STUDIES ON THE BIOLOGY AND CONTROL OF LYGUS OBLINEATUS (SAY)

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A CRITICAL STUDY OF THE FACTORS EFFECTING THE PHYSICAL CHEMICAL DETERMINATION OF VIT AMINS D IN OILS

by Manly Joy Pov/elJ.

A THESIS Subm itted t o t h e G raduate School of M ichigan S ta te College o f A g ric u ltu re and Applied S cience i n p a r t i a l f u lf illm e n t of th e req u ire m e n ts f o r th e degree of DOCTOR OF PHILOSOPHY

Department o f C hem istry Michigan S ta te C ollege - 1950 -

ACKNOWLEDGMENT The w r ite r w ishes t o express h i s appre c ia ti o n t o Dr. D. T . .Ewing f o r h i s h e lp f u l su g g e stio n s and guidance d u rin g t h e co u rse o f t h i s in v e s tig a tio n anc t o th e Parke Davis Company f o r t h e fe llo w sh ip making t h s work p o s s ib le .

COOT ENTS

I.

II.

Page I n tr o d u c tio n A. In tro d u c to r y S t a te m e n t . . . . ................................. 1 ............................... 1-5 B. L it e r a t u r e R e v ie w C. Statem ent o f O bject ............ ............................................ 6 E x p erim ental A. Equipment and R e a g e n ts .................................. 7-9 B. P rocedure 1. P ro ced u res S tan d ard ize d f o r t h i s Study a . S a p o n if ic a tio n and E x t r a c t i o n ..........10 b . D ig ito n in P r e c i p i ta t io n T reatm ent . . . . 11 c . HC1 T reatm ent ................................................11 d . S u p e r f i l t r o l Chrom atographic S teps . . . 11-12 e . Alumina Chrom atographic S t e p s ...... 12-15 2 . E xperim ental S tu d ie s Made. a . S tu d ie s Made on U n treated O il Solu­ tio n s of Irra d ia te d E r g o s te r o ls * 14 b . tStudies Made on S ap o n ified O il Solu­ t i o n s o f I r r a d i a t e d E r g o s t e r o l s ...... 15 1 . A bsorption Curve of Nonsaponif ia b le F r a c tio n ......................15 2 . Antimony T r ic h lo r id e C o lo ri­ m e tric Method .................................. .... 15 c . S tu d ie s on Removal o f In te i-fe rin g S u b stan ces by Chemical Means or Chromatography ........................................... 16 1 . S in g le S u p e r f i lt r o l Chromato­ g ra p h ic S tep U ltr a v io le t Ab­ s o rp tio n Curve Method ............ 16 2* M a te ria l Removed by HC1 T r e a t­ m ent, S u p e r f i l t r o l Chromato­ g rap h ic S te p , an.d D ig ito n in T reatm ent .................................... .. 17 '3*. Two Chrom atographic S tep U ltr a ­ v i o le t A bsorption Curve Method - Using S u p e r f i l t r o l and Alumina Columns ....................... 18-19

III.

R e s u lts .......................................................................................... 20-42

4

CONTENTS

Page IV.

D isc u ssio n A. Development o f S ta n d a rd iz ed P rocedures 1 . S a p o n ific a tio n and ED etraction . . . . 2. D ig ito n in Treatm ent 3. HC1 T reatm ent . . . . . . 4 . Large S u p e r f i l t r o l Column C h a re c te ris tic s ;..... 5 . Alumina Column Chara ct e r i s t i c s .................. 6 . Small S u p e r f i l t r o l Column C h a ra c te ri s t i c s ...................... B.

C onversion F a c to rs Used

43-53 43 44 44 45 46-51 51-52 52-55

V.

Summary .............................................

54-56

V I.

L it e r a t u r e C ited .........................

57-5S

V II.

Graphs and frav d n g s ...................

60-85

1

A C r i t i c a l Study o f th e F a c to rs E ffe c tin g t h e P h y s ic a l Chemical D eterm in atio n o f V itam ins D i n O ils .

The r e s u l t s o f a stu d y made upon methods f o r rem oving su b sta n c es which i n t e r f e r e in t h e p h y s ic a l chem ical d e te rm in a tio n o f v ita m in s D i n o i l s a r e g iv e n .

Two methods a p p lic a b le t o h ig h -p o te n cy i r r a d i a t e d

e r g o s te r o ls i n o i l a re d e s c rib e d .

A t h i r d method which can be a p p lie d

t o o i l s o lu tio n s o f i r r a d i a t e d e r g o s te r o ls , and p o s s ib ly t o m u ltip le v ita m in o i l s o lu tio n s , having p o te n c ie s a s low a s 2,000 t o 3,000 Dg u n i t s p e r gram i s a ls o g iv e n . V arious p h y s ic a l chem ical methods f o r d e te rm in in g v ita m in s D have been proposed i n t h e l i t e r a t u r e .

A g re a t m a jo rity o f t h e s e , however,

a re lim ite d i n a p p lic a tio n because th ey can be used o nly f o r c e r t a i n ty p e s o f s o lu tio n s o r f o r lim ite d c o n c e n tra tio n ra n g e s . a r e lie d t o only a few o i l s .

O thers were

A c r i t i c a l e v a lu a tio n o f some o f th e s e

m ethods, t h e r e f o r e , seems d e s i r a b l e . S e v e ra l i n v e s ti g a t o r s have attem pted t o d eterm in e v ita m in s D q u a n ti t a t iv e l y by u sin g t h e a b s o rp tio n maxima o f t h e u n tre a te d s o lu tio n a t 265 mu (2 2 , 27, 55, 4 4 ).

T h is method h ss been used s u c c e s s fu lly

only in c a se s where t h e s o lu tio n s analysed were th o s e c o n ta in in g v i t a ­ mins D i n t h e p ure s t a t e o r a s t h e i r r a d i a t e d p ro v ita m in .

The p resen ce

*

o f b y -p ro d u c ts o f i r r a d i a t i o n d e c re a se s t h e accuracy o f th e s e m easure­ m ents c o n s id e ra b ly .

F u rtherm ore, i f th e v ita m in s a re d isso lv e d i n a

v e g e ta b le o r f i s h l i v e r o i l , a s th e y u s u a lly a r e , some means f o r c o r­ r e c tin g f o r t h e a b s o rp tio n o f t h e solvent i t s e l f must be p ro v id ed .

2

C o lo rim e tric methods have a ls o been proposed i n which i n t e n s i t y measurements o f a c o lo r, produced b; t d ir e c t a d d itio n o f a rea g en t t o t h e v ita m in s o lu tio n , serv e a s a qn a n ti t a t iv e e s tim a tio n o f t h e amount o f v itam in D p re s e n t (17, 18, 55, 5 7, 59, 40, 41, 4 2 ).

Maiy o f th e s e

re a g e n ts , how ever, a re not s p e c if ic f o r v ita m in s D and g iv e c o lo r r e ­ a c tio n s w ith p ro v ita m in s , o th e r i r r a d i a t i o n p ro d u c ts , o r compounds p resen t i n t h e o i l i n which t h e v ita m in i s d is s o lv e d (7 , 8, 20, 50, 5 6 ). The reag en t proposed by Brockmann and Chen (4) has b een s tu d ie d q u ite e x te n siv e ly and s e v e ra l m o d ific a tio n s have been suggested i n o rd e r t o in c re a s e i t s s e n s i t i v i t y and s t a b i l i ty (26, 52, 5 8 ).

N ie ld , R u s s e ll,

and Zimmerli (2 6 , 48) found t h a t when a c e ty l c h lo rid e i s added t o th e antimoqy t r i c h l o r i d e re a g e n t of Bro ckmann and Chen, in c re a s e d s e n s it i v i t y r e s u lte d and t h a t s t e r o l s gs ve a n e g lig ib le r e a c t i o n .

S te r o ls

w ith double bonds i n t h e s id e c h a in showed no r e a c tio n w h ile th o s e w ith double bonds i n t h e r in g s tr u c tu r e s gave th e fo llo w in g a b s o rp tio n c o e ffic ie n ts ; 1 double bond

E ( l $ , leu .)

2 .2 3 500 mu

2 double bonds

E (1$, lcrr .)

7

$ 515 mu

D«? and Dg

E (1$, lcc .)

1800

@ 500 mu

V itam in A a ls o .h a s been shown t o re a c t v.'ith t h e Brockmann and Chen reag en t and m ust, t h e r e f o r e , f i r s t be removed (1 1 ). S e v e ra l methods have been proposed i n v;hich i n t e r f e r i n g su b stan ces such as v ita m in A and s t e r o l s have been removed from f i s h l i v e r o i l s by chem ical means o r fre e z in g b e fo re e stim a tin g t h e amount o f v ita m in s D p re se n t from t h e a b so rp tio n maxima a t 265 mu o r c o lo r im e tr ic a lly . S te r o ls have fre q u e n tly been removed by p r e c i p i t a t i o n w ith d ig ito n in

i

3

(32, 46) o r by fre e z in g (1 5 ).

Ma l e i c anhydride has a ls o b een suggested

f o r t h e remove! o f v ita m in A (15 , 2 3 ). C hrom atographic pro ced u res have been by f a r th e most s u c c e s s f u l means o f s e p a ra tin g v ita m in A ard carcrtenoids from v ita m in s D.

S e v e ra l

a d so rb e n ts such a s alum ina ( 3 , £5, 34, 4 5 ), c h a rc o a l (2 2 ), Montana e a r th (4 6 ), t r i- c a l c i u m phosphat e (24) and a m ixture o f m agnesia w ith diatom aceous e a rth (9) have beer . su g g e ste d . With few e x c e p tio n s, a l l c f t h e proposed methods above were a p p lie d t o o n ly a sm all number of o i l s t nd i n most c a se s could not be used f o r s o lu tio n s o f i r r a d i a t e d e r g o s t e r o l s . In th e s e l a b o r a to r i e s , Ewing and Tompkins (13) se p a ra te d v ita m in A from t h e n o n sa p o n ifia b le f r a c t i o n o f s ix te e n (16) f i s h l i v e r o i l s by chrom atographic a d s o rp tio n on S u p e r f i l t r o l from h e x a n e -e th e r-a lc o h o l s o lu tio n .

A fte r t h e rem oval o f s t e r o l s w ith d ig ito n in , t h e m odified

re a g e n t o f N ie ld , R u s s e ll, and Zimmerli was used t o determ in e t h e v i t a ­ mins D p r e s e n t.

Ewing, K in g sle y , Brown, and Emmett (12) m odified t h i s

p ro ced u re by r e p la c in g t h e s t e r o l p r e c i p i t a t i o n ste p w ith a n o th e r chro­ m atographic s te p i n which t h e st e r o l s were se p a ra te d from v ita m in s D from a b e n z e n e-S k e lly so lv e s o l u t io n u sin g S u p e r f i l t r o l a s t h e a d so rb e n t. F if ty - o n e f i s h l i v e r o i l s w ith p a te n c ie s ran g in g from 250 t o 325,000 u n its/g ra m were t e s t e d by t h i s method and f o r ty - f o u r o f t h e s e had an av erag e v a r i a t i o n of 5.8ja from .he b io a ssa y v a lu e . Hage (16) f u r t h e r sim p lif::.ed t h i s method e lim in a tin g t h e second chrom atographic s te p by s w irlin g t h e b e n z e n e-S k e lly so lv e s o lu tio n o f v i t a ­ mins D and s t e r o l s v dth t h e ad so rb en t in s te a d o f u sin g a packed column.

S e v e ra l i n v e s ti g a t o r s have t r i e d w ith l i t t l e su c ce ss t o apply th e method o f Ewing e t a l . t o s o lu tio n s o f i r r a d i a t e d e r g o s te r o ls (2 , 16, 4 7 ). Baker (2) how ever, observed t h a t t h e m a te r ia l e lu te d from t h e column i n th e f i r s t s te p o f t h i s p ro ced u re had an a b s o r p tio n curve s im ila r t o t h a t of v ita m in Dg when t h e o r i g i n a l sample was an i r r a d i a t e d e r g o s te r o l.

A

c o n s id e ra b le amount o f ex tra n eo u s a b s o rp tio n below 265 mu was observed, and a tte m p ts t o c o rr e c t f o r i t were u n s u c c e s s fu l. Pow ell (31) found t h a t by u sing a lo n g e r column o f th e ad so rb en t, which was prewashed w ith t h e s o lv e n t, t h e v ita m in Dg could be separated q u a n ti t a t iv e l y from crude i r r a d i a t e d e r g o s te r o ls i n v o l a t i l e so lv e n ts and th e chrom atographed m a te r ia l gave an a b s o r p tio n curve s im ila r t o t h a t o f a sta n d a rd c a l c i f e r o l .

T h is method was a p p lie d t o seven i r r a d i ­

a te d e r g o s te r o ls produced i n t h e la b o r a to r y , a s w e ll a s e ig h t commercial s o lu tio n s .

P o te n c ie s , c a lc u la te d from t h e a b s o rp tio n cu rv es of th e

chrom atographed s o lu tio n s , compared v e ry w e ll w ith t h e b io a s sa y values Yrtiich were a v a ila b le f o r f i v e d i f f e r e n t s o l u t io n s . B u lla rd (5) a p p lie d t h i s same s e p a ra tio n t o m ix tu res o f e rg o s te ro l and v ita m in Bg, end a s im ila r study was made by P in k e rto n (29) i n which a m o d ific a tio n o f t h e s o lv e n t, a s w ell a s t h e le n g th o f t h e column, was p roposed.

