I. THE CHROMATOGRAPHIC SEPARATION AND THE DETERMINATION OF VITAMIN D2 IN CORN OIL I I . THE CHROMATOGRAPHIC SEPARATION AND THE DETERMINATION OF VITAMINS D IN VARIOUS OILS CONTAINING VITAMIN A By Tom D . S c h la b a c h J r . A THESIS S u b m itte d to th e S ch o o l o f G rad u ate S tu d ie s o f M ich ig an S t a t e C o lle g e o f A g r ic u lt u r e and A p p lie d S cie n ce i n p a r t i a l f u l f i l l m e n t o f th e r e q u ir e m e n ts f o r th e d e g re e o f DOCTOR OF PHILOSOPHY D epartm ent o f C h e m istry 1952 7 i'f /y / „ I . THE CHROMATOGRAPHIC SEPARATION AND THE DETERMINATION OF VITAMIN D3 IN CORN OIL I I . THE CHROMATOGRAPHIC SEPARATION AND THE DETERMINATION OF VITAMINS. D IN VARIOUS OILS CONTAINING. VITAMIN A By ’.c' Tom D t S ch la b ach J r . AN ABSTRACT S u b m itte d to th e S ch oo l o f G rad u ate S tu d ie s o f M ich ig an S ta t e C o lle g e o f A g r ic u lt u r e and A p p lie d S c ie n c e 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 e g re e o f DOCTOR OF PHILOSOPHY DeiDartment o f C h e m istry Y ear 1992 r A pproved t ! Tom D . S c h la b a c h J r . THESIS ABSTRACT T ire d i f f i c u l t i e s and l i m i t a t i o n s a s s o c ia t e d w ith th e a v a i l a b l e p h y s i c o - c h e m i c a l methods f o r th e d e te r m in a tio n o f v ita m in s D i n v a r io u s o ils , e s p e c ia lly to t h e p r e s e n t in v e s tig a tio n . Two v ita m in se c o n d , m e th o d s a r e p ro p o sed ; th e f i r s t f o r th e d e te r m in a tio n o f D 3 , p r e s e n t as an i r r a d i a t e d e r g o s t e r o l , i n c o rn o i l , and th e fo r v ita m in th e d e te r m in a tio n o f v ita m in s D i n v a r io u s o i l s c o n ta in in g A . T ire m e th o d s pro p o sed u t i l i z e a tw o -s te p ch ro m a to g ra p h ic p r o c e s s f o r the s e p a r a t i o n s t i t u e n t 's . to r e m o v e seco n d , i n samples c o n ta in in g sm a ll am ounts o f v ita m in s D, le d o f v itam in D from th e o th e r n o n - s a p o n if ia b le o i l con­ I n t h e f i r s t s te p an a c t i v a t e d e a r th y S u p e r f i l t r o l , i s u se d v i t a m i n A, c a r o te n o id s , p ig m e n ts , and some s t e r o l s . s te p I n th e a n a c t i v a t e d alum ina i s u se d to remove v ita m in A decom posi­ t i o n p r o d u c t ( s ) , c e r t a in p o ly e n e s, and o th e r r e s id u e s from th e o i l and the S u p e r f i l t r o l a d s o rb e n t i t s e l f . The v ita m in D i s u l t i m a t e l y d e t e r m i n e d b y u t i l i z i n g i t s a b s o r p tio n maximum a t 265 mu a s a m easure of t h e a m o u n t p re s e n t. T he v ita m in f i r s t m eth o d has b een s u c c e s s f u lly a p p lie d to sam ples o f D 2 , p r e s e n t as an i r r a d i a t e d erg o s t e r o l , i n c o rn o i l c o n ta in in g as l i t , h i e a s 1*000 U .S .P . u n i t s o f v ita m in D2 p e r gram, o f o i l . The second, m e t h o d h a s been s u c c e s s f u lly a p p lie d to sam ples o f v ita m in s D in a v a r i e t y v ita m in o f o i l s c o n ta in in g as l i t t l e as 1*000 U .S .P . u n i t s o f ID p e r g ra m , and w here th e v ita m in A to D r a t i o d oes n o t ex ceed mt W Tom D. S ch la b a c h J r . The c h ro m a to g ra p h ic b e h a v io r o f v ita m in D2j v ita m in k } e r g o s t e r o l } c h o l e s t e r o l } th e n o n - s a p o n if ia b le f r a c t i o n o f c o rn o i l ^ and v a r io u s f i s h l i v e r o i l s have b een e x t e n s i v e ly s tu d ie d on b o th a d s o r b e n ts . - 2- ACKNOWLEDGMENT 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 ia ti o n to D r . D . T . Ewing f o r h i s e x p e r i­ en ced c o u n s e l and g u id a n ce d u rin g th e c o u rs e o f th is in v e s tig a tio n . G r a te f u l acknow ledgm ent i s a l s o made to P a r k e } D av is and Company f o r i t s f i n a n c i a l su p ­ p o r t o f t h i s work i n th e form o f a f e l l o w s h i p } and to D r. 0 . D, B i r d , f o r h i s many h e l p f u l s u g g e s tio n s . TABLE OF CONTENTS PAGE PART I — THE CHROMATOGRAPHIC SEPARATION AND THE DETERMINATION OF VITAMIN D2 IN CORN OIL INTRODUCTION................... 1 REAGENTS AND EQUIPMENT..................................................................................................... 3 PROCEDURE FOR THE DETERMINATION OF IRRADIATED ERGOSTEROL IN CORN O IL .................................................................................................................................... 6 RESULTS....................................................................................................................................... 9 DISCUSSION OF THE RESULTS.............................................................................................. 12 EXPERIMENTAL STUDIES ON FACTORS RELATED TO THE METHOD.............................. Ill SUMMARY.......................................................... 25 PART I I — THE CHROMATOGRAPHIC SEPARATION AND THE DETERMINATION OF VITAMINS D IN VARIOUS OILS CONTAINING VITAMIN A INTRODUCTION.................................................................... 26 REAGENTS AMD EQUIPMENT..................................................................................................... 28 PROCEDURE FOR THE DETERMINATION OF VITAMINS D IN THE PRESENCE OF VITAMIN A IN OIL SAMPLES......................................... 29 RESULTS.................................................................................................................................... 32 DISCUSSION OF RESULTS....................................................................................................... 37 EXPERIMENTAL STUDIES ON FACTORS RELATED TO THE METHOD.............................. ill ■SUMMARY.......................................................... 51 REFERENCES ......................................................................................................................... 52 PART I THE CHROMATOGRAPHIC SEPARATION AND THE DETERMINATION OF VITAMIN D2 IN CORN OIL 1 INTRODUCTION F o r s e v e r a l y e a r s w ork i n t h i s la b o r a t o r y h a s b een d i r e c t e d to w ard s th e d e te r m in a tio n o f v ita m in s D p r e s e n t i n v a r io u s o i l s u s in g ch ro m ato ­ g ra p h ic te c h n iq u e s to s e p a r a t e th e v ita m in D from o th e r n o n - s a p o n if ia b le o il c o n s titu e n ts . P r i o r to t h i s w ork two m ethods w ere p ro p o s e d : th e f i r s t by Ew ing, K in g s le y , Brown and Emmett { ? ) , and th e seco n d by Ew ing, P o w e ll, Brown and Emmett (1 0 ) . The l a t t e r m ethod was d e v e lo p e d to d e te rm in e th e amount o f v ita m in Do p r e s e n t i n sam ples o f i r r a d i a t e d e r g o s t e r o l i n co rn o i l . I t employed a s in g le c h ro m a to g ra p h ic s te p u s in g S u p e r f i l t r o l a s th e a d s o r b e n t . The u lt im a te a n a l y s i s o f v ita m in D2 was done u s in g th e an tim ony t r i c h l o r i d e c o lo r r e a c t i o n , and t h i s was com pared w ith th e ab so rb a n c y a t 265 mu as a m easure o f th e amount o f v ita m in D2 p r e s e n t . N e ith e r m ethod was com­ p l e t e l y s a t i s f a c t o r y w ith sam p les c o n ta in in g l e s s th a n 50^000 U .S .P . u n i t s o f v ita m in D2 p e r g ram . I n o r d e r to d e te rm in e th e v ita m in D2 p r e s e n t i n sam p les o f t h i s ty p e , c o n ta in in g , l e s s th a n th e above amount o f v ita m in D2 , a m o d if ic a ­ t i o n o f th e m ethod u t i l i z i n g th e a b so rb a n c y a t 265 mu was i n v e s t i g a t e d . The m ethod in v o lv e s a tw o -s te p ch ro m a to g ra p h ic p r o c e s s . I n th e f i r s t s te p a S u p e r f i l t r o l column i s u s e d to remove c a r o te n o id 's , p ig m e n ts , some s t e r o l s , and i r r a d i a t i o n p r o d u c ts o f e r g o s t e r o l o th e r th a n v ita m in D2 . The u se o f t h i s and s im i la r a d s o r b e n ts f o r l i k e s e p a r a t io n s h a s b een w e ll e s t a b l i s h e d - ( 2 , 9 j l 0 , l i t , 15)* and r e c e n t l y h a s been s u c c e s s f u l l y 2 employed, by J . G reen and c o -w o rk e rs ( l l ) , f o r th e d e te r m in a tio n o f v ita m in D2 i n c ru d e i r r a d i a t i o n m i x tu r e s , and v ita m in D3 i n some f i s h liv e r o i l s . The seco n d c h ro m a to g ra p h ic s te p u s e s an alu m in a colum n to remove c e r t a i n u n s a tu r a t e d compounds o f th e s q u a le n e t y p e , r e s i d u a l i m p u r i t i e s from th e S u p e r f i l t r o l a d s o r b e n t, and o th e r m a t e r i a l s p r e s e n t i n th e n o n s a p o n if i a b le f r a c t i o n o f v e g e ta b le o i l s . T h is a d s o rb e n t h a s b een u se d by p r e v io u s i n v e s t i g a t o r s f o r s i m i l a r s e p a r a t io n s ( 5 , 6 , 1 2 ) , b u t f o r th e m ost p a r t th e s e s t u d i e s have n o t d e a l t w ith th e q u a n t i t a t i v e a s p e c ts o f th e s e p a r a t i o n . Em ploying th e p ro c e d u re o u t l i n e d i n t h i s p a p e r , th e i n v e s t i g a t o r h a s d e te rm in e d , w ith i n th e l i m i t s o f e r r o r l i s t e d , th e amount o f v ita m in D2 p r e s e n t i n sam p les o f i r r a d i a t e d e r g o s t e r o l i n c o r n 'o i l con­ ta in in g as l i t t l e a s 1|000 U .S .P . u n i t s o f v ita m in D2 p e r gram . T h is m o d if ic a tio n o f p r e v io u s m ethods c o n s t i t u t e s an im p o r ta n t e x te n s io n o f th e low er l i m i t o f a p p l i c a b i l i t y from 50,000 U .S .P . u n i t s o f v ita m in D2 p e r gram down to th e above v a lu e o f J4OOO U .S .P . u n i t s p e r gram . 3 REAGENTS AND EQUIPMENT 1 . n-H exane i s o b ta in ed , i n an o p t i c a l l y p u re s t a t e b y t r e a t i n g a c o m m ercially a v a i l a b l e p e tro le u m f r a c t i o n c a l l e d " S k e lly s o lv e B" o b t a i n ­ a b le from th e S k e lly O il Company o f 3711 C a l i f o r n i a A venue, C h ic a g o , I l l i n o i s a s n o te d b elo w . The b o i l i n g ra n g e sh o u ld be 6 3 -6 8 ° C . The m a t e r i a l i s p a s s e d th ro u g h a colum n o f a c t i v a t e d s i l i c a g e l k cm. i n d ia m e te r by 73 cm. lo n g . The f i r s t 30 0 -6 0 0 ml o b ta in e d i s k e p t f o r u se and sh o u ld have an ab so rb an cy v a lu e o f l e s s th a n 0 .0 3 3 a t 230 mu a s m easu red a g a i n s t a i r . The s i l i c a g e l i s a c t i v a t e d by h e a tin g 2h h o u rs i n a m u ffle f u r n a c e a t 230- 233° C , and may b e r e a c t i v a t e d a f t e r u se by w ashing o u t th e ad so rb ed o rg a n ic m a t e r i a l w ith d i s t i l l e d w a te r , d r y in g , and r e a c t i v a t i n g . 2 . A b so lu te a lc o h o l o f s u f f i c i e n t o p t i c a l p u r i t y i s o b ta in e d by t r e a t i n g each l i t e r o f a b s o lu te C .P . e t h y l a lc o h o l w ith 20 g. C .P . p o ta ss iu m h y d ro x id e and 10 g. C . P . s i l v e r n i t r a t e e i t h e r f o r 2+8 h o u rs w ith f r e q u e n t s h a k in g , o r f o r 8 h o u rs u n d e r a r e f l u x . I t is d is tille d from an e f f i c i e n t colum n, d is c a r d i n g th e f i r s t 30 ml and l a s t 130 m l. The d i s t i l l a t e i s d r ie d u s in g am algam ated aluminum f o i l w hich i s l e f t i n c o n t a c t w ith i t f o r 2+8 h o u rs o r 8 h o u rs u n d e r a r e f l u x . t i l l e d from an e f f i c i e n t column a s i n th e f i r s t s t e p . I t is d is ­ I t sh o u ld have an a b so rb an cy v a lu e o f l e s s th a n 0 .073 a t 230 mu and l e s s th a n 0 .0 2 3 a t 260 mu. h 3 . A lc o h o lic p o ta ss iu m h y d ro x id e f o r s a p o n i f i c a t i o n i s made up a t th e tim e o f u s e by a d d in g 28 g . / l o f C .P . p o ta ss iu m h y d ro x id e to th e above a lc o h o l to make a 0.5N s o l u t i o n . li. A nhydrous e t h e r i n an o p t i c a l l y p u re s t a t e i s o b ta in e d b y d i s ­ t i l l i n g a b s o lu te C ,P . d i e t h y l e t h e r from a m ix tu re o f anhy d ro u s C ,P . sodium s u l f i t e and sodium h y d ro x id e i n an e f f i c i e n t colum n j u s t b e f o r e use. The p ro d u c t s h o u ld have an a b so rb a n c y v a lu e o f l e s s th a n 0 .2 0 a t 230 mu and l e s s th a n 0 .0 2 5 a t 260 mu. 5 . The S u p e r f i l t r o l a d s o rb e n t was o b ta in e d from th e F i l t r o l C o r p o r a tio n , 315 N. 5 th S t r e e t , Los A n g e le s , C a l i f o r n i a . N ot a l l l o t s o b ta in e d w ere s a t i s f a c t o r y and a t t e n t i o n i s c a l l e d to th e d is c u s s io n o f a d s o rb e n ts i n t h i s p a p e r . 