13 Hi-Eff 17
( cont. )


5a 53
Functional -
Group Free Acet TMSi Free Acet TMSi


17S-ol
17p-ol / Androstane
3p,17p-diol / 3p-o1
3-one,17p-ol / 3-one

17a-ol
3-one,17a-ol / 3-one

36-ol
3B-ol / Androstane
3B,17p-diol / 17p-ol
3p-ol,17-one / 17-one

3a-ol
3a,17p-diol / 17p-ol
3C-ol,17-one / 17-one

17-one
17-one / Androstane
3p-ol,17-one / 3p-ol
3,17-dione / 3-one

3-one
3-one / Androstane
3-one,17p-ol / 17p-ol
3,17-dione / 17-one


0.44
0.68
0.69


0.38
0.63
0.62


0.03
0.57
0.20


0.71
0.67
0.70


0.61 0.57 0.15


0.45
0.69
0.71


0.40
0.64
0.67


0.61
0.59
0.64


0.20
0.54
0.21


0.67 0.57 0.08


0.08
0.61
0.26


0.39
0.35
0.48


0.64 0.54 0.48
0.66 0.59 0.09


0.37
0.64
0.67


0.46
0.71
0.76


0.64




0.70


0.56




0.63


0.35
0.33
0.50


-0.01
0.45
-0.18


0.38 0.38 0.42
0.40 0.36 -0.06


0.65
0.74
0.67


0.43
0.42
0.46


0.80




0.47
-


0.48




0.44
-


- 80 -






TABLE 13-NGSeb 3%
(/cont. )


5a 5p
Functional --
Group Free Acet TMSi .Free Acet TMSi
.^


176-ol
17p-ol / Androstane
3p,17p-diol / 3p-ol
3-one,17p-oi / 3-one

17--ol
3-one,17a-ol / 3-one


30-ol / Androstane
3p,17p-diol / 17p-ol
3p-ol,17-one / 17-one

3a-ol
3c, 17p-diol / 17p-ol
3a-ol,17-one / 17-one

17-one
17-one / Androstane
3p-ol,17-one / 3p-ol
3,17-dione / 3-one

3-one
3-one / Androstane
3-one,17p-ol / 17p-ol
3,17-dione / 17-one


0.72
0.71
0.73


0.69
0.67
0.69


0.35
0.32
0.34


0.66 0.64 0.28


0.72
0.71
0.73


0.72
0.70
0.71


0.37
0.34
0.37


0.68 0.58 0.19
0.67 0.62 0.17


0.64
0.65
0.67


0.73
0.74
0.76


0.63




0.73


--
0.64




0.73


1.02
0.71
0.72


1.00
0.65
0.67


0.69 0.62 0.17


0.68
0.36
0.42


0.66
0.32
--m


0.39 0.37
0.40 0.39


0.94
0.69
0.67


0.72
0.42
0.44


0.39


- 81 -.


0.34


II






TABLE 13 NGSeb 1%
( cont. )


Functional 5_____
Group Free Acet TMSi Free Acet TMSi

178-ol
17p-o1 / Androstane 0.53 0.52 0.25 0.82 0.82 0.51
3p,17p-diol / 3p-ol 0.65 0.22 0.28 0.64 -- -.06
3-one,17p-ol / 3-one 0.67 0.64 0.30 0.67 0.66 0.32

17a-ol
3-one,17a-ol / 3-one .64 .58 .18


3B-ol / Androstane 055 0.56 0.28 0.51 0.53 0.52
3p,17p-dio1 / 17p-ol 0.67 0.26 0.32 0.33 -.05
3p-ol,17-one/ 17-one 0.68 0.68 0.35 0.35 0.34 0.03

3a-ol
3a,17p-diol / 17p-ol 0.61 0.21 0.18 0.36 0.02 0.01
3a-ol,17-one / 17-one 0.62 0.58 0.19 0.37 0.36 0.03

17-one
17-one / Androstane 0.46 -- -- 0.75 -- --
3p-ol,17-one / 3p-ol 0.58. 0.58 0.53 0.59 0.5.6 0.2-7
3,17-dione / 3-one 0.61 0.63 --- --

3-one
3-one / Androstane 0.55 -- -- 0.54 --
3-one,17p-ol / 17p-ol 0.69 0.67 0.60 0.39 0.38 0.35
3,17-dione / 17-one 0.71 0.42 -- -


- 82 -


_ _:_ ~_ I__ __ I















S..
N Nr r ur 0O
go C4 % %0 cJi c4 10 Ci
*S S? o
00 C4 C4 C4 C4 0 0








1 J I a ,



Co'09 0 %.0 %0 r- i CI -c t M


o o


%C
0
.1-

0 % I- I



4) to 01 00 co --t o
-5 0r. l1 t. vn .
>e w u O L r4 ci rn e' r. -1
a) 0i 0d OC14 C4 Ci C4i
0 .


