A CRITICAL STUDY OF
THE CHROiiATOSRa PHIC SEPii.Rii.TION OF
CALCIFEROL FROL ERG-OSTEROL

Jean B ullard B u rn ett

AN ABSTRACT
S u b m itte d to th e School of G raduate S t u d i e s of M ichigan
S ta te C ollege o f A g r i c u l tu r e and A p p lie d Science
in p a r t i a l f u l f i l l m e n t of the re q u ire m e n ts
f o r the degree of
DOCTOR OF PHILOSOPHY
D epartm ent of Chem istry
1952
A p p r o v e d _____

// 6

a~^—
Art

Jean 3 . B u rn e tt

AN ABSTRACT
The chroma “t o g r a p h i c
o f pure c a l c i f e r o l
p l i s h e d by u s i n g

s e p a r a t i o n of s y n t h e t i c m ix tu r e s

f ro m p u r e e r g o s t e r o l h a s been accom­

an

eth er

o r e t h e r - h e x a n e s o l v e n t and

an alumina or s u p e r f i l t r o l a d s o r b e n t .
t o be the b e s t s o l v e n t

E t h e r was f o u n d

a n d e lu e n t f o r the accom plishm ent

o f the s e p a r a t i o n

b y m e a n s of a n a l u m i n a a d s o r b e n t .

V/hen s u p e r f i l t r o l

was u s e d as th e a d s o r b e n t , e t h e r

hexane in the r a t i o

o f 1 : 5 p a r t s b y volume was f o u n d t o

be a very s a t i s f a c t o r y
In o b t a i n i n g

a

s o l v e n t f o r the s e p a r a t i o n .

c o m p l e t e s e p a r a t i o n o f t h e two

compounds, the w e i g h t o f
t o which i t was

p a.ek ed

v e r y im p o r t a n t.

and f r e e from a n y

th e ad so rb en t and the h e i g h t

in

a lu m in a

t h e two compounds ,

-

t h e c h r o m a t o g r a p h i c t u b e were
g a v e a v e r y good s e p a r a t i o n of

t h e r e s u l t a n t f o rm o f e a c h b e i n g pur e
o th e r m aterial.

However,

the l e n g t h ­

e n i n g of th e c o l u m n a n d t h e r e s u l t i n g b r o a d e r b a n d i n g
o f each compound w i t h i n
undesirable.

t h e column were f o u n d t o b e

S u p e r f i l t r o l gave a v e r y s h a r p s e p a r a t i o n

much more q u i c k l y

a n d o n a very much s h o r t e r c o l u m n .

However, a c o n v e r s i o n o f
irrelevant s u b s t a n c e s
while c a l c i f e r o l

and

t h e e r g o s t e r o l i n t o two

t o o k p la c e o n th e a d s o r b e n t a n d ,
e r g o s t e r o l were o b t a i n e d f r e e

from

J e a n B. B u r n e t t

each o th er,

n e i t h e r compound, was f o u n d i n t h e e l u a t e

c o m p l e t e l y f r e e f r o m one o r b o t h o f t h e i r r e l e v a n t
m ateria ls.

T h i s o b s e r v a t i o n le ad , t o a p a t t e r n a n a l y s i s

o f th e c o n v e r s io n p ro d u c ts and as a r e s u l t ,
fractio n s

c o u l d b e a n a l y z e d by a s y s t e m o f s i m u l t a n e o u s

lin e a r equations.

A lso,

the c o n c e n tr a tio n of c a l c i f e r o l

or e r g o s te r o l in the t o t a l e lu a te ,
thereof,

the e lu a te

o r any f r a c t i o n

c o u l d b e d e t e r m i n e d ©Xd c t l y .

The r e l a t i v e a n d t o t a l c o n c e n t r a t i o n s o f c a l c i f e r o l
and e r g o s te r o l i n i t i a l l y
very l i t t l e

p l a c e d on e i t h e r a d s o r b e n t h a d

e f f e c t on t h e t o t a l c a l c i f e r o l r e c o v e r y .

H owev er , b o t h t h e s e p a r a t i o n o f t h e two compounds a n d
the

t o t a l e r g o s t e r o l r e c o v e r y were i n f l u e n c e d c o n s id e r a b l y

by t h i s

property.

ACKNOWLEDGEMENT
The a u t h o r w i s h e s t o e x p r e s s h e r s i n c e r e
a p p r e c i a t i o n t o D o c t o r D. T. E w in g , P r o f e s s o r
o f P h y s i c a l C h e m i s t r y , a n d D o c t o r V. G. G r o v e ,
P r o f e s s o r of M a th e m a tic s , f o r t h e i r guidance and
a s s i s t a n c e th ro u g h o u t th e d u r a t i o n o f t h i s work.
The a u t h o r i s a l s o i n d e b t e d t o t h e members o f t h e
G raduate C ouncil f o r th e g r a n t of a f e l lo w s h i p
which a id e d i n th e c o n t i n u a t i o n of t h i s work.

INTRODUCTION
The s t u d y o f t h e c h r o m a t o g r a p h i c s e p a r a t i o n o f
pu re c a l c i f e r o l

( v i t a m i n Dp) f r o m p u r e e r g o s t e r o l h a s

been of c o n s id e ra b le

in te r e s t in recen t y ears.

t h r o u g h a more c r i t i c a l

in v e s tig a tio n of th is

f u r t h e r i n f o r m a t i o n c a n be f o u n d r e l a t i v e

Perhaps
sep aratio n

t o t h e more

c o m p le x s e p a r a t i o n o f c a l c i f e r o l f r o m i r r a d i a t e d ,
e rg o ste ro l or r e la te d

co m p o u n d s.

