7$ '/V t kem*i f ' r' i / CHAOMAT0 GRAPHIC BEHAVIOR, SOLUBILITIES, AND PARTITION RATIOS OF ERGOSTEROL AND CALCIFEROL IN SOLVENT MIXTURES F r e d e r ic k J . M i l l e r , J r . AN ABSTRACT S u b m itted to th e School o f G raduate S tu d ie s o f M ich ig an S ta te C o lle g e o f A g r ic u lt u r e and A p p lie d .Science i n p a r t i a l f u l f i l l m e n t o f th e re q u ire m e n ts f o r th e d eg ree o f DOCTOR OF PHILOSOPHY D epartm ent o f C h em istry Y ear Approved 1952 }M F r e d e r ic k J . M i l l e r , J r . THESIS ABSTRACT The c h r o m a t o g r a p h i c b e h a v io r o f h e x a n e -d io x a n e m ix tu re s on S u p erf i l t r o l i s d i s c u s s e d a n d th e a d s o r p tio n o f d io x a n e from liexane s o l u t i o n s on S u p e r f 'i l t r o l , c a l c u l a t e d from ch ro m a to g ra p h ic s t u d i e s , i s v e r i f i e d by data o b t a i n e d b y t h e n o rm al m ethod f o r d e te rm in in g a d s o r p tio n from so lu tio n . C h r o m a to g r a p h ic b e h a v i o r o f ergo s t e r o l ancl c a l c i f e r o l on S u p e rf i l t r o l w ith e l u t i n g and an e q u a t i o n i s s o l v e n t m ix tu re s o f d io x a n e and hexane i s d is c u s s e d d e d u c e d w hich r e l a t e s th e a d s o r b a te r e t a i n e d by th e cliro m ato g rap h ic c o lu m n t o th e q u a n tity o f th e a d s o r b e n t, to th e amount of ad so rb ate e n t e r e d o n t h e colum n, and to th e volume o f s o l u t i o n con­ ta in in g th e a d s o r b a t e b e f o r e a d s o r p tio n . of c a lc ife ro l fro m c a lc if e r o l f r o m e r g o s t e r o l i s d is c u s s e d and an 81% r e c o v e ry o f p u re a 3'0 - 5>0 m ix tu r e o f c a l c i f e r o l and e r g o s t e r o l i s demon­ strated. u t i l i z i n g hexane e l u t i n g The clirom ato g ra p h ic s e p a r a t i o n s u p e r f i l t r o l as th e a d s o r b e n t and a 1 . 96% d io x a n e i n s o l v e n t m i x tu r e . S o lu b ilitie s a re g i v e n f o r e r g o s t e r o l and c a l c i f e r o l i n e th a n o l- w ater m ix tu re s a n d f o r e r g o s t e r o l in h e x a n e -e th a n o l m ix tu r e s and in hexane-dioxane m i x t u r e s . solvent m i x t u r e s i s The s o l u b i l i t y o f n o n - e l e c t r o l y t e s i n b in a r y d i s c u s s e d and th e s o l u b i l i t y o f e r g o s t e r o l and c a lc if e r o l i n e t h a n o l - w a t e r m ix tu re s i s shown to be e x p la in a b le by an equation d e r i v e d fro m r e g u l a r s o lu ti o n th e o r y . - 1 - F r e d e r ic k J . M i l l e r , J r . P a r t i t i o n r a t i o s a re l i s t e d f o r e r g o s t e r o l and c a l c i f e r o l i n th e two p h a s e , l i q u i d - l i q u i d s y s te m , h e x a n e -m e th a n o l-w a te r. The change i n th e p a r t i t i o n r a t i o s w ith th e amount o f w a te r i n th e s y s te m , i s d i s ­ cu ssed a s i s th e p o s s i b i l i t y o f s e p a r a t in g e r g o s t e r o l and c a l c i f e r o l by c o u n t e r - c u r r e n t e x t r a c t i o n p ro c e d u re s u t i l i z i n g a l i q u i d - l i q u i d system c o n s i s t i n g o f hexane and 95% m e th a n o l. ACKNCRLEDUMENT The a u th o r w ish e s to e x p re s s h i s s in c e r e a p p r e c i a t i o n to P r o f e s s o r D. 'I. Ewing f o r h is e x p e rie n c e d c o u n s e l and g u id an ce d u r in g th e c o u rs e o f t h i s i n v e s t i g a t i o n . G r a te f u l acknow ledgm ent i s made to th e G rad u ate S c h o o l, M ich ig an S ta t e C o ll e g e , f o r i t s g r a n t o f a G rad u ate C o u n c il F e llo w s h ip and to th e C h em istry D epartm ent f o r i t s f i n a n c i a l a s s i s t a n c e i n th e form o f a G raduate A s s i s t a n t sliip and a R e se a rc h F e llo w s h ip sp o n so red by th e S t a t e 's S m e ltin g and R e f in in g Company, Lim a, O h io . Acknowledgment i s a ls o inaoe to D r. 0 . D. B ir d o f P a rk e , D av is and Company f o r many v a lu a b le s u g ­ g e s tio n s and to M iss H . LeDuc f'o r t e c h n i c a l a s s i s t a n c e w ith th e i n f r a r e d a o s o r p tio n a n a l y s e s . TABLE 01' CONTENTS PAGE INTRODUCTION............................................................................................................................ 1 EXPERIMENTAL............................................................................................................................ 3 'M a te r i a ls ....................................................................................................................... D e te rm in a tio n o f th e C h ro m ato g rap h ic B e h a v io r o f D io x an eriexane M ix tu re s on S u p e r f i l t r o l ................................................................ The D e te rm in a tio n o f D ioxane in th e P re s e n c e o f Hexane by I n f r a r e d A bsorbancy M easu rem e n ts.............................................................. The D e te rm in a tio n o f th e C h ro m ato g rap h ic B eh av io r o f C a l c i ­ f e r o l and E r g o s te r o l on S u p e r f i l t r o l ..................................................... S o l u b i l i t y D e te r m in a tio n s .................................................................................. P a r t i t i o n R a tio D e te r m in a tio n s ....................................................................... D ata and R e s u l t s ....................................................................................................... 3 DISCUSSION................................................................................................................................ U 5 6 6 7 7 18 The C lirom atographic B eh av io r o f D ioxane-H exane M ix tu r e s ................ 18 The C liroiriatographic B eh av io r o f E r g o s te r o l and C a l c i f e r o l on S u p e r f i l t r o l i n th e P re s e n c e o f M ix tu re s o f D ioxane and H exane......................................................................................................................... 21 The C h ro m ato g rap h ic S e p a r a tio n o f C a l c i f e r o l from E r g o s t e r o l . . 27 Summary........................................................................................................................... 28 The S o l u b i l i t y o f N o n - e le c tr o ly te s i n S o lv e n t M ix tu r e s .................. 29 The S o l u b i l i t y o f E r g o s te r o l and C a l c i f e r o l i n S o lv e n t Mixtures.................................................. 3>b The E f f e c t o f th e A d d itio n o f W ater on th e P a r t i t i o n R a tio s o f E r g o s te r o l and C a l c i f e r o l i n th e L iq u id -L iq u id S ystem : He xane -Me thano 1 3o Summary........................................................................................................................... 37 LITERATURE CITED.................................................................................................................. 