C o rre c tio n f o r ex tran eo u s a b s o rp tio n due t o re s id u e s con­

ta in e d i n t h e a d so rb en t was made e it h e r by making a b la n k ru n o r mathe­ m a tic a lly from t h e e x tin c tio n v a lu e s f o r t h e chrom atographed s o lu tio n measured a t 230 and 265 mu. The problem o f d e te rm in in g v ita m in s D i n t h e p resen ce o f o th e r p ro d u cts o f i r r a d i a t i o n i s a r e l a t i v e l y sim ple m a tte r , when th e y a re d is s o lv e d i n v o l a t i l e s o lv e n ts .

The s e p a r a tio n becomes much more com-

p le x , however, when s o l u t i on i s made i n v e g e ta b le o r f i s h l i v e r o i l s , The n o n sa p o n ifia b le f r a c t i on of both v e g e ta b le and f i s h l i v e r o i l s have been found t o c o n ta in squalene ty p e compounds which have been i s o l a t e d a s t h e h y d ro c h lo rid e by s e v e ra l in v e s ti g a t o r s (1 4 , 1 9 ).

I n ad­

d i t i o n t o t h i s , l a r g e amounts o f s t e r o l s , pigm ents, and p o s s ib ly o th e r f a t so lu b le v ita m in s may be p r e s e n t.

A p a r t i a l s e p a r a tio n of th e s e

compounds from t h e n o n sa p o n ifia b le f r a c t io n o f v e g e ta b le o i l s such a s c o rn , o l iv e , and wheat germ o i l , has been o b ta in e d u sin g chrom atographic te c h n iq u e s (10, 14, 455). From t h i s review i t becomes evident t h a t any s u c c e s s fu l s p e c tro photom etric method f o r det erm ining v ita m in s D must in c lu d e one o r more o f th e fo llo w in g : — (a)

method f o r s e p a ra tin g t h e v ita m in s D from

i n t e r f e r i n g s u b s ta n c e s , such a s : (1) S u b stan ces prest?nt i n th e o i l o r so lv e n t i n which th e v ita m in i s d is s o lv e d (e ,g ., s t e r o l s , c a ro te n o id s , pigm ents, o th e r f a t so lu b le v ita m in s

squalene ty p e compounds, snd s a p o n ifia b le

m a te r ia l) • (2) I n t e r f e r i n g sub sjtances in tro d u c ed by t h e method i t s e l f ( e . g . , so lv e n t re s id u e ^ , re s id u e s e lu te d from ad so rb en t o f chrom ato­ graph colum n). (3) O ther p ro d u c ts pf i r r a d i a t i o n p ro c e ss by which v ita m in D was produced ( e . g . , lu m is te r o l, t o x i s t e r o l , s u p r a s te r o l, and unco n v erted ergosl > e ro l)• (b) A re a g e n t which i s very n e a rly s p e c if ic f o r v ita m in s D i n t h e p resen ce o f o th e r su b s ta n :e s which were not p re v io u s ly removed.

With t h i s i n mind, a stu d y was made t o determ ine what method o r methods were most e f f i c i ent i n s e p a ra tin g v ita m in s D from i n t e r f e r i n g su b sta n c es w ith an eye t oward developing a method which co u ld be eq u ally ad ap ted t o b oth h ig h - and low -potency f i s h l i v e r o i l s , m u lti­ v ita m in s o l u t io n s , and i r r a d i a t e d e r g o s te r o ls i n v e g e ta b le o i l s .

7 EQUIPMENT AND REAGENTS

S u p e r f i l t r o l Columns. — (a) Large S ize - A column o f S u p e r f i lt r o l 18 mm. i n d iam ete r (u n le ss othervd.se s p e c ifie d ) and 9 cm. i n le n g th i s u sed .

For t h e f i s h l i v e r o i l s , t h e le n g th i s 11 cm.

pared by t h e method o f Ewing et a l . (1 2 ).

I t i s p re­

The packed column must be

th o ro u g h ly washed w ith t h e chrom atographic developing s o lu tio n (ap­ p ro x im ately 60 m l.) b e fo re t h e sample i s added. (b) Sm all S ize - Enough S u p e r f i l t r o l , packed m erely by ta p p in g and ap p ly in g 10 cm. d i f f e r e n t i a l i n p re s s u re , i s used t o p rep are a column 8 mm. i n d iam eter and 4 cm. i n le n g th .

T h is column i s prewashed

w ith 8 m l. c f t h e chrom atographic developing s o lu tio n b e fo re t h e sample i s added. Alumina Columns. — Four gm. o f alum ina a re packed s im ila r t o th e sm all S u p e r f i l t r o l columns above.

T h is column must be prewashed w ith 10 m l.

e th e r b e fo re a d d itio n o f th e sam ple. S p ec tro p h o to m e ters. — E ith e r a Beckman spectrophotom eter (Model DU) equipped vdth a hydrogen d isc h a rg e tu b e and 1 cm. q u a rtz c e l l s o r an o p tic a l system c o n s is tin g o f a Bausch & Lomb s e c to r photom eter and a medium q u a rtz sp e ctro g rap h i s used f o r a b so rp tio n measurements i n t h e u ltra v io le t. F o r m easurements i n t h e v i s i b l e ran g e, a Bausch and Lomb v is u a l sp ectro p h o to m eter equipped w ith a M a r tin 's p o la riz in g u n it and 1 cm. g la s s c e l l s i s employed.

EQUIPMENT AND REAGENTS

E th y l A lcohol* —

E th y l A lcohol (95$) i s p u r i f ie d by t r e a t i n g v d th a

s i l v e r oxide p r e c i p i t a t e , d e c a n tin g , end d i s t i l l i n g .

Twenty gm. o f

potassium h y d ro x id e and 10 gm. o f s i l v e r n i t r a t e p e r 2 l i t e r s o f a lc o h o l a re added t o form t h e s i l v e r o x id e . A lco h o lic Potassium H ydroxide. — F ourteen gm. o f high gruae potassium hydroxide a re d is s o lv e d i n 500 m l. o f p u r if ie d 95$ e th y l a lc o h o l. E th y l E th e r. — A.nhydrous e th e r ( c .p .) i s p u r if ie d by d i s t i l l i n g over c ry s ta llin e fe rro u s s u lfa te .

I t should be f r e e o f p ero x id e s.

S k e lly s o lv e . — S k e lly so lv e B i s f r a c tio n a te d and t h a t p o rtio n from 65°68°C i s saved and f u r t h e r p u r i f ie d by p a ssin g i t th ro u g h a tv ;o -fo o t column o f s i l i c a g e l which h a s been a c tiv a te d by h e a tin g © 250°C f o r tv/o h o u rs.

F r a c tio n s which tra n s m it, t o 220 mu a re c o lle c te d end u se d .

A

commercial g rad e o f hexane may b e s u b s titu te d f o r t h e S k e lly s o lv e B i f o f s u f f i c i e n t p u r i ty . T ran sm issio n cu rv es o f a l l t h e p u r if ie d s o lv e n ts (F ig . 1) were used a s a c r i t e r i o n f o r t h e i r p u r i ty . Developing S o lu tio n s f o r S u p e r f i l t r o l Columns. — (a) Large Columns T h is s o lu tio n i s made up from t h e p u r if ie d re a g e n ts d escrib ed above by ta k in g 50 p a r t s o f hexane o r S k e lly s o lv e , 10 p a r t s o f anhydrous e th y l e th e r , and 1 p a rt o f a b so lu te e th y l a lc o h o l. (b) Sm all Columns - The so lv e n t m ix tu re, 50 p a r t s hexane o r S k e lly s o lv e , and 10 p a r ts anhydrous e th y l e th e r , which was proposed by P in k e rto n (29) i s used fo r t h i s s iz e column o f a d so rb e n t.

9 EQUIPMENT AND REAGENTS

D eveloping S o lu tio n s f o r Alumina Columns. T h is d ev elo p in g s o lu tio n i s composed of 1 p a rt o f hexane or S k e lly s o lv e , end 1 p a r t anhydrous e th y l e th e r. Alumina. — High g ra d e alum ina ( l e s s th a n 80 mesh) o f chrom atographic q u a lity i s employed. D iprjtonin S o lu tio n . — A one per c en t s o lu tio n o f com m ercial D ig ito n in i n 95% e th y l a lc o h o l i s used as a p r e c i p i t a t i n g a g e n t. HC1. — A c y lin d e r o f com m ercially p rep a red anhydrous HC1 i s equipped v d th a t r a p and a sm all j e t f o r b u b b lin g t h i s gas i n t o s o lu tio n s . C hloroform . — Sm all amounts o f a lc o h o l a r e removed from c .p . c h lo ro ­ form by washing seven tim e s vdth an equal volume o f d i s t i l l e d w a ter. I t i s th e n d rie d o v e r anhydrous sodium s u l f a t e , d e ca n te d , and d i s t i l l e d ; t h e f i r s t and l a s t 10% o f th e d i s t i l l a t e a re d is c a rd e d . Antimony T r ic h lo r id e R eagent. — T h is rea g en t i s p rep a red fre s h f o r each d a y 's ru n .

E ig h teen gm. o f c . p . antimony t r i c h l o r i d e a re d isso lv e d

i n 100 m l. o f p u r if ie d chloroform .

A fte r t h i s s o lu tio n i s f i l t e r e d , two

m l. o f r e d i s t i l l e d a c e ty l c h lo rid e a r e added.

i

10

PROCEDURES STANDARDIZED FOR THIS STUDY

S a p o n ific a tio n and E x tr a c tio n . — O il sam ples a re weighed in to sm all g la s s c a p su le s and th e n p laced i n 125 m l. Erlenm eyer f l a s k s c o n ta in in g 10 m l. o f a lc o h o lic potassium hydroxide f o r each gram o f sample. A short-stem m ed fu n n e l i s th e n p la c e d i n t h e neck o f th e f la s k and th e sample i s s a p o n ifie d i n a w ater b a th a t 70°C. from l / 2 t o one hour o r u n t i l s a p o n if ic a tio n i s com plete.

Then 20 m l. o f w ater a re added t o

th e sa p o n ifie d s o lu tio n and t h e n o n s a p o n ifia b le f r a c t i o n i s e x tra c te d i n a s e p a ra to ry fu n n e l, u sin g one 40-ral. follow ed by t h r e e 20-ml. p o rtio n s o f e t h e r .

The combined e th e r e x tr a c t s a re washed w ith a t l e a s t

fo u r 50-m l. p o r tio n s o f w ater or u n t i l t h e e th e r-w a te r in te r f a c e i s c le a r and t h e w ater la y e r i s not a lk a lin e t o p h e n o lp h th a le in .

The f i r s t two

w ashings a re made w ithout shaking t o prev en t t h e fo rm a tio n of an em ulsion. The washed e th e r e x tr a c t i s th e n f i l t e r e d th ro u g h an anhydrous sodium s u l f a t e f i l t e r pad i n to a 125 m l. Erlenm eyer f l a s k .

A fte r th e

s e p a ra to ry fu n n e l i s r in s e d and t h e f i l t e r pad washed w ith 10 m l. of e th e r , t h e combined e th e r p o rtio n s a re evaporated t o d ry n ess, u sin g g e n tle s u c tio n and a w ater b a th a t about 50°C. D ig ito n in T re a tm e n t. — The n o n sa p o n ifia b le f r a c t i o n i s ta k e n up i n 10 m l. o f 95j6 e th y l a lc o h o l.

Twelve m l. o f 1% d i g it o n i n s o lu tio n a re added

and t h e m ix tu re i s h e a te d f o r two h o u rs a t 70°C.

The p r e c ip ita te d

s t e r o l s a re th e n f i l t e r e d o f f and t h e f i l t r a t e c o lle c te d along vdth two 2-m l. washes o f c o ld e th y l a lc o h o l.

An a l t e r n a t i v e procedure i s used,

i f a chrom atographic s te p fo llo w s, i n which no f i l t r a t i o n i s made but t h e a lc o h o l i s e v ap o rated o f f and t h e dev elo p in g s o lu tio n added d i r e c t l y

11 PROCEDURES STAMDAUDIZED FOR THIS STUDY

D ig ito n in T re a tm e n t. — (continued) - t o t h e r e s id u e .

I n t h i s procedure

th e column o f a d so rb e n t se rv e s as t h e f i l t e r . ■ HC1 T re a tm e n t. —

The re s id u e from t h e p rec ed in g s te p i s ta k e n up in

10 m l. hexane o r p u r i f ie d S k e lly so lv e B. ly th ro u g h t h i s s o lu tio n f o r one h o u r.

Anhydrous HC1 i s bubbled slow­ Any squalene h y d ro ch lo rid e

c r y s t a l s which form a r e th e n removed by a p rocedure s im ila r t o t h a t o f t h e d i g it o n i n p r e c i p i t a t e above. Chrom atographic P rocedure f o r Large S u p e r f i l t r o l Columns. —

The r e s i ­

due i s ta k e n up i n 10 m l. o f th e chrom atographic d eveloping s o lu tio n . T h is i s allow ed t o p a ss th ro u g h t h e p re p a re d 9 - o r 11-cm. column of S u p e r f i l t r o l which h a s been p re v io u s ly washed w ith 40 t o 60 m l. o f th e d ev elo p er and h a s not been allow ed t o become d ry .

A 10-cra. d i f f e r e n t i a l

i n p re s s u re i s m ain tain ed th ro u g h o u t t h e whole p ro ce d u re. The f l a s k i s r in s e d w ith t h r e e t o f i v e m l. o f t h e developing s o lu ­ t i o n , which i s added a t once t o t h e column.

By means o f a short-stem m ed

s e p a ra to ry fu n n e l which i s f i t t e d t o t h e t u b e , th e dev elo p in g s o lu tio n i s added t o t h e column drop by drop u n t i l t h e v ita m in Dr> has passed th ro u g h t h e a d so rb en t column.

I n t h e a u t h o r 's e x p erien c e t h i s sep ara­

t i o n i s com plete when t h e low est v i s i b l e band re a c h e s th e bottom o f t h e column. The f i l t r a t e from t h e column i s th e n ev ap o rated t o dryness u sin g s u c tio n and & h o t w ater b a th (about 5 0 °C .).

