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<, can be se p a ra te d t o some e x te n t from
th e i n t e r f e r i n g m a te r ia ls i n co rn o i l .

T h is i s shown i n F ig s . 16 and 17.

A tte n tio n must be c a l le d , however, t o t h e f a c t t h a t t h e S k e lly so lv e de­
v e lo p e r does not c a rry th ro u g h a l l t h e i n t e r f e r i n g su b sta n c es o f c o rn o i l
w ithout u sin g e x c e s s iv e ly l a r g e amounts o f s o lv e n t.

T h is i s evidenced by

th e shape o f t h e a b s o rp tio n c u rv es f o r t h e f i r s t and l a s t f r a c tio n s o f t h e
e lu a te i n F ig . 17.
E ffe c t o f changing le n g th o f column. — Very l i t t l e change i n t h e ab­
s o rp tio n cu rv e o f t h e e lu a te i s o b tain ed when th e le n g th o f th e alum ina
column i s v a r ie d .

I f t h e n o n sa p o n ifia b le f r a c t i o n o f c r y s t a l l i n e Dg i n

corn o i l i s t r e a t e d i n t h e same manner a s i n p a rt (1) u s in g 30-ral. S k e lly so lv e a s t h e d ev elo p er and d is c a rd in g t h e f i l t r a t e u i p t o t h a t p o in t, t h e
e lu a te o b ta in e d , by p a ssin g 30 m l. e th e r th ro u g h v a rio u s le n g th s o f a lu ­
mina colum ns, gave t h e fo llo w in g e x tin c tio n s :
Wt. i n grams
o f Alumina
lolumn

Wave Length in mu
235

245

255

262

264

266

270

280

290

500

1
2
5
4
6

.547
.567
.558
.516
.351

.584
.395
.383
• 559
.373

.428
.452
.428
.411
.422

.452
.456
.448
.448
.442

.456
.458
.450
.450
.442

.455
.455
.447
.448
.439

.439
.459
.429
.451
.423

.544
.547
.354
.338
.334

.212
.214
.204
.207
.202

.109
.109
.099
.100
.096

DISCUSSION
R e s u lts in t h i s t a b l e in d ic a t e t h a t t h e Dp i s very t i g h t l y h e ld
by t h e a d so rb e n t.

The f a c t t h a t Sudan I I I , having a d s o rp tio n c h a r a c te r ­

i s t i c s s im ila r t o Dg, rem ains n e a r t h e to p o f th e column throughout t h e
development o f t h e chromatogram a ls o te n d s t o confirm t h i s .

A d d itio n of

amounts a s h ig h a s 100 m l. o f S k e lly s o lv e t o t h e column f a i l e d t o c a rry
th ro u g h any o f t h e v ita m in Dp i n to t h e e lu a te .

For t h i s rea so n no t h r e s h ­

old volumes u sin g t h i s so lv e n t were determ ined f o r t h e column.
I f t h e S u p e r f i l t r o l chrom atographic s te p , u sin g t h e long 9-cm. column,
p reced es t h e alum ina chrom atographic p ro ce d u re, v ery l i t t l e d if f e r e n c e i s
observed i n th e r e s u l t s , ' except t h a t s l i g h t l y l a r g e r a b s o rp tio n i s e x h ib ite d
i n t h e £550 mu re g io n when lo n g e r alum ina columns a re u se d .

T h is i s shov/n i n

t h e fo llo w in g t a b l e :
7ft ./g ra m s
o f Alumina
in Column

Wave Length i n mu
255

1
2
4

.348
.402
.398

£45

£55

£62

£64

266

270

£80

£90

500

.593 .442
.423 .459
.423 .461

.458
.473
.472

.453
.471
.470

.448
.463
.462

.428
.441
.438

.322
.528
.314

.197
.194
.179

.103
.098
.086

These r e s u l t s were a ls o confirm ed when s o lu tio n s o f c r y s t a l l i n e Dc in a l K>

cohol were s u b s titu te d i n p la c e o f t h e sa p o n ifie d co rn o i l s o lu tio n s .
S ince com plete s e p a r a tio n i s not achieved u sin g S k e lly s o lv e as t h e s o lv e n t,
v a rio u s so lv en t m ix tu re s were t r i e d .
(b)

With Hexane - A lcohol M ixtures As t h e D eveloping S o lu tio n . -T h e

e f f e c t o f adding sm all amounts o f a lc o h o l t o S k e lly s o lv e and u sin g t h e
m ix tu re a s a d e v elo p in g s o lu tio n f o r t h e alum ina chrom atographic ste p i s
g r a p h ic a lly shovm i n F ig . 18.