36 INTRODUCTION 1 INTRODUCTION The u s e o f m ixed p o la r - n o n p o la r s o lv e n ts i n th e c h ro m a to g ra p h ic s e p a r a t io n o f c a l c i f e r o l , v ita m in D2 j from i r r a d i a t e d e r g o s t e r o l has r e c e iv e d much a t t e n t i o n i n t h i s l a b o r a t o r y d u r in g r e c e n t y e a r s . C a rls o n ^ found t h a t e r g o s t e r o l was more s t r o n g ly ad so rb ed on S u p e r f i l t r o l th a n c a l c i f e r o l was from m ix tu re s o f hexane and d i e t h y l e t h e r . P in k e r to n p ob­ t a i n e d a s e p a r a t i o n o f c a l c i f e r o l from e r g o s t e r o l on S u p e r f i l t r o l u s in g a m ix tu re o f hexane and d i e t h y l e t h e r a s an e l u t i n g s o l v e n t . B u rn e tt^ found a s a t i s f a c t o r y s e p a r a t i o n o f c a l c i f e r o l from e r g o s t e r o l on an " a c ti v a te d " alu m in a u s in g a m ix tu re o f hexane and d i e t h y l e t h e r as th e e lu tin g s o lv e n t. O th er s t u d i e s ^ in v o lv in g th e u se o f m ix tu r e s o f h ex an e e th a n o l and d i e t h y l e t h e r a s e l u t i n g s o lv e n ts and S u p e r f i l t r o l as th e a d s o rb e n t have b e e n more o r l e s s in c o n c lu s iv e . I t w i l l be n o te d t h a t i n e v e ry in s ta n c e th e s o lv e n t m ix tu re has in v o lv e d th e u se o f d i e t h y l e t h e r as th e p o la r c o n s t i t u e n t o r a s one o f th e p o la r c o n s t i t i i e n t s , and t h a t e tiie r i s a somewhat u n d e s ir a b le s o lv e n t to h a n d le j due to i t s v o l i t i l i t y and c o n s e q u e n tly due to th e d i f f i c u l t i e s e n c o u n te re d i n m a in ta in in g a c o n s ta n t c o m p o sitio n m ix tu re o f s o l v e n t s . I n v e s t i g a t i o n s w ere t h e r e f o r e .u n d e rta k e n to a s c e r t a i n th e f e a s i b i l i t y o f s u b s t i t u t i n g a l e s s v o l a t i l e p o la r s o lv e n t f o r th e d i e t h y l e t h e r . The i n v e s t i g a t i o n s u n d e rta k e n in c lu d e d th e d e te r m in a tio n o f th e ch ro m ato g rap h ic b e h a v io r o f e r g o s t e r o l and c a l c i f e r o l i n h ex an e -d io x an e m i x tu r e s , th e d e te r m in a tio n o f th e s o l u b i l i t y o f e r g o s t e r o l and c a l c i f e r o l in e t h a n o l- w a te r m ix tu r e s , th e d e te r m in a tio n o f th e s o l u b i l i t y o f e r g o s t e r o l i n h e x a n e -e th a n o l and h e x a n e -d io x a n e m i x t u r e s , and th e d e te r m in a tio n o f p a r t i t i o n r a t i o s o f e r g o s t e r o l and c a l c i f e r o l i n t h e two p h a s e , l i q u i d l i q u i d sy ste m ; h e x a n e , m e th a n o l, w a t e r . EXPERIMENTAL 3 EXPERIMENTAL M a te r ia ls E th a n o l was t r e a t e d to remove a ld e h y d e s by th e m ethod d e s c r ib e d by Ivildem an^ wM ch c o n s i s t s o f an a l k a l i n e s i l v e r n i t r a t e o x id a tio n o f th e ald e h y d e s t o th e a c i d s . A f te r d i s t i l l a t i o n , th e e th a n o l was f u r t h e r t r e a t e d w ith f r e s h l y am algam ated alum inium to remove w a te r a c c o rd in g to th e m ethod d e s c r ib e d b y W is lic e n u s and K aufm ann,° fo llo w e d by r e d i s t i l l a ­ tio n . M e th a n o l, C . P . an h y d ro u s g ra d e , was d i s t i l l e d , t r e a t e d b y r e f lu x i n g w ith p o ta ssiu m h y d ro x id e o v e r alum inium c h ip s to remove a ld e h y d e s , and re d is tille d . Hexane was o b ta in e d by c a r e f u l l y f r a c t i o n a t i n g a com m ercial p r o d u c t, ta k in g th e f r a c t i o n b etw een 67 and 69° C a s h e x a n e . To remove u n s a tu r a te d h y d ro c a rb o n s , th e d i s t i l l a t e was p a sse d th ro u g h an a c t i v a t e d s i l i c a g e l colum n, h cm. i n d ia m e te r b y 75 cm. i n l e n g t h . The hexane was ch ro m ato ­ graphed u n t i l i t had an a b so rb a n c y o f l e s s th a n 0 .0 1 5 a 't 230 mu. m easured a g a in s t w a te r and showed no e v id e n c e o f benzene i n th e s p e c tru m . p -D io x a n e , p r a c t i c a l g r a d e , was p u r i f i e d by th e method d e sc rib e d , by F i e s e r ,^ fo llo w e d by r e d i s t i l l a t i o n . The e r g o s t e r o l , o b ta in e d from W in th ro p C hem ical Company, was r e ­ c r y s t a l l i z e d from a 50-50 ( v /v ) m ix tu re o f 95/S e th a n o l and th io p h e n e f r e e b e n ze n e, a s d e s c r ib e d by H u b er, Ew ing, and K r ig e r .^ The r e s u l t i n g e r g o s t e r o l had an a b so rb a n c y w h ich a g re e d w ith t h a t r e p o r t e d by H uber, e +t ao-,l . 8 C a l c i f e r o l , W intlirop C hem ical Company (hlil!?£C) p u r e s y n t h e t i c v i t a ­ min Da , was u sed w ith o u t f u r t h e r tr e a tm e n t and had an a b s o rb a n c y i n ag reem en t w ith t h a t r e p o r te d by H uber, e t a l . 8 The c a l c i f e r o l and e r g o s t e r o l w ere s to r e d u n d er re d u c e d p r e s s u r e a t -1 0 ° C i n an atm o sp h ere o f C 02 . S u p e r f i l t r o l Ho. 63 was u s e d a s th e a d s o r b e n t th ro u g h o u t t h i s i n ­ v e s tig a tio n . D e te rm in a tio n o f th e C h ro m ato g rap h ic B e h a v io r o f D ioxane-H exane M ix tu re s on . o u p e r f i l t r o i A d s o rp tio n tu b e s were p r e p a r e d by s e a l i n g a s h o r t p ie c e o f 3 * . I. D. P y re x g la s s tu b in g to one end o f a c o n v e n ie n t l e n g th o f 7 Mm. I . D. P y re x g la s s tu b in g and by s e a l i n g a 15 cm. le n g th o f 35 mm. I . D. P y rex g la s s tu b in g to th e o p p o s ite e n d . These a d s o r p tio n tu b e s were p acked to a h e i g h t of 6 cm. w ith 2 g . o f S u p e r f i l t r o l Ho. o3 fo llo w in g th e m ethod d e s c r ib e d b y Sw ing, K in g s le y , Brown, and Sm m ett. 9 V ario u s s o lv e n t m ix tu re s o f hexane and d io x a n e w ere ch ro m ato g rap h ed and e l u a t e f r a c t i o n s w ere c o l l e c t e d w ith th e a i d o f a T ech n ico n A utom atic F ra c tio n C o lle c to r. The e l u a t e f r a c t i o n s w ere a n a ly s e d to d e te rm in e th e am ount o f d io x a n e by in f r a r e d , a b so rb a n c y m easurem ents u t i l i z i n g a Beckman IR -2 (N o. 108) i n f r a r e d s p e c tr o p h o to m e te r , as d e s c r ib e d b e lo w . 5 T1le D e te rm in a tio n o f D ioxane i n th e P re s e n c e o f Hexane by I n f r a re d Abso rb a n c y M easurement s D ioxane was fo u n d to have a d i f f e r e n t a b so rb a n c y a t 1 1 .3 $ m ic ro n s th a n d o es h e x a n e . A s e r i e s o f known c o m p o s itio n m ix tu re s o f d io x a n e and h ex an e were u s e d to e s t a b l i s h a c a l i b r a t i o n o f th e a b so rb a n c y a g a i n s t th e c o n c e n tr a ti o n o f d io x a n e i n h e x a n e . T h ese r e s u l t s a r e shov/n in T able I . TABLE I INFRARED ABSORPTION DATA FOR DIOXANE I N HEXANE SOLUTIONS AT 1 1 .3 $ MICRONS A bsorbancy Volume % Dioxane i n Hexane In te n s ity 0 ( I o = 9 1 .8 ) -------- 0 .1+98 7 k .7 0 .0 8 9 0 .9 9 1 6 1 .0 0 .1 7 7 1.2 2 $ 3 .$ 0 .2 3 k 1 .6 3 I46.6 0 .2 9 k 2 .kk . 3U.0 0.U31 I l 0g . ( l 0/ l ) A greem ent w ith B e e r 's Law was found th ro u g h o u t th e c o n c e n tr a tio n ra n g e 0 to 2 .$ $ d io x a n e in h e x a n e . ( F ig . l ) . A ll su b se q u e n t a n a ly s e s w ere c a r r i e d o u t w i t h i n th e s e ab so rb an cy l i m i t s by d i l u t i o n o f an a l i q u o t o f th e more c o n c e n tr a te d s o l u t i o n . 