,, i I so a 5

.I0 0 0 0 0. -
-'-

d 00 %
d 0 t 0 n l ln ON 0 %
0 (D c, *0 0 0 0 '



03 W 0
-0000 000



0
g 0


p1 0




0 I a 0 0 0 0





41 r-% 04-
-4 0 0 ? .


1 c 1 ca C11


7fa ?f ci n
gr v? -? -? ? ^<


- 83 -








TABLE 15 SE-30 37.a

Ratio of Relative Retention Times of Derivatives to Free Steroids

Steroids Acetate TMSi TFA

5a-Androstane -

5p-Androstane -

5C-A-17p-ol 1.22 1.02

5p-A-17p-ol 1.27 1.19

5a0-A-17-one -

5p-A-17-one -

5a-A-3p-ol 1.25 1.09 0.75

5p-A-3p-ol 1.22 1.09 0.75

5a-A-3-one -

5p-A-3-one -

5a-A-3a, 17p-diol 1.65 1.09 0.52

5p-A-3a,17p-diol 1.69 1.09 0.63

5a-A-3p,17p-diol 1.68 1.17 0.62

5p-A-3p,17p-diol 1.75 1.14 -

5a-A-3a-ol,17-one 1.13 0.91 -

5p-A-3a-ol,17-one 1.20 0.98 0.60

5a-A-3p-ol,17-one 1.89 0.99 0.54

5p-A-3p-ol,17-one -

5a-A-3-one,17p-ol 1.30 1.09 0.60

5p-A-3-one,17p-ol 1.28 1.09 0.61

5c-A-3-one, 17a-ol 1.32 1.07 -

5p-A-3-one, 17a-ol 1.14 0.90 0.62

50-A-3,17-dione -

5-A-3,17-dione -


-84 -








TABLE 15 XE-60 3%
(Cont.)


Steroids Acetate TMSi

5a-Androstane

5p-Androstane -

5a-A-17p-ol 1.18 0.63

5p-A-17p-ol 1.17 0.58

5a-A-17-one -

5p-A-17-one -

5a-A-3p-ol 1.08 0.59

5p-A-3p-ol 1.11 0.60

5a-A-3-one -

5p-A-3-one

5a-A-3a, 17p-diol 1.23 0.29

5p-A-3a.,17p-diol 1.16 0.30

5a-A-3p, 17p-diol 1.17 :0.30

5p-A-3,p17p-diol 1.22 0.33

5a-A-3a-ol,17-one 1.07 0.44

5P-A-3a-ol,17-one 1.13 0.50

5a-A-3p-ol,17-one 1.16 0.55

5p-A-3p-ol,17-one 1.51 0.43

5c-A-3-one, 17p-ol 1.09 0.51

50-A-3-one, 17p-ol 1.04 0.48

5a-A-3-one, 17a-ol 1.14 0.57

5p-A-3-one,17a-ol 1.00 0.43

5a-A-3,17-dione -

5p-A-3,17-dione




85 -








TABLE 15 Hi-Eff 3%c
(Cont.)


Steroids Acetate TMSi TFA

5a-Androstane -

50-Androstane -

S5a-A-17p-ol 0.82 0.31 0.23

5p-A-17p-ol 0.86 -

5a-A-17-one

5p-A-17-one -

5a-A-3p-ol 0.95 0.32 0.27

5p-A-3p-ol 0.93 0.25

5a-A-3-one -

5p-A-3-one -

5a-A-3, 17p-diol 0.63 0.07 0.07

5p-A-3a,17p-diol 0.73 0.10 0.08

5a-A-3p,17p-diol 0.76 0.10 0.06

5p-A-3, 17p-diol 0.69 0.08 0.07

5a-A-3a-ol,17-one 0.70 0.20 0.21

5p-A-3a-ol,17-one 0.80 0.29 0.17

5a-A-3p-ol, 17-one 0.87 0.30 0.17

5p-A-3p-ol,17-one -

5a-A-3-one, 17p-ol 0.84 0.28 0.27

5p-A-3-one,17p-ol 0.82 0.27 0.27

5a-A-3-one, 17a-ol 0.76 0.25 -

5p-A-3-one,17a-ol 0.73 0.20 0.20

5a-A-3,17-dione -

5p-A-3,17-dione -


- 86 -


C__ ____








TABLE 15 Hi-Eff 1%d
(Cont.)


Steroid Acetate THSi TFA

55-Androstane

5p-Androstane -

5a-A-17p-ol 0.88 0.39 0.23

5p-A-17p-ol 0.79 0.31 -

5Sa-A-17-one -

5p-A-17-one -

5c-A-3p-ol 0.89 0.43 0.27

5p-A-3p-ol 0.90 0.40 0.25

55a-A-3-one .