In th e course of a s p e c tr o o h o to m e tr ic i n v e s t i g a t i o n
of an e l u a t e ,

w h i c h h a d b e e n o b t a i n e d f ro m a c h r o m a t o g r a m

i n which th e a d s o r b e n t ,

so lv en t,

and e l u e n t had been

s e l e c t e d s o as t o r e t a i n t h e e r g o s t e r o l a n d e l u t e o n l y
the c a l c i f e r o l ,

i t was o b s e r v e d t h r t t h i s e l u a t e

a s u b s ta n c e f o r e i g n to th e i n i t i a l
c a l c i f e r o l and pure e r g o s t e r o l .
f o re ig n substance

contained

s o l u t i o n o f pure

The p r e s e n c e o f t h i s

i n t h e e l u a t e h a s c a u s e d much

s p e c u l a t i o n an d f u r t h e r i n v e s t i g a t i o n .
C a r l s o n ^ n o t e d t h a t when e t n y l a l c o h o l a n d d i e t h y l
e t h e r r i c h s o l v e n t s were p a s s e d th ro u g h an a d s o r p t i o n
colum n c o n t a i n i n g s u p e r f i l t r o l ,
a substance,

the f i l t r a t e

contained

a p p a r e n t l y e l u t e d from th e s u p e r f i l t r o l ,

which gave i t a d e n s i t y a p p r e c i a b l y g r e a t e r t h a n
expected.
C oncurrently,

Baker^, u s in g the s p e c tro p h o to m e te r

t o a n a l y z e a n e l u a t e w h i c h was e x p e c t e d , t o c o n t a i n o n l y
calcifero l,

o b serv ed t h a t c a l c i f e r o l and a n o th e r

s u b s t a n c e were p r e s e n t .

Both h ad an a b s o r p t i o n sp e c tru m

in the u l t r a v i o l e t reg io n .

He c o n c l u d e d t h a t t h i s

i n t e r f e r i n g s u b s t a n c e was e l u t e d f r o m t h e s u p e r f i l t r o l
and t h a t a c o n s t a n t c o r r e c t i o n f a c t o r f o r i t

c o u l d be

o b t a i n e d by r u n n i n g a b l a n k a l o n g w i t h e a c h s a m p l e .

He

a l s o d i d some w o r k w i t h a l c o h o l i c s o l u t i o n s o f p u r e
e r g o s t e r o l and c a l c i f e r o l and n o t i c e d t h a t ,

u sing

h e x a n e - d i e t h y l e t h e r - e t h y l a l c o h o l a s a s o l v e n t an d
eluent,

e r g o s t e r o l was a d s o r b e d by a c t i v a t e d b e n t o n i t e

c l a y a n d c a l c i f e r o l p r o c e e d e d on t h r o u g h t h e c o l u m n .
How ev er , h e d i d no q u a n t i t a t i v e
q u a l i t a t i v e work a l o n g t h i s

or even co n clu siv e

lin e.

The s t u d y o f t h e unknown s u b s t a n c e w h i c h was
t h o u g h t t o b e e l u t e d f r o m t h e s u p e r f i l t r o l was f u r t h e r
c o n s i d e r e d by B u llard -^.

She f o u n d t h a t t h e m e th o d o f

c a rry in g a c o r r e c tio n f a c to r b lan k alo n g w ith each
sample gave q u a n t i t a t i v e r e s u l t s .

If the c a l c i f e r o l

c o n c e n t r a t i o n i n t h e o r i g i n a l s o l u t i o n was s u f f i c i e n t l y
larg e,

t h e c o r r e c t i o n f a c t o r was e l i m i n a t e d by a d e q u a t e

d i l u t i o n of the r e s u l t a n t e l u a t e .

The s o l v e n t a n d

e l u e n t u s e d f o r t h i s work was n - h e x a n e - d i e t h y l e t h e r e t h y l a l c o h o l i n th e r e l a t i o n s h i p of 50- 1 0-1 r e s p e c t i v e l y
by v o l u m e .

An e x a m i n a t i o n o f s e v e r a l s i m i l a r b i n a r y an d

-2-

t e r n a r y s o l v e n t s was wade i n an a t t e m p t t o f i n d a
s o l v e n t which would e l i m i n a t e

the e lu t i o n of t h i s

f o r e i g n m a t e r i a l fro m t h e c o l u m n a n d p e r h a p s l e a d t.o
a n e v e n more c o m p l e t e
More r e c e n t l y ,

c a lc if e r o l recovery.

i n 1948,

P in k e rto n ^ found the

t e r n a r y ' s o lv e n t of hexane - d i e t h y l e th e r - a lc o h o l
t o he more c o m p l e x t h a n n e c e s s a r y .

A satisfa cto ry

s e p a r a t i o n of c a l c i f e r o l from e r g o s t e r o l u s in g s u p e r f i l t r o l
a s a n a d s o r b e n t was a c c o m p l i s h e d by e i t h e r a h e x a n e e t h e r o r benzene - e t h e r s o lv e n t i n the r a t i o
r e s p e c t i v e l y by v o l u m e .

of 5 :1

E t h e r seem ed t o be a v e r y

im p o rta n t c o n s t i t u e n t of every b in a ry and te r n a ry
s o lv e n t exam ined.

No s a t i s f a c t o r y

sin g le

s o l v e n t was

fo u n d w hich would g iv e th e d e s i r e d s e p a r a t i o n .
ad d itio n ,

In

a r a t h e r t h o r o u g h s t u d y was ms.de w i t h r e s p e c t

t o t h e s e p a r a t i o n o f c a l c i f e r o l fro m e r g o s t e r o l u s i n g
s u p e r f i l t r o l ana the b in a r y s o lv e n t hexane - e t h e r .
The e l u a t e was c o r r e c t e d f o r t h e i n t e r f e r i n g s u b s t a n c e
e l u t e d f r o m t h e s u p e r f i l t r o l by d e t e r m i n i n g c e r t a i n
c o n s t a n t r a t i o s b e t w e e n e x t i n c t i o n v a l u e s a t two
d i f f e r e n t wave l e n g t h s

f o r b o th the i n t e r f e r i n g m a t e r i a l

and th e c a l c i f e r o l .
The r a t e s o f m i g r a t i o n o f compounds on a n a d s o r b e n t
a r e i n f l u e n c e d by the p o l a r i t y o f th e s o l v e n t an d e l u e n t .
5
Cnen s t u d i e d t h e e l u t i o n o f v i t a m i n Dp a n d v i t a m i n D-^
f ro m a n a l u m i n a column i n w h i c h v i t a m i n Dg p r e c e d e d

-3-

vitam in

when a p o l a r s o l v e n t was u s e d ,

p o l a r it y of the s o lv e n t d ecrease d ,
was r e v e r s e d .

and as th e

the o rd e r of e l u t i o n

S im ila r ly , v ita m in s D could p a r t i a l l y

b e s e p a r a t e d f r o m v i t a m i n A a n d e r g o s t e r o l on a n
a lu m in a column.