0.6 4 0.2 0.0 0 0 .5 VOLUME 1.0 1 .5 2 .0 DIOXANE IN HEXANE FIGURE 1 IN FR A R ED ABSORBANCY C A L IB R A T IO N CURVE USED TO DETERMINE THE AMOUNT OF DIOXANE IN ELUATE SA M PLE S. 2 .5 The D e te rm in a tio n o f th e C h ro m a to g rap h ic B e h a v io r o f C a l c i f e r o l and E r g o s te r o l on S u p e r f i l t r o l S u p e r f i l t r o l colum ns were p r e p a r e d a s d e s c r ib e d above and were "prew ashed11 w ith 25 m l. o f th e d e s i r e d e l u t i n g s o lv e n t m ix tu r e . E r g o s te r o l a n d /o r c a l c i f e r o l i n two m l. o f t h e d e s ir e d e l u t i n g s o lv e n t was e n te r e d on th e S u p e r f i l t r o l colum ns and was fo llo w e d by an e x c e ss o f th e e l u t i n g s o lv e n t . S u c c e s s iv e one m l. p o r t i o n s o f th e e l u a t e w ere c o l l e c t e d and an aly zed by d e te rm in in g th e u l t r a - v i o l e t a b s o rb a n c y w ith th e a id o f a Beckman D. U. (Ho. 316) s p e c tr o p h o to m e te r . S o l u b i l i t y D e te rm in a tio n s F ig u re 2 shows th e a p p a r a tu s u se d to o b ta in th e s a t u r a t e d s o lu ti o n sam ples f o r th e s o l u b i l i t y m easurem ents . These d e p ic te d " E q u ilib riu m tu b e s " , w hich a r e m o d if ic a tio n s o f th o s e d e s c r ib e d by C ra ig and P o s t , ^ were immersed i n a c o n s ta n t te m p e ra tu re w a te r b a th c o n t r o l l e d to - 0 .05° . A p p ro x im ately 1 m l. p o r ti o n s o f th e d e s ir e d s o l v e n t , were sh ak en w ith ex c e ss s o l u t e , e r g o s t e r o l o r c a l c i f e r o l , f o r t e n h o u r s . The " e q u ilib r iu m tu b e s " w ere in v e r t e d to f i l t e r th e s a t u r a t e d s o l u t i o n s in to th e t a r e d 5 m l. v o lu m e tric f l a s k s . The s a t u r a t e d s o l u t i o n sam ples were w eig h ed , d i l u t e d w ith e t h a n o l, and th e u l t r a - v i o l e t a b s o r p tio n sp ectru m o f th e d i l u t e d sam ples d e te rm in e d w ith a Beckman m odel D. U . (#316) s p e c tr o ­ p h o to m e te r. e q u a t io n s : The s o l u b i l i t i e s w ere c a l c u la te d u s in g t h e f o llo w in g A BSORBENT COTTON PYREX G L A S S TUBE 5 ML. VOLUMETRIC F L A S K MICRO F I L T E R FUNNEL FYREX G L A SS V IA L RUBBER S T O P P E R FIG U R E 2 APPARATUS USED TO O B TA IN THE SATURATED S O L U T IO N S FO R S O L U B IL IT Y D E T E R M I N A T IO N S . where Ce and Cc e q u a l th e s o l u b i l i t y o f e r g o s t e r o l and c a l c i f e r o l r e s p e c t i v e l y i n grarns p e r 1000 grams o f s o l u t i o n , A e q u a ls th e ab so rb a n c y d eterm in e d f o r th e s a tu r a t e d s o l u t i o n s , n e q u a ls th e w e ig h t o f th e s a t u ­ r a te d s o l u t i o n s i n grains, 2 3 .3 a nd 3 6 .8 e q u a l th e a b so rb a n c y o f an e th a n o l s o l u t i o n c o n ta in in g one gram p e r 1000 'grams o f s o l u t i o n f o r e r g o s t e r o l and c a l c i f e r o l r e s p e c t i v e l y . The ab so rb a n c y v a lu e s were m easured a t wave le n g th s 282 mu. and 263 mu. f o r e r g o s t e r o l and c a l c i f e r o l re s p e c tiv e ly . P a r t i t i o n R a tio D e te rm in a tio n s Hexane was e q u i l i b r a t e d w ith th e v a r io u s m ix tu re s o f m eth an o l and w a te r u n t i l e q u a l volum es o f th e two p h a s e s , n o n -p o la r and p o l a r , w ere o b ta in e d . The v a r io u s p h a se s w ere s e p a r a te d and d iv id e d i n t o . 10 m l. p o r ti o n s and e r g o s t e r o l a n d /o r c a l c i f e r o l w ere added t o th e n o n -p o la r p hases. These s o l u t i o n s in th e n o n -p o la r p h a s e s w ere s u b s e q u e n tly e q u i l i ­ b r a te d w ith th r e e s u c c e s s iv e t e n m l. p o r t i o n s o f th e p o l a r p h a se s and th e c o n c e n tr a tio n a f t e r e a c h e q u i l i b r a t i o n was d e te rm in e d from th e u l t r a - v i o l e t ab so rb an cy a t 282 mu. f o r e r g o s t e r o l and a t 265 mu. f o r c a l c i f e r o l . D ata and. R e s u lts The s o l u b i l i t y d a t a f o r e r g o s t e r o l and c a l c i f e r o l i n e th a n o l- w a te r m ix tu re s a t 1 5 ° , 2 5 ° , and 35° C a r e l i s t e d i n T a b le s I I tliro u g h V I I . 8 TABLE I I THE SOLUBILITY OF ERGOSTEROL IN ETHANOL-WATER MIXTURE AT 15° ± 0 .0 5 ° C VJt . % E th an o l 100 it it W t. S a t . S o lu tio n Sample C* to 0 .6 7 7 1 0 .6 9 7 5 0 .7 5 2 6 A bsorbancy S o lu b ility g p e r 1000 g s o l u t i o n A O bserved 5U.0 8 8 .2 56 .2 3 .2 1 3 .0 3 3 .2 2 A verage 3 .1 5 6 0 .1 ii u it 0.673U 0 .6 0 6 5 0 .8 7 2 5 0 .7 8 3 8 3 .8 9 5 .9 5 ' 5 .3 3 5 .5 7 0 .2 5 8 0.2 6 3 0 .2 6 3 0.2 5 5 6 9 .0 If It 0.751)4 0.73)41 0 .7 6 3 9 1 .3 2 I .23 1 .1 9 0.0753 0.0723 0.0 6 7 0 0.0 7 1 5 6ii. 5' tt tt 0 .8 2 1 9 0 .6 5 0 2 0 .8 9 2 5 0 .7 7 6 0 .7 6 7 0 .8 5 3 0.0507 0.0 3 9 3 0.0507 0.0502 S9.U n 0 .8 9 2 3 0 .6 8 7 7 0 .5 8 5 0 .5 1 5 0 .0 2 1 5 0.0 2 0 1 0 .2 5 5 0.0207 vmsmvim 9 TABLE I I I THE SOLUBILITY OF ERGOSTEROL IN ETHANOL-WATER MIXTURES AT 25° + 0 .0 5 ° C Wt . Jo E th a n o l 100 ii it ii n it 9 1 .0 ti ii tt n it 8 2 .0 n tr 81.7 mi ii ti 7 0 .3 ii tt ti 6 9 .0 it ti 59.7 ii ti n VJt. S a t . S o lu tio n Sample g A bsorbancv ti S o lu b ility g p e r 1000 g s o l u t i o n A O bserved 0.7011) 0 .7 0 1 3 0 .7 9 1 5 0.7601; 0.7617 0.71)51 7 7 .1 77.U 86.3 83.7 8 2 .5 8 1 .0 1|.73 1) .75 1) .70 5 .7 5 1). 68 I4.69 0.9627 0.8 7 0 5 0 .8 9 0 2 0.91)72 0 .7 3 8 5 0.7902 38.1) 3l).2 3 5 .0 3 6 .5 3 0 .3 3 2 .0 1 .7 2 1 .6 9 1 .6 9 1 .6 6 1.7 7 1 .7 5 1 0 .9 1 0 .3 1 0 .0 0 .5 5 6 0.5 5 5 0.51)0 0.81;57 0.8021; 0 .7 9 7 6 ' 0.6 2 6 7 0 .7231) 0 .7 6 2 6 0 .6 3 0 6 7 .7 8 8 .9 6 9 .2 6 8 .1 1 0.5 3 2 0 .5 3 5 0.521) 0 .5 5 3 0.7629 0 .8 1 0 8 0.8 2 2 1 0 .8 1 7 3 2 .9 9 3 .9 7 3 .0 8 3 .0 7 0.161) . 0 .1 5 8 0.1 6 1 0.1 6 2 1.6 6 5 1 1.681)8 0.7921) 3.1)2 3 .2 0 1.61; 0 .0 8 8 5 0.0817 0.0892 1.0551) 0 .8 9 1 2 0 .9 1 9 7 0 . 9)438 0 .3 2 1 0 .2 3 6 0.3 0 7 0 .2 9 5 0 .0131 0 .0115' 0 .011)1 0.0135 A verage 1) .71 1 .7 1 0 .5 5 0 0 .5 3 5 0 .1 6 1 0 .0 8 6 5 0.0131 10 TABLE IV THE SOLUBILITY OF ERGOSTEROL IK ETHANOL-.-JATER MIXTURES AT 35° t 0 .0 5 ° C w t. % E th a n o l 100 n n u ¥ t . S a t. S o lu tio n Sample g 0 .6693 0.61*58 0 .6 1 3 8 0.6 1 3 9 A bsorbancy A S o lu b ility g p e r 1000 g s o lu ti o n O bserved 10.6 103 9 9 .2 9 8 .6 11*.0 11*. 1+ 1 5 . 1* 15.1* 6 .8 3 6.87 6 .9 5 6.93 0 .8 9 0 0 .8 5 5 0 .8 8 1 0 .8 7 9 A verage 6 .9 0 81.9 1! tt 11 0.6736 0 . 726L1 0.7 3 3 3 0.7332 66.3 ft It 0 .801*1* 0.7 3 3 9 0.199b 2 .7 9 2 .5 6 2 . 6b 0.11*9 0.11*5 0 .1 6 3 68,1 it it ti 0.7083 0.6973 0 . 711*2 0 .3 9 6 0 1 .7 7 1 .8 2 1 .6 2 1 .5 8 0 .1 0 8 0 .1 1 2 0 .1 1 0 0 .1 1 A 59.7 tt u tt 0.6913 0 .7 1 1 3 0 .7 2 6 5 0 .7 2 8 6 0.9U 0 0 .9 2 1 1 .0 1 0 .9 6 7 0 .0581* 0 .0 5 5 7 0 .0 6 0 2 0.0572 53.6 tt n 0.9711 1.0231 1 .0 3 3 6 0 .6 2 6 0 . 67)4 0 .6 7 5 0 .0 2 7 8 0 .0 2 8 5 0 .0 2 8 3 0.0 2 8 2 53.2 it it 1 .9 5 3 9 2 .01*31 2.0382 1.1*9 1 .6 1 1.1*6 0 .0 3 2 0 0.0 3 2 9 0 .0 3 0 8 0 .0 3 1 9 89.7 tt it tt 1.9855 1.8933 1 .9 7 6 5 2.07814 0 .9 1 1 O .8I4O 0 .8 9 7 0 .9 2 9 0.0198 0 .0 1 9 1 0 .0 1 9 6 0.0 1 9 2 88,2 1.71*12 1 .6 7 ill 0 .6 6 1 0.61*9 0 .0 1 6 3 0 .0 1 6 7 ii 0 .8 7 8 0 .11*6 0 .1 1 1 0 .0 5 8 0 0.019A 0.0165 11 THE SOLUBILITY OF CALCIFEROL IN ETHANOL-V/ATEA MIXTUhES AT 15° + 0 .0 5 ° C Vvt , /o E th an o l A bsorbancy A 0 .7 0 2 1 0 .7 0 1 6 0 .7 0 9 3 1190 1190 1210 79 .9 n it 0 .7 6 1 3 0 .7 3 9 0 0 .7 8 0 6 285 276 286 66.1 0 .7 1 6 1 0 .7 2 3 1 . 0 .7 0 6 0 90.0 ii ti 6 0 .7 6 0 .2 6 0 .0 O bserved A verage 6 6 .2 6 6 .3 6 6 .6 6 6 .6 1 0 .5 1 0 /2 9.'90 1 0 .2 1 .5 6 1 .5 1 1 .5 6 0.1 3 8 69.6 it n 0 .8 3 7 6 0 .7 3 3 3 0.7892 6 .2 0 3 .8 5 3 .9 9 0 .1 3 3 0 .1 6 2 0.137 37.6 0.8 6 2 9 0 .6 3 7 0 0 .7 2 9 0 .7 3 0 0.0162 0.0297 ii on ii ii S o lu b ility g p e r 1000 g s o l u t i o n 1—1 w t. S a t . S o lu tio n Sample oc 0 .0 2 3 0 12 TABLE VI THE SOLUBILITY OF CALCIFEROL IN ETHANOL-L'ATER MIXTURES AT 25° + 0 .0 5 ° C wt . % E th a n o l W t. S a t. S o lu tio n Sample F S o lu b ility g p e r 1000 g s o l u t i o n A bsorbancy A O bserved A verage 65 .1 tl tt 0 .5 8 2 1 o .5510 0.5 7 8 2 921 863 912 6 3 .1 6 2 .5 6 2 .8 6 2 .8 7 5 .3 ' tr 0.6 8 1 2 o .5 6 o i 0 .5 7 2 8 267 212 222 1 0 .7 10 .7 1 0 .6 10.7 6 2 .8 it tt 0 .7 3 2 9 0.7)458 0 .7 1 9 6 U6.7 tt tt 0.6095 0.5 8 1 5 0 .6 1 9 9 3 .6 2 3 .6 2 3 .6 7 3 9 .6 tt tt 0.6722 0 . 