The re s id u e i s ta k e n up i n

a b s o lu te a lc o h o l and t h e e x tin c tio n a t 265 mu i s measured on t h e Beckman q u a rtz sp e c tro p h o to m e te r.

12 PROCEDURES STANDARDIZED FOR THIS STUDY Chrom atographic P rocedures f o r Sm all S u p e r f i l t r o l Columns* — The r e s i ­ due i s ta k e n up i n t h r e e m l. o f t h e chrom atographic developing s o lu tio n . T h is i s added t o t h e prewashed S u p e r f i l t r o l column which has not been p e rm itte d t o become d ry . The f l a s k i s rin s e d v d th t h r e e m l. o f t h e developing s o lu tio n which i s added t o t h e column.

A fte r t h i s h as passed through th e column,

an a d d itio n a l f i v e m l. o f t h e developing s o lu tio n i s added t o develop t h e chromatogram. The f i l t r a t e from t h e column i s th e n evap o rated t o d ry n e ss, u sin g su c tio n and a hot w ater b a th (about 5 0 °C .). Alumina Chrom atographic S te p . — The re s id u e from t h e f i r s t chromato­ graph i s ta k e n up i n t h r e e m l. o f t h e developing s o lu tio n .

T h is i s

allow ed to f i l t e r th ro u g h t h e prewashed alum ina column ?;hich has not been p erm itted t o become d ry .

A 1-cm. p re s s u re d i f f e r e n t i a l i s main­

ta in e d th ro u g h o u t t h e p ro ce d u re. A t h r e e m l. r in s e i s th e n added t o t h e column.

The procedure

v a r ie s th e n , depending upon t h e ty p e o f sam ple. — (a) I r r a d ia te d E rg o s te ro ls o r D2 i n Corn O i l . - An a d d itio n a l f iv e m l. of t h e develop­ in g s o lu tio n i s passed th ro u g h t h e column and a l l t h e f i l t r a t e up t o t h i s p o in t i s d is c a rd e d .

The Dg i s e lu te d from th e column w ith 15 ml.

e th e r and i t s e x tin c tio n i n t h e d eveloping s o lu tio n a t 265 mu i s determ ined by means o f t h e sp e ctro p h o to m e te r. (b) M u ltiv itam in P repar a tio n s o r F is h L iv e r O i l s . — An a d d itio n a l f iv e m l. o f th e developing s o lu tio n i s passed th ro u g h th e column and a l l t h e f i l t r a t e up t o t h i s p o in t i s d is c a rd e d .

The D i s e lu te d from th e

13 PROCEDURES STANDARDIZED FOR THIS STUDY

column w ith an a d d itio n a l 12 m l. o f t h e developing s o lu tio n and i t s e x tin c tio n a t 265 mu i s determ ined d i r e c t l y by means o f t h e sp e c tro ­ photom eter.

EXPERIMENTAL STUDIES S tu d ie s Made on U n treated O il S o lu tio n s o f I r r a d i a t e d E r g o s te r o ls . — From t h e a b s o rp tio n curve o f a t y p i c a l c o rn o i l d is s o lv e d in p u r if ie d a lc o h o l (F ig . 2 ) , i t i s obvious t h a t due t o t h e e x c e ssiv e ly h ig h r a t i o o f o i l t o v ita m in p re se n t i n a sample (ev en th o s e having a potency o f 1,000 ,0 0 0 u n i t s p e r gram or more) i t i s im p r a c tic a l t o attem p t t o e s t i ­ mate t h e v ita m in D potency d i r e c t l y from t h e a b s o rp tio n curve o f th e sample d isso lv e d i n a lc o h o l o r s im ila r s o lv e n ts .

For t h i s re a so n , no

stu d y o f t h i s ty p e was made. An a tte m p t, however, was made t o c o rr e c t f o r t h e e x tin c tio n due t o th e co rn o i l p re se n t i n a g iv e n sam ple.

T h is was done by d is s o lv in g an

e q u al amount o f c o rn o i l i n t h e same volume o f t h e so lv e n t a s t h a t o f th e o i l b e in g t e s t e d .

The e x tra n eo u s a b s o rp tio n e x h ib ite d by th e o r ig in a l

v ita m in s o lu tio n th e n , v/hen ru n w ith t h e c o rn o i l s o lu tio n i n t h e so lv e n t c e l l , should b e a u to m a tic a lly c o rre c te d f o r . P ro ced u re. — .100 t o .120 gm. o f t h e i r r a d i a t e d e r g o s te r o l i n o i l i s d is s o lv e d i n 50 ml. o f p u r i f ie d a lc o h o l and p la c e d i n th e s o lu tio n c e l l . An eq u al weight o f co rn o i l i s d is s o lv e d i n 50 m l. o f a lc o h o l and placed i n t h e so lv e n t c e l l .

The e x tin c tio n a t 265 mu i s th e n measured by t h e

sp ect rophotom et e r . The paten cy o f t h e o r i g in a l o i l i n v ita m in Dg u n i t s p er gram i s determ ined by c a lc u la tin g t h e E (l$ , ICm.) o f t h e sample @ 265 mu and m u ltip ly in g by 86,960.

T h is f a c t o r i s o b ta in e d by d iv id in g th e number of

v ita m in Dg u n i t s p e r gram o f t h e sta n d a rd c a l c i f e r o l (40,000,000) by t h e E (l$ , 1cm.) a t 265 mu, which i s 460. A t y p i c a l a b s o rp tio n cu rv e i s shown i n F ig . 3.

EXPERIMENTAL STUDIES S tu d ie s f^Iade on S a p o n ifie d O il S o lu tio n s o f I r r a d i a t e d E r g o s te r o l. Assuming t h a t t h e s a p o n ifia b le m a te r ia l in corn o i l i s la r g e ly re s p o n s ib le f o r t h e e x tra n e o u s a b s o rp tio n o r c o lo r r e a c tio n produced when an i r r a d i a t e d e r g o s te r o l in o i l i s s tu d ie d , t h e fo llo w in g stu d y was made: P ro c e d u re , — The n o n sa p o n ifia b le f r a c t io n of th e o i l s o lu tio n of i r r a d i a t e d e r g o s te r o l i s o b ta in e d by c a rry in g a sample o f t h e o r i g in a l o i l th ro u g h t h e s a p o n if ic a tio n and e x tr a c tio n p ro ced u re d e sc rib e d above. The r e s id u e i s th e n t r e a t e d by one o f t h e fo llo w in g m ethods: (a) A bsorption Curve o f N o nsaponifiable F r a c tio n . - The re s id u e i s ta k e n up in a lc o h o l and i t s a b s o rp tio n curve i s ru n d i r e c t l y .

The po­

te n c y o f t h e o r i g in a l o i l i s th e n c a lc u la te d by m u ltip ly in g th e E(lj«, 1cm.) a t 285 mu by th e f a c t o r 83,360.

A t y p i c a l curve i s shown i n F ig . 4 .

(b) Antimony T r ic h lo r id e C o lo rim e tric Method. — The r e s id u e from t h e e th e r e x tr a c t i s ta k e n up in 10 m l. o f th e p u r if ie d chloroform .

To

one m l. o f t h i s s o lu tio n , 10 m l. o f th e antim ony t r i c h l o r i d e rea g en t a re added.

A fte r 30 seconds* s w irlin g , a 1-cm. c e l l i s f i l l e d and th e ex­

t i n c t i o n a t 500 mu i s measured in e x a c tly t h r e e m inutes from t h e tim e th e re a g e n t was f i r s t added, u sin g t h e Bausch & Lomb v is u a l sp ectro p h o ­ to m e te r . The potency o f t h e o r i g i n a l o i l i n v ita m in Pg u n i t s p e r gram i s th e n determ ined by c a lc u la tin g t h e E (l;t, 1cm.) from th e e x tin c tio n a t 500 mu and m u ltip ly in g by t h e f a c to r 19,300 as determ ined by Ewing e t a l (1 2 ). An attem p t t o c o rre c t f o r th e n o n sa p o n ifia b le p a r t o f corn o i l , as w ell a s t h e s a p o n ifia b le p o r tio n , was made by d is s o lv in g an equal amount o f t r e a t e d corn o i l i n t h e same volume o f so lv e n t as t h a t o f th e t r e a t e d

16 EXPERIMENTAL STUDIES ir r a d i a t e d e r g o s te r o l, and p la c in g i t i n t h e so lv en t c e l l .

The

r e s u ltin g a b so rp tio n curve should th e n be a u to m a tic a lly c o rre c te d fo r th e ex tran eo u s a b s o rp tio n due t o co rn o i l . P ro ced u re. — .120 gm. o f t h e i r r a d i a t e d e rg o s te r o l in o i l i s sa­ p o n ifie d and t h e n o n s a p o n ifia b le f r a c t i o n e x tr a c te d .

An equal amount

o f corn o i l i s c a r r ie d th ro u g h a s im ila r p ro ced u re.

The c o rre c te d ab­

s o rp tio n curve i s o b tain ed by d is s o lv in g each o f th e above i n 50 ml. a lc o h o l, p la c in g t h e t r e a t e d Dg s o lu tio n i n th e s o lu tio n c e l l , and com­ p erin g i t w ith t h a t o f t h e t r e a t e d c o rn o i l p laced i n t h e so lv e n t c e l l . P a te n c ie s a re th e n e v a lu a te d by m u ltip ly in g t h e E(l/£, 1cm.) @ 265 mu by th e f a c to r 8 6,360.

See F ig . 5 f o r a t y p i c a l cu rv e .

S tu d ie s Made on Removal o f I n t e r f e r i n g S ubstances by Chemical Means o r Chromatography. —

S ince i t h as b een shown t h a t t h e n o n sa p o n ifia b le

f r a c t io n o f v e g e ta b le and f i s h l i v e r o i l s c o n ta in s t e r o l s and squalene ty p e compounds and t h a t th e y can be removed, a t l e a s t i n p a r t , by t r e a t ­ ment w ith d ig ito n in , anhydrous HC1, o r chrom atography, a study was made t o determ ine i f th e s e p re lim in a ry tr e a tm e n ts , s e p a ra te ly o r combined, would in c re a s e t h e accuracy o f th e patency e v a lu a tio n s . (a)

Chrom atographic S e p a ra tio n A tta in e d by Use o f a S in g le Column

o f S u p e r f i l t r o l . — The s e p a r a tio n of v ita m in Pg from crude I r r a d ia te d e r g o s te r o ls i n v o l a t i l e s o lv e n ts which was s u c c e s s fu lly o b tain ed by Pow ell (5 1 ), u sin g a s u p e r f i l t r o l column and so lv e n t m ix tu re , hexane, e th e r and a lc o h o l, was a p p lie d t o o i l s o lu tio n s a s fo llo w s:

17 EXPERIMENTAL STUDIES P ro c ed u re . — A one-gm. sample o f t h e v ita m in s o lu tio n i s saponi­ f ie d , e x tr a c te d , and chrom atographed u sin g a 9-cm. column o f S u p e r f i l t r o l ( la r g e columns) acco rd in g t o t h e sta n d a rd ise d p ro ced u res above. The a b s o rp tio n curve o f th e t r e a te d sample should have a maxima a t £65 mu s im ila r t o th a t e x h ib ite d by pure c a l c i f e r o l (F ig . S ) .

The po­

te n c y o f t h e sample i s th e n determ ined by m u ltip ly in g t h e E (l$ , 1cm.) of th e sample a t £65 mu by t h e f a c to r 8 6 ,9 6 0 .

A t y p i c a l curve o b tain ed fo r

low -potency o i l s i s shown i n F ig . 7. (b)

S e p a ra tio n Achieved by Combined Chemical and Ch r o mat onr a oh i c

T rea tm en t. — T h is study was confined t o corn o i l s o lu tio n s o f i r r a d i a t e d e r g o s te r o l o r c r y s t a l l i n e Dg having a p o t e n c y o f approxim ately 1 0 , 0 0 0 u n i t s p e r gram. Procedure.

--

One gm. samples o f t h e s o lu tio n a re t r e a t e d s e p a ra te ly

u sin g s ta n d a rd is e d p ro ced u res i n t h e o rd e r d e sc rib e d below : I r r a d i a t e d E r r o s te r o l in Corn O il (#75978) Sample No.

No. o f T reatm en ts

1

1

S a p o n ific a tio n & e x tr a c tio n .

2

?-

S a p o n ific a tio n & e x tr a c tio n , chromato­ graphed th ro u g h 9-cra. column o f Super­ filtro l.

2

2

S a p o n ific a tio n & e x tr a c tio n , HC1 Treatm ent,

^

3

S a p o n ific u tio n & e x tr a c tio n , chrom ato­ graphed th ro u g h 9-cm. column o f Super­ f i l t r o l , HC1 tr e a tm e n t.

®

3

S a p o n ific a tio n & e x tr a c tio n , HC1 t r e a t ­ m ent, chrom atographed th ro u g h 9-cm. column o f S u p e r f i l t r o l .

O rder o f T reatm en ts

EXPERIMENTAL STUDIES C r y s t a ll i n e Dg i n Corn O il Sample No. -

No. of T reatm en ts

O rder o f T reatm en ts

I

1

S a p o n ific a tio n & e x tr a c tio n .

II

2

S a p o n ific a tio n & e x tr a c tio n , d ig ito n in t r e a tm e n t.

Ill

3

S a p o n ific a tio n & e x tr a c tio n , d ig ito n in tr e a tm e n t, chrom atographed through 9-cm column o f S u p e r f i l t r o l .

The a b so rp tio n curve o f t h e re s id u e o b tain ed from t h e above sam ples, t r e a te d a s d e sc rib e d , i s ru n .