U sing a s u p e r f i l t r o l column,

she

found t h a t a h e x a n e - d i e t h y l e t h e r s o l v e n t gave a
b e t t e r recovery of vitam in s D than did a hexane d ie th y l eth er - eth y l alcohol so lv en t.
I t i s the purpose of t h i s

i n v e s t i g a t i o n to study

the chrom atographic s e p a r a tio n of s y n th e tic m ixtures
o f pure c a l c i f e r o l from pure e r g o s t e r o l u s i n g a
d i e t h y l e t h e r o r d i e t h y l e t h e r - hexane s o lv e n t and
an a lu m in a o r s u p e r f i l t r o l a d s o r b e n t and t o examine
the i n t e r f e r i n g m a te r ia l in the
s u p e r f i l t r o l column.

e l u a t e from a

MATERIALS AND EQUIPMENT
The c a l c i f e r o l u s e d i n t h i s

s t u d y was a p u r e

s y n t h e t i c c r y s t a l l i n e v i t a m i n Dg, e a c h gram c o n s i d e r e d
t o c o n t a i n a minimum o f 4 0 , 0 0 0 , 0 0 0 U . S . P . u n i t s .

This

was o b t a i n e d fro m t h e S p e c i a l m a r k e t s D i v i s i o n o f t h e
W i n t h r o p C h e m i c a l Company,

In c .,

New Y o r k , N. Y . . a n d

was u s e d w i t h o u t f u r t h e r p u r i f i c a t i o n .
A good commercial g ra d e o f f i n e

cry stallin e

e r g o s t e r o l was o b t a i n e d f r o m t h e M o n t r o s e C h e m i c a l
Company, Newark, N. J . a n d was r e c r y s t a l l i z e d f r o m a
m i x t u r e o f one p a r t o f a b s o l u t e e t h y l a l c o h o l t o two
p a r t s o f b e n z e n e by v o l u m e .

The e r g o s t e r o l c r y s t a l s

were f i l t e r e d b y s u c t i o n , w a s h e d w i t h m e t h a n o l , a n d
d r y i n g was c o m p l e t e d i n a vacuum a t room t e m p e r a t u r e .
I t was n e c e s s a r y t o f u r t h e r p u r i f y a C . P . g r a d e
of a b s o lu te e th y l a lc o h o l in o rd e r to use i t as a
s o lv e n t f o r the c r y s t a l l i n e
erg o stero l.

forms o f c a l c i f e r o l and

Twenty grams o f p o t a s s i u m h y d r o x i d e a n d

t e n g r a m s o f s i l v e r n i t r a t e w er e a d d e d t o one l i t e r
o f t h e a l c o h o l a n d w i t h o c c a s i o n a l s h a k i n g t h i s was
a l l o w e d t o s t a n d f o r one w e e k .

The a l c o h o l was

d e c a n t e d a n d d i s t i l l e d from a l l g l a s s a p p a r a t u s .
d istilla te

The

was c o l l e c t e d i n 100 m i l l i l i t e r f r a c t i o n s

a n d f o r u s e was r e q u i r e d t o t r a n s m i t down t o

-5-

230 m i l l i m i c r o n s

( t r a n s p a r e n c y a b o v e 90 p e r c e n t ) a s

m easured on t h e s p e c t r o p h o t o m e t e r . .
A p p r o p r i a t e a m o u n t s o f sodium, h y d r o x i d e a n d a n h y d r o u s
s o d iu m s u l f i t e w e r e a d d e d t o t h e a m o u n t o f a n h y d r o u s
d i e t h y l e t h e r (C.P..) r e q u i r e d f o r im m ediate u s e .
d i s t i l l a t i o n from a n a l l g l a s s a p p a r a t u s ,
t r a n s m i t down t o 2 30 m i l l i m i c r o n s

A fter

th e e t h e r must

( t r a n s p a r e n c y above

70 p e r c e n t ) a s m e a s u r e d on t h e s p e c t r o p h o t o m e t e r i n
o r d e r t o be u s e a b l e . ,
S k e l l y s o l v e B, a c o m m e r c i a l p r e p a r a t i o n o f n - h e x a n e ,
was p u r i f i e d by p a s s i n g i t

th ro u g h a l a r g e Tsw ett tu b e,

24 in c h e s lo n g and 1 .5 in ch es
freshly activ ated s ilic a g el.

in diam eter,

containing

The p e r c o l a t e f r a c t i o n s

t o be u s e d m u s t t r a n s m i t down t o 230 m i l l i m i c r o n s
( t r a n s p a r e n c y a b o v e 96 p e r c e n t ) a s m e a s u r e d o n t h e
s p e c t r o p h o t o m e t e r a n d m u s t show no a b s o r p t i o n due t o
benzene.

The u s e d s i l i c a g e l may be r e p u r i f i e d a n d

r e a c t i v a t e d by w a s h i n g i t f r e e o f h e x a n e w i t h d i s t i l l e d
w ater, allow ing i t
it

to a i r dry ,

and then f u r t h e r d ry in g

i n a n o v e n a t 2 5 0 ° C . f o r a t l e a s t 24 h o u r s .
The a d s o r b e n t s u s e d were a c t i v a t e d a l u m i n a a n d

su p erfiltro l.