6 . A lum ina was o b ta in e d from The Aluminum Company o f A m erica, C hem icals D i v i s i o n , P i t t s b u r g h , P e n n s y lv a n ia as an a c t i v a t e d alu m in a Grade E-20 ( o r F -2 0 ) . O th e r a c t i v a t e d a lu m in as have a l s o been u se d s a tis fa c to rily . 7. C ry s ta llin e v ita m in D3 , 9$% p u re oh th e b a s i s o f th e E ( l $ , l e q u a l to lj.65 a t 265 mu f o r p u re v ita m in Da , was o b ta in e d from cm.) th e W in th ro p C hem ical C o ., I n c , , New Y o rk , N. Y .) and u se d a s r e c e iv e d . 8 . C r y s t a l l i n e e r g o s t e r o l o b ta in e d from th e M ontrose C hem ical Company was u s e d a s r e c e iv e d . 9 . The a b s o r p tio n s p e c tr a w ere a l l o b ta in e d u s in g a model DU Beckmann S p e c tro p h o to m e te r eq u ip p e d w ith a h ydrogen so u rc e and em ploying 1 cm. q u a rtz c e l l s . The s e n s i t i v i t y was a d ju s te d so t h a t th e s l i t v a r ie d from ab o u t 1 mm. a t 230 mu to 0 ,U mm. a t 290 mu. 5 10. The a d s o r p tio n tu b e s employed c o n s i s t o f a c o n v e n ie n t le n g th (2 0 -3 0 cm .) o f 8 mrn. i n t e r n a l d ia m e te r P y re x g la s s tu b in g to w h ich a s h o r t p ie c e o f 6 mm. P y rex g l a s s tu b in g h a s b e e n s e a le d a t one e n d . T h is ty p e o f tu b e i s u se d i n b o th ch ro m a to g ra p h ic s t e p s . 6 PROCEDURE FOR THE DETERMINATION OF IRRADIATED ERGOSTEROL IN CORN OIL The p ro c e d u re r e q u ir e s t h a t c lo s e a t t e n t i o n be p a id to d e t a i l i n o r d e r to o b ta in a c c u r a te and r e p r o d u c ib le r e s u l t s . H owever, an e x ­ p e r ie n c e d o p e r a t o r can ru n s i x to e i g h t sam ples i n a d a y . 1 . A w eighed one gram sam ple o f th e o i l i s p la c e d i n a 125 m l. E rlenm eyer f l a s k and 10 m l. o f a l c o h o lic p o ta s s iu m h y d ro x id e i s a d d ed . The sam ple i s s a p o n if ie d on a w a te r b a th a t 70° C f o r 30 m in u te s t o one hour w ith o c c a s io n a l s w i r l i n g . f o r com plete s a p o n i f i c a t i o n ) . (The fo rm er tim e i s g e n e r a lly s u f f i c i e n t A s h o r t stem f u n n e l p la c e d i n th e m outh o f th e f l a s k s e r v e s a s a c o n d e n s o r. 2 . When s a p o n i f i c a t i o n i s co m p lete th e f u n n e l and s i d e s o f th e f l a s k a re w ashed down w ith 20 m l. o f w a t e r . The n o n - s a p o n if ia b le p o r ti o n o f th e o i l i s e x t r a c t e d w ith one 1*0 m l . , fo llo w e d b y th r e e 20 m l. p o r t i o n s , o f e t h e r . The com bined e t h e r e x t r a c t s a re w ashed w ith f i v e 50 m l. p o r ti o n s o f w a te r ( th e f i r s t th r e e w ith o u t sh a k in g to a v o id th e fo rm a tio n o f an e m u ls io n ) . The i n t e r f a c e .sh o u ld be c l e a n , and th e aqueous la y e r s h o u ld n o t be a l k a l i n e to p h e n o lp h th a le in a t t h i s p o i n t . The w ashed e t h e r e x t r a c t i s d r i e d by f i l t e r i n g th ro u g h a fu n n e l c o n t a in ­ in g 10 g . o f an h y d ro u s C. P . sodium s u l f a t e . The s e p a r a to r y f u n n e l i s r i n s e d w ith 5 m l. o f e t h e r and th e f u n n e l c o n ta in in g th e d ry in g a g e n t i s r i n s e d w ith 25 m l . o f e t h e r i n sm a ll p o r t i o n s . 3 . The sam ple i s th e n e v a p o ra te d to d ry n e s s on a w a te r b a th a t ab o u t 1*5° C -under re d u c e d p r e s s u r e (30-1*0 mm o f m ercury) . 7 1*. The d ry r e s id u e i s ta k e n up i n 3 m l. o f a m ix tu re o f $0 h ex an e-1 0 e t h e r b y volume ( v / v ) . T h is i s added to th e S u p e r f i l t r o l column w hich h a s b e e n p r e v io u s l y t r e a t e d a s n o te d b e lo w . The S u p e r f i l t r o l column i s p ack e d to a h e ig h t o f ig. cm. (am o u n tin g to a b o u t 3 g .) b y th e m ethod o f Ewing e t a l . (9 ) . The colum n i s th e n prew ashed w ith 13 m l. o f 30 h ex an e -1 0 e t h e r ( v /v ) u n d er a s u f f i c i e n t p r e s s u r e d i f f e r e n t i a l so t h a t th e r a t e o f flo w i s c lo s e to 1 m l ./ 2 .3 m in. The e l u a te to t h i s p o in t i s d is c a r d e d , and j u s t b e f o r e th e to p o f th e colum n becomes d r y th e r e s i d u e , d is s o lv e d i n th e above s o l v e n t , i s added to th e colum n. Then th e f l a s k i s r i n s e d w ith 3 m l . o f t h i s s o l ­ v e n t w h ich i s added to th e colum n i n a s i m i l a r m anner. F i n a l l y , 10 m l. o f th e above s o lv e n t i s added to th e column to c o m p le te ly e l u t e th e ad so rb ed v ita m in D2 . The e n t i r e e l u a t e o b ta in e d i s e v a p o ra te d to d r y ­ n e ss u n d e r th e c o n d itio n s n o te d i n s te p ( 3) . 3. F o r th e alu m in a s t e p , lg g . o f a c t i v a t e d alu m in a i s added to th e a d s o r p tio n column and p ack ed b y m e re ly ta p p in g th e s id e s o f th e column w hich i s m a in ta in e d u n d e r s l i g h t l y re d u c e d p r e s s u r e . The ad ­ s o rb e n t i s s ta n d a r d iz e d b y p re w a sh in g th e colum n w ith 20 m l. o f s o lv e n t c o n ta in in g 0 .3 3 $ a lc o h o l i n e t h e r ( v /v ) a t a flo w r a t e o f 1 ml./igO s e c . T h is flo w r a t e i s m a in ta in e d th ro u g h o u t th e d ev elo p m en t o f th e ch ro m ato ­ gram . F o llo w in g t h i s , 3 m l. o f hexane i s added to th e column to remove th e p rew ash . N e x t, th e d ry r e s id u e from th e S u p e r f i l t r o l s te p i s d i s ­ s o lv e d i n 3 m l . o f hexane and added to th e colum n, fo llo w e d by 3 m l. o f hexane u se d t o r i n s e th e f l a s k . The column i s d ev elo p ed w ith 12 m l. o f a m ix tu re o f 70 e th e r - 3 0 hexane ( v / v ) . The e l u a t e to t h i s p o in t i s 8 The v ita m in D2 i s f i n a l l y e l u te d w ith 30 m l. o f a m ix tu re d is c a rd e d . o f 0 .3 3 $ a lc o h o l i n e t h e r ( v / v ) . 6. s te p ( 3) . The e l u a t e i s e v a p o ra te d to d ry n e s s u n d er c o n d itio n s n o te d i n The d ry r e s id u e i s d is s o lv e d i n a known amount o f n -h ex an e su ch t h a t th e a b s o r p tio n maximum a t 263 mu w i l l a p p e a r on th e d e n s it y s c a le somewhere betw een 0 .2 3 0 and 0 .I4.3 0 , w hich em braces th e ra n g e o f maxium s e n s i t i v i t y o f th e in s tr u m e n t. To c o n v e rt th e a b so rb an cy v a lu e s to U ,S J? . u n i t s o f v ita m in D2 p e r gram th e below r e l a t i o n s h i p i s u s e d . The symbol E i s u se d to r e p r e s e n t th e ab so rb a n c y v a l u e , E °b 5 ‘ 263 mu x 1Q£________________= cone . (gram s o i l / 1 0 0 ml s o l 'n ) U .S .P . u n i t s o f D3 p e r gram o f o il. The c o n v e rs io n f a c t o r o f 1 0 0 ,0 0 0 h as been a r r i v e d a t b y an e x te n ­ s iv e s tu d y c o v e rin g many d i f f e r e n t o i l s , and h as b een fo u n d to g iv e a c l o s e r co rre sp o n d e n c e betw een th e amount o f D2 p r e s e n t , i n gram s, and th e b i o l o g i c a l e f f ic a c y o f th e v ita m in th a n th e v a lu e p r e v io u s ly pub­ lis h e d (1 0 ). The c o n v e rs io n f a c t o r u sed i n t h i s p a p e r i s b a se d on th e cod l i v e r o i l s ta n d a r d o f U .S .P . X I I I . L im ite d d a t a o b ta in e d u s in g th e U .S .P . XIV v ita m in D s ta n d a r d , w hich i s v ita m in D ~, i n d i c a t e s t h a t a c o n v e rs io n f a c t o r o f a b o u t 93 ,3 0 0 s h o u ld be u s e d . I t i s a d v is a b le to ru n th e a b s o r p tio n cu rv e from 230 mu to 290 mu s in c e a la r g e amount o f a b s o r p tio n i n th e r e g io n o f 230 mu to 230 mu i n d i c a t e s q u a l i t a t i v e l y t h a t th e d e te r m in a tio n i s p ro b a b ly i n e r r o r p o s i t i v e l y , e s p e c i a l l y w here s m a ll am ounts o f D2 a r e in v o lv e d i . e . , 3000 to I4.OOO U .S .P . u n i t s o f v ita m in D2 p e r gram o r 6 .3 x 10 5 g . to 8 .6 x 10“e g. 9 RESULTS The r e s u l t s o b ta in e d a r e g iv e n f o r v a r io u s d i l u t i o n s o f a com­ m e r c ia l ly p r e p a r e d i r r a d i a t e d e r g o s t e r o l i n co rn o i l w hich was v e ry c a r e f u l l y b io a s s a y e d a s c o n ta in in g $0 0 ,0 0 0 U .S .F . u n i t s o f v ita m in D2 p e r gram. T h is p o te n c y was a l s o checked s e v e r a l tim e s u s in g th e method o f Ewing e t a l . (1 0 ) i n an u n d ilu te d form and gave an av era g e p o te n c y o f ls-7U,000 U .S .P . u n i t s o f v ita m in D2 p e r gram. upon th e fo rm er v a l u e . The r e s u l t s a r e b a se d The d i l u t i o n s o f th e above o i l were made by w e ig h t u s in g a c o m m ercially a v a i l a b l e v e g e ta b le o i l (M a z o la ). The r e s u l t s a r e l i s t e d i n T a b le I . V alues u n d e r th e h e a d in g C a lc u la te d B io a s s a y , U .S .P . u n i t s o f v ita m in D2/ g . o i l a re b a s e d upon th e above b io a s s a y v a l u e , and th e n c a l c u la te d from th e w e ig h t o f t h i s o i l p r e s e n t i n a g iv e n w eig h t o f c o rn o i l . I n th e c o u rse o f t h i s w ork two m ark ed ly d i f f e r e n t alu m in a ad­ s o r b e n ts w ere u s e d , eac h r e q u i r i n g d i f f e r e n t s o lv e n t system s f o r th e s e p a ra tio n . H ow ever, th e r e s u l t s o b ta in e d f o r b o th w ere v e ry s i m i l a r and th e d a ta have b e e n p l o t t e d to g e t h e r . TABLE I C a lc u la te d B io a s s a y , U .S .P . u n i t s ox v i t a min D2/ g . o i l O bserved U .S .P . o f P e r C en t Mean v ita m in D2/ g . o i l D e v ia tio n D e v ia tio n a s c a l c u l a t e d from in P er C ent th e E ( l $ ,lc in .) a t ________________________________ 263 mu____________________________________________________ / 1 2 3 1*1*,ooo n si 36,800 39,600 3 8 , 1*00 -1 6 .2 -1 0 .0 - 1 2 .6 1* 5 23,620 I! ft 23,360 21,1*30 21,600 2 8 ,2 0 0 21,300 - 1 6 .1 - 1 5 .6 +10.0 - 8 .6 12 , 1*20 ii n 1 2 ,1 3 0 II II II 10,630 it 11,030 10 ,3 0 0 10,850 1 0 , 1*00 1 0 ,3 0 0 1 0 ,5 0 0 1 1 ,3 0 0 10,600 9,950 - 1 1 .0 -1 5 .5 - 1 2 .6 -11*. 