An i n d ic a t i o n , as t o 'what ty p e of compound

i s removed by each s te p , i s o b ta in e d by s u b tra c tin g t h e a b so rp tio n curves of sam ples r e p r e s e n tin g two ( 2 ) p ro ce d u res made in t h e same o rd e r but d i f f e r in g by one ( 1 ) a d d itio n a l tr e a tm e n t. A bsorption curves o f m a te r ia l removed by t h e v a rio u s tre a tm e n ts a re shown in F ig s . 8 , 9, and 10. - (c) S e p a ra tio n O btained Usinn S e p a ra te S u p e r f i l t r o l and Alumina Columns. — A p a r t i a l s e p a ra tio n o f s t e r o l s and squalene ty p e compounds has been o b tain ed by s e v e ra l i n v e s ti g a t o r s u sin g alum ina columns (10, 14, 4 3 ).

A chrom atographic p ro c e d u re , t h e r e f o r e , was employed in which th e

v ita m in s P a re f i r s t s e p a ra te d from v ita m in s A, c arat.e n o id s, pigm ents, and o th e r p ro d u c ts o f i r r a d i a t i o n , by u se o f t h e P in k e rto n m o d ific a tio n of th e S u p e r f i l t r o l column d e sc rib e d above (sm a ll colum ns). P a re co n tain ed in t h e e lu a te from t h i s column.

The v itam in s

Any rem aining s t e r o l s ,

squalene ty p e compounds, and p o s s ib ly v ita m in E a re th e n se p ara ted from th e v ita m in s P by use o f an alum ina column.

I n t h i s s te p th e v itam in s D

a re h eld on t h e column and th e n must be e lu te d t o be rec o v e re d .

19 EXPERIMENTAL STUDIES

P ro c ed u re . — One gm. samples o f th e v ita m in s o lu tio n a re saponi­ f ie d , e x tra ct ;ed, and chrom atographed th ro u g h s sm all S u p e r f i lt r o l column a s de scribed in t h e sta n d ard ize d pi’o ced u res.

The r e s id u e i s th e n

adsorbed upo n a column o f alum ina u sin g t h e sta n d a rd iz e d procedure c o rresponding t 3 th e ty p e o f v itam in s o lu tio n bein g t e s t e d .

The potency

o f t h e o r i g in a l sample i s o b tain ed by m u ltip ly in g th e E(l/o, 1cm.) a t £65 mu o f t h e e lu te d v ita m in s D by t h e f a c to r 100,000.

A bsorption

curves o b ta i ned f o r b oth ty p e s of v ita m in s o lu tio n s a t v a rio u s potency l e v e ls a re s io '.’t* i n F ig s . 11 and IE .

1

20 RESULTS

For each method a tte m p te d , c a lc u la te d potency v a lu e s a re compared t o b io a sse y v a lu e s ra n by t h e IT.S.P. p ro ce d u re.

A ll anim al t e s t s were

run a t two o r t h r e e l e v e l s , 15 t o 20,t a p a r t , a n d -th e U .S .? . re fe re n c e o i l was used a s th e s ta n d a rd .

As no attem pt was made t o i n te r p o la te

between t h e b io a s sa y l e v e l s , some of th e d is c re p a n c ie s between t h e p h y si­ c a l chem ical and t h e b io lo g ic a l* d a ta may be due t o t h e 15 t o 20% range a t which t h e sam ples were t e s t e d . The v a lu e s o b tain ed from t h e anim al t e s t s re p re s e n t th e h ig h e s t b io lo g i c a l potency t h a t could be obtained from t h e l e v e ls a t . which -t h e samples were t e s te d .

For exam ple, th e data, .of a ■sample t e s t e d a t 1,200,000

and 1 , 0 0 0 ,0 0 0 u n i t s p e r gram might in d ic a te t h e m a te r ia l t o be s l i g h t l y l e s s th a n 1 ,2 0 0 ,0 0 0 u n i t s p e r gram but above 1 ,0 0 0 ,0 0 0 u n i ts p e r gram.

In

t h i s c ase t h e a ssa y would be re p o rte d a t 1 . 000,000 u n i t - p i r gram, although th e -m a te r ia l might 'a c tu a lly c o n ta in 1,1 0 0 ,0 0 0 o r 1,150,000 u n its p e r gram. Theru aeu :u to be very l i t t l e c o r r e la tio n V.-et wr-on th e t r u e potency v r j u r s and t h e rb; o ri'tio ri curve o f th e u n tre a te d sam ples. (T able I ) .

Po-

t '-p cier c a lc u lc .ti d f o r 15 d i f f i - n n t sam pler u sin g th e a b so rp tio n curves of th e u n tre a te d o i l , o b tain ed w ith corn o i l o f th e same c o n c e n tra tio n i n t h e so lv e n t c e l l , showed an a v e ra g e v a r i a ti o n o f 55.2;* from th e b io a ssa y v a lu e . Only two o f t h e s e d i f f e r e d from th e b io a s sa y v a lu e by l e s s th a n 20$. S a p o n if ic a tio n does not seem t o in c r e a s e t h e accu racy o f d e term in in g v ita m in s P p re s e n t i n a n o i l by use of t h e i r a b s o rp tio n c u rv e s.

For sa ­

p o n ifie d o i l s hav ing b io e s sa y v a lu e s , t h e a b s o rp tio n curves o b tain ed w ith a lc o h o l i n t h e so lv e n t c e l l invariably'- gave potency v a lu e s which were high (T abic I I ) .

E ig h t o f t h e 15 o i l s t e s t e d had b io a s sa y v alu es and showed an

21

T ab le I .

Sample No.

POTENCIES OF IRRADIATED ERGOSTEROLS IN OIL AS DETERMINED BI THEIR ABSORPTION CURVES IN ALCOHOL V.HEN COMPARED TO A SOLUTION OF CORN OIL IN THE SOLVENT CELL.

E ( l £ , 1cm.) '3 265 mu

C a lc u la te d ®2 N n its/G .

B ioassay U .S .P . Eg U n its/G .

64184

2.14

183,075

250,000

64824

1 .7 7

153,901

250,000

65104

5.75

326,062

250,000

65464

2 .2 2

148,129

200,000

67784

1 .8 3

158,249

250,000

68564

1 .3 6

118,252

250,000

69934

3.10

269,545

250,000

70514

2.66

230,417

200,000

72114

2.76

239,982

300,000

73554

2 .7 0

234,765

180,000

77424

1 .1 9

105,470

500,000

80454

2.49

215,505

300,000

87114

24.29

2,112,015

1,200,000

88875

8.19

712,120

1,200,000

9.1095

5 .1 8

520,501

400,000

22 T able I I .

Sample No.

POTENCIES OF IRRADIATED ERGOSTEROLS IN OIL AS DETERMINED FROM THE ABSORPTION CURVE OF THEIR NONSAPONIFIABLE FRACTION IN ALCOHOL.

E (1$, 1cm.) @ 265 mu

C a lc u la t ed Do U n its/G .

B ioassay U .S .P . Dg U nits/G ,

BalOS

22.0 24.8

1,912,900 2,156,360

CPS

20.7 22.5

1,799,865 1,956,375

DPS

15.8 14.6

1,199,910 1,269,470

FPS

1 2 .7

1,104,265

5772

4 .7 4 .9

408,665 426,055

200,000

64824

4.20

365,190

250.000

65104

4.65

404,317

250.000

65464

5 .3 2

445,184

200.000

M9025

7.50

652,125

B11299

19.8

1,721,610

525.000

65624

1.15

99,992

48,000

64184

5.75

526,062

250.000

Syn 2

8.9

775,855

i

RESULTS average v a r i a ti o n of 127ft.

The r e s u l t s a r e h ig h , p ro b ab ly , due t o

p resen ce o f so lv e n t r e s id u e s o b ta in e d from th e e x tr a c tio n p ro ced u re. P la c in g an equal amount o f t h e n o n sa p o n ifia b le f r a c t i o n o f co rn o i l in t h e so lv e n t c e l l , a s t h a t o f t h e v ita m in s o lu tio n i n t h e s o lu tio n c e l l , te n d s t o c o rre c t f o r r e s id u e s o b ta in e d from th e e x tr a c tio n pro ce­ d u re , but t h e c a lc u la te d p a te n c ie s s t i l l vary c o n sid e ra b ly from t h e b io ­ assay v a lu e s .

The r e s u l t s from f iv e d i f f e r e n t samples l i s t e d i n T able

I I I show an av erage v a r i a t i o n o f 24.5ft and a s ix th sample v;hich was not averaged w ith t h e group v a rie d 254ft from t h e b io a s s a y . T ab le IV g iv e s t h e p o te n c ie s o f v a rio u s i r r a d i a t e d e r g o s te r o ls i n corn o i l as determ ined by t h e antimony t r i c h l o r i d e c o lo rim e tric method a p p lie d t o t h e n o n s a p o n ifia b le f r a c t io n o f t h e sam ple.

The f i r s t p a rt

o f T ab le IV i s made up o f v a lu e s o b ta in e d f o r h ig h -p o te n cy i r r a d i a t e d e r g o s te r o ls i n co rn o i l , and t h e second p a r t c o n s is ts o f v a lu e s f o r lowpotency sam ples i n c o rn o i l c o n ta in in g around 10,000 u n i t s p e r gram. These were o b ta in e d on t h e open m arket. Out o f t h e 51 h ig h -p o te n cy o i l s assayed by t h e antim ony t r i c h l o r i d e c o lo r im e tr ic method, 10 o i l s showed a d if f e r e n c e from t h e b io a s sa y of more th a n 25ft.

The maximum d if f e r e n c e shown by t h i s method was 57.0ft,

w hile t h e average v a r i a ti o n o f a l l t h e o i l s t e s t e d was 15.8ft.

The maxi­

mum v a r ia tio n shown when t e s t i n g s ix low -potency o i l s o f about 10,000 v ita m in pg u n i t s p er gram by t h i s method was 21.4ft.

The average d i f ­

fe re n c e was 16.4ft. The s e p a ra tio n obtained, when a s in g le column o f S u p e r f i lt r o l i s employed, i s r e a d i ly shown by T ab les V, V I, and V II.

Comparison of th e

24

T able I I I .

POTENCIES OF IRRADIATED ERGOSTEROLS AS DETERMINED FROM THE ABSORPTION CURVE OF THEIR NONSAPONIFIABLE FRACTION YiHEN COMPARED TO AN ALCOHOL SOLUTION OF THE NONSAPONIFIABLE Fraction of corn o i l .

Sample No.

E (1$, 1cm.) Q 265 mu

C a lc u la te d Dg U n its/G .

B ioassay U .S .P . Dg U n its/G .

65104

2.879

250,529

Syn 2

1.553

135,055

65464

5.171

275,718

200,000

67784

5.458

300,675

250,000

69954

4.058

552,843

250,000

70514

7.715

670,645

200,000

250,000

25 T able IV. Sample Ho.

POTENCIES OF IRRADIATED ERGOSTFROLS AS DETERMINED BY ANTIMONY TRICHLORIDE COLORIMETRIC METHOD. E ( l $ , len t.) © 500 mu

C n lc u la te d Dg U n its/G .

B ioassay U .S .P . Dg U n its/G .

H igh-Pot encv Samples i n Corn O il 1 ,5 9 2 ,6 8 8 1 ,5 5 8 . 720 Av. 1 ,5 7 5 ,7 0 4

1 , 200,000

Av.

72.16 70.40 71.28

1,1 0 5 ,9 6 0 1 ,1 5 7 ,9 2 8 Av. 1,1 2 0 ,9 4 4

1,200,000

Av.

57.20 58.96 58.08

1 .3 5 8 .7 2 0 1 .3 5 8 .7 2 0 Av. 1 .5 5 8 .7 2 0

1,200,000

Av.

70.40 70.40 70.40

1015

56.76

1 ,0 9 5 ,4 6 8

1,00 0 ,0 0 0

1755

52.80

1 ,0 1 9 ,0 4 0

1,000,000

7505

942,612 1 .0 0 6 .2 0 2 Av. 974,407

1,200,000

Av.

4 8 .8 4 52.1450.49

305,712 531.188 351.188 322,696

600,000

Av.

15.84 17.16 17.16 16.72

87114

88875

S7195

89025

Av.

94455

25.76

458,568

600,000

25446

Av,.

492,556 501,028 484.022 4 8 4 .0 2 2 490,402

525,000

Av.

25.52 25.96 25.08 25.08 25.41

Av .

471,306 420, 354446.000 445,887

500,000

Av.

24.42 21.78 25.10 23.10

428,846

600,000

6105

4985

oo oo

• f». fv

26 T ab le ! Sample No.

i

(C o n tin u e d ). E ( l £ , 1cm.) 0 500 mu

7705 Av.

20.46 20.68 20.57

C alculated. Dg U n its/G .

B ioassay U .S .P . Dg U nits/G .

594,878 599.124 Av. 597,001

600,000

456,000

525,000

19656

22.60

92545

577,894 556.664 Av. 367,279

525,000

Av.

18 .5 8 18.48 18.53

484,044 475.552 Av. 479,798

515,000

Av.

25.08 24.64 24.86

492,536 488,290 471,506 501,028 501,028 505,274 509,520 496,792 501,028 509.520 Av. 497,652

500,000

Av.

25.52 25.50 24.42 25.96 25.96 2 6 .1 8 26.40 25.74 25.96 2 6 .4 0 2 5 .7 7

459,000

525,000

458,568 455.092 Av. 445 , 850

525,000

25476

25456

21226

23.75

21376

25.76 22.44 25.3.0

Av. 21986

26.10

504,000

525,000

25196

25.3.0

484,000

525,000

91095

23.76 25.76 25.76

458,568 458.568 Av. 458,568

400,000

Av. 25496

21.54

412,000

450,000

8645

1 5.40

297,500

525,000

T able IV* Sample No*

(C ontinued) .