A pproxim ately 10,000 u n i t s of Dg i n one m l.

S k e lly s o lv e were added t o t h e wet alum ina column, and f iv e m l. p o rtio n s of

48
DISCUSSION

th e d ev elo p in g s o lu tio n were used t o develop t h e chromatogram, saving
t h e co rresp o n d in g f r a c t i o n s o f th e f i l t r a t e .

The approxim ate th r e s h o ld

volumes f o r each so lv e n t m ix tu re were determ ined try p l o tt i n g t h e ex­
t i n c t i o n o f t h e f i l t r a t e f r a c t i o n @ 265 mu a g a in s t t h e number o f t h e
f ra c tio n .

Any g re a t in c r e a s e i n e x tin c tio n a t 265 mu in d ic a te d t h e

p resen c e o f Dg i n t h e f i l t r a t e and i n a l l c a se s i f a la r g e in c re a s e
o c c u rre d , t h e com plete a b s o rp tio n curve resem bled t h a t o f p ure Dp.
No f u r t h e r work was done u sin g S k e lly s o lv e -a lc o h o l m ix tu res a s a
so lv e n t b e cau se i t was found t h a t i n t e r f e r i n g su b sta n c e s o f co rn o i l
were a ls o c a r r ie d i n to t h e f i l t r a t e along w ith th e Dg.
(c)

With S k e llv s o lv e -E th e r M ixtures As th e D eveloping S o lu tio n . —

The approxim ate th r e s h o ld volume f o r t h e alum ina column, u sin g S k e lly s o lv e - e th e r m ix tu res a s t h e s o lv e n t, was determ ined i n t h e same manner
as f o r t h e S k e lly s o lv e -a lc o h o l m ix tu res i n p a r t (b) •
F ig . 19.

T h is i s shown i n

The sm all in c r e a s e i n e x tin c tio n i n t h e f i r s t f r a c t i o n in d i­

c a te s t h a t a r e s id u e i s e lu te d from t h e alum ina.

A b so rp tio n cu rv es of

t h e r e s id u e e lu te d from a fo u r gm. alum ina column by su c c e ssiv e fiv e -m l.
p o r tio n s o f e th e r i s shown i n F ig . 20.
i s re q u ire d t o red u ce t h i s t o a minimum.

Approxim ately 10 m l. o f e th e r
The c h a r a c t e r i s t i c s o f a p re ­

washed column o f alum ina were th e n d ete rm in e d .
A s p e c ia l a p p a ra tu s w ith which f r a c t io n s o f t h e p e rc o la te could be
ta k e n w ithout d is tu r b in g t h e e q u ilib riu m o f t h e column was c o n stru c te d
(F ig . 2 1 ).

A pproxim ately 10,000 u n i ts o f c r y s t a l l i n e Dg i n t h r e e m l. o f

1 :1 S k e lly s o lv e - tth e r s o lu tio n was added t o a fo u r gm. column o f alum ina

DISCUSSION
which had been prewashed w ith 10 m l. o f e th e r .

The a b s o rp tio n cu rv es

o f t h e v a rio u s f r a c t io n s ta k e n a re shown i n F ig . 22.
From t h e s e cu rv es i t a p p ea rs t h a t t h e f i l t r a t e o b ta in e d up t o t h e
p o in t v.'here e ig h t m l. o f d ev elo p in g s o lu tio n h a s p assed th ro u g h t h e
column, may be d isc a rd e d w ithout lo s s o f v ita m in Dp.