5p-A-3-one .

5a-A-3a,17p-diol 0.70 0.27 0.07

5p-A-3a,17p-diol 0.79 0.33 0.08

5a-A-3p,17p-diol 0.79 0.33 0.05

5p-A-3p 17p-dio1 0.76 0.29 0.06

5a-A-3a-ol,17-one 0.84 0.27 0.21

5p-A-3a-o1,17-one 0.89 0.34 0.17

5a-A-3p-ol,17-one 0.91 0.35 0.17

5p-A-3p-ol,17-one -

5a-A-3-one,17p-ol 0.85 0.32 0.27

5p-A-3-one,17p-ol 0.87 0.32 0.27

5a-A-3-one, 17a-ol 0.92 0.35 -

35-A-3-one, 17a-ol 0.79 0.26 0.20

50-A-3i17-dione -

5p-A-3,17-dione


- 87 -


_ I


I --








TABLE 15 NGSeb 3%e
(Cont.)




Steroids Acetate TMSi TFA

5a-Androstane -

5p-Androstane -

5a-A-17p-ol 0.94 0.43 0.34

5p-A-17p-ol 0.95 0.38

5a-A-17-one -

5p-A-17-one -

5a-A-3p-ol 1.00 0.45 0.38

5p-A-3p-ol 0.97 0.34

5a-A-3-one -

5p-A-3-one -

5a-A-3, 17p-diol 0.74 0.14 0.13

5p-A-3a,17p-diol 0.91 0.17 0.16

5c-A-3, 17p-dio 0.91 0.18 0.17

5p-A-3p,17p-diol 0.86 0.15 0.15

5a-A-3a-ol,17-one 0.88 0.32 0.37

5p-A-3a-ol,17-one 0.98 0.40 0.41

5a-A-3p-o, 17-one 0.95 0.43 0.45

5p-A-3p-ol,17-one -

5a-A-3-one,17p-ol 0.91 0.41 0.45

5p-A-3-one,17p-ol 0.90 0.41 0.44

5a-A-3-one,17a-ol 0.95 0.42

51-A-3-one,17a-ol 0.86 0.30 0.24

5a-A-3,17-dione -

5p-A-3,17-dione -


- 88 -


1








TABLE 15 NGSeb 1f


Steroids

5a-Androstane

5p-Androstane

S5a-A-17p-ol

5B-A-17p-ol

5a-A-17-one

5p-A-17-one

5a-A-3p-ol

5p-A-3p-ol

5a-A-3-one

5p-A-3-one

5a-A-3a,17p-dio 1

5p-A-3a,17p-diol

5a-A-3p, 17p-diol

5p-A-3p, 17p-diol

5a-A-3a-ol, 17-one

5P-A-3a-ol,17-one

5a-A-3p-ol,17-one

5p-A-3p-ol,17-one

5a-A-3-one, 17p-ol

50-A-3-one,17p-ol

5a-A-3-one,17a-ol

5p-A-3-one,17a-ol

5a-A-3,17-dione

5R-A-3,17-dione

Retention time of


Acetate


0.98

0.10





1.02

1.05





0.39

0.46

0.38



0.92

0.99

1.02

0.97

0.93

0.97



0.89


Cholestane: a12-14,


b6-9, c6-8, d5-7,


TMSi


-

0.52

0.49





0.54







0.19

0.22

0.23

0.21

0.37

0.46

0.48

0.48

0.43

0.44



0.35


e-11, f0 minutes.


- 89 -


I IIIL~IL~










TABLE -16


The Average Change in Retention Time Occurring in Derivative Formation,


( rderiv/ free x 100 = r',% )


Acetate


Column


SE-30


XE-60


Hi-Eff


NGSeb


37.


3%


8B 3%


37.


Mono

123


110


83


93


TMS i


Di

169


120


70


85


Mono

102


53


27


41


Di

115


30


9


16


Mono

69


- .90 -


TFA


Di

59


----------


TMSi


Acetate


S.1-C











TABLE 17


Values of T at 2600 Based on Measurements Obtained with

3% SE-30 and 3% XE-60 Columns

t' t'
( T XE-60 SE-30
t
SE-30




Functional Group 5a 5p

17p-ol 0.88 1.14

17-one 1.03 1.26

3p-ol 1.09 0.83

3-one 1.33 1.13

3a, 17p-diol 2.09 3.08

3p,17p-diol 2.21 2.19

3a-ol,17-one 2.92 3.04

3p-ol, 17-one 2.82 -

3-one,17p-ol 3.38 3.52

3-one,17a-ol 2.63 3.04

3,17-dione 3.55 3.98


- 91 -


I I I


~---