6 -1 5 .1 -1 3 .8 - 7 .1 - 0 .3 - 6.1* 9,200 8,700 8,01*0 M 8,350 8,100 8,580 7,1 0 0 7,100 7,0 7 0 7,200 7,920 - 9 .1 - 7.0 + 6 .5 -1 1 .7 -1 1 .7 -1 2 .0 -10.1* - 1.1* 6 7 8 9 10 11 12 13 lh 15 16 17 18 19 20 21 22 23 2b tt It It C o n tin u e d n e x t page TABLE I - (continued) Sample Number , C a lc u la te d B io a s s a y , U .S . P . u n i t s o f v i t a ­ min D2/ g . o i l 25 26 27 28 29 30 31 6,160 ,tt 5,980 32 33 3k 35 36 37 38 5,0 7 5 it ii n it 11 If 5,0 7 0 5 ,oiiO U,U5o It,000 39 lto la hZ h3 2,980 Ik hS Lt6 2 ,6 3 0 hi U8 h9 50 t! 11 II It ii 2,100 1,U30 II II (I Mean D e v ia tio n i n P e r C ent O bserved U .S .P . o f v ita m in D2/ g . o i l a s c a l c u l a t e d from th e E ( l $ ,lc m .) a t 265 mu P e r C ent D e v ia tio n 5,U90 5,8 1 0 6,180 5,9Uo 6,3 6 0 5,3 0 0 5,o o o -1 0 .8 - 5 .6 + 3 .3 - 0 .6 + 6 .3 - ll.lt -1 6 .3 - 5 .0 It, 800 it, 920 5,2 0 0 I t,370 5 ,5 0 0 It, 300 It ,170 - 5 . It - 3 .0 + 2 .3 -1 3 .8 + 9.2 - 3 .3 + I t.3 - l.it 3,680 3,780 3,370 2 ,7 9 0 2,970 +23.5 +26.8 +13.8 - 6 .3 - 0 .3 +11. it 3,3 5 0 3 ,5 8 0 2 ,8 0 0 +27.3 +36.0 +33.2 +32 .1 1 ,8 9 0 1 ,9 0 0 2,0 5 0 2,320 +32 .2 +32.8 +lt3.3 +62.2 +1+2 .6 12 DISCUSSION OF THE RESULTS The r e s u l t s g iv e n a re r e p r e s e n t a t i v e a ls o o f many o th e r d e te r m in a ­ t i o n s w hich w ere made b u t n o t l i s t e d . I n th o s e c a s e s w here th e e r r o r was a b n o rm a lly l a r g e , and th e c a u se o f e r r o r c o u ld n o t be s p e c i f i c a l l y d e te rm in e d , th e v a lu e s have b een in c lu d e d to i n d i c a t e w hat m ig h t be e x ­ p e c te d i n a c e r t a i n number o f c a s e s . The d a ta h a s b e e n p l o t t e d i n F ig u r e 1 w hich g iv e s th e dependence o f th e mean d e v i a tio n on th e v ita m in D2 c o n c e n tr a tio n p e r gram o f o i l . An e x a m in a tio n o f th e r e s u l t s shows t h a t th e r e i s a c e r t a i n l e v e l below w hich th e m ethod i s o f l i t t l e v a l u e . T h is l e v e l o c c u rs somewhere i n th e neig h b o rh o o d o f lj.,000 U .S .P . u n i t s o f v ita m in D2 p e r gram . Below t h i s th e mean d e v i a tio n becomes i n c r e a s i n g l y more p o s i t i v e a s th e am ount o f v ita m in D2 p r e s e n t p e r gram d e c r e a s e s . The e x p la n a tio n f o r t h i s b e h a v io r i s to be fo u n d i n th e f a c t t h a t a c e r t a i n s m a ll and r e l a t i v e l y c o n s ta n t amount o f e x tra n e o u s m a t e r i a l i s c a r r i e d th ro u g h w ith th e f i n a l v ita m in D2 c o n ta in in g e l u a te . The c o n t r i b u t i o n o f t h i s m a t e r i a l , to th e t o t a l , o b serv ed a b so rb an cy o f th e v ita m in D2 c o n ta in in g e l u a t e , becomes i n c r e a s i n g l y more a p p a re n t a s th e amount o f v ita m in D2 p r e s e n t d e c r e a s e s . T h is can a ls o be s e e n from t y p i c a l a b s o r p tio n 'cu rv es g iv e n in F ig u r e 2 f o r th e in d i c a t e d sam ples l i s t e d i n T a b le I , w h ich had v a ry in g amounts o f vitam in. D2 . C oupled w ith t h i s p o s i t i v e e r r o r i s th e f a c t t h a t a c e r t a i n sm a ll amount o f l o s s i s in v o lv e d i n h a n d lin g th e sam ple and on th e colum ns; 30 o20 UJ 0 6000 1 2,000 1 8 ,0 0 0 2 4 ,0 0 0 U.S.P. UNITS OF VITAMIN Dz PER GRAM OF OIL FIGURE I 0 .3 4 ABSORBANCY 0.3 0 0 .2 6 0 .2 2 28 230 240 250 260 270 WAVELENGTH , MU FIGURE 2 280 290 th e l a t t e r l o s s h av in g b een d e te rm in e d f o r p u re v ita m in D2 on b o th colum ns. The r e c o v e ry from th e S u p e r f i l t r o l column d e s c r ib e d i s 98% and from th e alu m in a colum n i s 96-98%. The two e r r o r s t h e n , o p p o s ite i n s i g n , ap p ea r to c o u n te r - b a la n c e one a n o th e r i n th e r e g io n o f a b o u t lj.,000 U .S .P . u n i t s o f v ita m in D3 p e r gram . As th e amount o f v ita m in D3 p r e s e n t i n c r e a s e s , th e mean d e v i a tio n ap p ro a c h e s a l i m i t i n g v a lu e o f - 1 0 .1 $ . The a v e ra g e mean d e v i a tio n f o r a l l d e te r m in a tio n s above 1|.,000 U .S .P . u n i t s i s -7.9% . Hi EXPERIMENTAL STUDIES ON FACTORS RELATED TO THE METHOD I n th e c o u rs e o f d e v e lo p in g t h i s m ethod i t was n e c e s s a r y and d e s i r ­ a b le to s tu d y th e e f f e c t o f v a r io u s f a c t o r s upon th e c h ro m a to g ra p h ic p ro cesses u sed . T hese co m p rised th e e f f e c t o f s o lv e n t c o m p o sitio n and a c t i v i t y o f th e a d s o r b e n ts (a n d i n th e c a s e o f alu m in a a c o n v e n ie n t m ethod o f s ta n d a r d i z a t io n ) upon t h e c h ro m a to g ra p h ic b e h a v io r o f c o rn o i l , c r y s t a l l i n e v ita m in D2 and e r g o s t e r o l . The e x p e rim e n ts were c a r r i e d o u t a t room te m p e r a tu r e , and w ith in th e norm al ra n g e o f v a r i ­ a t i o n , t h i s f a c t o r d id n o t n o t i c e a b l y a f f e c t th e c h ro m a to g rap h ic p ro c e s s e s em ployed. G en eral Remarks C o n cern in g th e S u p e r f i l t r o l A d so rb e n t The S u p e r f i l t r o l u se d was s e l e c t e d from v a r io u s a v a i la b l e l o t s , and. i n g e n e r a l th e w h ite r g ra d e s seem to be more e f f e c t i v e f o r ‘th e s e p a r a t io n d e s ir e d th a n th e d a r k e r g r a d e s . A tte m p ts to s ta n d a r d iz e th e m a t e r i a l b y h e a t i n g , o r by w ash in g fo llo w e d b y d r y in g and h e a tin g y ie ld e d an a d s o rb e n t w hich was u n s u i t a b l e f o r u s e . The a d s o r b e n t was u s e d a s r e c e iv e d . W ith c o n tin u e d p re w a s h in g , th e l e v e l o f a c t i v i t y o f th e a d s o rb e n t i s d e c r e a s e d , b u t a c e r t a i n amount o f p re w a sh in g i s r e q u ir e d to remove m a t e r i a l p r e s e n t on th e a d s o rb e n t when r e c e iv e d w hich g iv e s r i s e to e x tra n e o u s l i g h t a b s o r p ti o n . P r e f e r a b l y , t h i s p rew ash in g s o lv e n t sh o u ld have th e same c o m p o sitio n as th e e l u a n t , and a ls o be o f com parable volum e. 15 W ith th e m ethod o f p a c k in g u s e d , i f th e am ount o f a lc o h o l o r o th e r h ig h ­ l y p o la r component i n th e prew ash becomes to o l a r g e n o t o n ly i s th e a c t i v i t y o f th e a d s o r b e n t d e c r e a s e d , b u t a ls o s w e llin g o f th e a d s o rb e n t o c c u rs w hich r e t a r d s th e r a t e o f f lo w . B e h a v io r o f th e M o n -S a p o n ifia b le F r a c ti o n o f C orn O il On th e S u p e r f i l t r o l Column U nder V a rio u s C o n d itio n s F o r t h i s s tu d y , one gram sam ples o f two r e p r e s e n t a t i v e s c o r n o i l s w ere p re p a re d f o r ch ro m a to g rap h in g on th e S u p e r f i l t r o l colum n a c c o rd in g to th e p ro c e d u re g iv e n . B o th co rn o i l sam p les gave s i m i l a r r e s u l t s and th e y a r e d is c u s s e d t o g e t h e r . The f a c t o r s w hich w ere v a r ie d w e re , f i r s t l y th e c o m p o sitio n o f th e e l u a n t , and s e c o n d ly th e amount o f e l u a n t u sed to p rew ash th e colum n b e f o r e c h ro m a to g ra p h in g . E f f e c t o f V ary in g th e C o m p o sitio n o f th e E lu a n t U sin g 15 m l. o f e l u a n t to prew ash th e colum n and v a r y in g th e con­ c e n t r a t i o n o f e t h e r i n t h i s e l u a n t from 50 hex an e-1 0 e t h e r ( v /v ) to 50 h ex an e-30 e t h e r ( v / v ) , i t was fo u n d t h a t th e b an d in g o f th e zone ( a s r e g a r d s where th e maximum amount o f m a t e r i a l i s rem oved) on th e column was n o t c h an g e d , b u t a s th e e t h e r c o n c e n tr a tio n in c r e a s e d th e t o t a l amount o f m a t e r i a l e l u t e d was in c r e a s e d . P a r t o f t h i s in c r e a s e d amount o f m a te r ia l e l u te d was r e s id u e from th e S u p e r f i l t r o l i t s e l f w hich c a n n o t be c o m p le te ly rem o v ed . T y p ic a l a b s o r p tio n c u rv e s o b ta in e d f o r s u c c e s s iv e e l u a t e f r a c t i o n s o f th e n o n - s a p o n if ia b le p o r t i o n o f c o rn o i l u s in g 50 h ex an e-1 0 e t h e r ( v /v ) a r e shown i n F ig u re 3 w here th e f i r s t f i v e f r a c t i o n s a r e 3 m l. i n volume and th e l a s t i s 5 m l. i n volum e. o 1.0 2 < CD CC 8 0.8 m < 0.6 0.4 0.2 230 240 250 ' 260 270 WAVELENGTH, MU FIGURE 3 280 290 ____ ^ 16 The c h ro m a to g rap h ic b e h a v io r o f th e n o n - s a p o n if ia b le p o r t i o n o f c o rn o i l on S u p e r f i l t r o l u s in g 50 hex an e-1 0 e t h e r (v /v ) was a l s o com­ p a re d w ith th e r e s u l t s u s in g 50 h ex an e-1 0 e t h e r - 1 a lc o h o l ( v / v ) . The r e s u l t s in d i c a t e d t h a t th e ty p e o f m a te r ia l e l u t e d , a s ju d g ed b y com­ p a r is o n o f th e a b s o r p tio n c u rv e s o b ta in e d , w ith b o th s o lv e n t system s was v e ry s i m i l a r . The b a n d in g o f th e zone ( a s r e g a r d s w here th e maximum amount o f m a t e r i a l i s rem oved) a ls o was s im i la r f o r b o th s o lv e n t s y s te m s . H owever, u s in g 50 h ex an e-1 0 e th e r ( v / v ) 60- 70$ o f th e m a t e r i a l g iv in g r i s e to th e a b s o r p tio n o f l i g h t i s removed a s c o n t r a s t e d to o n ly 20- 30$ rem oval when 50 h e x a n e -10 e t h e r -1 a l c o h o l ' { v / v ) i s u s e d . E f f e c t o f V ary in g th e Amount o f Prew ash F o llo x d n g t h e above w h ich was done in a l l c a s e s u s in g 15 m l. o f p rew ash , th e amount o f p rew ash was re d u c e d to 3 m l. w h ich was j u s t s u f f i c i e n t to w et th e S u p e r f i l t r o l colum n. The a b s o r p tio n c u rv e s ob­ ta in e d f o r v a r io u s f r a c t i o n s o f th e e l u a te a r e q u ite s i m i l a r to th o s e o b ta in e d above, an d th e b a n d in g o f th e zone a l s o rem ain ed s i m i l a r . The amount o f m a te r ia l g iv in g r i s e to th e a b s o r p tio n o f l i g h t , o b ta in e d i n th e e l u a t e , was somewhat l e s s th a n when 15 m l. o f p rew ash was u s e d . H owever, when su ch a sm all amount o f prew ash i s u s e d , i t i s i n s u f f i c i e n t to remove c e r t a i n l i g h t a b s o rb in g m a te r ia l p r e s e n t i n t h e S u p e r f i l t r o l its e lf. C hrom atograph ic B e h a v io r o f V ita m in P 2 and Ergo s t e r o l o n th e S u p e r f i l t r o l Column U nder V a rio u s C o n d itio n s A t th e same tim e as th e n o n - s a p o n if ia b le f r a c t i o n o f th e c o rn o i l was s tu d ie d , i t was n e c e s s a r y to exam ine th e ch ro m a to g ra p h ic b e h a v io r o f 17 v ita m in D2 and e r g o s t e r o l , u n d er s i m i l a r c o n d i t i o n s , on th e S u p e r f i l t r o l colum n. These r e s u l t s w ere o b ta in e d u s in g th e p u re c r y s t a l l i n e m a t e r i ­ a l s w hich were ta k e n up i n th e a p p r o p r ia te s o lv e n t system and c h ro m a to ­ graphed d i r e c t l y . U sin g SO h ex an e-1 0 e t h e r ( v /v ) th e t h r e s h o l d volume f o r v ita m in D2 was ab o u t 3 m l. and th e maximum am ount was rem oved i n th e t h i r d 3 .m l. p o rtio n c o lle c te d . W ith SO h ex an e-1 0 e t h e r - 1 a lc o h o l ( v / v ) , th e t h r e s ­ h o ld volume was th e sam e, b u t th e maximum amount o f m a t e r i a l was removed i n th e second 3 m l. p o r t i o n . T h is b e h a v io r was th e same w h e th e r th e amount o f prew ash was 3 m l. o r I S m l. From t h i s i t can b e co n clu d ed t h a t th e c h ro m a to g rap h ic b e h a v io r o f b o th th e n o n - s a p o n if ia b le f r a c t i o n o f th e c o rn o i l and v ita m in D2 a r e a p p ro x im a te ly th e same f o r v a rio u s am ounts o f p rew ash . I n th e c a s e o f e r g o s t e r o l h ow ever, i t was n o te d t h a t i f o n ly 3 m l. o f p rew ash was u s e d none o f th e e r g o s t e r o l added to th e column was e l u t e d u s in g e i t h e r e l u a n t . I f I S m l. o f p rew ash was u se d ab o u t 1 0 .6 / o f th e e r g o s t e r o l i n i t i a l l y p r e s e n t ( 0 .0 00083 g .) was e l u t e d w ith th e D2 c o n ta in in g e l u a t e when SO h ex an e-1 0 e t h e r (v /v ) was u se d a s compared w ith 3 7 .2 / when 5>0 hex an e-1 0 e t h e r - 1 a lc o h o l (v /v ) was u s e d . A summary o f th e above r e s u l t s , in c lu d in g th o s e f o r c o r n o i l , a r e g iv e n i n F ig u r e s U and S w here I S m l. o f p rew ash was u s e d . I n F ig u re U a summary o f th e c h ro m a to g rap h ic b e h a v io r o f ( a ) v ita m in D2 , (b ) th e n o n - s a p o n if i­ a b le f r a c t i o n o f c o rn o i l , and (c ) e r g o s t e r o l on th e S u p e r f i l t r o l column u s in g SO hex an e-1 0 e t h e r ( v /v ) i s shown, and i n F ig u re S th e summary i s g iv e n f o r th e s e same m a t e r i a l s u s in g SO h ex an e-10 e t h e r -1 a lc o h o l ( v / v ) . 