The a l u m i n a was t h e A l c o a b r a n d ,

G ra de F - 2 0 , m a n u f a c t u r e d by t h e Aluminum Ore Company,
East S t.

Louis,

111.

The s u p e r f i l t r o l ,

fin e ly divided a c tiv a te d b e n to n ite
the F i l t r o l C orporation,

c l a y p r e p a r e d by

Los A n g e l e s ,

-6-

SF 6 3 , was a

C alif.

C hro m ato g ra p h ic tu b e s of d i f f e r e n t l e n g t h s were
n e c e s s a r y when u s i n g t h e d i f f e r e n t a d s o r b e n t s .
in v e s t i g a t i o n u s in g alum ina,

For the

the le n g th of the a d s o rb e n t

c o n t a i n i n g s e c t i o n o f t h e t u b e was a b o u t 47 c e n t i m e t e r s ;
and f o r th e study u s in g s u p e r f i l t r o l ,
s e c t i o n was 17 c e n t i m e t e r s .

the le n g th o f t h i s

In both cases,

the d iam eter

o f t h e s e c t i o n o f t h e t u b e w h i c h h e l d t h e a d s o r b e n t was
7 .5 m i l lim e te r s and th e d ia m e te r of th e low er c o n s t r i c t e d
p o r t i o n o f t h e t u b e was 3 . 7 m i l l i m e t e r s .
When t h e v o l u m e o f t h e e l u a t e p e r f r a c t i o n a n d t h e
num ber o f f r a c t i o n s r e q u i r e d h a d b e e n d e t e r m i n e d ,

they

were c o l l e c t e d i d e n t i c a l l y w ith a T echnicon A u to m atic
F r a c t i o n C o l l e c t o r made by t h e T e c h n i c o n C h r o m a t o g r a p h y
C o rp oration,

New Y o r k , N. Y.

The r a t e o f e l u t i o n c o u l d

b e c o n t r o l l e d by v a r y i n g t h e p r e s s u r e a t t h e t o p o f t h e
c h r o m a t o g r a p h i c c o lu m n u s i n g a c a r b o n d i o x i d e s o u r c e a n d
a n a d j u s t a b l e m ercury manometer.
A b s o r p t i o n m e a s u r e m e n t s w e r e made w i t h a Beckman
Q uartz S p e c tro p h o to m e te r,
Technical L a b o ra to rie s,

Model DU , made by t h e N a t i o n a l

South P asadena,

a b s o r p t i o n c e l l s w e r e made o f s i l i c a ,

C alif.

The

th e t h ic k n e s s of

e a c h b e i n g 1 .0 0 0 ± 0 .0 0 2 c e n t i m e t e r .
A l l c o m p u t a t i o n s were c a r r i e d o u t u s i n g a c o m p l e t e l y
a u t o m a t i c F r i d ^ n U l t r a - m a t i c C a l c u l a t o r made by t h e F r i d e n
C a l c u l a t i n g M ach in e C o . ,

In c.,

San Leandro,

C alif.

EXPERIMENTAL PROCEDURE
From s t o c k a l c o h o l i c s o l u t i o n s o f c a l c i f e r o l a n d
erg o stero l,

w h i c h w e r e made u p f r o m t h e c r y s t a l l i n e

o f t h e com pounds,

forms

the d e s ire d q u a n t i t i e s fo r each in v e s t­

i g a t i o n were v o l u m e t r i c a l l y

t a k e n . . The s t o c k s o l u t i o n o f

c a l c i f e r o l c o n t a i n e d 0 . 0 1 7 4 gram/ 1 0 0 m i l l i l i t e r s

of

a b s o lu te e t h y l a lc o h o l, and t h a t of e r g o s t e r o l c o n ta in e d
0 . 0 2 4 4 gram/ 1 0 0 m i l l i l i t e r s

of a b so lu te e th y l a lc o h o l.

Having d e te r m in e d th e amount o f c a l c i f e r o l o r
e r g o s t e r o l o r c o m b in a tio n o f t h e s e to be i n v e s t i g a t e d ,
t h i s q u a n t i t y was t a k e n f r o m t h e s t o c k s o l u t i o n s a n d
u s i n g a warm w a t e r b a t h was t a k e n t o d r y n e s s u n d e r
reduced p re s s u re .
m illiliters

The s a m p l e was t h e n t a k e n up i n t h r e e

of a hexane o r e t h e r - hexane s o lv e n t .

The a d s o r p t i o n co lum n was p r e p a r e d by p l a c i n g a
s m a l l wad o f c o t t o n a t t h e t o p o f t h e c o n s t r i c t e d p o r t i o n
o f the ch rom atographic tube as a s u p p o rt f o r the
adsorbent.

The a l u m i n a was w e i g h e d o u t t o t h e n e a r e s t

gram a n d t h e n w h i l e g e n t l y t a p p i n g a r o u n d t h e s i d e s o f
the tube,

t h e a d s o r b e n t was i n t r o d u c e d i n t o t h e t u b e , .

The h e i g h t t o w h i c h t h e a l u m i n a came was t h e n m e a s u r e d .
I f s u p e r f i l t r o l was u s e d a s
w e i g h e d o u t gram by g r a m .
e a c h gram i n t o t h e t u b e ,

the a d s o rb e n t,

i t was

A f te r the i n t r o d u c t i o n of
t h e s u p e r f i l t r o l was v e r y

firm ly

packed w ith th e a i d of a l a r g e g l a s s ro d

f l a t t e n e d on o n e en d t o a d i a m e t e r j u s t s l i g h t l y l e s s
th an t h a t of the tu b e .

The h e i g h t o f t h e a d s o r b e n t was

then m easured to h e lp in s u r e r e p r o d u c i b l e hand p a c k in g .
The c h r o m a t o g r a p h i c t u b e was t h e n p o s i t i o n e d i n
the f r a c t i o n a t o r .