Av.

14. C8 14.08 14.50 14.08 14.52 14.08 14.31

Av.

11.22 11.22 11.22

Av.

9.46 9.46 9.46

Av.

12.98 1 2r98 12.98

Av.

14.08 13.86 15.97

Av.

13.42 15.42 15.42

Av.

15.86 15.64 13.64 15.71

Av.

10.12 10.12. 10.12

Av.

12.10 12.54 12.52

78174

04614

77424

80454

79404

85024

64184

64824

65104

67784

C a lc u la te d Drf5 U n its/G .

E (1/j , 1cm.) ©'500 mu

9.46 9.46

Av.

271,744 271,744 275,990 271,744 280,236 271.744 274,867

Av.

216,546 216.5*6 216,546

Av.

182,578 182.578 182,5 78

Av.

250,514 250.514 250,514

Av.

271,744 267.498 269,621

Av.

259,006 259.006 259,006

Av.

267,498 ,2 6 3 ,2 5 2 265.252 264,667

Av.

195,516 195.516 195,516

Av.

255,550 242.022 237,776 182,578 182.578

28 T able IV Sample No.

(C ontinued) • E (3$, 1cm.) 3 500 mu

Ciilc u la te d U n its/G .

B ioassay U .S .P . Dg U nits/G .

186,824 186.824 Av. 186,824

250,000

Av.

9.68 9 .6 8 9 .6 8

191,070 195.316 Av. 193,193

250.000

Av.

9 .9 0 10.12 10.01

212,500 212.300 Av. 212,500

250.000

Av.

11.00 11.00 11.00

5305

10.23

197,459

250.000

80904

9.35

180,453

204.500

84174

10.34

199,562

204.500

65464

197,459 199.562 Av. 198,500

200.000

Av.

10.23 10.3.2 10.18

205,808 205,808 203.808 Av. 203,808

200,000

Av.

10.56 10.56 10,56 10.56

209,500 209.500 5, AV*,, 209,500

200,000

68564

69934

84944

70514

74294

10.85 1.0.85 Av. ^IQ.,85,,,,^^

73534

205,808 205,808 203.808 Av. 205,808

180,000

Av.

10.56 10.56 10.56 10.56

118,888 123,134 112,519 114,642 116,765 118.888 Av. 117,473

160,000

Av.

6.16 6.38 5 .8 3 5.94 6.05 6 .3,6 6 .09

42943

29 T able IV. (C o n tin u e d ). Sample No.

E (1$, 1cm.) @ 500 mu

C a lc u la te d Dg U n its/G .

B ioassay U .S .P . Dg U n its/G .

5.39 4 .8 4 5.06 5.06 5 .3 9 5.39 5.19

104,027 93,412 97,658 97,658 104,027 104.027 Av. 100,135

125,000

75584

8.14 8.14 Av. 8.14

157,102 157.102 Av. 157,102

100,000

18546

22.70

438,000

400.000

19196

20.45

395,000

450.000

20826

8.03

154,800

250.000

24646

23.10

446,000

450.000

24876

22.45

433,000

450.000

38153

Av.

T.ow-Pot encv Samples i n Corn O il 0855

0.594

11,464

14.000

0865

0.550

11,000

14.000

2985

0.605

12,000

14.000

0905

0.418

8,000

10.000

74334

0.450

8,775

10,000

89545

0.594

11,500

10,000

89555

0.489

9,500

10,000

T ab le V.

EXTINCTION RATIOS OF CRYSTALLINE CALCIFEROL AND CHROMATOGRAPHED SAMPLE IN ETHANOL.

w«ve L e n sth . Mu

E x tin c tio n R a tio s (£65 mu) T e s t M a te ria l C a lc if e r o l

240

76

° - 67

250

O’ 89

° * 86

260

° ’ 98

0 ,9 8

270

0*95

0 .9 8

280

O’ 76

° - 75

290

° ’ 47

0 ,4 5

500

° ’ 20

°*25

31 T able VI. Sample No.

POTENCIES OF IRRADIATED ERGOSTEROLS AS DETERMINED BY CHROMATOGRAPHIC ULTRAVIOLET ABSORPTION CURVE AT 265 MU. E (l$5, 1cm.) 265 mu

C a lc u la te d D0 U n its/G .

B io assay , U .S .P . D2 U n its/G .

Kl

H igh-P ctency Samples i n Com O il 97195 87114 5155 7305 1015 1755 4985 25446 2915 3265 0145 89025 91285 92345 94455 97025 98655 21226 21976 21986 24646 23196 6105 7705 0555 18546 99775 97775 3005 19196 24876 19196 78174 84163 80434 79404 85024 64184 64824 65104 67784 68564 69934

1 6 .7 7 1,4 5 8 ,0 0 0 17.89 1 ,5 5 5 ,0 0 0 1 5 .1 8 1 ,3 1 8 ,0 0 0 15.39 1 ,1 6 4 ,0 0 0 1 4 .0 7 1 ,2 2 1 ,0 0 0 11.89 1 ,0 3 3 ,0 0 0 5 .4 3 472,000 5 .5 1 479,000 4 .6 8 407,000 4.95 450,000 5 .0 4 437,500 4 .2 7 371,000 4.26 370,000 4 .1 1 557,000 5.16 448,000 6.09 529,000 5.70 495,000 5.52 479,000 4 .4 7 389,000 5.86 509,000 5 .0 4 438,000 4 .6 4 404,000 5.44 473,500 5 .1 3 446,500 4 .6 8 407,000 5.05 439,000 5 i 4 8 - ^ ^ ^ ^ ^ : 4 , 7 6 , 0 0 0 ^ ^ . . ^ . .. 5 .1 1 444,000 5 .9 1 513,000 4 .5 8 598,000 4 .5 4 395,000 5-.'26 456,000 3.22 280,000 3.09 268,500 2.75 259,000 3.35 , 281,000 2.96 * 257,000 2.69 254,000 2.60 226,000 2 .7 4 238,000 2 .2 8 198,000 2.25 195,500 2.60 226,000

1,200,000 1,200,000 1,200,000 1,200,000 1,000,000 1,000,000 600,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 500,000 600,000 475,000 400,000 450,000 400,000 450,000 450,000 450,000 330,000 330,000 500,000 275,000 275,000 250,000 250,000 250,000 250,000 250,000 250,000

32

T ab le V I. (C o n tin u ed ).

Sample No.

E (1 %, Icra.) 265 mu

C a lc u la te d Dg U n its/G .

B io assay , U .S .P. Dg U n its/G .

H igh-Potency Samples i n Corn O il 5305 20826 80904 84174 65464 70514

2.43 1.81 2.52 2.60 2 .4 8 2.46

211,000 157,500 219,000 226,000 216,000 214,000

250,000 250,000 204,500 204,500 200,000 200,000

H igh-Potency I r r a d i a t e d E rg o s te ro ls i n F is h L iv e r O il

74254 66 844 71064 20206 25646

2 .5 7 2 .4 2 2.32 2 .9 7 2.02

.

223,000 210,500 202,000 258,300 175,800

250,000 200,000 200,000 300,000 200,000

33

T ab le V II.

POTENCIES OF IRRADIATED ERGOSTEROLS AS DETERMINED BY CHROMATOGRAPHIC ULTRAVIOLET ABSORPTION CURVE AT 265 MU

Sample No.

E (1%, lcm .) 265 mu

C a lc u la te d Dg U n its/G .

B ioasaay, U .S .P . Dg U nits/G .

Low-Potency I r r a d i a t e d E r g o s te ro ls i n O il 0855

0.296

25.800

14.000

0865

0.236

20,520

14.000

0905

0.196

17,070

10.000

2985

0.262

22.800

14.000

39597

0.234

20,570

10.000

89535

0.153

13,300

10,000

Y/hite Lab. Dg i n Corn O il

0.511

44,400

40.000

ADMA D g i n Corn O il

0.565

26,650

15.000

V io s te r o l i n Sesame O il

0.272

23,600

10.000

RESULTS

a b s o rp tio n cu rv es o f e th a n o l s o lu tio n s of c r y s t a l l i n e Dg and of t h e chrom atographed sample i s w e ll shown i n T ab le V.

The e x tin c tio n r a t i o s

f o r g iv en w avelengths a re ta b u la te d a c c o rd in g t o t h e method of O ser, Llelnick, and Pader (2 8 ). Of 49 i r r a d i a t e d e r g o s te r o ls i n c o rn o i l assayed by t h e chromato­ g rap h ic u l t r a v i o l e t a b s o rp tio n curve method, only 8 d if f e r e d from th e b io a s sa y v a lu e s by more th a n 25%. (T able V I).

The maximum % d iff e r e n c e

from th e b io a s sa y f ig u r e s was S6.9, and t h e average v a r i a t i o n of a l l t h e o i l s ru n by t h i s method was 1 4 .5 /j. The r e s u l t s o b ta in e d when u sin g t h e chrom atographic method f o r f iv e h ig h -p o te n cy i r r a d i a t e d e r g o s te r o ls , i n f i s h l i v e r o i l c o n ta in in g v i t a ­ min A, o re ta b u la te d in th e second p a rt o f T ab le V I.

The c lo se agreement

o f th e s e r e s u l t s w ith t h e b io assay v a lu e s in d ic a te d t h a t t h e chrom atographic method might a ls o be a p p lic a b le t o f i s h l i v e r o i l s f o r t i f i e d vdth i r r a d i a t e d ergo s t e r o l .

However, not enough o i l s of t h i s ty p e were t e s t e d t o recommend

u sin g t h i s method f o r them . When t h e chromet ogre phi c u l t r a v i o l e t a b s o rp tio n curve method i s ap­ p lie d t o low -potency o i l s , in v a r ia b ly th e p o te n c ie s calcu late d , v d l l be h ig h . T h is i s shown i n T a b le V II.

The average d e v ia tio n from t h e b io a ssa y v alu e

o f n in e d i f f e r e n t o i l s t e s t e d was 65.5%.

T h is m ethod, t h e r e f o r e , can be

a p p lie d only t o h ig h -p o te n cy o i l s . The most s u c c e s s fu l method f o r d e te rm in in g t h e p o te n c ie s of low valued o i l s was t h e two chrom atographic s te p u l t r a v i o l e t a b so rp tio n curve method.

P o te n c ie s e v a lu a te d f o r b o th i r r a d i a t e d e rg o s te r o ls and m u ltip le

v itam in s o lu tio n s a re given i n T ab le V I I I .

Out o f s ix i r r a d i a t e d e rg o s te ro ls

35 T ab le V I I I .

POTENCIES OF IRRADIATED ERGOSTEROLS AND MULTIPLE VITAMIN SOLUTIONS AS DETERMINED BY TITO CHROMATOGRAPHIC STEP UITRAVIOLET ABSORPTION CURVE METHOD. B ioassav

Sample No.

Type

E (1>S, 1cm.) 265 mu

U .S .P . Do U n its/G .

U .S .P . V it. A U n its/G .

881,000

1,000,000

...........

0339

Irra d ia te d E rg o s te ro l i n Corn O il

97559

Irra d ia te d Ergosfcerol i n Corn O il

8.46

846,000

1,000,000

...........

98449

Irra d ia te d Ergosfc e ro l i n Corn O il

4 .1 4

414,000

464,000

...........

5299

Irra d ia te d E rg o st e ro l i n Corn O il

4 .4 4

444,000

404,000.......................

96599

Irra d ia te d E rg o st e ro l i n Corn O il

1*40

140,000

151,000

...........

2519

Irra d ia te d Ergosfcerol i n Corn O il

0.0807 0.0797

8,070 7.970 Av. 8,020

11,250

...........

Syn I I I

C r y s ta llin e Dg i n Corn O il

0.lG2'5'^'''’'';‘'''''^ ''l6 7 '2 S 0 0.0900 9.000 Av. 9,615

9,680*

......

0899

57786

8 .8 1

C a lc u la te d Do U n its/G .

Irra d ia te d E rg o s te ro l in f i s h l i v e r o il

1 .7 5

175,000

250,000

33,800

Nat o la

0.115

11,500

11,000

55,000

* T h e o r e tic a l potency c a lc u la te d on b a s is o f vreignt o f c r y s t a l l i n e Dg p resen t i n sam ple.

36

T able V I I I .

(C o n tin u ed ). B ioassay

Sample Mo.

Type

E (1%, 1cm.) 265 mu

High D O il • (M ostly D „) 6

0.102 0.098

H a liv e r O il

4969 6299

0399

37756

U .S .? . Dg U .S .? . V it. A U n its/G . U n its/G .

10,220 9.800 10,010

10,630

12,200

0.170

17,000

10,000

60,000

N atola

0.222

22,200

11,000

59,800

O il Mix fo r N atola (75)5

0.0492 0.0588 0.0582

4,000

19,860

Av.

4,920 5,880 5.820 5,540

Av.

2,460 2,760 2.940 2,720

D r,, 2 5 $ D g)

ABDEC

C a lc u la te d Do U n its/G .

M u ltip le V ita ­ min S o lu tio n i n P o ly e th y le n e g ly c o l.

Av.

0.0246 0.0276 0.0294

1,590 (L abel claim )

7,950

37 RESULTS

m e one c r y s t a l l i n e Eg s o lu tio n i n co rn o i l , only one showed a v a r ia tio n from t h e b io a s sa y v a lu e above

and. t h a t was 2 8 .7 ^ .

t i o n from t h e b io a ssa y o f a l l seven sam ples was H : .l l/ j .

The average d e v ia ­ These o i l s

ranged from p a te n c ie s o f 1,000,000 down t o 9,680 u n i ts p e r gram and, a s shown i n T ab le IX, t h e method i s r e l i a b l e f o r o i l s having a potency as low a s £,000 - 5,000 u n its /g ra m .