70 50 40 30 PER CENT RECOVERED 60 20 2 4 6 8 FRACTION NUMBER ( EACH FRACTION FIGURE 4 - 3 ML.) 70 60 50 30 20 2 4 6 FRACTION NUMBER ( EACH FRACTION = 3 ML.) FIGURE 5 ifl 18 R ecovery o f V itam in Da on th e S u p e r f i l t r o l Column The r e c o v e ry o f v ita m in D2 from th e S u p e r f i l t r o l column u s in g th e p ro c e d u re g iv e n i s 9&%-. The re c o v e ry was o b se rv e d to d e c re a s e s l i g h t l y as th e amount o f v ita m in D2 p r e s e n t d e c r e a s e d . The a b o v e , and th e e f f e c t o f S u p e r f i l t r o l column le n g th a re d is c u s s e d more f u l l y i n P a r t I I , d e a lin g w ith th e d e te r m in a tio n o f v ita m in s D i n p re s e n c e o f v ita m in A i n c o rn o i l and some f i s h l i v e r o i l s . Summary and C o n c lu sio n s C o n cern in g th e E x p e rim e n ta l S tu d ie s Made on th e S u p e r f i l t r o l Column S in c e th e b an d in g o f th e z o n e , f o r b o th th e v ita m in D2 and th e s a p o n if ia b le f r a c t i o n non- o f co rn o i l , does n o t v a r y a p p r e c ia b ly u n d e r d i f f e r e n t e l u a n t c o m p o sitio n s o r am ounts o f p re w a sh , and s in c e th e more p o la r e l u a n ts g iv e r i s e to a g r e a t e r amount o f e x tra n e o u s l i g h t a b s o rp ­ t i o n , th e n th e l e a s t p o l a r e l u a n t w hich w i l l q u a n t i t a t i v e l y e l u t e th e v ita m in D2 i n a r e a s o n a b le volume would, be th e m ost d e s i r a b l e e l u a n t to u s e . F u r t h e r , th e above b e h a v io r i s an in c r e a s e i n column n o t a l t e r e d a p p r e c ia b ly w ith l e n g t h , w h ile th e r e c o v e ry o f v ita m in D2 was o b serv ed to d e c re a s e s l i g h t l y w ith in c re a s e d colum n le n g th ; h e n c e , th e s h o r t e s t column com m ensurate w ith th e d e s ir e d s e p a r a tio n sh o u ld b e u s e d . A ls o , t h i s i s d e s ir a b l e s in c e t h e r e i s a c e r t a i n amount o f r e s id u e p r e s e n t on th e S u p e r f i l t r o l a d s o r b e n t i t s e l f w hich m ust be removed w ith th e p rew ash in g s o l v e n t . G eneral Remarks on A lum ina A d so rb e n ts I n 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 , a s was n o te d in th e r e s u l t s , two m a rk e d ly d i f f e r e n t a lu m in a a d s o rb e n ts w ere u s e d . The f i r s t , f o r 19 w hich th e p ro c e d u re i s g iv e n , was i n i t i a l l y more h ig h l y a c t i v a t e d th a n th e s e c o n d , and a ls o i t p o s s e s s e d a l a r g e r av era g e p a r t i c l e s i z e , . B o th have b een s u c c e s s f u l l y u s e d . The f i r s t h a s th e a d v an tag e o f e a se o f c o n t r o l l e d d e a c t i v a t i o n , and th e second h a s th e ad v an tag e o f e a s e o f c o n t r o l l e d r a t e o f flo w due to i t s s m a lle r p a r t i c l e s i z e . The d if f e r e n c e in s u r f a c e a r e a i s n o t s u f f i c i e n t , f o r th e s iz e o f th e colum n u s e d , to be an im p o rta n t c o n s i d e r a t i o n . I n b o th c a s e s t r a c e c o n d itio n s a re p r e s e n t a s f a r a s th e v ita m in D2 i s c o n c e rn e d . T h is i s deduced from th e b an d in g o f th e v ita m in D2 zone w hich i s c h a r a c t e r i s t i c f o r t r a c e co n ­ d i t i o n s ( 17) . The C h ro m ato g rap h ic B eh av io r o f th e N o n -S a p o n ifia b le F r a c t i o n o f C orn O il on th e A lum ina Column One gram sam ples w ere u se d i n a l l c a s e s , and th e n o n - s a p o n if ia b le f r a c t i o n o f c o rn o i l was f i r s t ch ro m ato g rap h ed on th e S u p e r f i l t r o l column u s in g th e p ro c e d u re d e s c r i b e d . The e l u a te n o rm a lly e l u t e d w ith th e v ita m in D2 was ta k e n f o r ch ro m a to g rap h in g on th e U g . alu m in a colum n. E f f e c t o f V ary in g th e C o m p o sitio n o f th e D ev elo p in g S o lv e n t U sin g th e more a c t iv e alu m in a a d s o r b e n t, w hich h ad b een p rew ashed i n a l l c a s e s w ith 10 m l. o f e t h e r , a b s o r p tio n c u rv e s o f f r a c t i o n s o f th e e l u a t e w ere o b ta in e d u s in g p u re e t h e r , 70 e t h e r - 30 hexane ( v / v ) , and 30 e t h e r - 30 hexane ( v /v ) . The r e s u l t s i n d i c a t e d t h a t th e b an d in g , o f th e zone f o r th e c o rn o i l r e s id u e re m a in s a p p ro x im a te ly c o n s ta n t o v er th e ra n g e o f s o lv e n t c o m p o sitio n s u s e d . The t o t a l amount o f m a te r ia l 20 e l u t e d , how ever, in c r e a s e s somewhat w ith an in c r e a s e i n th e e t h e r co n ­ c e n t r a t i o n o f th e s o l v e n t , b u t t h i s in c r e a s e i s n o t n e a r l y a s m arked as i t i s w ith th e S u p e r f i l t r o l colum n. u s in g th e l e s s a c t i v e a lu m in a . T h is same b e h a v io r was o b se rv e d F u rth e rm o re , th e b a n d in g o f t h i s m a t e r i a l , and th e amount e l u t e d , w ere n o t a f f e c t e d a p p r e c ia b ly w ith an in c r e a s e i n th e le n g th o f th e colum n. T h a t i s , th e a b s o r p tio n c u rv e s o f th e f r a c t i o n s o b ta in e d and th e t o t a l amount rem oved rem ain ed v e ry s im ila r. H ow ever, th e same f a c t o r s h ad a v e ry m arked e f f e c t on th e ch ro m a to g ra p h ic b e h a v io r o f v ita m in D2 on th e alu m in a colum n. C h ro m ato g rap h ic B eh av io r o f V ita m in Da ( P r e s e n t i n Sam ples o f I r r a d i a t e d E r g o s te r o l i n C orn O il) on th e A lum ina Column One gram sam ples o f i r r a d i a t e d e r g o s t e r o l i n c o rn o i l c o n ta in in g 2$,000 U .S .P . u n i t s o f v ita m in B2 p e r gram w ere c a r r i e d th ro u g h th e S u p e r f i l t r o l s te p and th e n ch ro m ato g rap h ed on th e more a c t i v e a lu m in a colum n. E f f e c t o f V ary in g th e E lu a n t C o m p o sitio n The r e s u l t s o b ta in e d , to g e th e r w ith th e above f o r c o rn o i l , a re sum m arized i n F ig u r e 6 . T h is F ig u r e g iv e s a summary o f th e ch ro m ato ­ g ra p h ic b e h a v io r o f (A) v ita m in D2 , and (B) c o rn o i l r e s id u e on th e alu m in a column u s in g ( l ) p u re e t h e r , ( 2 ) 70 e t h e r -30 hexane ( v / v ) , and (3 ) 50 e th e r - 5 0 hexane ( v /v ) t h a t w hereas th e b an d in g a s th e e lu a n t. T h is shows q u it e c l e a r l y o f th e zone f o r th e c o rn o i l r e s id u e i s not a p p r e c ia b ly a f f e c t e d by ch an g e s i n th e e l u a n t c o m p o s itio n , th e b an d in g o f th e v ita m in D2 zone i s p ro fo u n d ly a f f e c t e d . As th e c o n c e n tr a tio n o f 60 RECOVERED 50 40 B-l B-2 B-3 PER CENT 30 20 A-1 A-2 10 A-3 2 4 6 FRACTION NUMBER ( E A C H FIGURE 6 8 10 12 FRACTION = 10 ML.) 21 e t h e r p r e s e n t i n th e e l u a n t i n c r e a s e s , t h e t h r e s h o l d volume f o r v ita m in D2 d e c r e a s e s , and th e t o t a l amount o f e l u a n t r e q u ir e d to e l u t e a l l o f th e v ita m in D2 a ls o d e c r e a s e s . T h is i s due i n p a r t to th e f a c t t h a t th e " t a i l i n g ” becomes more p ro n o u n ced f o r th e l e s s p o la r e l u a n t s . I t i s to be n o te d t h a t r e l a t i v e l y l a r g e volum es o f e l u a n t a re r e ­ q u ir e d w ith a l l th e e l u a n t c o m p o sitio n s g iv e n , and t h i s i s u n d e s ir a b l e s in c e i t in c r e a s e s th e p o s s i b i l i t y o f e l u t i o n o f m a t e r i a l g iv in g r i s e to e x tra n e o u s a b s o r p tio n o f l i g h t and i s u n n e c e s s a r i ly w a s te f u l o f s o lv e n t . I t was f o r th e s e r e a s o n s t h a t a s ta n d a r d i z a t i o n o f th e a c t i v i t y o f v a r io u s alu m in as was d e v elo p ed f o r t h i s m ethod which i s d is c u s s e d la te r. S o lv e n t Used to I n tr o d u c e th e Sample Onto th e Column S in ce th e ch ro m a to g rap h ic b e h a v io r o f v ita m in D2 on alu m in a i s g r e a t l y a f f e c t e d by th e c o m p o sitio n o f th e s o lv e n t u s e d , i t i s d e s i r a b l e to in tr o d u c e th e sam ple o n to th e column i n a s o lv e n t w hich h as l i t t l e o r no te n d e n c y to e f f e c t any developm ent o f th e chro m ato g ram . Hexane i s s a t i s f a c t o r y f o r t h i s p u rp o s e s in c e ev en 100 m l. o f i t w i l l n o t e l u t e any d e t e c ta b le amount o f v ita m in D2 . F u r t h e r , th e b a n d in g o f v ita m in D2 u n d e r th e s e c o n d itio n s i s s h a r p e r th a n when t h i s i s n o t d o n e. T h is i s due to th e f a c t t h a t 'w i t h o t h e r more p o l a r e l u a n t s , dev elo p m en t o f th e chrom atogram i s s t a r t e d ev en b e f o r e th e e n t i r e sam ple i s on th e colum n. S ta n d a r d i z a t io n o f th e A lum ina A d so rb en t S in ce th e a c t i v i t y o f d i f f e r e n t sam ples o f alu m in a v a r y ( o f te n o v e r a c o n s id e r a b le ra n g e ) i t was n e c e s s a r y to f i n d some c o n v e n ie n t 22 m ethod o f s ta n d a r d i z in g th e a d s o rb e n t w ith r e s p e c t t o : l ) th e th r e s h o l d volume o f v ita m in D2 , and 2) th e b a n d in g o f t h i s zone f o r a g iv e n s o l ­ v e n t s y s te m , r a t e o f flo w , and te m p e r a tu r e . I n g e n e r a l, f o r any g iv e n e l u a n t , th e more a c t i v e th e a d s o rb e n t th e g r e a t e r i s th e t h r e s h o l d volume f o r th e v ita m in D2 , th e g r e a t e r th e s p r e a d in g o f th e z o n e , and th e low er th e r e c o v e ry o f t h e v ita m in D2 from th e colum n. The c o m m e rc ia lly a v a i l a b l e alu m in a a d s o rb e n ts u s u a l l y ad so rb v ita m in D2 to o s t r o n g l y , and hence th e problem i s one o f s u i t a b l e d e ­ a c t i v a t i o n o f th e a d s o r b e n t . The m ethod em ployed was to p rew ash th e s e a d s o r b e n ts w ith a d e f i n i t e volume (20 m l.) o f m ixed e t h e r - a l c o h o l s o l v e n t s , in tr o d u c e p u re c r y s t a l ­ l i n e v ita m in D2 i n hexane o n to th e colum n, and th e n d e te rm in e i t s t h r e s h o ld volume u s in g 70 e t h e r - 30 hexane ( v /v ) to e l u t e th e v ita m in D2 . The r e s u l t s a r e shown i n F ig u re 7 . The d a t a a re r e p r o d u c ib le and th e o b se rv e d dependence o f th e t h r e s h o l d volume on th e c o m p o sitio n o f a given am ount o f p re w a sh in g s o lv e n t a llo w s f o r re a d y i n t e r p o l a t i o n . T h is b e in g th e c a s e , i t becomes q u i t e c o n v e n ie n t f o r any i n v e s t i g a t o r to s ta n d a r d iz e h i s a d s o r b e n t p r o v id in g th e l e v e l o f i n i t i a l a c t i v i t y i s n o t too l o w . I t i s d e s i r a b l e to s ta n d a r d iz e th e a b s o rb e n t so t h a t th e t h r e s h o l d volume i s 12-13 m l. u s in g 70 e t h e r - 30 hexane ( v / v ) , and so t h a t n o t more th a n 23-30 m l . o f th e e t h e r - a l c o h o l e lu a n t ( p r e f e r a b l y h a v in g th e same c o m p o sitio n a s th e p rew ash in g s o lv e n t) a r e r e q u ir e d to c o m p le te ly e l u t e th e v ita m in D2 . F u r t h e r , th e b a n d in g o f th e v ita m in D2 zone s h o u ld p o s s e s s th e c h a r a c t e r i s t i c shape f o r t r a c e c o n d itio n s w here th e BY VOLUME ETHER 0.0 0.8 LOG. ALCOHOL CONC. IN - .6 0 20 40 THRESHOLD VOLUME IN MLS FIGURE 7 23 le a d in g edge i s s t e e p . T h is can h e o b se rv e d i n F ig u r e 8 , w here th e c h ro m a to g ra p h ic b e h a v io r o f v ita m in D2 on th e alu m in a colum n, u s in g th e p ro c e d u re o u t l i n e d , h a s b een sum m arized. R ecovery o f V itam in P 2 from th e A lum ina Column U sing th e m ethod d e s c r ib e d i n th e p r o c e d u r e , th e re c o v e ry o f p u re v ita m in D2 was d e te rm in e d f o r sam ples c o n ta in in g v a r io u s am ounts o f t h i s m a te ria l. The r e c o v e ry i s 96-95%, and w ith in th e ran g e o f ab o u t 13,000 down to a b o u t 1^,000 U .S .P . u n i t s o f v ita m in D2 th e r e c o v e ry i s a p p ro x im a te ly in d e p e n d e n t o f th e amount p r e s e n t o r i g i n a l l y . F o r lo n g e r colum ns th e re c o v e ry f a l l s o f f , and th e r a t e o f f a l l i n g o f f i s g r e a t e r th a n t h a t o b se rv e d w ith th e S u p e r f i l t r o l column ( 1)4) . Summary and C o n c lu sio n s C o n cern in g th e E :xperim ental S tu d ie s Made on th e A lum ina Column The b a n d in g o f th e zone f o r th e c o rn o i l r e s i d u e , and th e t o t a l amount o f m a t e r i a l e l u t e d , i s n o t m a rk e d ly a f f e c t e d o v e r r a th e r .w id e ra n g e s o f s o lv e n t c o m p o s itio n , o r ch an g es i n th e column l e n g th . The same f a c t o r s p ro fo u n d ly a f f e c t th e ch ro m a to g ra p h ic b e h a v io r o f v ita m in D 2 . A s th e e t h e r c o n c e n tr a tio n i n h e x a n e - e th e r s o lv e n ts in c r e a s e s th e t h r e s h o l d volume f o r v ita m in D2 d e c r e a s e s , and th e t o t a l volume r e ­ q u ir e d to e l u t e th e v ita m in D2 i s d e c r e a s e d ( t h a t i s , th e b an d in g o f th e zone i s s h a r p e r ) . I n c r e a s e d a lc o h o l c o n c e n tr a tio n i n e th e r p ro d u ces r e s u l t s s i m i l a r to th o s e f o r e t h e r i n h e x a n e , b u t h e re th e a lc o h o l con­ c e n t r a t i o n r e q u ir e d to p ro d u ce th e same e f f e c t i s much l e s s . be c l e a r l y se e n by co m p ariso n o f F ig u r e s 6 and 8 . T h is can T h u s, a s r e g a r d s 24 RECOVERED 12 PER 16 CENT 20 8 4 1 2 3 4 5 6 FRACTION NUMBER ( EACH FRACTION = 3 ML.) FIGURE 8 2 h t h e i r a b i l i t y to move th e v ita m in D2 b an d th ro u g h th e colum n, a lc o h o l i s much more e f f e c t i v e th a n e t h e r w h ich , i n t u r n , i s much more e f ­ f e c t i v e th a n h e x a n e . 