6)431 0.6827 1 .7 1 1 .6 5 ■1 .7 3 6 8 .8 5 0 .3 6 9 .1 '■ 1 .8 1 1 .6 3 1 .8 5 1 .8 3 0 .1 5 3 0 .1 5 9 0 .1 5 2 0 .1 5 2 0.0 6 9 3 0 .0699 0 .0 6 8 9 0.069k 13 TABLE V II THE SOLUBILITY OF CALCIFEROL Bl ETHAMOL-WATER MIXTURES AT 33'° + 0 .0 3 ° C Vtf"b , /o E thanol 61.1 if ii 66.1 ii it 3 5 .0 II M 39.0 II It 31.3 n ii W t. S a t . S o lu tio n Sample R A bsorbancy A 0 .7 3 2 3 0 .7 5 3 0 O .76OI 1190 1190 1210 0.7 0 9 3 0.716ft 0 .7 1 0 9 i3 i 1 35 133 0 .6 7 2 8 0 .oil 61 0 .6 8 7 2 2 3 .3 23.ii 2 6 .1 S o lu b ility g p e r 1000 g s o l u t i o n O b serv ed A verage 3 3 .0 3 2 .8 3 3 .2 3 3 .0 3 .3 9 3 .3 2 3 .35 3 .3 3 1 .0 2 1 .0 7 1 .0 3 1 .0 3 0.1 1 3 0 .3 8 2 9 0 .3 9 9 9 0.3 6 9 2 2 .hi 2 .1 0 2 .37 0 .1 1 3 0 .1 1 3 0 .1 1 8 0.9667 1.0 2 3 7 0 .9 7 9 9 l.ii7 1 .3 1 l .ii-U 0 .0 3 9 9 0 .0 3 0 0 0.0397 0 .0 3 9 9 ' The s o l u b i l i t y d a ta f o r e r g o s t e r o l i n e th a n o l-h e x a n e and d io x a n e hexane m ix tu re s a t 23°C a r e l i s t e d in T a b le s V I I I and IX r e s p e c t i v e l y . lit TABLE V III THE SOLUBILITY OF ERGOSTEROL IB ETHANOL-HEXANE MIXTURES AT 25° - 0 .0 5 ° C Wt. % Hexane 100 St 1! Wt. S a t . S o lu ti o n Sample K 0.6989 0.8835 0 .8 5 9 8 A bsorbancy A 6 1 .5 3 9 .8 3 8 .6 S o lu b ility g p e r 1000 g s o l u t i o n O bserved Average 3 .5 7 3 .5 1 3 .6 1 3 .5 7 86.5 it n 0 .5 2 8 8 O.H275 0.8957 282 287 233 1 9 .8 2 0 .1 2 0 .2 78.9 it 0.5283 0.5567 329 380 2 6 .8 2 6 .3 6 9 .8 n it 0 .380 0 0.3517 0 .65 96 316 287 368 3556 3 5 .0 38.2 3 8 .9 62.9 ti • ti 0.6952 0.5732 0.5567 398 866 858 3 8 .6 3 5 .0 3 5 .2 3 8 .9 Ii5.6 n tt o .6988 0.5 6 0 9 0.5122 310 388 38.2 26.7 27 .8 2 8 .8 27 .6 3 3 .1 tt it 0.3778 0 .6 9 7 9 0.5 7 0 2 169 2 88 288 2 1 .7 2 1 .1 21 .7 2 1 .5 19. h it tt 0 .6 1 1 3 0 .6 7 8 0 0 .5 0 2 8 198 153 157' 1 3 .9 1 3 .9 1 3 .5 1 3 .8 Ik. h it tt 0.5911 0 .5 5 1 0 0 .5 8 0 9 110 9 0 .0 101 n .ji ii ii 0.3852 0 .8 0 1 0 0.3961 9 7 .7 108 100 0 (10056 E th a n o l - S e e T ab le I I I ) 7.92 7 .0 2 7 .8 8 1 0 .9 1 1 .1 1 0 .8 2 0 .0 2 6 .5 7.87 1 0 .9 8 .7 1 15 TABLE IX THE SOLUBILITY OF ERGOSTEROL IN DIOXANE-HEXANE MIXTURES AT 25° t 0 . 0 5 ° C W t. % hexane Wt . S a t . S o lu tio n Sample A bsorbancy A O bserved cr 100 79.6 11 tt 62 .0 it 52 .2 n 3 9 .5 ti 29 .0 n 0 (1002 Dioxane) S o lu b i ■ lily g p e r 1000 g s o l u t i o n See T ab le V I I I A verage 3 .5 7 0.26 02 0 .2 Uli 6 0 .39 10 201 178 2 Cii 31.0 31 .3 3 1 .1 0 .3 2 h 9 0 .1 9 5 5 399 21*0 52.7 52.7 52.7 0.31*76 0 .3 7 3 3 1*96 529 61.2 61.0 61.1 0.161*9 0.21 79 237 31U 61.7 62 .0 61.9 0.2091 0.3105 257 360 53.7 53.7 53.7 0 .1 9 5 1 0 .3 3 I k 0.1*31*2 li-tl 222 321 31.2 26.8 3 1 .6 31.1 3 0 .9 The p a r t i t i o n r a t i o s f o r e r g o s t e r o l and c a l c i f e r o l i n t h e v a r io u s two l i q u i d p h ase sy stem s c o n s i s t i n g o f v a r io u s m ix tu r e s o f hexane m ethanol and w a te r are. l i s t e d i n T a b le s X and X I. 16 TABLE X THE PARTITION RATIOS OF ERGOSTEROL IN VARIOUS HEXANE, METHANOL-WATER SYSTEMS Volume % W ater Added t o M ethanol C o n c en tratio n mg p e r 100 ml s o l u t i o n C ^ p o lar p o la r O bserved Average 8 9 .8 3 1 .9 1 9 .3 1 0 2 .6 3 7 .8 2 2 .3 0 .8 7 6 0 .8 7 6 0 .8 7 5 0 .8 7 6 10 2 .3 3 3 .2 2 7 .6 9 3 .3 3 9 .0 2 5 .3 1 .0 9 1 .0 9 1 .0 9 1 .0 9 ii ii 1 3 0 .0 8 8 .1 5 9 .8 61.9 3 1 .9 2 8 .5 2 .10 2 .1 0 2 .10 2 .1 0 3 n II 13.2.6 1 1 6 .0 9 3 .5 3 2 .6 2 6 .6 2 1 .6 3.37 3 .3 7 3 .3 7 3 .37 12 it 7 .8 0 7 .3 0 7.0 2 1 8 .6 1 8 .6 1 8 .6 1 8 .6 ii 1U5.0 1 3 7 .7 1 3 0 .7 20 it n 9 6 .9 9 3 .7 9 2 .6 2 .2 3 2 .1 9 2 .1 3 3 3 .2 3 3 .2 3 3 .3 33 .2 0 ii ii 1 ii ii 3 n o n - p o la r P a r t i t i o n R atio s ^ n o n -p o lar 17 TABLE XI THE PARTITION RATIOS OF CALCIFEROL IN VARIOUS HEXANE, METHANOL-WATEH SYSTEMS Volume % "Water Added to M ethanol C o n cen tratio n mg p e r 100 ml s o l u t i o n C n o n - p o la r p o la r P a rpt i t i o n R a t i o s n o n - p o la r up o la r Average O bserved 0 n ii 6 .9 3 3 .3 3 1 .6 0 7.147 3.59 1 .7 3 0 .9 2 8 0 .9 2 8 0 .9 2 6 0.927 1 it 7 .6 1 3 .9 8 2.0 8 6.95 3.6 3 1 .9 0 1 .1 0 1 .1 0 1 .0 9 1 .1 0 ii ii 9 .1 5 5 .6 9 3 .2 9 6.10 3 .6 6 2.1 9 1 .5 0 1 .5 0 1 .5 0 1 .5 0 It u 1 0 .5 7 .7 0 5 .6 5 3 .7 8 2.77 2 .0 3 2 .7 6 2 .7 8 2.7 8 2.78 2 0 .0 1 8 .6 1 7 .3 1 .6 6 1 .3 6 1.2lt 1 3 .9 1 3 .9 1 3 .9 13 .9 Hi.. 3 1 3 .9 1 3 .5 0 .3 9 6 0 .3 8 5 0 .3714 36 .1 36.1 3 6 .1 3 6 .1 ii 3 12 ii ii 20 ii ti DISCUSSION 18 DISCUSSION The C hrom atographic B e h a v io r o f Dioxane-H exane M ix tu r e s The a p p ro a c h t o c h ro m a to g ra p h ic e q u i l i b r i u m betw een t h e a d s o r b e n t and tiie v a r i o u s s o l v e n t m i x tu r e s o f d io x a n e and hexane was d e te rm in e d by f o ll o w i n g t h e c o n c e n t r a t i o n o f d io x a n e i n t h e e l u e n t . a r e l i s t e d i n Table X I I . In F i g . 3, t h e f r a c t i o n , c / c q , T hese d a t a w here c e q u a l s t h e c o n c e n t r a t i o n o f d io x a n e i n t h e sam p les o f t h e e l u a t e and cq e q u a l s t h e c o n c e n t r a t i o n o f d iox ane i n th e e l u e n t , h a s been p l o t t e d a g a i n s t t h e volume o f e l u a t e . I t w i l l be n o te d t h a t t h e s lo p e o f t h e c e n t e r p o r t i o n o f t h e v a r i o u s cu rv es d e c r e a s e s from l e f t to r i g h t . T h is i s i n t e r p r e t e d a s a d i f f e r e n c e i n th e r a t e o f a p p ro a c h t c c iiro m ato g rap h ic e q u i l i b r i u m f o r th e v a r i o u s s o l v e n t m i x tu r e s . The volume o f e l u a t e , w hich p r e c e d e s t h e s o l u t i o n o f t h e a d s o r b a t e i s c alled th e r e ta r d a ti o n v o lu m e ,^ v . The r e t a r d a t i o n volume i s measured a s t h a t volume o f l i q u i d w hich h a s emerged from t h e column t o t h e p o i n t where t h e a d s o r b a te a p p e a r s . I n t M s w ork, t h e ap p ro ac h t o ch ro m a to g ra p h ic e q u i l i b r i u m d i f f e r s f o r eac h s o l v e n t m i x t u r e , hence t h e p o i n t a t w hich th e a d s o r b a te f i r s t a p p e a r s i s c o n s i d e r e d n o t a t r u e measure o f th e r e t a r d a t i o n volume . However a u s e f u l v a l u e may be o b t a i n e d by assum ing i n s t a n t a n e o u s e s ta b l i s l i m e n t o f t h e e q u i l i b r i u m . For exam ple, t h e d a t a f o r th e e l u e n t c o n t a i n i n g Q,k9&% dioxane i n d i c a t e s t h a t t h e dioxane c o n c e n t r a t i o n i n c r e a s e s th r o u g h o u t a 9 m l. p o r t i o n o f t h e e l u a t e 19 TABLE X II THE CONCENTRATION OF VARIOUS ELUATE FRACTIONS OBTAINED BY CHROMATOGRAPHING VARIOUS SOLVENT MIXTURES OF DIOXANE AND HEXANE S o lv e n t v E l u a te 1 .0 1 .6 2 .0 2 .6 3 .0 3 .3 i+.o 1+.5 3 .° 5 .5 6 .0 0 .5 7 .0 7 .5 8 .0 6 .5 9 .0 9 .5 1 0 .0 1 1 .0 1 2 .0 1 3 .0 11+.0 1 5 .0 1 6 .0 17 .0 1 8 .0 1 9 .0 2 1 .0 21+.0 2 7 .0 30 .0 X C o n c e n t r a t i o n o f Dioxane Volume % 16.7% D ioxane 966% 3775% Dioxane D ioxane 0 .0 1 0 5 .5 5 1 6 .1 1 6 .7 16 .7 0 I7UB% Dioxane 0.991% 0~JM% Dioxane Dioxane 6.02 6 .9 5 16.7 179^ Dioxane 9 .0 9 9.0 9 0 0 .1 3 9 1 .5 6 3 .6 k 3 .7 b 3 .8 5 3 .8 5 3 .8 5 0 0.1 8 6 1.26 1.82 1.9 6 1 .9 6 0 ' 0.062 0.312 1.19 1.1+1+ 1.1+8 1.1+6 0 0.0901 0 .51+8 0.915 0.962 0.991 0 0 .00 69 0.991 0 .125 0 .1+67 0.1+98 0.1+98 1 .0 0 .3 .6 0 .4 0.2 0.0 0 6 18 12 U L . ELUATE 24 30 FIGURE 3 CHROMATOGRAPHIC BEH A V IO R OF LIO XA N E-H EX A N E MIXTURES ON SU PE R FIL TR O L. C EQUALS THE CONCENTRATION OF ELUATE S A M PL E S, C0 EQUALS THE CONCENTRATION OF SOLVENT M IXTURES ( 1 } 1 6 . 7 (2 ) 9 .0 9 (3) 3 .8 5 4 , (4) 1 .9 6 (5) 1 .4 8 < , (6) 0 .9 9 £ , AND ( 7 ) 0 . 4 9 8 $ DIOXANE IN H E X a N E . 20 b e fo re r e a c h i n g e q u i l i b r i u m , i . e . t h e i n c r e a s e t a k e s p l a c e from 15 m l. to 2b m l. o f e l u a t e . T h e r e f o r e , t h e r e t a r d a t i o n volume was d e te rm in e d as f o l l o w s : = 0.1*01 = a v e r a g e c / c 0 v a l u e f o r t h e t h r e e 3 m l. sam ples 3 a n a ly z e d w hich c o n t a i n e d some d io x a n e b u t l e s s t h a n th e e l u e n t . 9 = 3 .6 0 9 m l. = th e volume o f s o l u t i o n of th e e l u e n t c o n c e n - 0 . 1*01 t r a t i o n , w hich c o n t a i n s t h e t o t a l amount o f d io x a n e found i n t h e 9 m l . p o r t i o n from 15 m l , to 2h m l . o f e l u a t e . 2i* .0 - 3 . 