The column was w a s h e d w i t h a d e f i n i t e

a m o u n t o f e t h e r o r e t h e r - h e x a n e s o l u t i o n w h i c h was
d e t e r m i n e d by t h e t y p e a n d a m o u n t o f a d s o r b e n t .

Just

p r i o r t o t h e l a s t p o r t i o n o f w as h s o l u t i o n l e a v i n g t h e
to o of the a d s o rb e n t,
columno

t h e s a m p l e was i n t r o d u c e d o n t o t h e

The f r a c t i o n a t o r was i m m e d i a t e l y s e t i n t o

operation.

The f l a s k w h i c h h a d c o n t a i n e d t h e s a m p l e

s o l u t i o n was t h e n r i n s e d w i t h t h r e e m i l l i l i t e r s
p re v io u sly used s o lv e n t,

and a s t h e l a s t

sample s o l u t i o n d i s a p p e a r e d , i n t o
»
r i n s e s o l u t i o n was a d d e d t o t h e

p o rtio n of the

the a d s o rb e n t,
column.

of the

th is

The d e v e l o p e r ,

h av in g been p laced in a s e p a r a to r y fu n n e l a to p the
chrom atographic tube,
the r in s e

was i n t r o d u c e d o n t o t h e c olu m n a s

s o lu tio n d isa p p e a re d in to the a d s o rb e n t.

At

no t i m e a f t e r t h e i n i t i a l w e t t i n g o f t h e a d s o r b e n t w i t h
t h e was h s o l u t i o n was t h e t o p o f t h e co lu m n e v e r
p e r m i t t e d t o become d r y .

The same g l a s s d r o p p i n g t i p

(a

p i e c e o f t h e f r a c t i o n a t o r ) was u s e d f o r a l l e x p e r i m e n t s .
This a s s u r e d e l u a t e d ro p s of c o n s ta n t s i z e .
When a s u f f i c i e n t num ber o f f r a c t i o n s ,

having used

45 d r o p s = 1 f r a c t i o n — 1 m i l l i l i t e r ,

had been c o l l e c t e d ,

e a c h was t a k e n t o d r y n e s s u n d e r r e d u c e d p r e s s u r e a n d
u s i n g a warm w a t e r b a t h .

If a residue,

e v a p o r a tio n o f the e l u a t e a s i t
dropping t i p ,

c o l l e c t e d on th e

h ad form ed on th e t i p ,

w ith the develo p er,

due to a s l i g h t

i t was r e m o v e d

c o lle c te d in a t e s t tube, evaporated

t o d r y n e s s , a n d f u r t h e r t r e a t e d t h e same way a s a n y o t h e r
sam ple.

E a c h r e s i d u e was v o l u m e t r i c a l l y

four m i l l i l i t e r s

tak en up in

o f hexane and th e a b s o r p t i o n c u rv e of

t h e r e s u l t a n t s o l u t i o n d e t e r m i n e d on t h e s p e c t r o p h o ­
tom eter.
when i t s

A f r a c t i o n was f u r t h e r d i l u t e d w i t h h e x a n e
c o n c e n t r a t i o n was t o o g r e a t f o r t h e d e t e r m i n a ­

t i o n o f i t s a b s o r p t i o n s p e c t r u m t o b e made on t h e m o st
a c c u r a t e p o r t i o n of th e i n s t r u m e n t .
r e a d i n g s w er e t a k e n a t v a r i o u s

The e x t i n c t i o n

i n t e r v a l s o f one t o t e n

m i l l i m i c r o n s d e p e n d in g upon th e p o s i t i o n s

o f t h e maxima

a n d min im a o f t h e e l u a t e a b s o r p t i o n s p e c t r u m .

DISCUSSION
E r g o s t e r o l h a s t h e e m p i r i c a l f o r m u l a C2 QH4.4 O, w h i c h
h as been e s t a b l i s h e d by c a r e f u l a n a l y s i s

of d e r iv a tiv e s

c o n ta in in g h e te ro atom s.

C alcifero l,

d eriv ativ e of e rg o ste ro l,

a l s o has th e e m p ir ic a l form ula

CggH^O.

H owev er ,

an isom er and •

th e s t r u c t u r a l form ulas of e r g o s te r o l

and c a l c i f e r o l a re d i f f e r e n t

.

E r g o s te r o l has an

a n g u l a r m ethyl group betw een r i n g s A and B a t carbon
a t o m 10 a n d t h r e e d o u b l e b o n d s ,

one i n t h e s i d e

chain

b e t w e e n c a r b o n ato m s 22 a n d 23 a n d two c o n j u g a t e d o u b l e
bonds in t h e 5 ,6 - and 7 , 3 - p o s i t i o n s .

C a lc ife ro l has a

m e t h y l e n e g r o u p on c a r b o n a to m 10 a n d h e n c e f o u r d o u b l e
b o n d s , one i n t h e s i d e

c h a i n b e t w e e n c a r b o n a t o m s 22 a n d

23 a n d two d o u b l e b o n d s , w h i c h a r e i n c o n j u g a t i o n w i t h
th e double bond a t th e 7 , 8- p o s i t i o n ,
1 0 ,1 8 -p o sitio n s

in th e 5 , 6 - and

(see Figure 1 ).
21

21

12 19

12 19

20
:22

11

20
22

18
10

28

110

28

HQ

E rg o stero l

C alcifero l
Figure 1.

The p r e s e n c e o f f o u r d o u b l e b o n d s ,
are

conjugate,

double bonds,

in c a l c i f e r o l as

t h r e e of which

compared t o o n ly t h r e e

two o f w h i c h a r e c o n j u g a t e ,

in e rg o ste ro l

i n d i c a t e s t h a t c a l c i f e r o l may be more p o l a r t h a n
erg o stero l.