The a b so rp tio n curves o b tain ed i n a l l

ca se s resem ble t h a t o f pure c a l c i f e r o l and have very l i t t l e extraneous a b s o rp tio n i n t h e low u l t r a v i o l e t re g io n s . T h is method was a ls o a p p lie d t o v a rio u s ty p e s of m u ltiv ita m in so lu ­ tio n s .

R e su lts f o r th e s e v a rie d somewhat, but t h e g r e a te s t d iffe r e n c e s

seemed t o be e x h ib ite d by th o s e samples c o n ta in in g high amounts o f v i t a ­ min P.

The a b so rp tio n curves i n most c a s e s , however, were very s im ila r

t o t h a t e x h ib ite d by pure c a l c i f e r o l :

Of t h e seven d i f f e r e n t m u ltiv ita ­

min s o lu tio n s t e s t e d , ran g in g from £50,000 t o approxim ately 1,590 v i t a ­ min Dg o r Dg u n i t s p e r gram, an average d e v ia tio n o f 41.7;© was o b ta in e d . In most c a se s t h e c a lc u la te d v a lu e s were h ig h e r th a n t h e b io a ssa y v a lu e . Experim ents were made t o d eterm ine t h e r e p r o d u c ib ility of both t h e s in g le chrom atographic ste p u l t r a v i o l e t a b so rp tio n curve method and th e antimony t r i c h l o r i d e c o lo r im e tr ic method. T ab le X.

These r e s u l t s a re shown in

F iv e s e p a ra te samples of o i l £*65464 were ru n by t h e chromato­

g rap h ic u l t r a v i o l e t a b s o rp tio n curve method and t h e maximum d e v ia tio n from t h e av erag e v a lu e was 8.7;$.

Ten d if f e r e n t d e te rm in a tio n s f o r o i l

7ff:5456 by t h e antim ony t r i c h l o r i d e c o lo rim e tric method showed a maximum d e v ia tio n from t h e average o f 1 .6 $ .

I

38

T able IX.

DILUTION SERIES OF OIL #6105 SHOWING LIMIT OF ACCURACY OF TWO CHROMATOGRAPHIC STEP ULTRAVIOLET ABSORPTION CURVE METHOD.

Wt. o f Samples, Grains

T h e o r e tic a l Potency U .S .P . Dg Units/Gram

E ( l £ , 1cm.) 265 mu

C alculirt ed Potency Dg Units/Gram

1.0027

15,400

0.1272

12,720

1.0087

13,400

0.1207

12,070

1.0100

13,400

0.0945

9,450

1.0175

6,860

0.0606

6,060

1.0099

5,380

0.0354

5,540

1.0170

1,652

0.0252

2,320

59

T ab le X.

REPR0DUCI3ILITY FOR IRRADIATED ERGOSTEROLS IN CORN OIL.

E (1!&, lcra.)

C a lc u la te d Dg U n its/G .

B ioasaay Dg U nits/G .

Chrom atographic U ltr a v io le t A b so rp tio n Curve Method, O il 7f65464. 187,000 182,000 184,500 176,000 185.000

2.28 2.09 2.12 2.02 2.10 Av.

200,000

182,500

Antimony T r ic h lo r id e C o lo rim e tric Method, O il #25456 492,500 488,500 471,500 501,000 501,000 505,500 509,500 497,000 501,000 509.500

25.52 25.50 24.42 25.96 25.96 26.18 26.40 25.74 25.96 26.40 Av.

497,700

500,000

40 RESULTS R e lia b le d e te rm in a tio n s were o b ta in e d by t h e antim ony t r i c h l o r i d e c o lo rim e tric method w ith o i l s c o n ta in in g a s low a s 10,000 v ita m in Dg u n its p e r gram and t h e i n d ic a tio n s a re t h a t o i l s o f much low er potency can be e v a lu a te d s u c c e s s f u lly . I n o rd e r t o determ in e how sm all an amount o f sample may be employed and s t i l l an a c c u ra te potency d e te rm in a tio n can be o b ta in e d by t h e a n t i ­ mony t r i c h l o r i d e c o lo rim e tric method, v a rio u s amounts o f a n o i l c o n ta in ­ in g about 10,000 v ita m in Dg u n i t s p er gram were put th ro u g h th e p ro ced u re. The r e s u l t s , a s shown i n T a b le XI in d ic a t e t h a t sam ples c o n ta in in g as low as 500 v ita m in Dg u n i t s can be assayed w ith a f a i r d eg ree o f r e l i a b i l i t y . However, when working w ith v e ry sm all amounts of t h e sam ple, i t i s neces­ sa ry t o add t h e rea g en t d i r e c t l y t o th e e th e r e x tr a c t r e s id u e .

O rd in a ri­

l y , i n a l a r g e sam ple, t h e r e s id u e i s ta k e n up i n chloroform and a 1-m l. a li q u o t ,o f t h i s s o lu tio n i s mixed w ith 10 m l. o f r e a g e n t.

T hus, th e

c o n c e n tra tio n o f t h e rea g en t i n t h e s o lu tio n c e l l i s s l i g h t l y more d i lu t e th a n t h a t i n t h e so lv en t c e l l .

The d if f e r e n c e cannot be d e te c te d on t h e

v is u a l sp ectro p h o to m eter a t 500 mu, how ever, and no a p p re c ia b le e r r o r i s in tro d u c e d by adding th e re a g e n t d i r e c t l y t o t h e dry r e s id u e . Although v ery sm all amounts o f v ita m in Do may be measured a s i n d i f+t

c a te d by T a b le X I, th e antim ony t r i c h l o r i d e c o lo rim e tr ic method i s not recommended f o r o i l s c o n ta in in g below 10,000 v ita m in Dg u n i t s p e r gram. Corn o i l a lo n e a ls o e x h ib i t s , t o a s lig h t e x te n t, t h e same c o lo r r e a c tio n w ith t h e antim ony t r i c h l o r i d e rea g en t a s v ita m in Dg, th u s in tro d u c in g a n o th e r e r r o r i n t h e d e te rm in a tio n .

41

T ab le X I.

EFFECT OF MOUNT OF SAMPLE UPON ACCURACY OF ANTIMONY TRICHLORIDE COLORIMETRIC METHOD.

YJt. o f Sample, Grams

1.000

C a lc u la te d Dp U n its i n Sample (Based on B ioassay)

C a lc u la te d E (1$, 1cm.) Potency 500 mu Dg Units/Gram

14,000

0.606

11,690

0.4992

7,000

0.475

9,130

0.2514

3,520

0.542

10,480

0.1262

1,769

0.654

12,220

0.0640

897

0.676

15,050

0.0572

521

0.537

10,370

0.0144

202

0.389

7,510

I

RESUIJS

E xperim ents were a ls o made t o d eterm ine how low' valued an o i l can be d eterm ined a c c u ra te ly by means o f t h e two chrom atographic s te p u l t r a ­ v io le t a b s o rp tio n curve m ethod.

I r r a d i a t e d e r g o s te r o l ,£'6105, having a

potency o f ap p ro x im ately 500,000 u n i t s p e r gram, was d i lu t e d w ith co rn o i l t o g iv e sumples ra n g in g from 13,400 u n i t s t o 1,652 u n i t s p er gram. Potency v a lu e s o f t h e d ilu te d samples were th e n determ ined by t h i s method and t h e r e s u l t s a r e shown i n T a b le IX. th e t r e a t e d samples a re shovm i n F ig . 13.

The a b s o rp tio n curves o f

T hese r e s u l t s in d ic a te t h a t

o i l s c o n ta in in g a s low a s 2,000-3,000 u n i ts p e r gram can be ru n v/ith a f a i r d eg ree o f a cc u ra c y .

43 DISCUSSION

S in ce v a rio u s p ro ce d u res i n t h i s study have been s ta n d a rd iz e d , i t seems f i t t i n g t h a t a d is c u s s io n a s t o how th ey were developed, t h e i r e f­ f e c tiv e n e s s , and th e a f f e c t o f v a rio u s f a c t o r s upon each o f them should b e made. S a o o n ific at i o n and E x tr a c tio n . The s a p o n if ic a tio n and e x tr a c tio n pro ced u re used i s e s s e n t ia ll y t h e same as t h a t o f Ewing et a l (12) w ith t h e ex ce p tio n t h a t a s in g le 40 m l. follow ed by t h r e e 20 m l. p o r tio n s o f e th e r were used f o r e x tr a c tio n in s te a d o f f iv e 20 m l. p o rtio n s o f e th e r .

T h is change h elp ed t o prev en t em ulsions

anc f a c i l i t a t e d f a s t e r and c le a n e r s e p a ra tio n s o f t h e e th e r and aqueous la y e rs . I n s p i t e o f a l l a tte m p ts to o b ta in o p t ic a l l y pure s o lv e n ts , c o n sid e r­ a b le ex tran eo u s a b s o rp tio n i s in tro d u c ed from re s id u e s i n th e so lv e n ts used i n t h e s a p o n if ic a tio n nnd e x tr a c tio n p ro ce d u re.

T h is was r e a d ily

shown by ru n n in g 10 m l. a lc o h o l samples a s a blank th ro u g h th e s a p o n if i­ c a tio n and e x tr a c tio n p ro ced u re and o b ta in in g a b s o rp tio n curves f o r a d i f ­ f e r e n t one a t su c c e ss iv e s te p s o f t h e p ro c e d u re .

The a b s o rp tio n curves

a re a l l s im ila r to t h a t e x h ib ite d by th e re s id u e c o n ta in e d i n 100 m l. o f anhydrous e th y l e th e r ( F ig . 14) and acco u n ts i n p a rt f o r t h e e x tr a o r d i­ n a r ily h ig h r e s u l t s o b ta in e d f o r p o te n c ie s c a lc u la te d on t h e b a s is o f th e a b s o rp tio n cu rv es of s a p o n ifie d o i l s .

44 DISCUSSION P r e c i p i t a t i o n o f S te ro ls * The d i g ito n in p ro ced u re was developed by determ in in g e x p erim e n tally what volume o f 1$ d ig it o n i n i n e th a n o l i s re q u ire d t o p r e c i p it a te com pletely .015 gm. o f e r g o s te r o l i n t h e p re sen c e of 5,000 u n i t s of c r y s t a l l i n e Dg d is s o lv e d i n 5 m l. a lc o h o l.

T hese p ro p o rtio n s were used

t o approxim ate t h e r a t i o o f s t e r o l s t o Dg p re s e n t i n .5 gm. o f a 10,000 u n it o i l , assum ing co rn o i l c o n ta in s ap p ro x im ately

s te ro ls .

From

t h e cu rv es shown i n F ig . 15, seven m l. o f l £ d ig ito n in s o lu tio n i s s u f­ f i c i e n t t o p r e c i p i t a t e t h e s te ro .ls p re s e n t o r approxim at ely 14 ml. p e r gram o f co rn o i l .

I n o rd e r t o have a s lig h t excess o f d ig ito n in p re s e n t,

20 m l. o f d ig ito n in s o lu tio n p e r gram o f o i l was f i n a l l y decided upon. Very l i t t l e d e c re a se i n ex tran eo u s a b s o rp tio n i s a c tu a lly made by t h e d ig ito n in tre a tm e n t and su b sta n c es which a re p r e c i p it a te d e x h ib it o nly a g e n e ra l a b s o rp tio n a s shown i n F ig . 10.

T hese curves were ob­

ta in e d u sin g a c r y s t a l l i n e Dp s o lu tio n i n c o rn o i l having a t h e o r e t i c a l * pot ency o f 9,930 u n it s/g ram . HC1 T re a tm e n t. The HC1 tre a tm e n t i s e s s e n t i a l l y t h e same as t h a t used by H e ilb ro n , Kamm, & Owen (19) vdth t h e e x c e p tio n t h a t S k e lly s o lv e B i s used a s t h e s o lv e n t.

The m a te r ia l removed by t h i s t r e a t m e n t , when t e s t i n g an i r ­

r a d ia te d e r g o s te r o l i n co rn o i l , has maxima a t 260, 270, and 280 mu and ap p ears t o be p a r t l y v ita m in Dg (F ig . 8 ). was d is c o n tin u e d .

For t h i s re a so n , t h e procedure

DISCUSSION

Large S u o e r f i l t r o l Column C h a r a c t e r i s t i c s . The m a te r ia l removed by a s in g le S u p e r f i l t r o l chrom atographic s te p i s r e a d i ly shown i n F ig . 9 .

A bsorption maxima a t 250 and 280 mu stro n g ­

ly in d ic a te t h a t t o x i s t e r o l and lu m is te r o l a re adsorbed upon t h e column and t h e v ita m in Dg p a s s e s on th ro u g h in to t h e e l u a t e .

The procedure

used i n t h i s experim ent was t h a t d e sc rib e d by Pow ell (31) u sin g a 9-cm. column o f S u p e r f i l t r o l and a so lv e n t m ixture o f 50 p a r ts S k e lly s o lv e , 10 p a r t s e th e r , and 1 p a r t a lc o h o l. S ince d i g it o n i n tre a tm e n t appeared t o make l i t t l e d if f e r e n c e i n t h e a b s o rp tio n cu rve o f t h e t r e a t e d sample and t h e HC1 tre a tm e n t seemed t o remove p a rt o f th e v ita m in Dp a s w e ll a s im p u r itie s b o th wer e d isc a rd e d and chrom atographic methods were r e s o rte d t o c o m p lete ly . For low -potency o i l s a tw o s te p chrom atographic p ro cedure was decided upon.

A S u p e r f i l t r o l column was employed f o r rem oval o f v ita m in s

A, pigm ents, c a ro te n o id s and p ro d u c ts o f i r r a d i a t i o n o th e r th a n v ita m in Dg.

T h is v/as fo llow ed by an alum ina column t o s e p a ra te v ita m in s D from

sq u alen e ty p e compounds and p o s s ib ly v itam in E.