25 SUMMARY 1 . A p h y s ic o -c h e m ic a l method f o r th e d e te r m in a tio n o f v ita m in Ds . & y p r e s e n t a s an i r r a d i a t e d e r g o s t e r o l , i n c o rn o i l h a s been p ro p o s e d . The m ethod u t i l i z e s a tw o -s te p c h ro m a to g ra p h ic p r o c e s s f o r th e s e p a r a t i o n o f v ita m in D2 from o th e r n o n - s a p o n if ia b le o i l c o n s t i t u e n t s . The v ita m in D2 i s u l t i m a t e l y d e te rm in e d b y u s in g i t s a b s o r p tio n maximum a t 265 mu a s a m easure o f th e amount p r e s e n t . 2. The m ethod h a s been s u c c e s s f u l l y a p p l ie d to sam ples o f th e above ty p e c o n ta in in g a s l i t t l e gram o f o i l . a s IjOOO U .S .P . u n i t s o f v ita m in D2 p e r The a v era g e mean d e v ia tio n f o r a l l sam p les d e te rm in e d , w h ich c o n ta in a t l e a s t I4OOO U .S .P . u n i t s o f v ita m in D2 p e r gram o f o i l , i s -7 .$% . 3 . A c o n v e n ie n t m ethod f o r th e s ta n d a r d i z a t i o n o f v a r io u s a c t i v a t e d alu m in a a d s o rb e n ts h a s been d e s c r ib e d and s u c c e s s f u l l y u s e d . ll. The ch ro m a to g rap h ic b e h a v io r o f v ita m in D2 , e r g o s t e r o l , and th e nons a p o n ix ia b le f r a c t i o n o f c o rn o i l have b e e n s tu d ie d on b o th a d s o rb e n ts w ith r e s p e c t to re c o v e ry and b an d in g o f th e zone u n d e r a v a r i e t y o f c o n d itio n s . PART I I THE CHROMATOGRAPHIC SEPARATION AND THE DETERMINATION OF VITAMINS D IN VARIOUS OILS CONTAINING VITAMIN A 26 INTRODUCTION The m a j o r i t y o f th e m ethods p ro p o sed f o r th e d e te r m in a tio n o f v ita m in s D i n th e p re s e n c e o f v ita m in A i n v a r io u s o i l s have c e n te r e d around th e u s e o f th e antim ony t r i c h l o r i d e r e a g e n t ( 3 ,9 , 1 3 ,1 9 ) . These m ethods a r e n o t e n t i r e l y s a t i s f a c t o r y ( U^8 jlU ) i n some c a s e s due to th e n o n - s p e c i f i c i t y o f th e r e a g e n t. S t e r o l s , v ita m in A, and o th e r p o ly e n e s (7 ) p ro d u ce a c o lo r r e a c t i o n w ith th e r e a g e n t, and th e s e in t e r f e r e n c e s c a n n o t alw ay s be c o m p le te ly e l im i n a te d . I t was f o r th e s e re a s o n s t h a t th e f o llo w in g method was in v e s t i g a t e d f o r u se w ith such sa m p le s. The m ethod p ro p o sed f o r th e s e sam p les i s an e n la r g e d m o d if ic a tio n o f t h a t p ro p o se d f o r th e d e te r m in a tio n o f v ita m in D2 i n sam ples o f i r r a d i a t e d , e r g o s t e r o l i n c o rn o i l d is c u s s e d i n P a r t I . I t in v o lv e s a tw o - s te p ch ro m a to g ra p h ic s e p a r a t io n o f th e v ita m in s D from o th e r nons a p o n if i a b le o i l c o n s t i t u e n t s . The f i r s t s te p u s e s an a c t i v a t e d e a r t h , S u p e r f i l t r o l , to remove th e v ita m in A , c a r o t e n o id s , p ig m e n ts , some s t e r o l s , and p o s s i b l y c e r t a i n o th e r o i l com ponents. T h is , and s im i la r a d s o r b e n ts , have b een em ployed f o r t h i s p u rp o se by o th e r i n v e s t i g a t o r s ( 9 , 1 0 ) , and r e c e n t l y h as b een s u c c e s s f u lly u sed by G reen ( l l ) i n th e m ethod he p ro p o se d f o r th e d e te r m in a tio n o f v ita m in s D i n v a rio tis o i l s u s in g an io d in e t r i c h l o r i d e r e a g e n t f o r th e u lt im a te a n a l y s i s . The second, s te p employs an alum ina column to remove v ita m in A d e c o m p o sitio n p r o d u c t( s ) ( 1 , 7 , 1 2 ) , p o ly e n e s such as a q u a le n e ( 6 , l o ) , and c e r t a i n r e s id u e s from th e S u p e r f i l t r o l a d s o rb e n t i t s e l f (lit) . 27 F o llo w in g th e s e s e p a r a t io n s th e v ita m in D i s u l t i m a t e l y d e term in e d by u t i l i z i n g i t s a b s o r p tio n maximum a t 265 mu. F u r t h e r , i t s a b s o r p tio n spectrum o v e r th e r e g io n o f 230 to 290 mu s e r v e s a s an added c r i t e r i o n f o r th e d e g re e o f s e p a r a t io n a c h ie v e d by co m p ariso n w ith th e spectrum o f p u re v ita m in D2 o r D3 i n t h i s r e g i o n . M arked d i s t o r t i o n o f t h i s cu rv e b e in g i n d i c a t i v e o f a d e te r m in a tio n p ro b a b ly i n e r r o r c o n s id e r a b ly . The m ethod h as b e e n s u c c e s s f u l l y a p p l i e d , 'w i t h i n th e l i m i t s g iv e n , to sam ples o f c r y s t a l l i n e D2 and v ita m in A a c e ta t e i n co rn o i l , i r r a d i a t e d e r g o s t e r o l and v ita m in A p a lm ita te i n c o rn o i l , some f i s h l i v e r o i l s , and some m is c e lla n e o u s s a m p le s . 28 REAGENTS AND EQUIPMENT 1 . C r y s t a l l i n e v ita m in A a c e t a t e (p o te n c y g iv e n a s 2,9 0 6 ,0 0 0 U .S .P . u n i t s p e r g ram ), o b ta in e d from M erck , was u se d a s r e c e iv e d . 2 . A l i s t o f th e o t h e r r e a g e n ts , t h e i r p u r i f i c a t i o n , and th e e q u ip ­ m ent u se d w i l l be found i n P a r t I , d e a lin g w ith th e d e te r m in a tio n o f v ita m in D2 i n sam p les o f i r r a d i a t e d e r g o s t e r o l i n c o rn o i l . 29 PROCEDURE FOR THE DETERMINATION OF VITAMINS D IN THE PRESENCE OF VITAMIN A IN OIL SAMPLES 1 . The p ro c e d u re fo llo w e d i n p r e p a r in g th e sam ple f o r ch ro m a to g rap h ­ in g on th e S u p e r f i l t r o l colum n i s i d e n t i c a l w ith t h a t g iv e n i n P a r t I . From t h i s p o i n t on th e p ro c e d u re d i f f e r s and i s g iv e n h e r e a f t e r i n d e­ t a i l e d fo rm . 2 . S u p e r f i l t r o l Column S te p : P ro c e d u re A: F o r sam ples w here th e v ita m in A to D r a t i o does n o t exceed t e n to o n e. The t o t a l amount o f v ita m in A in tr o d u c e d o n to t h i s column s h o u ld n o t exceed 7 0 ,0 0 0 -7 5 ,0 0 0 U .S .P . u n i t s . T h is i s th e s a t u r a t i o n l e v e l o f th e a d s o r b e n t, and i f t h i s i s exceeded th e d e s ir e d s e p a r a t io n i s n o t com­ p le te ly e ffe c te d . T h is can b e q u a l i t a t i v e l y a s c e r t a in e d by n o tin g how f a r down th e colum n th e b lu e c o l o r a t i o n h as p r o g r e s s e d by th e en d o f th e e l u t i o n . C om plete t r a v e r s a l i n d i c a t e s t h a t th e s a t u r a t i o n l e v e l has b een ex ce ed ed . The d ry sam ple from s te p ( l ) i s ta k e n up i n 3 m l . o f a m ix tu re o f $0 hex an e - 10 e t h e r ( v / v ) . T h is i s th e n added to th e S u p e r f i l t r o l column w hich h as b een p r e v io u s ly t r e a t e d a s d e s c r ib e d i n th e fo llo w in g p a ra g ra p h s. The a d s o rb e n t i s p ack ed i n th e column to a h e ig h t o f 6 cm. u s in g th e m ethod o f Ewing e t a l . (9 ) . The column i s th e n prew ashed w ith 15 ml,, o f 50 hexane - 10 e t h e r ( v /v ) u n d er a s u f f i c i e n t p r e s s u r e d i f f e r e n t i a l so t h a t th e r a t e o f flo w i s c lo s e to 1 m l ./ 2 .5 m in. m a in ta in e d th r o u g h o u t. T h is r a t e o f flo w i s J u s t b e f o r e th e to p o f th e column becomes d ry th e sam p le, i n th e above s o l v e n t , i s added to th e colum n. The f l a s k i s 30 r i n s e d w ith 2 m l. o f th e above s o lv e n t w hich i s added to th e column i n a s i m i l a r m a n n e r. When t h i s l a s t a d d i t i o n has j u s t e n te r e d c o m p le te ly on to th e a d s o r b e n t, th e e l u a t e o b ta in e d th u s f a r i s d is c a r d e d . The v ita m in D i s e l u a te d w ith 15 m l. o f 50 hexane - 10 e th e r ( v / v ) . P ro c e d u re B: F o r sam ples w here th e v ita m in A to D r a t i o d o es n o t ex ceed f i v e to o n e . In t h i s case i t i s more c o n v e n ie n t, and d e s i r a b l e , to use th e 1| cm. S u p e r f i l t r o l colum n. The p ro c e d u re f o r th e S u p e r f i l t r o l s te p u s in g t h i s le n g th colum n i s i d e n t i c a l w ith t h a t g iv e n i n P a r t I . ' The t o t a l amount o f v ita m in A in tro d u c e d , onto t h i s column sh o u ld n o t ex ceed 1|0,000-14-5jOOO U .S .P . u n i t s , and th e m ethod f o r d e t e c t i n g t h i s i s d is c u s s e d u n d er (2A) . 3. T h e.sam p le i s e v a p o ra te d to. d ry n e s s on a w a te r b a t h a t a b o u t 1|.5° C u n d e r re d u c e d p r e s s u r e ( 3O-I1O mm. o f m ercury) , and th e d ry r e s id u e ta k e n up i n 3 iAL • o f h e x a n e . U. The alum ina column s te p i s th e same f o r sam ples t r e a t e d u n d e r e i t h e r s t e p (2A) o r (2B) . The alu m in a colum n i s p r e p a r e d by f i r s t ad d in g 0 .5 g . o f a c t i v a t e d alu m in a to i t , w hich h as a t l e a s t a m inus 160 mesh s i z e . T h is i s p ac k e d b y m e re ly ta p p in g th e s id e s o f th e column w hich i s m a in ta in e d u n d e r s l i g h t l y re d u c e d p r e s s u r e . Then 3 .5 g . ° f a c t i v a t e d alu m in a ( ty p e E-20 o r F -2 0 w hich a s co m m e rc ia lly a v a i l a b l e , h a s a minus 80 mesh s i z e ) i s ad d ed , and packed l i k e th e a b o v e . A column so p re p a re d y i e l d s r e a d i l y r e p r o d u c ib le r e s u l t s , and h as th e f u r t h e r ad v an tag e o f e a s e in c o n t r o l l i n g th e r a t e o f f lo w . 31 T h is colum n i s s ta n d a r d iz e d , a s d e s c r ib e d i n P a r t I , b y p rew ash in g w ith 10 m l. o f s o lv e n t c o n ta in in g 0.6% a lc o h o l i n e t h e r ( v /v ) a t a flo w r a t e o f 1 m l . / l . 3 m in . w h ich i s m a in ta in e d th r o u g h o u t. J u s t b e f o r e th e to p o f th e colum n becomes d r y , ^ m l . o f hexane i s added to remove th e p re w a sh . Then th e sam p le, d is s o lv e d i n 3 m l. o f h ex an e , i s added to th e colum n i n a s i m i l a r m anner; t h i s i s fo llo w e d b y th e a d d i tio n o f 3 m l. o f hexane u se d to r i n s e th e f l a s k . The column i s d e v e lo p e d w ith 12 m l. o f 70 e th e r - 30 hexane (v /v ) fo llo w e d b y 2 m l, o f 0 , 6 % a lc o h o l i n e th e r ( v /v ) . t h i s p o i n t i s d is c a r d e d . The e n t i r e e l u a te to The v ita m in D i s e l u t e d w ith 18 m l. o f 0.6% a lc o h o l i n e t h e r ( v / v ) . 