6 = 20 ,U m l . = t h e r e t a r d a t i o n volume. The r e t a r d a t i o n volumes t h u s d e te r m in e d f o r t h e v a r i o u s s o l v e n t m ix tu r e s o f d io x a n e and hexane a r e shown i n T ab le X I I I . TABLE X I I I THE CHROMATOGRAPHIC ADSORPTION OF DIOXANE FROM HEXANE SOLUTIONS OIM SUPERFILTROL S o lv e n t C om po sition Volume % Dioxane i n H Hexane . 0.1*98 0.991 1.1*96 1.9 6 0 3.65 0 9.090 16.670 R eta rd a tio n Volume m l. 20.1* 13.1* 9.60 7.69 b . o ’S 2.39 1 .3 5 S p ecific R eta rd a tio n Volume ml .p e r g . 1 0 .2 6.70 1*.80 3 .8 5 2.33 1 .2 0 0 .6 8 0 X IT] u l .p e r g 50. b 67.0 7 2 .0 7 7 .0 9 3 .0 1 1 9 .5 1 3 5 .0 21 The s i g n i f i c a n c e o f t h e s e " c a l c u l a t e d ” r e t a r d a t i o n volumes i s r e a d i l y seen from F i g . it, where t h e amount o f d io x a n e a d s o rb e d p e r gram o f S u p e r f i l t r o l was c a l c u l a t e d d i r e c t l y from th e s p e c i f i c r e t a r d a t i o n volum es. To ch ec k t h e v a l i d i t y o f th e c a l c u l a t i o n o f th e amount o f dioxane ad so rb ed p e r gram o f S u p e r f i l t r o l from t h e s p e c i f i c r e t a r d a t i o n volum es, th e a d s o r p t i o n o f d io x a n e from hexane by S u p e r f i l t r o l was d ete rm in e d by n o rm al e q u i l i b r i u m methods . These r e s u l t s a r e a l s o shown in F i g . I4 and s u b s t a n t i a t e th e v a l u e s d e te rm in e d fi'om th e r e t a r d a t i o n volumes i n t h e low c o n c e n t r a t i o n r e g i o n . The a d s o r p t i o n v a lu e s d e t e r ­ mined by normal e q u i l i b r i u m methods f o r s o l u t i o n s c o n t a i n i n g more th a n h i d i o x a n e , were u n r e l i a b l e due t o i n h e r e n t e r r o r s i n th e m ethod, normal e q u i l i b r i u m methods i n v o l v e s u b t r a c t i n g t h e c o n c e n t r a t i o n o f the s o l u t i o n a f t e r a d s o r p t i o n from th e c o n c e n t r a t i o n o f th e s o l u t i o n b e f o r e a d s o r p t i o n and f o r t h e s o l u t i o n s c o n t a i n i n g more th a n ii% d io x a n e in v o l v e s s u b t r a c t i n g one l a r g e number from a n o th e r l a r g e number. Since th e a d s o r p t i o n o f dioxane i s s m a l l , t h e a c t u a l e r r o r f o r such an a d s o r p ­ t i o n v a l u e becomes l a r g e . The d e t e r m i n a t i o n of t h e r e t a r d a t i o n vo lum es, however, were n o t s u b j e c t t o t h i s i n h e r e n t e r r o r and th e c a l c u l a t e d a d s o r p t i o n v a l u e s from s o l u t i o n s c o n t a i n i n g more t h a n l\$ dioxane were re p ro d u c e d w i t h h ig h p r e c i s i o n . The C h r o m a to g ra p h ic .B e h a v io r o f E r g p s t e r o l and C a l c i f e r o l on S u p e r f i l t r o l i n t h e P re s e n c e o f M ix tu r e s o f Dioxane and Hexane I n th e i n i t i a l p h ase o f t h i s s t u d y , th e a d s o r b a te was added t o th e ch ro m a to g rap h ic column i n a hexane s o l u t i o n from whicn b o th c a l c i f e r o l IS O 80 40 0 0 4 8 13 VOLUME 4, DIOXANE IN HEXANE 16 FIGURE 4 A DSORPTION OF DIOXANE FROM HEXANE S O L U T IO N S ON S U P E R F I L T R O L ; O CALCULATED FROM OHROMATOGRAPHIC RETARDATION VOLUMES AND • DETERMINED BY NORMAL E Q U IL IB R IU M METHODS. 22 and e r g o s t e r o l a r e s t r o n g l y a d s o r b e d on S u p e r f i l t r o l , ^ s i n c e s t r o n g a d s o r p t i o n y i e l d s a narro w i n i t i a l zone o f e r g o s t e r o l o r c a l c i f e r o l . S ubsequent e l u t i o n of t h e e r g o s t e r o l o r c a l c i f e r o l th u s e n t e r e d onto the S u p e r f i l t r o l column, i n d i c a t e d however t h a t t h i s p r o c e d u r e was n o t f e a s ib le f o r th e se a d s o r b a te s . From F i g ' s . 5 and 6 one can r e a d i l y s e e t h a t b o t h e r g o s t e r o l and c a l c i f e r o l undergo d e c o m p o s itio n d u r i n g th e a d s o r p t i o n or d e s o r p t i o n p r o c e s s e s on S u p e r f i l t r o l . Subsequent i n v e s t i ­ g a tio n i n d i c a t e d t h a t t h e s e a d s o r b a t e s could b e added t o t h e column i n a two j n l . p o r t i o n o f th e e l u t i n g s o l v e n t w i t h o u t s e r i o u s d e c o m p o sitio n bein g e v i d e n c e d . The e l u a t e c o n c e n t r a t i o n h i s t o r y d a t a a re l i s t e d i n T a b le s XIV and XT. The e l u t i o n o f e r g o s t e r o l a n d / o r c a l c i f e r o l p ro c e e d e d d i f f e r e n t l y fo r each d i f f e r e n t e lu e n t s o lv e n t m ix tu re. The c o n c e n t r a t i o n h i s t o r y c u r v e s f o r c a l c i f e r o l and f o r e r g o s t e r o l a re g iv e n i n F ig u r e s 7 and 8 re sp e c tiv e ly . I t w i l l be n o te d t h a t t h e g e n e r a l shape o f t h e s e c u rv e s u n d erg o es a change from a n a s y m e t r i c a l b e l l to a s y m e t r i c a l b e l l as th e c o n c e n t r a t i o n o f d io xane i n th e e l u t i n g s o l v e n t m ix tu re d e c r e a s e s . D e f a u l t ' s th e o r y " ^ and W e is s ' t h e o r y ^ o f chro m atog raph y e x p l a i n th e s e o b s e r v a t i o n s on t h e b a s i s o f a change i n t h e a d s o r p t i o n .isotherm of t h e s e a d s o r b a te s as t h e amount o f dioxane i n th e s o l v e n t m ix tu re c h a n g e s. Verrneulen and H i e s t e r ^ have shown t h a t th e v a r i a t i o n i n symmetry of t h e c o n c e n t r a t i o n h i s t o r y cu rv es i n th e c a s e ox io n -ex c h an g e clirom atographv i s e x p l a i n a b l e on t h e b a s i s o f v a r i a t i o n s i n th e e q u i l i b r i u m 1 .0 0.8 0.6 4 3 0 230 250 370 WAVELENGTH ( m u . ) 290 FIGURE 5 D EC O M P O SIT IO N OF ERGOSTEROL ON S U P E R F IL T R O L O PURE ERGOSTEROL AND ELUATE FR A C T IO N S OBTAINED WHEN ERGOSTEROL WAS ADDED TO A S U P E R F IL T R O L COL­ UMN IN HEXANE SOLUTION AND ELUTED WITH SOLVENT MIXTURE3 OF e 0 . 9 9 - % DIOXANE AND • 1 6 . 7 $ DIOXANE IN HEXANE. o.s 0 .4 o m 0.2 230 250 270 290 WAVELENGTH ( m u . ) 310 FIGURE 6 D E C O M PO SIT IO N OF C A L C IF E R O L ON S U P E R F I L T R O L . 0 PURE C A L C IF E R O L , AND ELUATE F R A C T IO N S O B TA IN ED WHEN C A L C IF E R O L WAS ADDED TO A S U P E R F I L T R O L COLUMN I N HEXANE S O L U T IO N AND ELUTED WITH SOLVENT MIXTURES OF • 3 . 8 5 4 DIOXANE AND 0 1 6 . 7 4 DIOXANE I N HEXANE. 23 TABLE XIV THE ELUATE CONCENTRATION HISTORY DATA FOR CALCIFEROL CHROMATOGRAPHED ON SUPERFILTROL WITH VARIOUS PilXTUrtES OF HEXANL-DIOXANE AS ELUTING SOLVENTS S o lv e n t H I. E lu a te 1 2 0 J 'h X - 9.096 Dioxane 0 0.606 1.006 0 .1 2 6 0 , 02oo o.oofcfc 7 6 9 10 11 12 13 lli 15 16 17 16 19 20 21 22 23 2L 25 26 27 Mg C a l c i f e r o l E l u te d P er Ml E lu a te 1.965b 0 . 9 9 1L 3 .X D ioxane Dioxane Dioxane 0 0 .0 3 6 6 0 .6 3 6 o . 616 0 .1 9 9 o .0366 0 .0 1 3 6 0 .0 0 6 4 0 0 .0 0 5 0 0.0 7 1 2 0 .5 6 0 0.6 2 2 0 .2 6 6 0 .0 6 7 0 0 .0 2 7 6 0 .0 1 2 6 ------- 0 0.0102 0 .1 2 1 u 0 .2 9 6 0 .3 1 6 0 .2 2 6 0 .1 3 1 6 0.067R 0.0 3 1 2 0.0146 o.h9H% Dioxane 0 0.00 66 0.0252 ‘ 0 .0 5 3 6 0 .0 6 7 8 0 .06 36 0.01+98 0.03UU 0.0221} 0.013U 0 .00 78 . 26 TABLE XV THE ELUATE CONCENTRATION HISTORY DATA FOR ERGOSTEROL CHROMATOGRAPHED ON SUPERFILTROL WITH VARIOUS MIXTURES OF HEXANEDIOXA&R lib El S o lv e n t .1. D.uat, ( 3 ) 1 . 9 6 4 , AND ( 4 ) 0 . 9 9 $ DIOXANE I N HEXANE. 33 p a r a m e te r . I n so f a r a s a d s o rp tio n , chromatography may be compared to io n -e x c h a n g e cliro m atog raph y, one m i g h t c o n s i d e r the change from a s y m e t r i c a l t o s y m e t r i c a l c o n c e n t r a t i o n h i s t o r y c u r v e s } as b e i n g i n d i ­ c a t i v e o f a n approach t o t r a c e c o n d i t i o n s a s t h e c o n c e n t r a t i o n of d io x a n e i n t h e e l u t i n g s o lv e n t d e c r e a s e s } s i n c e the amount o f c a l c i f e r o l or e r g o s t e r o l i n s o l u t i o n a t a given i n s t a n c e i s d e c re a s e d due t o s t r o n g e r a d so rp tio n . The t h r e s h o l d volumes f o r c a l c i f e r o l a h a fo r e r g o s t e r o l when e l u t e d by t h e s e s o l v e n t m ix tu re s have b e e n d eterm in e d by t h e u s u a l method con­ s i s t i n g o f an e x t r a p o l a t i o n of th e c e n t r a l p o r t i o n o f th e e l u t i o n c u rv e s ( F i g ' s . 