S in ce a d s o r p t i o n t a k e s p l a c e most r e a d i l y
Q
from n o n - p o l a r s o l v e n t s , c a l c i f e r o l , a s compared w i t h
ergosterol,

s h o u l d b e p r e f e r e n t i a l l y a n d more s t r o n g l y

a d s o r b e d from a n o n - p o l a r o r s l i g h t l y
D esorption,

the d isp la c e m e n t of the adsorbed substance

from t h e a d s o r b e n t ,

i s done by a n e l u e n t w h i c h i s more

polar than th e adsorbed su b stan ce.
as

polar so lv en t.

compared w i t h c a l c i f e r o l ,

T hus , e r g o s t e r o l ,

s h o u l d b e more r a p i d l y

d e s o r b e d by a s o l v e n t more p o l a r t h a n i t s e l f .

However,

t h i s was n o t f o u n d t o be t r u e when a l u m i n a o r s u p e r ­
f i l t r o l was u s e d a s t h e a d s o r b e n t a n d e t h e r o r e t h e r h e x a n e was u s e d a s t h e s o l v e n t o r e l u e n t .
v astly d iffe re n t s te r ic

Perhaps the

c o n f i g u r a t i o n s o f t h e two

compounds h a v e . a l t e r e d t h e a , n t i c i p a t e d p o l a r r e l a t i o n ­
s h i p s b e t w e e n them o r h a v e made t h e more o r l e s s
s t r o n g l y a d s o r b e d compound more a v a i l a b l e t o ■t h e a c t i o n
of the e lu e n t.

Hence,

p o l a r i t y may o n l y b e a g u i d e t o

t h e a c t i o n t a k i n g p l a c e w i t h i n a n a d s o r b e n t c o lu m n .
S o lv e n ts d i f f e r in p o l a r i t y and a d s o rb e n ts vary
in a c t i v i t y .
used in th is

C o n s id e rin g th e s o l v e n t s and a d s o r b e n ts
study, hexane,

eth er,

-12-

and a lc o h o l in c re a s e

i n p o l a r i t y and alum ina i s a l e s s a c t i v e a d s o r b e n t th a n
9
su p erfiltro l .
The w e t t i n g a n d w a s h i n g o f t h e a d s o r b e n t c o lu m n
jp u s t p r i o r t o t h e i n t r o d u c t i o n o f t h e s a m p l e s o l u t i o n
i s done w i t h t h e m o s t p o l a r s o l v e n t t o b e u s e d i n t h e
course o f the i n v e s t i g a t i o n .
a s o lv e n t of l e s s
the ad so rb en t.
o f th e sam ple,

The s a m p le i s d i s s o l v e d i n

or e q u a l p o l a r i t y and in t r o d u c e d onto

The d e v e l o p m e n t o f t h e co lu m n a n d e l u t i o n
o r any f r a c t i o n t h e r e o f ,

is c a rrie d out

w ith a s o lu tio n of equal or g r e a t e r p o l a r it y th an th a t
p r e v io u s ly used as the s o lv e n t f o r the sam ple.
The a b s o r p t i o n s p e c t r a o f c a l c i f e r o l a n d e r g o s t e r o l
10,11
vary w ith the s o lv e n t used
. T herefore, the
a b s o r p t i o n s p e c t r a of c a l c i f e r o l and e r g o s t e r o l i n h ex an e
w e r e d e t e r m i n e d o v e r t h e r a n g e o f 230 m i l l i m i c r o n s

to

300 m i l l i m i c r o n s u s i n g t h e s p e c t r o p h o t o m e t e r ( F i g u r e 2 ) .
T h r o u g h o u t t h e i n v e s t i g a t i o n h e x a n e was t h e s o l v e n t u s e d
f o r the f i n a l a n a ly s is because o f i t s g r e a t tra n sp a re n c y
over the u l t r a v i o l e t r e g io n and i t s

s t r a i g h t f o r w a r d and

r a p i d m e th o d o f p u r i f i c a t i o n .
THE USE OF ALUMINA. AS AN ADSORBENT
The s p a t i a l c o n f i g u r a t i o n a n d f o r c e s e x i s t i n g among
t h e atoms o f c a l c i f e r o l and e r g o s t e r o l e v i d e n t l y
n ecessitate

the use of q u ite a c t i v e a d so rb e n ts i n o rd e r

to o b t a i n a com plete o r even a p a r t i a l s e p a r a t i o n of th e

two compounds f r o m a s i n g l e s o l u t i o n .
A lu m in a i s o b t a i n a b l e
is

in a v e ry pure s t a t e and

such t h a t alm o st a l l of the i r r e l e v a n t m a te r ia l

r e m a i n i n g on t h e c o m m e r c i a l p r e p a r a t i o n c a n be r e m o v e d
b y p r e w a s h i n g t h e co lu m n w i t h t h e p r o p e r o r g a n i c
so lv en t.

U sin g v a r i o u s amounts o f e t h e r ,

depending

u p o n t h e l e n g t h o f t h e co lu m n , a s t h e p r e w a s h s o l v e n t ,
the

i r r e l e v a n t m a t e r i a l i n th e e l u a t e from th e a lu m in a

c o l u m n c a n be r e d u c e d t o l e s s t h a n 0 . 0 0 2 e x t i n c t i o n
u n i t s a t 264 m i l l i m i c r o n s .

To rem o v e t h e

p o ssib ility

o f any i n t e r f e r e n c e from a s o l v e n t r e s i d u e ,
in the re fe re n c e

the s o lu tio n

c e l l was made u p c o m p a r a b l e t o t h e

s a m p l e s o l u t i o n f r o m t h e c o lu m n .