5 . The e l u a t e i s e v a p o ra te d to d ry n e s s u n d e r th e c o n d itio n s s t a t e d i n s te p ( 3) , and th e d ry r e s id u e i s ta k e n up i n a known volume o f n hexane. I t s a b s o r p tio n sp ectru m o v e r th e ran g e 230-290 mu i s o b ta in e d . M arked d i s t o r t i o n o f t h i s a b s o r p tio n c u r v e , from t h a t f o r p u re v ita m in D2 o r D3, i s i n d i c a t i v e o f a d e te r m in a tio n p ro b a b ly i n e r r o r p o s i t i v e l y . To c o n v e r t th e ab so rb a n c y v a lu e a t 2o3 mu to U .S .P . u n i t s o f v ita m in D p e r gram o f o i l th e below r e l a t i o n s h i p i s u s e d . The symbol E i s em ployed to r e p r e s e n t th e a b so rb an cy v a lu e . E (O b serv ed ) 253 mu ^ 10&. _ u . s . P . u n i t s o f v ita m in c o n e . (gram s o il /1 0 0 m l. D p e r gram . s o lu tio n s ). 32 RESULTS The r e s u l t s a re t a b u l a t e d f o r th e fo llo w in g sam ple ty p e s : ( l ) c r y s t a l l i n e D2 and v ita m in A a c e t a t e i n c o rn o i l , (2 ) i r r a d i a t e d e r g o s t e r o l ( a s a c o m m e rc ia lly p r e p a r e d p ro d u c t) and v ita m in A p a lm ita te in c o r n o i l , ( 3) some f i s h l i v e r o i l s , and (h ) some m is c e lla n e o u s sa m p le s. F o r ( l ) a s to c k s o l u t i o n o f p u re c r y s t a l l i n e v ita m in D2 i n c o rn o i l was m ade. On th e b a s i s o f th e v ita m in D2 p r e s e n t i t had an E (1%,1 cm.) o f 0 . JL1J48 a t 263 mu, o r u s in g th e c o n v e rs io n f a c t o r o f 105 i t had a p o te n c y o f i].ii,800 U .S .P . u n i t s o f v ita m in D2 p e r gram . d e s i r e d c o n c e n tr a tio n u s in g c o rn o i l . I t was d i l u t e d to th e To th e s e d i l u t e d s o lu ti o n s c r y s t a l ­ l i n e v ita m in A a c e t a t e was added a s r e q u ir e d ; th e v ita m in A c o n te n t b e in g c a l c u l a t e d on th e b a s i s o f 3 .3 x 10-7 g. v ita m in A a c e t a t e e q u a l 1 . U .S .P . u n i t o f v ita m in A . F o r (2 ) an o i l c o n ta in in g 810,000 U .S .P . u n i t s o f v ita m in A p e r gram and 1 6 2 ,0 0 0 U .S .P . u n i t s o f v ita m in D2 p e r gram (b io a s s a y ) was d il u te d w ith c o rn o i l . F u r th e r w here i t was d e s ir e d to a l t e r th e A to D r a t i o e i t h e r i r r a d i a t e d e r g o s t e r o l o r c r y s t a l l i n e v ita m in A a c e t a t e was ad d ed . T a b le I l i s t s r e s u l t s u s in g p ro c e d u re (2 A ), and a ls o two sam ples d e te rm in e d u s in g an 8 cm. S u p e r f i l t r o l colum n. T ab le I I l i s t s r e s u l t s u s in g p ro c e d u re (2B) f o r th e S u p e r f i l t r o l s t e p . U n le s s o th e rw is e s p e c i f i e d a f t e r th e sam ple number a l l d e te rm in a ­ ti o n s w ere made on one gram s a m p le s . TABLE I Sample Number 1 2 3 1* U .S .P . u n i t s o f v ita m in A /g . o i l U .S . P . u n i t s o f v ita m in D /g . o i l R a tio U n its A U n its D O bserved U .S .P . u n i t s o f v ita m in D /g . o i l a s c a l c u la te d from th e E ( l $ , l c m .) a t 265mu P e r C en t D e v ia tio n Mean D e v ia tio n i n P e r C en t The below a re f o r sam ples o f i r r a d i a t e d e r g o s t e r o l and v ita m in A p a l m it a te i n c o rn o i l . 16,7 0 0 1 .8 - 6 .1 9,3HO 8,770 ii n It 8,6 1 0 - 6 .8 » ii 1 1 8,110 -1 3 .1 it It it 8,180 - 1 2 . 1* 8,950 5-0 ii n ii it a ii ii ii 6 ,0 7 0 7 .3 n n 5 6 7 8 1*1*, 700 9 10 11' 12 13 1*1*, 300 11* 15 16 17 69,300 ii 11 ft n ii II 1 1 ii n it u 6,610 1 1 n 1 1 ii n M 1 0 .5 n n ii - 9 .6 8,160 7,880 7,8 9 0 8 ,0 90 - 8 .7 -1 1 .9 -1 1 .7 - 9 .6 6,080 5 ,6 1 0 5,2 7 0 5,U90 6,0 1 0 0 .0 - 7 .6 - 1 3 .1 - 9 .5 - 1 .0 - 6.2 8,350 7,31)0 5,7 0 0 5,890 +2 6 .2 +1 1 .0 -1 3 .7 - 1 0 .8 + 3 .2 5,790 5 ,0 3 0 5 ,6 7 0 5 ,7 1 0 +11*.0 - 1 .0 +1 1 .8 +12.3 + 9 .3 6,030 5 , 1*20 5,1*70 - 2 .7 -1 2 .5 -1 1 .5 . - 8 .9 . - 1 0 . 1* The below a r e f o r sam ples o f c r y s t a l l i n e : D2 and v ita m in A a c e t a t e i n c o rn o i l . 18 19 20 21 1*8,7 0 0 22 23 2h 1*2,900 M n ii 5,080 9 .6 It n m ti It ii 6,200 6 .9 ti H n II n C o n tin u ed n e x t page < uU v, o w i l d _ !_ J. 1 W J Sample Number U .S .P . u n i t s o f v ita m in A /g . o i l U .S .P . u n i t s o f v ita m in D /g . o i l R a tio U n its A U n its D C .L .O .a C .L .0 . C .L .O . lj.2,i|00 " •' 8 ,3 i|0 5 .1 ft 11 II T .L .0 .b T .L .O . 30.000 ( e s tm 'd ) " 20 000 0399 C 0399 12 ,2 0 0 " O bserved U .S .P . u n i t s o f v ita m in D /g . o i l as c a l c u la te d from th e E ( l $ ,l c m .) a t 269 mu P e r C ent ' D e v ia tio n Mean D e v ia tio n i n P e r C ent 11 7,300 8,020 7 ,7 i|0 -1 0 .1 - 3 .8 - 7 .3 - 7 .1 --ir 20,580 20,160 + 2 .9 + 0 .7 + 1 .8 10,630 » 1 .1 n 10,600 9,930 - 0 .1 - 6 .7 - 3 .il ABDECd ( 2 .0 g .) 8,800 ABDEC « " ABDEC « " ABDEC " " 1,760 11 11 11 5 .0 n 11 11 2 ,0 0 0 2 ,1 6 0 1 ,6 2 0 2 ,1 7 0 +13.8 +22.8 - 7 .9 +23.3 +1 3 .0 ABD0L® (3 .0 g .) 9,630 ABDOL " » 1,100 5 .1 11 1 ,2 0 0 1 ,2 5 0 + 9 .1 +13.7 +11 .u a) b) c) d) e) it II Cod l i v e r o i l f o r t i f i e d w ith i r r a d i a t e d e r g o s t e r o l and v ita m in A p a lm ita te Tuna l i v e r o i l T iiis o i l c o n ta in s 73% U3 and 29% D2 ABDEC D rops: A P a rk e , D avis a n d 'C o . p ro d u c t w hich i s al w a te r m is c ib le m u ltiv ita m in p r e p a r a t io n A P a rk e , D avis and Co.. p ro d u c t s i m i l a r to th e above i n a c a p s u le fo rm . The below r e s u l t s were o b ta in e d u s in g an, 8 cm. S u p e r f i l t r o l column f o r sam ples o f i r r a d i a t e d e r g o s t e r o l and v ita m in A p a lm ita te i n c o rn o i l . 38 39 69,300 " 6,610 11 1 0 .5 11 5,9 8 0 6,170 -1 0 .9 - 6 .6 IlO ( 0 .1 g .) 6 lli,0 0 0 IfL » » Il2 " » h9,900 m n 1 2 .3 hh,100 1|2,200 Ho, 700 - 1 1 .6 -15.1; II II - 6.J4 - 8 .8 - 1 1 .1 V_U TABLE I I O bserved U .S .P . u n i t s P resen t R a tio o f v ita m in D /g . o i l a s c a l c u l a t e d from th e D e v ia tio n U n its A E ( l / , l c m . ) a t 265 mu U n its D The below a r e f o r sam ples o f i r r a d i a t e d e r g o s t e r o l and v ita m in A p a lm ita te i n c o rn o i l . 5 ,9 0 0 27,600 1 5 .0 - 6 .9 5 ,5 2 5 11 9,280 2 ltf ,li00 - 6 .9 8,6 7 5 n 0 .0 162,000 162,000 3 ( 0 .0 5 i?.) 810/000 The below a r e f o r sam ples o f c ry s 't a l l i n e Do and v ita m in A a c e t a t e i n c o rn o i l , 18,5 0 0 20 I4.OO 25,600 -9.3 1* 1 .3 11 11 US 17,000 5 - 1 3 .3 28,800 6 19,3 0 0 17,130 -1 0 .3 1 .5 II n ft 17,5Uo 7 - 9 .3 Sample Number U .S .P . -u n its o f v ita m in A /g . o i l 8 9 10 11 12 27,600 13 Ik U .S .P . u n i t s o f v ita m in D /g . o i l - 1 0 .5 12,0 0 0 13,7 0 0 2 .1 1 .9 9,650 1 0 ,1 0 0 10,100 1 0 ,6 5 0 1 3 ,2 0 0 14.6,000 h i ,800 12,200 11,1 5 0 3 .8 U.3 11,000 10,700 - 9 .9 - I4.0 - 6 .9 135,600 01 « 30,880 h.k 29 ,8 0 0 29,600 28,900 - 3 .5 - h.2 - 6 .5 - U.7 18 19 20 22,100 U, I1.90 k.9 It 11 II n 5,280 k .700 k ,600 +17.5 + li-.7 + 2 .5 + 8 .2 21 22 23 2h 28,800 6,1|00 6,100 U.5 li.7 6,100 5 ,5 0 0 6 ,3 0 0 6,500 + + - 1 .0 1 5 (0 .1 g .) 1 6 (0 .1 g ) 1 7 (0 .1 g .’) ii n 25,600 II If n ii II II 11,200 Mean D e v ia tio n i n P e r C ent n 11 n 2 .5 II II n 11 n n n n -1 3 .8 - 9 .8 - 9 .8 -1 1 .3 - 3 .6 - 9 .7 5 .0 9 .8 3 .3 7.U C o n tin u ed n e x t page Vj l ) vn. TABLE II - (Continued) Sample Number U .S .P . u n i t s o f v ita m in A /g . o i l 25 26 27 28 5 9 , 1+00 29 30 i|2 ,900 T .L .0 . #0899 30,0 0 0 ( e s tim 'd ) 33,800 U .S .P . u n i t s o f v ita m in D /g . o i l R a tio U n its A U n its D 1 0 ,8 5 0 5 .5 n II ii II it ii II ti n 6,200 6 .9 m n ii 20,000 250,000 — - O bserved U .S .P . u n i t s o f v ita m in D /g . o i l a s c a l c u la te d from th e E ( l $ , l c m .) a t 265 mu P resen t D e v ia tio n Mean D e v ia tio n i n P e r C ent 11,800 12,300 9,820 10,890 + 8.7 +13.3 - 9 .5 0 .0 + 3 .1 8,033 7,990 + 29.5 +28.7 +29.1 20,700 201,500 - 3 .5 -1 9 .3 OJ On 37 DISCUSSION OF RESULTS The l i s t e d r e s u l t s i n d i c a t e t h a t s e v e r a l f a c t o r s m ust be c o n s id e r e d an s p e c if y in g th e li m it s ' o f th e p ro p o se d m ethod. The s i m i l a r i t y o f th e r e s u l t s o b ta in e d f o r th e sam ples o f c r y s t a l l i n e D2 and v ita m in A a c e t a t e in c o rn o i l ^ and sam ples o f i r r a d i a t e d e r g o s t e r o l and v ita m in A p a i m it a te m c o rn o i l shows th a t t h i s d if f e r e n c e h as l i t t l e e ffe c t. The r e s u l t s o b ta in e d f o r b o th are d is c u s s e d t o g e t h e r . The V ita m in A to V itam in D R atio F o r a l l o f t h e sam ples d e term in e d t h e r e i s a d e f i n i t e dependence o f th e mean d e v i a tio n upon t h e v ita m in A to D r a t i o . Above ab o u t ii500 U .S .P . u n i t s o f v ita m in D p e r granij and b elow th e v ita m in A s a t u r a t i o n l e v e l f o r a g iv e n le n g th S u p e r f i l t r o l colum n } t h i s dependence i s a p p r o x i­ m a te ly in d e p e n d e n t o f th e a c t u a l amount o f v ita m in D p r e s e n t p e r gram . D ata f o r sample ty p e s ( l ) and (2 ) have b een p l o t t e d i n F ig u re 1 w hich shows th e dependence o f th e mean d e v ia tio n on th e v ita m in A to D r a t i o . In th e p ro c e d u re where a It cm. S u p e r f i l t r o l column i s u se d th e mean d e v i a tio n b e g in s to in c r e a s e r a p i d l y when th e v ita m in A .to D r a t i o e x ­ ceeds f i v e to o n e ; where a 6 cm. S u p e r f i l t r o l column i s u sed t h i s in c r e a s e o c c u rs a t a r a t i o o f te n to one; and where an 8 cm. S u p e r f i l t r o l column i s used t h i s would p resu m ab ly o ccu r somewhere i n th e r e g io n o f a f i f t e e n to one r a t i o . T h is dependence i s due to th e f a c t t h a t some d eco m p o sitio n p r o d u c t( s ) o f v ita m in A (1 ^ 7 ,1 1 ,1 2 ) i s c o l l e c t e d w ith th e v ita m in D e l u a te from th e 20 4 CM. COL. iu CL 6 CM. COL. lli -10 8 CM. COL. RATIO •. U.S.P UNITS OF VITAMIN A PER GRAM OIL ' U.S.P UNITS OF VITAMIN D FIGURE I 38 S u p e r f i l t r o l colum n. T h is m a t e r i a l p re c e d e s th e th e v ita m in D zone on th e alu m in a colum n, b u t a s i n c r e a s i n g l y l a r g e r am ounts o f t h i s m a t e r i a l a r e p r e s e n t i t te n d s to o v e rla p th e v ita m in D zone and some i s e l u te d w ith th e l a t t e r from th e alu m in a colum n. The lo n g e r S u p e r f i l t r o l colum ns a llo w a h ig h e r v ita m in A to D r a t i o to be t o l e r a t e d b eca u se th e v ita m in D can s t i l l be re c o v e re d n e a r l y q u a n t i t a t i v e l y from th e s e lo n g e r colum ns w h ile th e amount o f v ita m in A d e c o m p o sitio n p r o d u c t( s ) e l u te d w ith th e v ita m in D i s g r e a t l y re d u c e d . The V ita m in D C o n te n t P e r Gram o f O il The mean d e v i a tio n i s d ep en d en t upon th e amount o f v ita m in D p r e s e n t a t any g iv e n v ita m in A to D r a t i o r e g a r d l e s s o f how low t h i s r a t i o i s . T h is dependence h as been p l o t t e d i n F ig u r e 2 f o r sam ple ty p e s ( l ) and (2). A s i m i l a r dependence h as b e e n o b se rv e d f o r sam ples o f i r r a d i a t e d e r g o s t e r o l i n c o rn o i l . The mean d e v i a t i o n r i s e s v e ry r a p i d l y below 3000 U .S .P u n i t s o f v ita m in D p e r gram o f o i l , and below ab o u t IjOOO U .S .P . u n i t s o f v ita m in D p e r gram o f o i l th e m ethod i s o f l i t t l e v a l u e . T h is dependence i s due to th e s m a ll and r e l a t i v e l y c o n s ta n t amount o f e x tra n e o u s m a t e r i a l e l u te d w ith th e v ita m in D i n th e f i n a l c h ro m a to g ra p h ic s t e p . T h is e x tra n e o u s m a t e r i a l a r i s e s from r e s id u e s p r e s e n t i n th e n o n - s a p o n if ia b le p o r ti o n o f c o rn o i l . S in ce t h i s i s th e c a s e , i n th o s e o i l s where th e v ita m in D c o n c e n tr a tio n i s a p p r e c ia b ly g r e a t e r th a n th e above v a lu e s th e v ita m in D l i m i t can be lo w ered to ab o u t 3000 U .S .P . u n i t s o f v ita m in D. 4 CM. COL. o-IO 0 6 CM. COL. 4000 8000 1 2 ,0 0 0 16,000 U.S.P. UNITS OF VITAMIN D PRESENT PER GRAM OF OIL FIGURE 2 39 The V ita m in A S a tu r a t io n L ev el The S u p e r f i l t r o l colum ns u se d have a d e f i n i t e v ita m in A s a t u r a t i o n l e v e l above w hich n o t a l l o f th e v ita m in A p r e s e n t can be a d s o rb e d . T h is l i m i t i s ab o u t iiOjOOO-U^jOOO U .S .P . u n i t s o f v ita m in A on th e I4. cm. column and ab o u t 7 0 ,0 0 0 -7 5 ,0 0 0 U .S .P . u n i t s on th e 6 cm. colum n. Column L o s s e s U sing th e p ro c e d u re d e s c r ib e d v ita m in D2 h a s b een d e te rm in e d f o r th e re c o v e ry o f p u re c r y s t a l l i n e a l l o f th e colum ns u s e d . The re c o v e ry from th e It cm. S u p e r f i l t r o l column i s 9 8 $ , from th e 6 cm. column i s 96$, from th e 8 cm. column i s 9h%, and from th e alu m in a column i s 9 6 -9 8 $ . L im it a ti o n s Im posed Upon th e M ethod 1 . The sample s h o u ld c o n ta in a t l e a s t 1|,000 U .S .P . u n i t s o f v ita m in D p e r gram o f o i l . I f th e v ita m in D c o n c e n tr a tio n p e r gram e x ­ c e e d s t h i s v a lu e a lo w er l i m i t o f 3*000 U .S .P . u n i t s o f v ita m in D i s a p p ro a c h e d . 