9 and 10) . I t i s apparent (Table M il) t h a t t h e t h r e s h o l d volumes f o r c a l c i f e r o l and e r g o s t e r o l i> ic r e a s e with a d e c r e a s e i n t h e amount o f d io x a n e i n t h e e lu e n t s o l v e n t rnixtcures. table XVI THE THRESHOLD VOLUMES AMD THE RLCOV.iM OP CALCIFEROL AND ERGOSTEROL VJHEN CHROMATOGRAPHED M TTH VARIOUS MIXTURES OF RBXANE-DIOXAI'Jit ON SUPERFILTROL S o lv e n t C o m p o s itio n % D ioxane 9 .0 9 3.85 1 .9 6 0 .9 9 1 0.ii98 T h re s h o ld Volume C a lc ife ro l E rg o ste ro l 1 .3 2 3 .1 2 6 .0 5 1 0 .7 1 8 .8 1 .5 5 h .39 l O .5 2 3 .2 — — — % A d so rb a te R ecovered E rg o ste ro l C a lc ife ro l 9 5 .2 9 0 .0 6 3 .0 60 .8 17 .2 85.2 7 8 .0 Ii8.2 1 8 .6 ''The e q u ilib r iu m param eter depends u p o n the e q u i l i b r i u m exchange b e tw e e n a t r a c e io n and th e c a r r i e r ion a n d also d epend s upon t h e r e l a ­ t i v e c o n c e n t r a t i o n o f t h e trace i o n and t h e c a r r i e r i o n i n t h e ca s e o f io n -e x c h a n g e chrom atography. T h u s Vermeulen and H i e s t e r have shown t h a t t h e symmetry o f the c o n c e n t r a t i o n h is to r y c u r v e i s a c r i t e r i o n f o r t r a c e c o n d itio n . ELUTED 0.6 FRACTION 0.8 ADSORBATE 1 .0 0 .4 0.8 0. 0 0 4 8 18 16 ULo ELUATE 80 84 FIGURE 9 THE CHROMATOGRAPHIC EL U T IO N OF C A L C IFE R O L FROM SU PE R ­ F I L T R O L COLUMNS BY SOLVENT M IX TU R ES ( l ) 9 . 0 9 = Ks1/ * /rtitf. where K, oc . ^ + r } and. 7 are c o n s t a n t s . we o b t a i n t h e e x p ire ssio n , v0 ( 6) S u b s t i t u t i n g i n t o E q u a tio n ($) (M L .) VOLUMN THRESHOLD 0 .5 3 .0 4 .0 1.0 VOLUME ■S DIOXANE I N HEXANE 8.0 FIGU RE 11 THE THRESHOLD VOLUMES F O R ( 1 ) C A LC IFE RO L AND ( 3 ) ERGOSTEROL ON S U P E R F IL T R O L F 3R VARIOUS ELUENT MIXTURES OF DIOXANE IN HEXANE. 80 40 20 10 1.0 2 .0 4 .0 0 .5 V O L U M E $> DIOXANE IN HEXANE 8 .0 FIG U R E 1 2 T H E R E T E N T I O N O F ( l ) C A L C IF E R O L AND ( 2 ) E R G O S T E R O L B Y S U P E R F I L T R O L C0LUV.N3 'UHEN ELUTED BY S O L V E N T M I X T U R E S O F DIOXANE IN KSXANE. 27 l o g Ar = a l o g [Ks1/ * / m ^ f + / v 0] + b ( 7) which r e l a t e s t h e q u a n t i t y o f a d s o r b a t e r e t a i n e d by th e column t o t h e q u a n t i t y o f a d s o r b e n t , th e amount o f a d s o r b a t e e n t e r e d on th e colum n, and. t h e volume o f s o l u t i o n c o n t a i n i n g t h e a d s o r b a te b e f o r e a d s o r p t i o n . The C h rom ato graph ic S e p a r a t i o n o f C a l c i f e r o l from E r g o s t e r o l I t was a p p a r e n t , from th e p r e c e d i n g c o n s i d e r a t i o n s t h a t a q u a n t i t a ­ t i v e r e c o v e r y o f c a l c i f e r o l a n d / o r o f e r g o s t e r o l was n o t p o s s i b l e i f an " e q u il ib r iu m " e l u t i o n method were em ployed. e l u t i o n p r o c e e d u re was a t t e m p t e d . C o n s e q u e n tly , a two s t e p A f t e r e l u t i n g a column, to which e r g o s t e r o l had b een a d d e d , i n t h e norm al m anner, w i t h I4O m l. o f e l u t i n g s o l v e n t c o n t a i n i n g 0 . 99/ d io x a n e , t h e column was f u r t h e r e l u t e d w ith 10 m l. o f s o l v e n t c o n t a i n i n g 1 6 . 7 / d io x a n e . tiie e l u a t e t h u s o b ta in e d i s shown i n F i g . 13. The a b s o r p t i o n cu rv e f o r By com paring t h i s a b s o rp ­ t i o n c u rv e w i t h t h a t o f p u re e r g o s t e r o l ( F i g . 5 ) , i t i s a p p a r e n t t h a t t h e e r g o s t e r o l th u s t r e a t e d , has undergone d e c o m p o sitio n d u r in g th e p rocess o f a d so rp tio n or d e s o rp tio n . A tte m p ts t o overcome t h i s d i f f i ­ c u l t y were u n s u c c e s s f u l . The s e p a r a t i o n o f . c a l c i f e r o l from e r g o s t e r o l was i n v e s t i g a t e d u t i l i z i n g a s o l v e n t m ix tu re o f hexane and dioxane w hich would y ie ld , th e b e s t reco v e ry o f pure c a l c i f e r o l . A co m parison o f t h e c o n c e n t r a t i o n h i s t o r y c u rv e s f o r c a l c i f e r o l and f o r e r g o s t e r o l i n d i c a t e s t h a t t h e b e s t s o l v e n t m ix tu r e i s t h a t c o n t a i n i n g 1.96% d io xan e i n hexane. F i g . 1U, th e c o n c e n t r a t i o n h i s t o r y c u r v e s f o r e r g o s t e r o l and f o r In 240 360 380 300 330 340 WAVELENGTH ( m u . ) 360 380 400 430 FIGURE 13 A B S O R P T IO N CURVE TO R THE R E S I D U E D IO X A N E IN HEXANE FROM A S U P E R F I L T R O L TO E L U T IO N OF ERGOSTEROL BY A SOLVENT 8 1 . 4 % OF THE ERGOSTEROL WAS R E T A IN E D ELUTED BY A SOLVENT FIX T U R E CONTAINING 1 6 . 7 4 COLUMN WHICH HAD PREVIOUSLY EE EN SUBJECTED MIXTURE OF 0 . 9 9 % DIOXANE IN HEXANE WHERE BY THE COLUMN. 0 .3 ELUATE FRACTION ADSORBATE ELUTED PER ML. 0 .3 0.1 0 0 4 8 12 16 ML. ELUATE FIGURE 14 THE CONCENTRATION HISTORY CURVES F O R ( l ) C A L C IF E R O L AND ( 2 ) ERGOSTEROL OBTAINED BY EL U TIO N FROM S U P E R F IL T R O L BY A SOLVENT MIXTURE OF 1 . 9 6 $ DIOXANE IN HEXANE. 20 28 c a l c i f e r o l , o b t a i n e d by e l u t i o n w ith t h e s o l v e n t m ix tu r e c o n t a i n i n g 1.9 6 # d io x a n e i n h ex an e , a r e i l l u s t r a t e d . I f th e e l u t i o n o f c a l c i f e r o l o r e r g o s t e r o l w ere n o t a f f e c t e d by t h e p r e s e n c e o f t h e second a d s o r b a t e , one would e x p e c t th e r e c o v e r y o f p u re c a l c i f e r o l t o be 77.3%, s in c e e l u a t e f r a c t i o n s from 10 t o 12 m l. 'would c o n t a i n a m i x tu r e o f c a l c i f e r o l and e r g o s t e r o l . The r e c o v e ry o f p u re c a l c i f e r o l was fou nd to be d e ­ pendent, on t h e amount o f e r g o s t e r o l i n th e m ix tu r e t o be s e p a r a t e d . These o b s e r v a t i o n s a r e i l l u s t r a t e d i n F ig u r e 1 5. W hereas t h e r e c o v e r y of p u re c a l c i f e r o l d e c re a s e d w i t h an i n c r e a s e i n t h e amount o f e r g o s t e r o l i n th e m ix tu r e to be s e p a r a t e d , t h e t o t a l r e c o v e r y o f c a l c i f e r o l i n ­ c r e a s e d w i t h an i n c r e a s e i n t h e e r g o s t e r o l p r e s e n t i n t h e m i x t u r e . From E q u a tio n (5) i t c a n be shown t h a t an i n c r e a s e i n t h e t o t a l amount o f c a l c i f e r o l r e c o v e r e d sh ould be co u p led w i t h a d e c r e a s e i n t h e t h r e s h o l d volume. T h u s, t h e t h r e s h o l d volume f o r c a l c i f e r o l s h o u ld e x p e r ie n c e a d e c r e a s e w ith an i n c r e a s e i n t h e amount o f e r g o s t e r o l i n t h e m ix tu re to be s e p a r a t e d . I n F i g . 16 t h i s i s s e e n t c be t h e c a s e . • Summary 1 . p-D ioxane was d e te rm in e d i n th e p re s e n c e o f hexane by i n f r a r e d a b s o r p t i o n a t 1 1 .3 5 m icro n and agreem ent w ith B e e r ' s Law th r o u g h o u t t h e c o n c e n t r a t i o n ra n g e 0 t o 2.5% d io x a n e i n hexane was fo un d. 2 . An e q u i l i b r i u m betw een S u p e r f i l t r o l and e l u t i n g s o l v e n t m ix tu r e s o f d io x a n e and hexane was o b s e rv e d and t h e tim e o f e s t a b l i s h m e n t o f th e e q u i l i b r i u m was found t o be d ep en d e n t on t h e amount o f dioxane i n th e so lv e n t m ix tu re . 