An e x te n siv e study was

made t o d eterm in e t h e e f f e c t o f changing such f a c t o r s a s column le n g th , s o lv e n ts , and c o rn o i l c o n c e n tra tio n upon t h e d eg ree of s e p a ra tio n and potency e v a lu a tio n o f sam ples added t o each o f t h e two chrom atographic colum ns.

A ll t h e s e s tu d ie s were made u sin g s o lu tio n s o f c r y s t a l l i n e Dg

i n a lc o h o l o r corn o i l w ith t h e sam ples ran g in g i n potency v a lu e s from 10,000 - 20,000 u n i t s u n le s s o th e rw ise in d ic a te d . i n every c a s e , were o f 8-mm. in s id e d iam ete r.

The a d s o rp tio n columns,

46 DISCUSSIOH Alumina Column C h a r a c t e r i s t i c s . — (a) With S k e lly s o lv e a s th e s o lv e n t. S e p a ra tio n a c h ie v e d * — Yihen a hexane s o lu tio n o f t h e n o n sa p o n ifia b le f r a c t io n o f c r y s t a l l i n e Dg i n co rn o i l , having a potency o f approxim ately 20,000 u n i t s p er gram, i s added t o a s i x gm. column o f alum ina p re v io u s ly wefc w ith S k e lly s o lv e , t h e v ita m in D 960, -j»hich*,has

in , ^

t h i s study in which th e E ( l$ , 1cm.) @ 265 mu i s used a s a m easure o f t h e amount o f v ita m in s D p r e s e n t, i s o b ta in e d by d iv id in g t h e number o f v i t a ­ min Dg u n i ts p e r gram o f t h e sta n d ard c a l c i f e r o l (40,000,000) by th e E (l> , 1cm.) 265 mu, which i s 460.

Arnold ( l ) h a s in d ic a te d t h a t c a l ­

c i f e r o l c o n ta in s 49,000,DG0 u n i t s p e r gram.

Livingood (2 1 ), i n a s t a t i s t i ­

c a l study c a r r ie d on sim u lta n eo u sly i n th e s e l a b o r a to r i e s u sin g t h e s in g le chrom atographic s te p u l t r a v i o l e t a b s o rp tio n curve method fo r h igh-potency o i l s proposed i n t h i s in v e s ti g a t i o n , found t h a t t h e co n v ersio n f a c t o r should.

53 DISCUSSION

be more n e er 1 00,000.

F o r t h i s reaso n t h e 100,000 f a c t o r i s used

t e n t a t i v e l y f o r t h e Two Chrom atographic S tep U ltr a v io le t A bsorption Curve Method.

SUMMARY 1.

Very l i t t l e o r no c o r r e l a t io n can be mode between t h e u l t r a v i o l e t

a b so rp tio n c u rv es o f an u n tr e a te d o r s a p o n ifie d corn o i l s o lu tio n o f an i r r a d ie te d ergo s t e r o l and i t s v ita m in D potency v a lu e . 2.

Chemical tre a tm e n t o f t h e n o n sa p o n ifia b le f r a c t io n o f an i r r a d i ­

ate d e r g o s te r o l w ith d i g it o n i n o r anhydrous HC1 does not in c re a s e appre­ c ia b ly t h e accuracy o f t h e potency e v a lu a tio n of t h e o i l from i t s u l t r a ­ v i o le t a b so rp tio n c u rv e .

The anhydrous HCl p robably removes t h e v ita m in

Pr along vdth sq u alen e ty p e compounds. 3.

I n t e r f e r i n g su b sta n c e s can be s u c c e s s fu lly removed by chromato­

g rap h ic a d so rp tio n upon a S u p e r f i l t r o l column and an alum ina column. (a) V itam in A, c a ro te n o id s , pigm ents, anti, p o s s ib ly t o x i s t e r o l and lu m is te r o l a re removed by means o f a prewashed S u p e r f i l t r o l column u sin g e i t h e r a m ix tu re o f S k e lly s o lv e B and e th e r o r a S k e lly s o lv e -e th e r -a lc o h o l m ixture as t h e s o lv e n t.

The v ita m in s D

a re c o lle c te d i n t h e f i l t r a t e . ..(b) S olvent and S u p e r f i l t r o l r e s id u e s , end squalene ty p e com­ pounds can be removed by u se o f an alum ina column end a 1 :1 S k e lly s o lv e - e th e r m ix tu re a s t h e s o lv e n t.

The v ita m in s D a re re ta in e d

on t h e column and must be e lu te d v dth e th e r o r a d d itio n a l develop­ in g s o lu tio n . 4.

Two p h y s ic a l chem ical methods f o r d e term in in g t h e v ita m in D.,

co n te n t o f h ig h potency sam ples o f i r r a d i a t e d e rg o s te r o l i n corn o i l have been dev eloped.

55 StMJAEY

One m ethod, u s in g t h e u l t r a v i o l e t a b s o rp tio n curve o f t h e nonsaponif i a b l e f r a c t io n o f t h e o i l sample which had b een chrom atographed vdth S u p e r f i l t r o l t o s e p a r a te t h e im p u r itie s , gave an average v a r i a ti o n from th e b io a s s a y v a lu e o f 1 4 .5£ when 49 d i f f e r e n t o i l s were t e s t e d .

T h is

method i s recommended f o r o i l s c o n ta in in g 50,000 o r more v ita m in Dg u n its p e r gram, when one-gram sam ples a re u sed . A c o lo rim e ti’i c m ethod, u sin g t h e c o lo r r e a c tio n o f v ita m in Dr, o b ta in e d

by adding an antim ony t r i c h l o r i d e re a g e n t t o t h e n o n sa p o n ifia b le f r a c tio n o f t h e o i l sam ple, gave an av erag e v a r i a ti o n o f 1 5 .8> from t h e b io assa y v a lu e s o f 51 h ig h -p o te n cy o i l s and an average d if f e r e n c e o f IS .4/i from th e b io a s s a y v a lu e s o f s i r low -potency o i l s .

T h is method i s recommended

fo r o i l s o lu tio n s o f i r r a d i a t e d e r g o s te r o l, having a potency o f 10,000 v ita m in D^ u n i t s o r more p e r gram. 5.

A two chrom atographic s te p method f o r d eterm in in g t h e v ita m in D

c o n te n t o f b o th h ig h o r low potency i r r a d i a t e d e rg o s te r o ls in c o rn o i l end p o s s ib ly m u ltiv ita m in o i l s o lu tio n s has been developed. f r a c t i o n o f th e o i l i s firs t- chrom atographed u sin g e four-cra. S u p e r f i l t r o l column.

The v ita m in s D c o lle c te d i n th e

f i l t r a t e a re th e n adsorbed upon an alum ina column f o r f u r t h e r p u r i f ic a t i o n . The u l t r a v i o l e t a b s o rp tio n curve o f t h e e lu te d v ita m in s D i s th e n used t o e v a lu a te th e potency o f t h e o r i g i n a l o i l . The av erag e d e v ia tio n from t h e b io a sse y v a lu e f o r s i x irra d ia te d , e r g o s te r o ls and one c r y s t a l l i n e Dg s o lu tio n i n corn o i l v/as ir ..ll> »

These

f

l

56 SUHMAIff

ranged from 1 ,000,000 t o 9,680 u n it s /g r a n .

The method was shown t o be

a c c u ra te f o r o i l s having p o te n c ie s a s low as 2,000 - 3,000 u n its/g ra m d i lu t i n g a h ig h e r valued o i l t o t h i s ran g e. Sever: m u ltip le v ita m in s o lu tio n s t e s t e d by t h i s method ranging from £50,000 t o 1,590 u n its /g ra m showed an average v a r i a ti o n of 41 .7 $ .

57 LITERATURE REFERENCES 1

Arnold, A., P ro c . Soc. E x p t. B i o l . Med., 63, 230 (1946).

2

Baker, D. H ., ftf. S. t h e s i s , Michigan S t a t e C o lle g e, 1944.

•Z u

Brockmann, H ., Z. p h y s i o l . Chem., 241, 104 (1936).

4

Brockmann, H ., snd Chen, Y ., Z. p h y s io l. Chem., 241, 104 (1936).

5 B u lla r d , L. J . , Michigan S t a t e C o lle g e, M. S. T h e s is , (1945). 7

Campbell, J . A ., Anal. Chem. 20, 766-71 (1948).

8

C h riste n s o n , E ., Munch. Med. Wachechr., 75, 1883 (1928).

9

DeWitt, J . B ., and S u l l i v a n , M. X ., In d . Eng. Chem., Anal Ed., 18, 117-119 (1946).

10

Drummond, S in g e r, and McWalter, Bio chem. J . , 29, 457 (1935).

11

Emmerie, A., and E ekelen, M. van, Acta B re v ia Neerland. P h y s io l. Pharmacol. M ic r o b io l., 6,, 135 (1936).

12

Ewing, D. T . , K ingsley, G. V ., Brown, R. A ., and Emmett, A. D., In d . Eng. Chem., Anal. Ed.‘, 15, 301 (1943).

13

Ewing, D. T . , end Tomkins, F . , Michigan S t a t e C o lleg e, Ph. D. T h e s is (1942).

14

F i t e l s o n , Assoc, of O ff. Agr. Chem., 26, 506 (1943).

15

Gudlefc, I . , P ro c . S c i . I n s t . Vitamin Research U .S .S .R ., 3, No. 1, 55 (1941).

16

Hsge, J . , M. S. t h e s i s , Michigan S t a t e C o lle g e, 1943.

17

Halden, W., N a tu rw isse n sc h a fte n , 24, 296 (1956).

18

Halden, W., end T zo n i, H ., N ature, 157. 909 (1956).

19

H e ilb ro n , Kamm, and Owen, J . Chem. S o c .,

20

Levine, J . , Biochera* J . , 27, 2047 (1935).

21

Livingood, A ., Michigan S t a t e C o lle g e, T h e s is (Forthcom ing).

22

Marcussen, E ., Pansk. T i d s . Farm., 13, 141 (1939).

M ilas, N., H eggie, R ., and Raynolds, J . , Ind. Eng. Chen., Anal. E d ., 13, 227 (19 41). M i l le r , S. E ., ( t o G eneral M i l ls , I n c .) U. S. 2,179, 560 Nov. 14 (1939). M u ller, P aul B ., Helv. Chini. Acta .50, 1172-90 (1947). N ie ld , C ., R u s s e l l , W., and Zimmerli, A. J . , J . B io l. Chem., 156, 75 (1940). 01 K'nin, E . , P ro c . S c i . I n s t . Vitamin Research U .S .E .R ., 5, No. 1, 28-9 (1941). Oser, B. L ., M elnick, D ., and P ader, M., In d . Eng. Chera., Anal. E d ., 15, 717 (1943). P in k e rto n , R ., Michigan S t a t e C o lleg e, M. S. T h e s is (1948). P i r l o t , G. and R o u ir, E. V. (Univ. L iege, Belgium) B u ll. se e . chim. B eiges 56, 296-308 (1947). Pow ell, M. J . , Michigan S t a te C o lle g e, M. S. T h e s is (1946). Raoul, Y ., and Meunier, P . , Compt. r e n d . , 209. 546 (1939). R eerink , E . , and Wijk, A. van, Chen. Weekblad., 29, 645

(1932).

R i t s e r t , K ., M erck's J a h r e s b e r . , 52, 27 (1938). Robinson, F . , Chemistry & I n d u s t r y , 56, 191 (1937). R u tk o v sk ii, L. A ., Biokhiraiya, 5, 528-54 (1940) S c h a lte g g e r, Herm. , Helv. Chim.

Acta

29, 285-302 (1946).

S hantz, E ., M., I n d . Eng. Chen. Anal. E d ., 16, 179 (1944). Shear, M. J . , P ro c . Soc. E x p tl. B io l. Med. 23, 546 (1925). Sobel, A. E ., Mayer, A. Margot, and Kramer, B e n j., In d . Eng. Chem. Anal. Ed. 17, 160-5 (1945). Solyanikova, V. L ., Biokhiraiya, 4 , 483-91 (1939). S t o e l t z n e r , W., Munch, med. Yiochachr., 75, 1584 (1928). Thorbjamarson and Drummond, Analyst ,60, 23 (1935).

59 (44) Topelraann, H ., and Schuhknecht, W., Z. V ita m in fo rs c h ., 4, 11 (1955). (45) Windaus, A ., and Stange, Z. p h y s i o l . Chem., 244, 218 (1956). (46) W olff, L . , Z. V ita m in fo rs c h ., 7, 277 (1958).

L

(47) Young, R ., Michigan S t a t e C ollege, Ph. D. T h e s is (1945). (48) Zimmerli, A ., N ie la , C ., and R u s s e l l , W., J . B i o l . Chem., 148, 245 (1945).

100

90

Per Cent

T ransm ission

80

70 Oi o

60

50

40

50 200

210

220

250

240 250 260 Wave Length i n Mu.

270

F ig ure 1. - T ransm ission curves o f p u r i f ie d s o lv e n ts as compared t o w ater: solve B; 5, anhydrous e th y l e t h e r .

280

290

500

1, 95% e th a n o l; 2, S k e lly ­

30

40 fa

O

SO

20

200

F igure 2.

220

250

240

250 280 Y.ave Length i n Mu

Absorption Curve of Corn O il i n E thanol.

270

280

290

500

JL.UO

.90

.80

.70

.60

M > .50 o a .S .40 o c: •r-4

s .50

,

.SO

.10

0 :so Wave Length i n Mu. F igure 3.

•'80

Absorption Curve of I r r a d i a t e d E rg o s te r o l $69954 i n Ethanol Ran With an E thanol S o lu tio n o f Corn O il in t h e Solvent C e ll.