2 . The r a t i o o f v ita m in A to D ( i n term s o f U .S .P . u n i t s of each) s h o u ld n o t ex ceed te n to o n e. Where th e r a t i o does exceed t h i s v a lu e somewhat th e u se o f an 8 cm. S u p e r f i l t r o l column i s i n ­ d i c a t e d f o r h a n d lin g th e s e sa m p le s. 3 . The t o t a l amount o f v ita m in A in tr o d u c e d o n to th e S u p e r f i l t r o l colum n sh o u ld n o t exceed th e s a t u r a t i o n l e v e l n oted a b o v e . W ith in th e r e g io n s p e c i f i e d by th e s e l i m i t a t i o n s th e mean d e v ia tio n v a r i e s from -1 0 .5 $ to +9.3$ f o r a l l sam ple ty p e s d e te rm in e d , and th e Uo av era g e mean d e v i a tio n f o r th e s e d e te r m in a tio n s i s -k.2% . Some t y p i c a l a b s o r p tio n c u rv e s o b ta in e d f o r th e in d i c a te d sam p les i n T a b le I a r e g iv e n i n F ig u re 3 . A tte m p ts to a p p ly th e m ethod to h a l i b u t l i v e r o il s ,, w ith in th e above g iv e n l i m i t s , le d to r e s u l t s w hich were alw ay s a b o u t 10^000 u n i t s p e r gram h ig h . The a b s o r p tio n cu rv e s o b ta in e d e x h i b ite d a m arked d i s ­ t o r t i o n when com pared w ith th o s e f o r v ita m in D. The n a tu r e o f t h i s i n t e r f e r e n c e i s d is c u s s e d more f u l l y u n d e r th e e x p e rim e n ta l s t u d i e s . 0 .2 6 ABSORBANCY 0 .2 4 0.22 0.20 0.18 0.16 230 240 250 260 270 WAVELENGTH , MU FIGURE 3 280 290 ia EXPERIMENTAL STUDIES ON FACTORS RELATED TO THE METHOD The p r i n c i p l e s e s t a b l i s h e d , and th e c o n c lu s io n s r e a c h e d , i n th e e x p e rim e n ta l s t u d i e s c a r r i e d o u t on th e m ethod p ro p o sed f o r th e d e te rm in ­ a t i o n o f v ita m in D2 , i n sam ples o f i r r a d i a t e d e r g o s t e r o l , i n c o rn o i l a p p ly e q u a l ly w e ll to th e sam ples now u n d e r c o n s id e r a tio n . H owever, th e p re s e n c e o f v ita m in A, and c e r t a i n o th e r o i l com ponents, h e r e to f o r e a b s e n t, n e c e s s i t a t e a d d i t i o n a l s tu d y . S in ce th e p rim a ry s e p a r a t io n d e s i r e d , nam ely th e rem o v al o f v ita m in A from v ita m in D, i s e f f e c t e d on th e S u p e r f i l t r o l column t h i s s te p was s tu d ie d i n g r e a t e r d e t a i l . I t w i l l be d is c u s s e d f i r s t . The f a c t o r s s tu d ie d w ere s o lv e n t c o m p o s itio n , th e ch ro m a to g rap h ic b e h a v io r o f v ita m in D2 u s in g v a r io u s le n g th colum ns, and th e ch ro m ato ­ g ra p h ic b e h a v io r o f v ita m in A, h a l i b u t l i v e r o i l , and c h o l e s t e r o l . C hoice o f S o lv e n t C o m p o sitio n S tu d ie s made i n c o n n e c tio n w ith P a r t I i n d i c a te d t h a t , f o r a g iv e n l e n g th S u p e r f i l t r o l colum n, a s th e p o l a r i t y o f th e s o lv e n ts in c r e a s e d , th e amount o f e x tra n e o u s m a t e r i a l e l u te d w ith th e v ita m in D in c r e a s e d c o n s id e ra b ly . To see i f t h i s g e n e r a l i z a t i o n h e ld t r u e f o r sam ples c o n ta in in g v ita m in A as w e ll a s v ita m in D, one gram sam p les o f c o d , tu n a , and h a l i ­ b u t l i v e r o i l w ere ch ro m ato g rap h ed on I4 cm. S u p e r f i l t r o l columns u s in g 50 h ex an e -1 0 e t h e r ( v /v ) and 50 hexane-10 e t h e r - 1 a lc o h o l ( v / v ) . U sin g th e ab so rb a n c y a t 230 mu a s a m easure o f th e amount o f m a t e r i ­ a l e l u t e d , and com paring t h i s w ith th e s o lv e n t u se d f o r each o i l , th e below r e s u l t s were o b ta in e d . I n th e c a se o f cod l i v e r o i l 39% more m a t e r i a l was e l u te d u s in g 30 h ex an e-1 0 e t h e r - 1 a lc o h o l ( v / v ) . I n th e c a se o f tu n a l i v e r o i l i t was 120$ m o re, and w ith h a l i b u t l i v e r o i l i t was 3h% m ore. The same b e h a v io r i s o b s e rv e d w ith lo n g e r colum ns, and th u s i t may b e c o n c lu d e d t h a t when more p o l a r e l u a n ts a r e u sed th e e f f e c t i v e column le n g th i s d e c r e a s e d . F u r t h e r , th e b a n d in g o f th e zone ( a s r e g a r d s w here th e maximum amount o f m a t e r i a l i s rem oved) i s n o t a p p r e c ia b ly a f f e c t e d by t h i s change i n e l u a n t . F o r th e s e re a s o n s th e l e a s t p o la r e l u a n t w hich w i l l q u a n t i t a t i v e l y e l u t e th e v ita m in D ,.in a re a s o n a b le volume i s th e m ost d e s ir a b l e e l u a n t to u s e . The C h ro m ato g rap h ic B eh av io r o f V itam in Da on th e S u p e r f i l t r o l A d so rb e n t S in ce th e lo n g e r S u p e r f i l t r o l columns a r e c a p a b le o f a d s o rb in g more m a t e r i a l , f o r any g iv e n e l u a n t , i t was d e s i r a b l e to s tu d y th e chrom ato­ g ra p h ic b e h a v io r o f v ita m in D2 on v a r io u s le n g th co lu m n s, and a t th e same tim e d e te rm in e i t s re c o v e ry from th e s e co lu m n s. E q u al am ounts o f p u re c r y s t a l l i n e v ita m in D3 were ta k e n up in 30 hexane-10 e t h e r (v /v ) and clirom ato g rap h ed on v a r io u s le n g th colum ns, a l l o f w hich had been p rew ash ed w ith 13 m l . o f th e same s o l v e n t . r a t e o f flo w , 1 m l ./ 2 .3 m i n ., was th e same in a l l c a s e s . The The b an d in g o f th e zone u n d er th e s e c o n d itio n s i s shown i n F ig u re I4. f o r ( l ) a U cm ., (2 ) a 3 .3 cm ., ( 3) a 7 .3 cm.., and (i;) a 9 cm. S u p e r f i l t r o l column u s in g 30 h ex an e-1 0 e t h e r (v /v ) a s th e e l u a n t . The r e s u l t s i n d i c a t e t h a t as S60 40 20 a. 1 3 5 7 FRACTION NUMBER ( EACH FRACTION = 3 ML.) FIGURE 4 h3 th e colum n le n g th in c r e a s e s t h e th r e s h o ld volume f o r v ita m in D2 i n ­ c r e a s e s , and th e volume r e q u ir e d to c o m p le te ly e l u t e th e v ita m in D2 in c re a s e s . . The re c o v e ry o f p u re v ita m in D2 f o r v a r io u s le n g th S u p e r f i l t r o l colum ns u s in g 50 hex an e-1 0 e t h e r ( v /v ) i s g iv e n in T a b le I I I . c o n ta in e d I k ,520 U .S .P . u n i t s o f v ita m in D2 . TABLE I I I Column L en g th ( i n cm.) k P e r C en t V ita m in D2 R ecovered 98.35 97.80 98.35 5 97.25-. 6 96.15 95.85 7 9U.50 8 9k. 50 92 .80 9k. 50 9 92. Uo Average 98.16 96.00 93.93 The sam ple hh U sin g th e 6 cm. colum n th e e f f e c t , on th e r e c o v e r y , o f d e c r e a s in g th e am ounts o f v ita m in D2 p r e s e n t i s ta b u la te d b elo w . TABLE IV U .S .P . U n its o f D2 111,520 it, 620 3 ,0 1 0 , P e r C en t V ita m in D2 R ecovered A verage 9 6 .1 5 9 5 .8 5 9 6 .0 0 9 h .k 3 9 3 .1 6 9 3 .1 6 9 5 .1 2 93.97 9U.29 95 .2 2 9U.55 9 3 .5 5 9U.U0 9 5 .0 1 92 .1 8 9 6 .1 9 9U.65 91i . 5 l 1 ,1 1 0 I n s p e c tio n o f th e s e t a b l e s i n d i c a t e s t h a t th e re c o v e ry d e c re a s e s w ith in c r e a s e d column le n g th , and a ls o a p p e a rs to d e c re a s e somewhat w ith d e c r e a s in g am ounts o f v ita m in D3 . The l a t t e r dependence i s n o t a s e v i ­ d e n t due to th e f a c t t h a t , w ith v e ry sm a ll am ounts o f v ita m in D2 , r e s id u e from th e S u p e r f i l t r o l column i t s e l f may i n t e r f e r e ; even th o u g h b la n k s a r e ru n on a s e p a r a te column u n d er i d e n t i c a l c o n d itio n s a t th e same t i m e . I t may be c o n clu d ed t h a t th e l o s s p r o b a b ly o c c u rs from i r r e v e r s i b l e ad ­ s o r p tio n o f a s m a ll amount o f v ita m in E g. In a c t u a l o i l sam ples th e p re s e n c e o f com ponents w hich a re a d so rb e d more s t r o n g l y th a n v ita m in D make th e s e minimum r e c o v e ry v a lu e s . The C h ro m ato g rap h ic B e h a v io r o f V ita m in A Upon th e S u p e r f i l t r o l Column P u re v ita m in A a c e ta t e (0 .0 1 5 8 g .) was p r e p a r e d f o r ch ro m a to g rap h ­ in g on th e 6 cm. S u p e r f i l t r o l column a c c o rd in g to th e p ro c e d u re g iv e n in th is p ap er. The m a te r ia l was in tr o d u c e d on to th e column i n 3 m l. o f 50 h ex an e -1 0 e t h e r ( v / v ) , f o llo w in g w hich th e column was e l u te d w ith th e same s o l v e n t . The a b s o r p tio n cu rv es o f s u c c e s s iv e e l u a te f r a c t i o n s c o l l e c t e d a re shown i n F ig u re 5 w h e r e .f r a c t i o n ( l ) i s 6 m l. i n volume and th e re m a in d e r a r e 3 m l. i n volum e. T hese c u rv e s c l e a r l y i n d i c a t e t h a t some ty p e ( s ) of. v ita m in A d e c o m p o sitio n p r o d u c t i s e l u t e d , and t h i s m a t e r i a l i s c h a r a c t e r i z e d by an a b s o r p tio n maximum i n th e w a v ele n g th r e g io n o f 280-290 mu. These a b s o r p tio n c u rv e s a re s i m i l a r to th o s e ob­ ta in e d b y Embree and S hantz ( 7 ) , f o r su b v ita m in A and k i t o l . Green ( l l ) found i n h i s work w ith v ita m in A on f l o r i d i n e a r t h t h a t f r a c t i o n s p o s s e s s ­ in g a s i m i l a r a b s o r p tio n cu rv e w ere o b ta in e d upon e l u t i o n . As t h i s m a te r ia l i s f u r t h e r a l t e r e d i t s a b s o r p tio n maximum te n d s to s h i f t to s h o r te r w a v e le n g th s . T h is m a t e r i a l i s l e s s s tr o n g ly ad so rb ed th a n th e s t e r o l s , in c lu d in g v ita m in D2 , on b o th th e S u p e r f i l t r o l and th e alu m in a colum n, a s was a ls o o b se rv e d by th e above a u t h o r s . T h is m a te r ia l does n o t a p p e a r to be f u r t h e r a l t e r e d upon b e in g chrom atographed, on th e alu m in a column a s can be o b serv ed i n F ig u re 8 . U sing th e k cm. S u p e r f i l t r o l column s im i la r f r a c t i o n s a r e c o l l e c t ­ e d , and th e b an d in g o f th e zone f o r b o th th e h cm. and 6 cm. column i s s im ila r . 