84 £ CALCIFEROL RECOVERED 83 82 81 80 0 1 2 3 MG. ERGOSTEROL P E R 2 MG. C A LC IFE R O L 4 FIGURE 15 THE E r F E C T OF ERGOSTEROL ON THE RECOVERY OF C A LC IFE RO L FROM A 3 U P E R F I L T R O L COLUMN WHEN ELUTED BY A SOLVENT MIX­ TURE OF 1 . 9 6 4 DIOXANE IN HEXANE. 6.0 5 .9 3o 5 .8 5 .7 5 .6 5 .5 0 3 1 2 MS. ERGOSTEROL PEP. 2 MO. C A LC IFE RO L F I G U R E 16 THE EF FEC T OF ERGOSTEROL ON THE THRESHOLD VOLUME OF C A LC IFE R O L ELUTED FRCi! A 3UPERF 1L T R 0 L COLUMN BY A SOLVENT MIXTURE OF 1 . 9 6 $ DIOXANE IN HEXANE. 4 29 3 . The a d s o r p t i o n o f d io x a n e from hexane s o l u t i o n s by S u p e r f i l t r o l h a s been c a l c u l a t e d from th e c h ro m a to g rap h ic e q u i l i b r i u m s t u d i e s , and h as b e e n shown t o a g re e w i t h t h e a d s o r p t i o n curve d e te rm in e d b y norm al pro ced u re. 1+. The c h ro m a to g ra p h ic b e h a v i o r o f c a l c i f e r o l and e r g o s t e r o l w ith e l u t i n g s o l v e n t m i x tu r e s o f hexane and d io x a n e has been s t u d i e d and an e q u a t i o n h as b een deduced r e l a t i n g th e amount o f a d s o r b a te r e t a i n e d by t h e column t o t h e q u a n t i t y o f a d s o r b e n t , th e amount o f a d s o r b a t e , and th e volume o f t h e s o l u t i o n c o n t a i n i n g th e a d s o r b a t e b e f o r e a d s o r p t i o n . 3 . The amount o f e r g o s t e r o l p r e s e n t i n a m ix tu r e o f c a l c i f e r o l and e r g o s t e r o l was found t o i n f l u e n c e t h e r e c o v e r y o f p u re c a l c i f e r o l . The t o t a l r e c o v e r y o f c a l c i f e r o l was found, to b e i n c r e a s e d by an in c r e a s e o f e r g o s t e r o l i n such a m i x t u r e . 6. The s e p a r a t i o n o f c a l c i f e r o l from e r g o s t e r o l h a s been shown to be d e p en d e n t on t h e amount o f e r g o s t e r o l i n th e m ix tu r e to be s e p a r a t e d . A m ix tu r e c o n t a i n i n g 2 mg. o f b o th c a l c i f e r o l and. e r g o s t e r o l was chromato graphed t o g iv e an 81% r e c o v e r y o f p u re c a l c i f e r o l . The S o l u b i l i t y o f N o n - e l e c t r o l y t e s i n S o lv e n t M ix tu r e s S o l u b i l i t y s t u d i e s o f n o n - e l e c t r o l y t e s i n s o l v e n t m ix tu r e s h as b een t h e s u b j e c t o f a r e c e n t p ap er b y Gordon and S c o t t ‘S i n w hich t h e y showed t h a t r e g u l a r s o l u t i o n t h e o r y ^ p r e d i c t s enhanced s o l u b i l i t y where th e s o l u b i l i t y p a ra m e te r o f th e s o l u t e i s betw een th o s e o f t h e s o l v e n t s . was th e p u rp o s e o f t h i s s tu d y t o show how th e t h e o r y , w i t h a few added a s s u m p ti o n s , may be u s e d t o e x p l a i n o t h e r th a n enhanced s o l u b i l i t y . It 30 The r e g u l a r s o l u t i o n t h e o r y of H ild e b r a n d , -| O a p p l i e d to t h e s o l u ­ b i l i t y o f s o l i d s i n l i q u i d s , r e l a t e s t h e s o l u b i l i t y to th e thermodynamic p r o p e r t i e s o f th e p u r e com ponents, i . e . t o th e m e l t i n g te m p e r a t u r e o f t h e s o l u t e , th e h e a t o f f u s i o n o f th e s o l u t e and t h e " s o l u b i l i t y p a r a ­ m e te rs " o f t h e s o l u t e and s o l v e n t . The s o l u b i l i t y p a ra m e te r i s d e f in e d a s t h e s q u a r e r o o t o f th e " i n t e r n a l p r e s s u r e " o r " c o h e s iv e e n e r g y d e n s it y " and f o r a giv en s u b s t a n c e , i s u s u a l l y c a l c u l a t e d from th e m o lar volume and th e m o lar en erg y o f v a p o r i z a t i o n , 19 th u s ( 8) The i d e a l s o l u b i l i t y , i n mole f r a c t i o n X1 , i s given by t h e e q u a t io n l o g X1 (9) where Z^Iix i s i t s h e a t o f f u s i o n and Tm i s i t s m e l t i n g te m p e r a tu r e . I f t h e s o l u t i o n i s n o t i d e a l , t h e d e v i a t i o n s from i d e a l i t y a r e r e l a t e d t o t h e p a r t i a l m olal h e a t o f m ix in g A H , , o f t h e s u p e rc o o le d l i q u i d s o l u t e w ith t h e s o lv e n t l o g Xx = l o g X1 -. -A Hi 2 .3 HT (10) ( 10) The t h e o r y f u r t h e r r e l a t e s iAH-^ to t h e m o la l volume V^, th e volume f r a c t i o n o f th e s o l v e n t A h x = where th e s u b s c r i p t s (J)0 , and th e s o l u b i l i t y p a r a m e te rs cT, o3 ( - So) 2 ( 11) and o prefer t o t h e s o l u t e and s o l v e n t r e s p e c t i v e l y . 31 F or a t h r e e component system i n v o l v i n g two s o l v e n t co m p on ents} t h e th e o ry 20 l e a d s t o e q u a t i o n s which a r e i d e n t i c a l w i t h E q u a tio n s (10) and (11) i f $q i s ta k e n as t h e t o t a l volume f r a c t i o n of s o l v e n t s 2 and 3 and S q i s t a k e n as t h e volume f r a c t i o n avera g e o f s ° = S3 . <1 2 > I f t h e volume f r a c t i o n s a r e c o n s i d e r e d p r o p o r t i o n a l t o th e con­ c e n t r a t i o n s o f components th e n 413 L2 x3 ( 13 ) - c. ana $2 = kX3j 4 a = kX3 J 4 2^ 3 = k ( x 2 + X3) (16) where X2 and X3 a r e th e mole f r a c t i o n s o f t h e s o l v e n t components and k i s a c o n s t a n t c o n s id e r e d in d e p e n d e n t o f t h e c o n c e n t r a t i o n of t h e s o l v e n t c;rr.ronent,s . S u b s t i t u t i n g i n t o n q u a n ic n (12 ) f o r 3} (j)3 and (J;2 + (f)3 S; = » S x3 j±2 • a 3 C o n s id e r rh e c a s e where t h e s o l u t e i s b u t s l i g h t l y s o l u b l e th ro u g h o u t —n 3.J—L Oi tU— it S_ = t V“It d-’i , §3x 2 + U7 1 P e A2 diiO -A;; *r 1 2 = -i- • S3x ... (it) = 4!2 + 4>3 = i:(^2 + * 3 ) = 09) acc o r a i r r uo ucue t i o n s ( I t ) ana (1 ?) i n f o e q u a t i o n ( 1 1) and _ . - / / •r 2\ ~ v i ) £ +D a3+aA s> -V2(V w Si - D- . , 2 r i / ,-v-.\ u o ; S j2 S ) - S, S2 4 S, S3- S 3 S 3) d' - - k 2( S _ 2 3 S , ! - Z § 3 S 3 ) I-• A*o — A-a “ A-bA5, . ; e r » r j - QT*~- = -V3 [ a ' + ( o ' + a ' ) X2 - d 'X 2Xa j (21) 3-,- d e f i n i t i o n ri:s s o l u b i l i t y p a r a m e te r e q u a ls th e sq u a re ro o i e ner gy o f i n t e r a c t i o n (,-psr c c -) • 33 (22) S u b s t i t u t i n g C1/ 2 f o r S t h e c o n s t a n t d ' becomes cl = - k 2(C22 + C33 - 2C321/ 3C331/ 2 ) and th e term CZ2X^ Z C333*/3 d e q u a ls C2 3 .^ (23) Thus E q u a tio n (23) becomes = - k 2(C22 + C33 - 2G23) (23) I f t h e e n e rg y o f i n t e r a c t i o n betw een u n l i k e s o l v e n t m o lecu le s i s con­ s i d e r e d t h e a r i t h m e t i c mean o f t h e e n e r g i e s ox i n t e r a c t i o n between l i k e m o l e c u le s , i t i s a p p a r e n t t h a t d ' e q u a l s 0 and E q u a tio n A h x = - Vx ( a ' + b % ) „ (21) becomes (23) S u b s t i t u t i n g E q u a tio n (23) i n t o E q u a tio n (1 3 ) and assum ing t h a t Yx i s in d e p e n d e n t o f th e c o n c e n t r a t i o n o f t h e s o l v e n t components and i s e s s e n t i a l l y c o n s t a n t , th e e q u a t io n l o g Xx = a b a + bX3 (26) = il o g a.x + V7a ' 2.3RT p r e d i c t s a n e x p o n e n t i a l change of th e s o l u b i l i t y w i t h t h e change o f th e s o l v e n t c o m p o sitio n .'" * I t s h o u ld be n o te d t h a t E q u a tio n (26) i s i n e s s e n t i a l l y t h e same form as t h a t of S t r o m b e r g , ^ which was d e r iv e d on t h e b a s i s o f Z h u k hov itsky *s r e g u l a r s o l u t i o n t h e o r y . 