That i s ,

i f a one

m i l l i l i t e r s a m p l e was t a k e n u s i n g e t h e r a s t h e e l u e n t ,
e v a p o r a t e d t o d r y n e s s , a n d t h e r e s i d u e t a k e n up i n
hexane,

t h e n one m i l l i l i t e r o f e t h e r was e v a p o r a t e d

to d ry n ess,

the re s id u e

( i f any)

t a k e n u p i n t h e same

a m o u n t o f h e x a n e , a n d t h i s was u s e d a s t h e r e f e r e n c e
s o l u t i o n f o r t h a t sam ple.
Since th e e l i m i n a t i o n of any i n t e r f e r i n g m a t e r i a l s
i n t h e f i n a l a n a l y s i s was q u i t e

com plete, a l l of th e

r e s u l t a n t a b s o r p t i o n c u r v e s were f o u n d t o b e e i t h e r p u r e
calcifero l,
these.

pure e r g o s t e r o l ,

or a l i n e a r co m bination of

The c o m b i n i n g o f L a m b e r t ' s a n d B e e r ' s la w s i n t o

a s in g le a b s o rtp io n e q u a tio n gives
V

= lo fL ilo 1
lc

-14-

1 1

w h e r e k i s t h e e x t i n c t i o n c o e f f i c i e n t a t a g i v e n wave
length, A,

I Q is the

i n t e n s i t y of th e t r a n s m i t t e d l i g h t ,

I is th e i n t e n s i t y o f the i n c i d e n t l i g h t ,
th ic k n e s s of the s o lu t i o n ,
of the s o lu tio n .

1 i s the

and c is the c o n c e n tra tio n

U sing t h i s

fundam ental r e l a t i o n s h i p ,

t h e a d d i t i v i t y o f t h e a b s o r p t i o n c u r v e s o f two o r more
s u b s t a n c e s i n s o l u t i o n i s d e v e lo p e d by H a r r i s and
Thimann

12

.

I f c^ a n d c 0 a r e t h e unknown c o n c e n t r a t i o n s

o f two s u b s t a n c e s i n a s o l u t i o n o f o n e c e n t i m e t e r
thickness,

k^ a n d kg t h e i r e x t i n c t i o n c o e f f i c i e n t s

at

a wave l e n g t h A , k-j^ a n d kg t h e i r e x t i n c t i o n c o e f f i c i e n t s
a t some o t h e r wave l e n g t h A 1, a n d D a n d D* t h e d e n s i t i e s ,
log (I / I ) ,

o f t h e m i x t u r e a t t h e two wave l e n g t h s ,

then

k l c l + k 2 c2 = D
and

k i ci + k 2 c 2 = D '

therefore

c-, =

kpD'

- k 'D
—

k {k 2 -

and

kj^kg

k.,ID 1 - kjD
1
Co2 = — ----------- =■
k-j^kg - k-[kg

a n d t h e u n i t s o f c c a n be d e t e r m i n e d a n d a d j u s t e d i n
acco rd an ce w ith k.

As t h e c r i t i c a l p o i n t s f o r t h e

c a l c u l a t i o n of the c o n te n t o f each f r a c t i o n ,

t h e maxima

o f c a l c i f e r o l a t 264 m i l l i m i c r o n s and t h a t o f e r g o s t e r o l

-1 5 -

a t 281 m i l l i m i c r o n s w e r e u s e d .

Having found th e c o n t e n t

of each f r a c t i o n to he a l i n e a r

com bination of c a l c i f e r o l

and e r g o s t e r o l and knowing th e c o n c e n t r a t i o n of b o t h
compounds p l a c e d on t h e c o lu m n ,

th e r e l a t i v e and e x a c t

a m o u n t s o f e a c h c a n be c o m p u t e d .
V a ria tio n of E luent P o la rity
As t h e e t h e r c o n t e n t (by v o l u m e ) o f t h e e l u e n t i s
increased,-

th e t o t a l amount o f b o t h c a l c i f e r o l and

e r g o s t e r o l e l u t e d from th e column i s
as

can be s e e n f r o m T a b l e I .

a little

increased .

increased,

H e n c e , b o t h compounds a r e

more e a s i l y d e s o r b e d a s

e lu e n t is

slig h tly

the p o l a r i t y of the

C alciferol is

somew hat more

c o m p le te ly d e s o r b e d from t h e column t h a n i s e r g o s t e r o l .
A t t h e same t i m e ,

t h e p e r c e n t s e p a r a t i o n o f t h e two

compounds r e m a i n s r e l a t i v e l y
rates
it

co n stan t.

Therefore,

the

f o r w h i c h e a c h compound i s a d s o r b e d - d e s o r b e d a s

p r o g r e s s e s downward on t h e a d s o r b e n t m u s t b e p r o ­

p o r t i o n a t e l y e q u a l as th e p o l a r i t y of the s o lv e n t
increases.

S i m i l a r p r o p e r t i e s a r e o b s e r v e d when t h e

l e n g t h o f t h e a l u m i n a c o lu m n i s v a r i e d .

Hhen t h e

co lum n i s 7 . 8 c e n t i m e t e r s l o n g (by w e i g h t ,
and th e e l u e n t i s

co mposed o f e t h e r a n d h e x a n e i n

e q u a l p a r t s by v o lu m e ,
8 2 /0

4 grams)

90% o f t h e c a l c i f e r o l a n d

of the e r g o s t e r o l a r e re c o v e re d w h ile ab o u t

the c a l c i f e r o l and

of

of the e r g o s t e r o l a r e re c o v e re d

-16-

in the pure s t a t e .

When t h e e l u e n t i s p u r e e t h e r ,

96% o f t h e c a l c i f e r o l a n d 90% o f t h e e r g o s t e r o l a r e
r e c o v e r e d w h ile a b o u t 32% of th e c a l c i f e r o l and 33% of
the e r g o s t e r o l a r e re c o v e re d in th e pure s t a t e
Figure 3 ).

When tiie c o lu m n i s

(by w e i g h t ,

6 grams) and th e e l u e n t i s

e t h e r and hexane in th e r a t i o
90% o f t h e c a l c i f e r o l ,
in the pure s t a t e ,

15.6 c e n tim e te r s

calcifero l,
state,

long

co mposed o f

o f 7 t o 3 p a r t s by v o l u m e ,

a b o u t 46% o f w h i c h i s o b t a i n e d

and 63% o f th e e r g o s t e r o l , a b o u t

46% o f w h i c h i s o b t a i n e d i n t h e p u r e s t a t e ,
As t h e e l u e n t i s

(see

in c r e a s e d to pure e t h e r ,

are recovered.