The Dg can be

q u a n tita tiv e ly re c o v ere d from t h e alum ina column a t t h i s p o in t by
e lu tin g w ith 15 m l. e th e r .

The recovery o f t h e Dp (10,000 u n its )

from th e column i s shown by com parison of t h e a b s o rp tio n curves i n
F ig . 23 o f an u n tr e a te d sample and a sample c a r r ie d th ro u g h t h e p ro ­
cedure u sin g e ig h t m l. o f t h e 1 :1 developing s o lu tio n follow ed by e lu ­
t i o n w ith 15 m l. o f e th e r .
I n o rd e r t o d eterm ine i f t h i s same s e p a ra tio n and rec o v ery could
be o b tain ed f o r c r y s t a l l i n e Dp i n corn o i l , a s o lu tio n was p rep a red
t o c o n ta in 9,680 u n i t s Dp p er gram.

The n o n sa p o n ifia b le p o r tio n o f a

1.000 gram sample o f t h i s s o lu tio n was chrom atographed th ro u g h S uperf i l t r o l and th e n added t o a fo u r gm. alum ina column a s above.

Ab­

s o r p tio n cu rv es o f v a rio u s f r a c t io n s o f t h e f i l t r a t e from t h e alum ina
column a re shown i n F ig . 24.

H ere a g a in , i t a p p ears t h a t t h e f i l t r a t e

may be d isc a rd e d up t o t h e p o in t where e ig h t m l. o f d eveloping s o lu tio n
h a s passed th ro u g h t h e column.
Two a d d it i o n a l sam ples were ru n d is c a rd in g 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, and e lu tin g t h e Dp from t h e column w ith 15 m l. o f e th e r .
The reco v ery o f t h e Dr, i s q u a n ti t a t iv e a s shown by t h e fo llo w in g t a b l e :

50
DISCUSSIOK

Sample
1
2

E (1 %, lcrn.)
.1025
.0900

C a lc u la te d B
U nits/gm .

T h e o re tic a l
Dg Unit a/gm.

10,230
9,000
Av. 9,615

9,680

A s im ila r study was mede v.ith a N a to la o i l and a b s o rp tio n cu rv es
o f v a rio u s f r a c t i o n s o f t h e f i l t r a t e from t h e alum ina column a r e shown
in F ig . 25.

From t h i s s e t o f curves i t a p p ea rs a ls o t h a t e ig h t m l. of

d e v elo p er may be passed th ro u g h t h e column and t h e f i l t r a t e d isc a rd e d
v ath o u t l o s s o f v ita m in s B.

E lu tio n w ith e th e r i s not p r a c t i c a l , how­

e v e r, sin c e e x tra n e o u s m a te r ia l i s e lu te d a f t e r 9 - 1 2 m l. o f a d d itio n a l
d e v elo p er h as p assed th ro u g h .
To check t h e r e s u l t s from th e s e c u rv e s, 1 /2 m l. of each o f th e s e
f r a c t io n s was t r e a t e d vdth antim ony t r i c h l o r i d e re a g e n t and i t s e x tin c ­
t i o n a t 500 mu was m easured on t h e sp e c tro p h o to m e te r.

R e s u lts were as

fo llo w s:

F iltra te
F r a c tio n

Bog I Q/ l
(j? 500 mu

Remarks

1
2
5
4

.2 7
.2 7
.25
.58

C h erry -re d
C h e rry -re d
C h e rry -re d
T y p ic a l B0

c o lo r produced
c o lo r produced
c o lo r produced
orange c o lo r produced

5
6
7

.40.
.3 7
.22

T y p ic a l
T y p ic a l
T y p ic a l

Dg orange c o lo r produced

8

.2 2

T y p ic a l

Dg orange c o lo r produced

tv

Dg orange c o lo r produced
Dg orange c o lo r produced

51
DISCUSSION
Thus, b o th th e u l t r a v i o l e t a b so rp tio n cu rv es and t h e c o lo r r e a c tio n vdth
antimony t r i c h l o r i d e rea g en t o f th e s e f r a c t io n s in d ic a te com plete sepa­
r a tio n o f t h e v ita m in Dg from o th e r i n t e r f e r i n g compounds.
Small S u p e r f i l t r o l Column C h a r a c te r is tic s :
The s e p a ra tio n achieved by a six-cm . S u p e r f i l t r o l column fo r both
f is h l i v e r o i l s and i r r a d i a t e d e rg o s te r o ls u sin g a S k e lly s o lv e -e th e r r-lcohol m ix tu re a s a so lv en t h as been d isc u sse d by v a rio u s a u th o rs (£ , I t ,
12, 16, 4 7 ) .

Pow ell (51) found t h a t by in c re a s in g t h e le n g th t o 9-cm,

and prew ashing t h e column vdth t h e solvent m ixture a q u a n tita tiv e separa­
t io n o f v ita m in Dg could be o b tain ed from i r r a d i a t e d e r g o s te r o ls in vo la­
t i l e s o lv e n ts .

The lo n g e r column was th e n a p p lie d t o high-potency i r r a ­

d ia te d e r g o s te r o ls i n corn o i l f o r t h i s stu d y .

T h is column, however,

r e q u ir e s la r g e amounts of s o lv e n ts end ad so rb en t so a study was made t o
d eterm in e i f a sm a lle r d iam ete r column (8 mm.) could be used.
P in k e rto n (£9) h as q u a n tita tiv e ly se p a ra te d c a l c i f e r o l , from sim ple
^^^^efci<r-'m ixtm ree>hi,’;'’t h r<rJvit'63mihry*fvith th e p ro v itam in e r g o s te r o l, by
means o f a sh o rt four-cm . column of S u p e r f i l t r o l .

A m ixture of 50 p a rts

S k e lly s o lv e and 10 p a r t s e th e r was used as a s o lv e n t.

Since no v i s i b l e

bands appear on t h i s column, a fix e d amount o f developing s o lu tio n (8 m l.)
must be added a f t e r t h e sample which i s ta k e n up in th r e e ml. o f th e
s o lu tio n i s added t o t h e column.

A m athem atical means of c o rr e c tin g fo r

r e s id u e s from S u p e r f i l t r o l and so lv e n ts was employed.
I n o rd e r t o compare th e 8-mm. d ia m e te r, long and short. S u p e r f i lt r o l
colum ns, t h e n o n sa p o n ifia b le f r a c t io n of one gm. o f an ir r a d i a t e d

52
DISCUSSION
ergo s t e r o l i n c o rn o i l c o n ta in in g approxim ately 500,000 u n it s p e r gram
(zl'6105) was c a r r ie d th ro u g h t h e i r s p e c if ic p ro c e d u re s.

T h is was f o l ­

lowed in each c ase by t h e r e g u le r alum ina chrom atographic s te p u sin g 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 a b s o rp tio n cu rv es o f th e

f i l t r a t e from each column a re shown i n F ig . 26.

I t i s q u ite ev id en t

from th e s e cu rv es t h a t w hile t h e 9-cm. column r e t a i n s alm ost 50% of
th e Dg, l i t t l e o r no Dg i s r e ta in e d by t h e sh o rt column and a good sepa­
r a t i o n i s a ch iev e d .

The sh o rt four-cm . column was, t h e r e f o r e , adopted

f o r t h e Two Chrom atographic Step U l t r a v i o le t A bsorption Curve Method.
Some d is c u s s io n of t h e v a rio u s c o n v ersio n f a c t o r s used i n t h i s study
seems d e s ir a b le .
For t h e antim ory t r i c h l o r i d e c o lo r im e tric method t h e f a c to r 19,500
proposed by Evdng et a l (12) was used and o tho ro u g h d is c u s s io n a s t o
how i t was o b tain ed i s g iv en by th e s e a u th o rs .

T h is f a c to r was a ls o used

su c c e s s fu lly by Pow ell (51) when a p p lie d t o 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 s o lv e n ts .
«>-^,--T-he-€actor-,;.86> 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