2.4 2.0 z 1.6 0.8 0.4 230 240 250 260 270 WAVELENGTH , MU FIGURE 5 280 290 I I t i s t h i s m a te r ia l w hich i s r e s p o n s ib le f o r th e l i m i t a t i o n im posed upon th e m ethod by th e v ita m in A to D r a t i o f o r a g iv e n le n g t h S u p erf i l t r o l colum n. C h ro m ato g rap h ic B eh av io r o f H a lib u t L iv e r O il On th e S u p e r f i l t r o l A d so rb e n t I t was n o te d p r e v io u s l y t h a t h a l i b u t l i v e r o i l s c o u ld n o t be s u c c e s s f u l l y d e te rm in e d u s in g t h i s m ethod. T h is o i l was s tu d ie d v e ry th o ro u g h ly in an e f f o r t to e lim in a te th e cau se o f i n t e r f e r e n c e . T hese a tte m p ts , based, upon c h ro m a to g rap h ic s e p a r a t io n were u n s u c c e s s f u l b eca u se th e c h ro m a to g ra p h ic b e h a v io r o f th e i n t e r f e r i n g m a t e r i a l i s n e a r l y i d e n t i c a l w ith t h a t o f v ita m in D2 u n d er w id e ly d i f f e r i n g c o n d i t i o n s . Changes i n column le n g t h , o r s o lv e n t s y ste m s , on b o th S u p e r f i l t r o l and a lu m in a , f a i l e d to e f f e c t a s e p a r a t io n . I n F ig u re o a b s o r p tio n c u rv e s a re g iv e n f o r s u c c e s s iv e e l u a t e f r a c t i o n s c o l l e c t e d from a 1; cm. S u p e r f i l t r o l column u s in g £0 hex an e-1 0 e th e r ( v /v ) as th e e l u a n t ; each f r a c t i o n i s 3 m l . i n volum e. A one gram sam ple o f t h i s o i l , c o n ta in in g 55,000 A .u . / g and 1250 D . u . / g . , had b een c a r r i e d th ro u g h th e 6 cm. colum n p ro c e d u re p r i o r t o th e above t r e a t m e n t . E x am in atio n o f F ig u r e 6 shows th e p re s e n c e o f v ita m in A decom posi­ t i o n p ro d u c t( s) and c o rn o i l r e s id u e i n f r a c t i o n s ( l ) and ( 2 ) , a s w i l l be found i n a l l o f th e s e sam p les. F r a c ti o n s ( 3 ) , ( b ) , and (5 ) show th e a b s o r p tio n cu rv e o f th e i n t e r f e r e n c e . I t i s c h a r a c te r i z e d by a s p l i t maximum a t 260 and 268 mu, and r a p i d l y f a l l s o ff to a low v alu e beyond 270 mu. 2.8 ABSORBANCY 2.4 .0 .6 .2 0.8 5 230 240 250 260 270 WAVELENGTH,MU FIGURE 6 280 290 hi The p re s e n c e o f t h i s m a te r ia l w i l l c a u se a m arked d i s t o r t i o n in th e a b s o r p tio n cu rv e o f th e f i n a l v ita m in D c o n ta in in g e l u a t e i n th e r e g io n o f 260-270 mu. B ecause o f t h i s f a c t , unknowns w h ich a re h a l i b u t l i v e r o i l s may be r e a d i l y re c o g n iz e d . C h ro m ato g rap h ic B eh av io r o f C h o le s te r o l on th e S u p e r f i l t r o l A d so rb e n t The n o n - s a p o n if ia b le p o r t i o n o f many f i s h l i v e r o i l s c o n ta in c h o l e s t e r o l , and h a l i b u t l i v e r o i l s c o n t a in r a t h e r la r g e am ounts o f t h i s m a te ria l. I t h as been r e p o r te d t h a t c h o l e s t e r o l can be tra n s fo rm e d to o th e r s t e r o l p r o d u c ts when a s o lu ti o n o f i t i s h e a te d i n th e p re s e n c e o f a c t i v a t e d e a r t h s w hich have b een a c id t r e a t e d (18) . I t seemed d e s ir a b l e to i n v e s t i g a t e th e ch ro m a to g rap h ic b e h a v io r o f c h o l e s t e r o l on th e 6 cm. S u p e r f i l t r o l a d s o rb e n t i n l i g h t o f th e r e s u l t s o b ta in e d f o r h a l i b u t l i v e r o ils . A sam ple o f c h o l e s t e r o l (0 .1 1 2 6 g .) was p r e p a r e d f o r ch ro m ato g rap h ­ in g on t h i s column a c c o rd in g to th e p ro c e d u re g iv e n , and i t s a b s o r p tio n cu rv e i n $0 hexane-10 e th e r ( v /v ) was o b ta in e d . A s i m i l a r sample was . t r e a t e d a s th e ab o v e , th e n ch ro m a to g rap h ed , and i t s a b s o r p tio n cu rv e com pared w ith th e a b o v e. The r e s u l t s a r e shown i n F ig u re 7 . Curve ( l ) i s th e o r i g i n a l sam ple o f c h o l e s t e r o l i n 10 m l. o f £0 hex an e-1 0 e t h e r ( v / v ) , cu rv e (2 ) i s th e sample a f t e r s a p o n i f i c a t i o n and b e f o r e chrom ato­ g ra p h in g i n th e same s o lv e n t and a t th e same c o n c e n tr a ti o n , and cu rv e ( 3) i s th e t o t a l e l u a te o b ta in e d from th e colum n i n th e same s o lv e n t a t th e same c o n c e n tr a ti o n . A m arked g e n e ra l i n c r e a s e i n th e a b s o r p tio n cu rv e fo llo w in g th e f i r s t tr e a tm e n t i s o b s e rv e d , b u t no m arked f u r t h e r change was o b se rv e d i n th e a b s o r p tio n cu rv e o f th e m a te r ia l e l u te d from th e colum n. 0 .5 6 0.4 8 0 .4 0 i0 .3 2 2 0 .2 4 0.16 0.0 8 230 240 250 260 270 WAVELENGTH , NIU FIGURE 7 280 290 i hB T h is th e n te n d s to e lim in a te th e p o s s i b i l i t y o f th e tr a n s f o r m a tio n o f c h o l e s t e r o l a s b e in g th e s o u rc e o f th e i n t e r f e r e n c e n o te d when h a l i b u t l i v e r o i l s a re d e te rm in e d . Summary and C o n c lu sio n s C o n cern in g S tu d ie s Made on th e S u p e r f i l t r o l A d so rb en t F or a g iv e n column l e n g t h , a s th e p o l a r i t y o f th e e l u a n t i n c r e a s e s , th e t o t a l amount o f e x tra n e o u s m a t e r i a l ( p r e s e n t i n th e n o n - s a p o n if ia b le ■ f r a c t i o n o f c o r n , and f i s h l i v e r o i l s ) e lu te d i s i n c r e a s e d . The e f f e c t o f th e s e more p o l a r e lu a n ts i s to d e c re a s e th e e f f e c t i v e column le n g th as f a r a s th e s e p a r a t io n o f v ita m in D from th e s e com ponents i s c o n c e rn e d . Thus th e l e a s t p o l a r e lu a n t w liich w i l l q u a n t i t a t i v e l y e l u t e th e v ita m in D, i n a r e a s o n a b ly sm all volum e, i s th e m ost d e s ir a b l e e l u a n t to u s e . I n sam ples c o n ta in in g v ita m in A some d eco m p o sitio n p r o d u c t( s ) i s e l u te d from th e colum n. S im ila r to th e m a t e r i a l s d is c u s s e d above, th e ban d in g o f t h i s a l s o i s n o t a p p r e c ia b ly a l t e r e d by changes i n e l u a n t com­ p o s i t i o n o r column le n g th . The b an d in g o f th e v ita m in D z o n e , h ow ever, i s a f f e c t e d b o th b y changes i n e l u a n t c o m p o sitio n and colum n le n g th . F u r th e r , th e re c o v e ry o f v ita m in D i s a ls o a f f e c t e d by th e s e c h an g e s, p a r t i c u l a r l y ch an g es i n column le n g t h . T h is p resu m ab ly b e in g due to th e i r r e v e r s i b l e a d s o r p tio n o f a sm a ll amount o f th e v ita m in D . I n th e c a s e o f h a l i b u t l i v e r o i l s an a d d i t i o n a l i n t e r f e r e n c e was fo u n d , and t h i s i n t e r f e r e n c e co u ld n o t be e f f e c t i v e l y removed by ch ro m ato ­ g ra p h ic m e an s. T h is i n t e r f e r e n c e i s c h a r a c t e r i s t i c of h a l i b u t l i v e r o i l s and can be r e a d i l y r e c o g n iz e d . cod o r tu n a l i v e r o i l s . A s i m i l a r i n t e r f e r e n c e was n o t found w ith A tte m p ts to a s s o c ia t e t h i s i n t e r f e r e n c e w ith some h9 tr a n s f o r m a tio n o f c h o l e s t e r o l on th e a d s o rb e n t i n d i c a te d t h a t t h i s was p ro b a b ly n o t r e s p o n s ib le f o r th e o b serv ed i n t e r f e r e n c e . From t h i s i t m ust be s t a t e d t h a t c a re s h o u ld be ta k e n in a p p ly in g th e m ethod to f i s h l i v e r o i l s n o t c o v e re d in t h i s p a p e r . The A lum ina Column S tep In P a r t I th e m ethod f o r s ta n d a r d iz in g th e alum ina a d s o rb e n t was g iv e n . F u r th e r s t u d i e s , d e a l t w ith i n th e a b o v e , i n d i c a te d t h a t th e b an d in g o f th e zone f o r th e c o rn o i l r e s id u e was n o t g r e a t l y a f f e c t e d by changes i n th e s o lv e n t sy stem . H owever, in m arked c o n t r a s t to t h i s , th e ban d in g o f th e v ita m in D zone was p ro fo u n d ly a f f e c t e d by th e s e c h a n g e s. T h e r e f o re , i t was n e c e s s a r y to d e term in e w h e th e r o r n o t th e v ita m in A d e co m p o sitio n p r o d u c t( s ) w ould be e l u t e d w ith th e corn o i l r e s id u e u s in g 70 e th e r - 3 0 hexane ( v /v ) as th e d e v e lo p in g s o lv e n t. A b so rp tio n c u rv e s o b ta in e d f o r s u c c e s s iv e e lu a te f r a c t i o n s ta k e n from th e alu m in a 'column f o r a t y p i c a l sample a r e shown i n F ig u re 8 . T his sam ple c o n ta in e d 1 9 ,300 D2 u . / g . and 28,800 A u . / g . C u rv es ( l ) th ro u g h (Li) show th e a b s o rp ­ t i o n c u rv e s o f th e m a te r ia l removed i n s u c c e s s iv e 3 m l. f r a c t i o n s u s in g 70 e th e r -3 0 hexane (v /v ) as th e e l u a n t , curve (3 ) shows th e m a te r ia l removed u s in g 2 m l . o f 0 . 6 / a lc o h o l i n e t h e r ( v / v ) , and c u rv e s (6 ) th ro u g h (1 0 ) show th e m a te r ia l removed i n s u c c e s s iv e 3 m l. f r a c t i o n s o f th e above s o lv e n t; t h i s b e in g th e v ita m in D2 . From th e s e c u rv e s i t can be se e n t h a t b o th are removed u s in g 70 e t h e r -30 hexane (v /v ) a s th e d e v e lo p in g s o lv e n t , fo llo w in g w hich th e v ita m in D can be e l u te d u s in g 0 . 6 / a lc o h o l i n e t h e r (v /v ). I f a l a r g e amount o f th e v ita m in A d eco m p o sitio n p r o d u c t( s ) i s 1.4 5 0 .8 0.6 0.4 0.2 230 240 250 260 270 WAVELENGTH, MU FIGURE 8 280 290 5o p r e s e n t i t te n d s to o v e rla p th e v ita m in D z o n e , and some o f i t i s e lu te d w ith th e l a t t e r from th e colum n. T h is o c c u rs i n sam ples where th e v ita m in A s a t u r a t i o n l e v e l f o r th e S u p e r f i l t r o l column h as been ex ce ed ed , and i s f u r t h e r e v id e n t i n th e s c a t t e r o f th e r e s u l t s o b ta in e d , f o r such s a m p le s . 51 SUMMARY 1 . A p h y s ic o -c h e m ic a l m ethod f o r th e d e te r m in a tio n o f v ita m in s D i n a v a r i e t y o f o i l s c o n ta in in g v ita m in A has b e e n p ro p o s e d . I t h a s b een a p p lie d s u c c e s s f u l l y , w ith in th e l i m i t s o f e r r o r g iv e n , to sam ples o f c r y s t a l l i n e v ita m in D2 and v ita m in A a c e ta te i n co rn o i l , i r r a d i a t e d e r g o s t e r o l and v ita m in A p a l m it a te i n co rn o i l , cod and tu n a l i v e r o i l s , a s w e ll a s some m is c e lla n e o u s sam p les. 2 . The m ethod i s s u i t a b l e f o r sam ples c o n ta in in g a t l e a s t lj.000 U .S .P . u n i t s o f v ita m in D p e r gram o f o i l , and w here th e v ita m in A to D r a t i o does n o t exceed, t e n to o n e . F o r sam ples w here t h i s r a t i o i s exceeded somewhat th e u se o f an 8 cm. S u p e r f i l t r o l colum n i s in d i c a t e d f o r h a n d lin g th e s e sa m p le s. W ith in th e above l i m i t s th e mean d e v ia tio n v a r ie s from -1 0 .5 $ to +9.3%, and th e average mean d e v ia tio n f o r a l l sdch sam ples d e te rm in e d i s -k.2%. 3 . The ch ro m a to g rap h ic b e h a v io r , and re c o v e ry , o f v ita m in D3 on v a rio u s le n g th S u p e r f i l t r o l columns h as b een s tu d ie d ,. . The ch ro m ato g rap h ic b e h a v io r o f v ita m in A, c h o l e s t e r o l , an d v a rio u s f i s h l i v e r o i l s have a ls o b een s tu d ie d on th e S u p e r f i l t r o l column. The i n t e r f e r e n c e p r e s e n t i n h a l i b u t l i v e r o i l h as been d is c u s s e d , and s tu d ie s on cod and tu n a l i v e r o i l s have i n d i c a t e d t h a t no such i n t e r ­ fe re n c e i s e n c o u n te re d in th e s e o i l s . 52 REFERENCES 1. B a ru a , R . K ., and M orton, R. A ., Biochem . J . , 55 308 (,19b 9 ) . 2. B ro c k le s b y , H. N ., and M oore, L . 513 (1933) . P . , C o n tr . C an. B i o l . F i s h . , 7 3 . Brockm ann, H ., and Chen, I . H ., Z . p h y s i o l . C hem ., 25 l 10 5 (1936) . 5 . Cox, H. E . , A n a ly s t, 75 521 (1950) . 5 . Drummond, J . C . , e t a l . , B iochem . J . , 29 P t . 1 , 556 (1 9 3 5 ). 6. Drummond, J . C . , e t a l . , A n a ly s t, 71 251 (1956) . 7. Embree N. D and S h a n tz , E . M ., J . (1 9 5 3 ). .. Am. Chem. Soc . , 65 9 0 6 , 910 ~ 8 . Em merie, A ., and E e k e le n , M. v a n , A cta B re v ia N e e rla n d . P h arm aco l. M ic r o b i o l., 6 133 (1 9 3 6 ). 9. 10. Ew ing, D. P h y s io l. T . , e t a l . , I n d . E ng. Chem ., A n a l. E d . , 15 301(1 9 5 3 ). Ewing, D. T . , e t a l . , A n a l. C hem ., 20 317 '(1958) . 1 1 . G reen, J . , B iochem . J . , 59 5 5 , 253 ( I 9 5 l) . 12 . G ridgem an, N. T . , e t a l . , A n a ly s t, 73 662 (1 9 5 8 ). 1 3 . N ie ld , C . H ., e t a l . , J . B i o l . Chem., 136 73 (1950) . 1 5 .. P o w e ll, M. J . , PhD. T h e s is , M ich ig an S ta t e C o lle g e (1950) . 1 5 . Sw ain, L . A ., F i s h e r i e s R e se a rc h B oard C a n . , P r o g r e s s R e p o rts of P a c i f i c C o a s t S t a t i o n s , No. 5 9 , 1 3 . 1 6 . T h o rb ja rn a rs o n , T . , £ t a l . , A n a ly s t, 60 382 (1 9 3 5 ). 1 7 . V erm eulen, T . , and. H i e s t e r , N. K ., In d . E ng. C hem ., 55 636 (1952) . 1 8 . Y oder, L . , J . B i o l . Chem ., 116 71 (1 9 3 6 ). 1 9 . Z im m e rli, A. J . , e t a l . , J . B i o l . Chem., 158 255 (1 9 5 3 ).