I

1.50

1.40

1.S0

1.20

230 F igure 4 .

240

250

270 260 Wave Length i n Mu.

280

300

Absorption Curve o f I r r a d i a t e d E rg o s te ro l #65624 i n Ethanol A fter S u p o n if ic a tio n .

i

64

E xtinction ,

Log I q/ I

1 .00

230 F ig u re 5.

240

250

260 270 Wove Length i n Mu.

260

290

500

Absorption Curve o f S a p o n ifie d I r r a d i a t e d E rg o s te r o l #69934 i n E thanol Ren With an E thanol S o lu tio n o f S ap o n ified Corn O il i n t h e Solvent C e ll .

65

Log I Q/ l

.50

E x tin ctio n ,

1.00

40

30

.10

240

250

£60 270 Wave Length i n Mu.

280

w s

"300

Figure 6 . - A bsorption curve o f o i l /JB-11299 in ethanol a f t e r s a p o n if ic a tio n and chrcmatograpning through column of S u p e r f i l t r o l : 1, t r e a t e d o i l 5-11299; 2, sta n d ard c a l c i f e r o l .

66 1 .0 0

.90

.80

.70

.60

E x tin c tio n

.50

.40

.30

20

.10

230 F ig u re 7.

240

250

260 270 Wave Length in Mu.

260

290

300

A bsorption Curve of Low Potency I r r a d i a t e d E rg o s te r o l £0905 A fter C arrying I t Through t h e S in g le Chromatographic Step U l t r a v i o le t A bsorption Curve Method. ( S u p e r f i l t r o l Column).

67 1.30

.90

.60

.70

E xtin ctio n,

Log I q/ I

.60.

.40

.53

.10

250

240

250

260 270 Wave Length in Mu.

500

F ig ure 8. - C a lc u la te d a b s o rp tio n curve o f m a t e r i a l removed from corn o i l s o l u t i o n of i r r a d i a t e d e r g o s t e r o l £73978 by HC1 tre a tm e n t: 1, O il £73978 a f t e r s a p o n i f ic a t i o n ; 2, O il £73978 a f t e r s a p o n i f i ­ c a t io n and HC1 tr e a tm e n t; 3, curve 1 minus curve 2 ( m a te r ia l removed by HC1 t r e a t m e n t ) .

68 1.0 0

80

H s 50

250

240

250

270 260 Wave Length i n Mu.

280

290

500

Figure 9. - C a lc u la te d a b so rp tio n curve of m a t e r i a l removed from corn o i l s o l u t io n of i r r a d i a t e d e r g o s te r o l £75976 by a column of Super­ f i l t r o l : 1, O il £75978 a f t e r s a p o n i f ic a t i o n ; 2, O il £75978 a f t e r s a p o n i f ic a t i o n and. chromatographing with a column o f S u p e r f i l t r o l ; 5, curve 1 minus curve 2. ( m a t e r i a l removed by S u p e rfiltro l).

i

69 1.00

.90

.60

.70

50

E xtinction,

Log I 0/ l

60

240

250

260 270 Wave Length i n Mu.

300

F ig u re 10. - Absorption curve o f m a t e r i a l removed from corn o i l s o l u t io n of c r y s t a l l i n e v itam in Pg by d i g i t o n i n t r e a tm e n t: 1, Dg s o lu tio n a f t e r s a p o n i f i c a t i o n ; 2, Dg s o l u t i o n a f t e r s a p o n i f ic a t i o n and d i g i t o n i n tr e a tm e n t; 5, curve 1 minus curve 2. ( m a te r ia l removed by d i g i t o n i n ) »

i

70 1.00

.90

.60

•rl

•H .50

.10

2 6 0 2 7 0 Wave Length in Mu.

280

290

300

F ig u re 11. - Absorption cu rv es o f i r r a d i a t e d e r g o s t e r o l s i n o i l a f t e r t r e a t ­ ment by t h e Two Chromatographic Step U l t r a v i o l e t Absorption Curve method: 1, O il #0339, 1,000,000 u/gm .j 2, O il #96559, 151,000 u/gm .; 5, O il #98449, 464,000 u/gm .; 4 , O il #'2319, 11,250 u/gm.

71 1.00

.80

.70

.60

.50 o .40

.50

.20

.10

250

240

250

260 £70 Wave Length i n Mu.

280

£90

500

F igure 12. - A bsorption curves of v a rio u s m u ltip le vitam in s o lu tio n s a f t e r tr e a tm e n t by t h e Two Chromatographic Step U l t r a v i o le t Absorp­ t i o n Curve method: 1, ABDEC, m u ltip le v ita m in s o lu tio n i n e th y le n e glycol} 2, H a liv e r O il #57756} 5, N atola #57786} 4, High D o i l (m ostly Ds ) #0399} 5, O il mix f o r Natola (75£ Dg, 2b% D j #6299. o

.

72 1.0 0

.90

.60

.70

in c tio n , Log I Q/ i

.60

.50

.40

.1 0

230

240

250

260 270 Wave Length i n Mu.

23U

300

F ig u re 13. - A bsorption curves o f v a rio u s d i l u t i o n s o f i r r a d i a t e d e r g o s t e r o l #6105 with corn o i l which were t r e a t e d by t h e Two Chromatographic Step U l t r a v i o l e t A bsorption Curve method. D ilu tio n s ran were; (1) 13,400 Dg u/gm .; (2) 6,660 Dg u/gra.; (3) 3,380 Dg u/gra.; (4) 1,652 Dg u/gm.

72 1.00

.90

.60

.70

60

M

•N

r; o

•H 40 o c •H w

230

240

250

260 270 Wave Length i n Mu.

■300

F ig u re 13. - A bsorption curves o f v a rio u s d i l u t i o n s o f i r r a d i a t e d e r g o s t e r o l #6105 vdtn corn o i l v\hich vere t r e a t e d by t h e Two Chromatographic Step U l t r a v i o l e t A bsorption Curve method. D ilu tio n s ran were: (1) 13,400 Dg u/gm .; (*2) 6,660 Dg u/gm .; (3) 3,380 Dg u/gra.; (4) 1,652 Dg u/gm.

1.4

Log I Q/ l

1.2

E x tin ctio n ,

e*

210

220

250

240

250

260 270 Wave Length i n Mu.

280

290

300

310

Figure 14. - Absorption curve o f blank sample (10 ml. eth anol) a t t h e end o f v a rio u s s te p s i n t h e saponi­ f i c a t i o n p ro c e d u re ;: ( 1 ) a f t e r s a p o n if ic a tio n and e x tr a c t i o n ; ( 2 ) a f t e r s a p o n if ic a tio n , e x tr a c t i o n , and washing; (3) a f t e r s a p o n if ic a tio n , e x tr a c t i o n , washing, and d rying through NS2 SO4 ; (4) r e s id u e from 100 ml. e th y l e th e r (a n h y d .).

74 .50

.45

.40

E x tin c tio n ,

Log

I Q/ l

.35

.15

.05

240

250

2 6 0 2 7 0 V/ave Length i n Mu.

280

Figure 15. - A bsorption curves of an e r g o s t e r o 1 - c a l c i f e r o l m ixture a f t e r t r e a t i n g with v a rio u s amounts o f 1£> a i g i t o n i n . Amounts °*'e * (1) u n tr e a te d s o l u t io n ; (2) 2 m l.; (3) 3 m l.; (4) 4 m l.; (5) 6 m l.; ( 6 ) c a lc u la te d curve o f c a l c i f e r o l p r e s e n t.

75 1.00

.60

.70

.60

.50 JH O H

to O

.40 c o •H o c •M

20

.10

50

250

260 270 Weve Length i n Mu.

280

290

500

F igure 16. - Absorption cu rv es of v a rio u s f r a c t i o n s of t h e n o n sap o n ifiab le p o rtio n of c r y s t a l l i n e Bg in corn o i l ta k e n as f i l t r a t e from 6 gram alumina column using S k e ily s o lv e as t h e s o lv e n t. Volumes f o r each f r a c t i o n a re l i s t e d in o rd er tak e n : (1) 4 m l.; (2) 4 m l.; (3) 4 m l.; (4) 5 m l.; (5) 5 ml.

i

76 1 .00

.90

.80

70

.60

n c tic n

til o .j

.50

40

50

.20

.10

0 250

250

260 Wave Length i n .Mu.

260

290

300

F ig ure 17. - A bsorption cu rv es of v a rio u s f r a c t i o n s of t h e n o n sa p o n ifia b le p o r t io n of c r y s t a l l i n e Dg in corn c i l ta k e n as e lu n te from 6 grams alumina column usin g S k e lly s o lv e as t h e s o lv e n t. Volumes f o r each f r a c t i o n a re l i s t e d i n o rd e r ta k e n : ( l ) 5 m l.; (2) 5 m l.; (3) 5 m l.; [4) 5 m l.; (5) 5 m l.; (6) 5 ml.

265 mu.

3.0

E xtin ctio n ,

Log I Q/l

2 .0

-o -3

1.0

P e r c o la te F r a c tio n Number (5 ml. each) F igure 16. - Approximate t h r e s h o ld volume of 4 gram alumina column f o r vitam in D0 u sing v a rio u s Skeilyso lv e -a ic o h o l m ixtures a s t h e s o lv e n t. S k e lly s o lv e -a lc o h o l r a t i o s used were: ( 1) 100: 1; (2) 50:1; (3) 5 0 :3 ; (4) 50:5; (5) 5 0 :8 .

50

40 lO

-aO C 20

•rl

- .1 0 P e r c o la te F r a c tio n Number (5 ml. each) F ig ure 19. - Approximate th r e s h o ld volume o f 4 gram alumina column f o r vitam in Dg using v a rio u s b kellysolvee th e r m ixtures a s t h e s o lv e n t. S k e lly s o lv e - e tn e r r a t i o s used were: (1) 50:1; (2) 50:3; (3) rrv .c .

f

a

\

C A .Q .

{

(&\

RlliSO .

M

•H

250

240

£50

£60 270 Wave Length i n Mu.

£80

£90

500

F ig ure £0. - Absorption curve of r e s id u e e lu te d from 4 gram alumina column by su c c e ss iv e 5 ml. p o rtio n s o f e th e r : 1, f i r s t p o rtio n ; 2> second p o r tio n ; 3 , t h i r d p o r t io n .

80

\

j

m

A - Small V ia ls Used As R eceivers. B - C a rria g e For V ia ls . ( S lo ts For V ia ls Centered on C irc le of Radius R.) C - Chromatograph Tube - 8 mm. diam eter. D - C otton Plug. E - Large Rubber Stopper. F - Base o f Brotvn B o t t l e .

To A sp ira to r

F ig u re £1.

S p e c ia l Apparatus Designed For C o lle c tin g F ra c tio n s From Chromatograph Column Without D istu rb in g E quilibrium .

81 1.00

.50

E x tinction,

Log l Q/ i

.60

.30

10 .10

230

250

260 Wave Length i n Mu.

300

Figure 22. - A bsorption cui'ves of v a rio u s f r a c t i o n s of 1 0 ,0 j 0 u n i t s c r y s t a l l i n e D ta k e n from alumina column. Volumes f o r each f r a c t i o n ere Listed in o rd e r ta k e n : (1) 3 m l.; (2) 5 m l.; (3) 3 m l.; (4) 2 m l.; (5) 2 m l.; (6) 2 m l.; (7) 2 m l.; (8) 2 m l.; (9) 2 m l.; (10) 2 m l.; (11) 2 ml.

82 1 .00

E xtinction.

Log I q/ I

.80

.40

20

250

260 270 Wave Length i n Mu.

280

290

300

F ig u re 23. - Recovery o f c r y s t a l l i n e Dg from alumina column: 1, u n tre a te d Dg s o l u t io n i n 1 :1 S k e i ly s o lv e - e t h e r ; 2, same Dg s o l u t io n c a r r i e d thro ugh alumina chrom atographic procedure.

85

1.00

.90

.80

.70

E xtinction,

Log I q/ I

.60

.50

.40

.30

20

.10

230

240

250

260 270 Wave Length i n Mu

280

290

300

F ig u re 24. - Absortition cu rv es o f v a rio u s f r a c t i o n s of t h e n c n s a p o n ifis b le p o r tio n o f c r y s t a l l i n e Dg i n corn o i l (9,660 u/gm.) t a k e n from t h e alumina column. ( S u p e r f i l t r o l ste p p re c e d in g ). Volumes f o r each f r a c t i o n a r e l i s t e d i n o rd er tak en : (1) 2 m l . ; (2) 2 m l.; (3) 2 m l.; (4) 2 m l.; (5) 3 m l.; (6) 3 m l.; (7) 3 m l.; (6) 3 ml.

Extinction,

Log

I 0/i

84

.10

250

260 '275" Wave Length in Mu.

F i g u r e 25. - A bsorption cu rv es of v a rio u s f r a c t i o n s o f t h e nonsaponifi&ble p o rtio n o f Natoln o i l ta k e n from t h e alumina column. (Superf i l t r o l s te p p re c e d in g ). Volumes f o r each f r a c t i o n a re l i s t e d i n o rd e r ta k e n : (1) 2 m l.; (2) 3 m l.; (3) 3 m l.; ( 4 ) 2 m l.; (5) 2 m l . ; (6) 2 m l.; (7) 5 m l.; (8) 3 ml.

85 1 . 10

1.00

ion

60

40

30

10 ?50

260 270 Y.ave Length in Hu.

ZOO

F ig u re 26. - Comparison of f i l t r a t e from 4 cm. and 3 cm. S u p e r f i l t r o l Columns using t h e n o n s a p o n ifia b le f r a c t i o n of 1 gram of I r r a d i a t e d e r g o s t e r o l #6105 as t h e s o l u t e : 1, f i l t r a t e from 9 cm. column; 2, f i l t r a t e from 4 cm. column.

I