3h The S o l u b i l i t y o f E r g o s t e r o l and C a l c i f e r o l i n S o lv e n t M ix tu r e s . E r g o s t e r o l was fo u n d t o have a g r e a t e r s o l u b i l i t y i n h ex an e -d io x an e m ix tu r e s and i n h e x a n e - e th a n o l m i x t u r e s , th a n i t does i n e i t h e r p u r e hexane, p u r e d io x a n e , o r p u re e t h a n o l . The dioxane m i x tu r e s was fo u n d t o be betw een maximum s o l u b i l i t y i n h ex ane- ItO and $ 2%by w e ig h t hexane and i n h e x a n e - e th a n o l m i x tu r e s was fo u n d t o be betw een 63 and. 69% b y w e ig h t h e x a n e . (T a b le XVII) . The t h e o r y p r e d i c t s t h i s enhanced s o l u b i l i t y where t h e s o l u b i l i t y p a r a m e te r o f t h e s o l u t e i s b etw ee n t h o s e t h e maximum s o l u b i l i t y where &x = o f t h e two s o l v e n t s and p r e d i c t s Since t h e s o l u b i l i t y p a ra m e te r s f o r hexane and dioxane a re 7 .3 and 1 0 .0 r e s p e c t i v e l y , 2 '$ and s in c e e r g o s t e r o l shows enhanced s o l u b i l i t y i n h e x a n e -d io x a n e m i x t u r e s , t h e s o l u b i l i t y p a r a m e te r f o r e r g o s t e r o l m ust be betw een t h e s e v a l u e s . A lso , s in c e e r g o s t e r o l e x p e r ie n c e s enhanced s o l u b i l i t y i n h e x a n e - e th a n o l m i x t u r e s , i t s s o l u b i l i t y p a ra m e te r m ust be betw een th o s e o f hexane and e th a n o l. F u r t h e r , s in c e t h e s o l t i b i l i t y o f e r g o s t e r o l i s n e a r l y t h e same i n hexane and i n e t h a n o l b u t much g r e a t e r i n d io x a n e , one can assignr e l a t i v e v a l u e s t o e r g o s t e r o l and e t h a n o l , th u s ^hexane = 7 . 3 < S e rg o ste ro l ^ ^ e t h a n o l < 1 0 ,0 = ^ d i o x a n e . The s o l u b i l i t y o f b o th e r g o s t e r o l and. c a l c i f e r o l i n e t h a n o l—w a te r m ix tu re s was found t o d e c r e a s e e x p o n e n t i a l l y w i t h an i n c r e a s e o f w a te r ( F i g . 17) and f o ll o w s th e form o f E q u a tio n ( 2 6 ) . A p p a re n tly , t h e r e f o r e , th e a s s u m p tio n s u s e d i n th e d e r i v a t i o n o f E q u a tio n (26) may be con.sic.ered 35 TABLE XVII THE SOLUBILITY OF ERGOSTErtOL IN MIXED SOLVENTS AT 25° S o lv e n t M ix tu r e Wt . % S o lv en t Component Y 0 9 .0 11.1+ 11+.1+ 16 .0 16.3 19.1+ 25.0 29.7 33.1 35.0 39.5 55.6 50.3 52. 2 62 .0 62.9 69.6 78.9 79.8 86.5 100 Y E th a n o l w a te r ± 0.05°C S o lu b ility g p e r 1000 g s o l u t i o n E th a n o l Hexane Dioxane Hexane 8 .71 3 0 .9 8 .7 1 1 .7 1 1 0 .9 7.87 0 .5 5 0 0.535 1 3 .8 53.7 0.16 1 2 1 .5 0 .0 8 6 5 61.9 2 7 .6 0 .0 1 3 1 61.1 52.7 38 .9 38.9 26.5 31.1 2 0 .0 3.57 3.57 v a l i d f o r t h i s s o l v e n t s y s te m } and t h e en erg y o f i n t e r a c t i o n betw een u n l i k e m o le c u le s o f e t h a n o l and w a te r may be c o n s id e r e d a p p r o x im a te ly e q u a l to t h e a r i t l r m e t i c mean o f th e e n e r g i e s o f i n t e r a c t i o n betw een l i k e m o le c u le s o f e t h a n o l and o f w a t e r . 100 1 0 .0 1 .0 0 0 .10 0.0 1 50 40 60 70 80 SO 100 WT. PERCENT ETHANOL IN ETHANOL-WATER MIXTURES FIGURE 17 THE S O L U B IL IT Y BEHAVIOR OF ( 1 ) C A LC IFE R O L AND ( 3 ) ERGOSTEROL IN ETHANGL-WATER F I X T U R E S AT 3 5 ° C . The E f f e c t o f th e A d d i t i o n o f 'Water on th e Pa r t i t i o n R a t i o s o f E r g o s t e r o l and C a l c i f e r o l i n t h e L i q u i d - L i q u i d System ; Hexane-Hethanol"! P a r t i t i o n r a t i o s o f b o t h e r g o s t e r o l and c a l c i f e r o l were fou nd to be in d e p e n d e n t o f t h e i r c o n c e n t r a t i o n s o v e r t h e c o n c e n t r a t i o n ran g e s t u d i e d . The p a r t i t i o n r a t i o s , however a r e i n f l u e n c e d b y th e a d d i t i o n o f w a t e r to th e h e x a n e -m e th a n o l s y s te m . C o n s id e r i n g t h e p a r t i t i o n r a t i o s as b e i n g t h e r a t i o betw een t h e c o n c e n t r a t i o n o f e r g o s t e r o l or c a l c i f e r o l i n th e n o n - p o l a r p h ase and th e c o n c e n t r a t i o n i n th e p o l a r p h a s e , one can r e a d i l y s e e t h a t an i n c r e a s e o f w a t e r to th e h ex an e-m eth an o l system c a u s e s an i n c r e a s e i n t h e s e r a t i o s , i . e . th e w a t e r may be s a i d t o " f o r c e " t h e e r g o s t e r o l o r c a l c i f e r o l from t h e p o l a r phase i n t o th e n o n -p o la r p h a s e . I t was a l s o fo u n d t h a t th e change i n t h e p a r t i t i o n r a t i o s of e r g o s t e r o l and c a l c i f e r o l w i t h t h e a d d i t i o n o f w a te r t o t h e hexanem ethanol s y s te m , was n o t e q u i v a l e n t , i . e . th e p a r t i t i o n r a t i o s o f e r g o s t e r o l i n c r e a s e d more r a p i d l y w i t h th e a d d i t i o n o f w a te r th a n d id th e p a r t i t i o n r a t i o s o f c a l c i f e r o l . T h is f a c t l e a d to a c o n s i d e r a t i o n o f th e problem o f s e p a r a t i n g c a l c i f e r o l from e r g o s t e r o l by c o u n t e r current d is tr ib u tio n . A cco rd in g t o C r a i g ‘S t h e r a t i o Ka / l t 0 i s a m easure o f th e e a s e o f s e p a r a t i n g component a from component b by l i q u i d - l i q u i d e x t r a c t i o n m ethods. The s e p a r a t i o n f a c t o r , ^ = Ke/Kc , where K0 and Kc e q u a l t h e p a r t i t i o n r a t i o s f o r e r g o s t e r o l and c a l c i f e r o l r e s p e c t i v e l y , i s p l o t t e d a g a i n s t t h e amount o f w a t e r added t o th e m e th an o l phase i n F i g . 18. I t i s a p p a r e n t t h a t t h e system p ro d u ced by a d d in g %% w a te r SEPARATION FACTOR {& ) 1.6 1 .4 1.2 1.0 0 4 8 16 12 VOLUME # WATER IN METHANOL F I G U R E 18 V A RIA TIO N IN THE S E PA R A T IO N FACTOR FO R C A LC IFE R O L AND ERGOSTEROL W ITH THE QUANTITY OF WATER IN THE METHANOL WHICH WAS EQ U IL IB R A T E D WIT:: HEXANE TO FORM THE TWO PHASE L I Q U I D - L I Q U I D SYSTEM 20 ( b y Volume) t o the m e th an o l p h a s e y i e l d s th e b e s t s e p a r a t i o n p o s s i ­ b ilitie s Summary 1. The s o l u b i l i t y of e r g o s t e r o l i n d io x a n e-h ex an e m ix tu r e s and e t h a n o l- b e x a n e m i x t u r e s has b e e n d e te rm in e d and. e x p l a in e d on th e b a s i s of e x i s t i n g r e g u la r s o l u t i o n t h e o r y . 2. The m ix tu re s a t s o l u b i l i t y of e r g o s t e r o l an d c a l c i f e r o l i n e t h a n o l - w a t e r th re e t e m p e r a t u r e s has b e e n d e te rm in e d and an e x p l a n a t i o n o f t h e i r s o l u b i l i t y b e h a v i o r h a s been d e r iv e d from th e e x i s t i n g r e g u l a r so lu tio n th e o ry . 3. An a p p ro x im a tio n o f t h e s o l u b i l i t y p a r a m e te rs f o r e t h a n o l and f o r e r g o s t e r o l has b e e n made , based o n t h e s o l u b i l i t y b e h a v io r o f e rg o ste ro l. ip The p a r t i t i o n r a t i o s o f e r g o s t e r o l and c a l c i f e r o l i n t h e two p h ase l i q u i d - l i q u i d system , hex an e -m eth a n o l have been d e te rm in e d show­ i n g the ciiange in t h e i r p a r t i t i o n r a t i o s w ith a change i n t h e amount o f w a te r ad d ed to the system . 27. v C a l c u l a t i o n s , based, on the w ork o f Bush and Densen'” i n d i c a t e s t h a t a 5 0 -5 0 m ix tu re of e r g o s t e r o l an d c a l c i f e r o l would y i e l d , a f t e r 100 e x t r a c t i o n s , a m ix tu re c o n t a i n i n g a p p r o x im a te ly 85% e r g o s t e r o l and a m ixture c o n t a i n i n g ap p ro x im ate ly 85% c a l c i f e r o l u s i n g t h i s sy stem . LITERATURE CITED 38 LITERATURE CITED 1 . 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Monograph h o . 17 R e i n h o l d , - 1 8 , Chapter X X I II . •- 1 8 , Chapter X I I , p . 201. L . J . and S c o t t , ft. L . , l o c . c i t . e 1 8 , Chapter X I , p . 180. ■C, A . G Zhur. F i z . Khim., 2 3 , 962 (I 9 h 9 ) . C . A. b h , i (1950). ' — J tS k ^ n o f o bysi00Cll3in- U* H* S - S * *■*, 508 (19UU), . 3 9 , U7oo2 (.19U5-). — :e 1 8 , Appendix I . C . and C r a i g , D . , i n V Jeissberger, e d . . "T ech niq ue o f r a n i c C hem istry," I n t e r s c i e n c e P u b lis h e r s ', In c New York Y. 19 50, Vol. i l l , C h a p te r IV. * 3