94% o f t h e

a b o u t 51% o f w h i c h i s o b t a i n e d i n t h e p u r e

a n d 67% o f t h e e r g o s t e r o l ,

o b ta in e d in the pure s t a t e ,

a b o u t 49% o f w h i c h i s

a re re c o v e re d (see F ig u re 4 ) .

T h u s , a n i n c r e a s e i n t h e l e n g t h o f t h e a d s o r b e n t colum n
i n c r e a s e s t h e p e r c e n t s e p a r a t i o n of t h e c a l c i f e r o l from
t h e e r g o s t e r o l by q u i t e a n a p p r e c i a b l e a m o u n t , b u t t h i s
i s a c c o m p l i s h e d a t t h e e x p e n s e o f t h e t o t a l a m o u n ts o f
c a l c i f e r o l a n d e r g o s t e r o l r e c o v e r e d w h i c h show a s l i g h t
decrease.
T h e re i s a c h a n g e i n t h e r a t e o f e l u t i o n o f c a l c i f e r o l
a n d e r g o s t e r o l when t h e p o l a r i t y o f t h e e l u e n t i s a l t e r e d ,
a s w o u ld b e e x p e c t e d ( T a b l e I I ) .
the e lu e n t,

When e t h e r i s u s e d a s

t h e c a l c i f e r o l a n d e r g o s t e r o l a r e e l u t e d fro m

t h e colum n more t h a n t w i c e a s f a s t a s when e t h e r - h e x a n e

i n e q u a l p a r t s by v o l u m e ,

is used.

A t t h e same t i m e ,

t h e v o lu m e o f e l u e n t n e c e s s a r y t o e l u t e

the

and e r g o s t e r o l from t h e a lu m in a i s g r e a t l y

calcifero l
in c re a s e d as

the e t h e r c o n te n t of th e e lu e n t i s d e c r e a s e d .
increase

This

i n v olu m e o f e l u e n t i s a c c o m p a n i e d by a

b r o a d e n i n g of t h e r a n g e i n which t h e e l u t i o n t a k e s
place and a c o n c u r re n t in c r e a s e

i n th e number of

f r a c t i o n s c o n ta in in g b o th c a l c i f e r o l and e r g o s t e r o l .
This band w id e n in g does n o t le n d i t s e l f a d v a n ta g e o u s ly
t o a s u b s e q u e n t s e p a r a t i o n o f t h e two c o m p o u n d s .
C o n s i d e r i n g t h e 7 . 8 c e n t i m e t e r a lu m in a column an d t h e
use of th e hexane - e t h e r e l u e n t ,
volume,

i n e q u a l p a r t s by

c a l c i f e r o l a p p e a r s a n d t e r m i n a t e s i n f r a c t i o n 37

and 60 r e s p e c t i v e l y , w h i l e e r g o s t e r o l i s
f r a c t i o n s 46 t h r o u g h 7 2 .

Hence,

p resen t in

t h e r e a r e 15 f r a c t i o n s

i n which c a l c i f e r o l and e r g o s t e r o l a r e t o g e t h e r .
p u r e e t h e r i s uhe e l u e n t u s e d ,

c a lc if e r o l appears in

f r a c t i o n s 15 t h r o u g h 26 a n d e r g o s t e r o l i s
f r a c t i o n s 20 t h r o u g h 3 5 •
many f r a c t i o n s

Thu s,

When

found in

t h e r e a r e only h a l f as

i n w h i c h t h e two compounds a p p e a r s i m u l ­

ta n e o u sly (F igure 5)-

T a k i n g t h e 1 5 .6 c e n t i m e t e r c o lu m n

f o r com parison, h a v in g used the e t h e r - hexane e l u e n t i n
the r a t i o

o f 7 : 3 p a r t s by v o l u m e ,

c a lc ife ro l is present

i n f r a c t i o n s 40 t h r o u g h 6 l a n d e r g o s t e r o l i s f o u n d i n
f r a c t i o n s 51 t h r o u g h 7 8 .

T h i s i s a n i n t e r v a l o f 11

fractio n s

i n w h i c h h o t h compounds o c c u r .

e th e r as the e lu e n t,

calcifero l fa lls

Using pure

in fra c tio n s

29

t h r o u g h 42 a n d e r g o s t e r o l o c c u r s i n f r a c t i o n s 37 t h r o u g h
56.

This i s an i n t e r v a l of 6 f r a c t i o n s

compounds a r e f o u n d ( F i g u r e 6 ) .
co lumn o f g i v e n l e n g t h ,

i n which b o t h

T herefore,

for a

the in c re a s e in e th e r co n te n t

of the e lu e n t n o t only h a s te n s the e l u t i o n of b o th
c a l c i f e r o l and e r g o s t e r o l b u t l e s s e n s th e i n t e r v a l in
w h i c h b o t h compounds a r e e l u t e d .
t h e compounds a r e e l u t e d l a t e r ,
in terv al

F o r a l o n g e r co lu m n ,
n a t u r a l l y , b u t the

i n w h i c h b o t h a p p e a r i s l e s s o r a t l e a s t no

g r e a t e r th a n t h a t f o r th e s h o r t e r column.
Because t h e r e i s l i t t l e

d if f e r e n c e in the p e rc e n t

s e p a r a t i o n o f t h e two compounds when a n e t h e r - h e x a n e
e l u e n t is u sed and s in c e t h e r e i s a d e c id e d i n c r e a s e i n
t h e r a n g e i n w h i c h b o t h compounds a p p e a r t o g e t h e r when
e t h e r - hexane is the e lu e n t used,

p u r e e t h e r was

s e l e c t e d a s t h e e l u e n t t o be u s e d f o r t h e f u r t h e r
i n v e s t i g a t i o n o f t h e c h a n g e o f colum n l e n g t h a n d t h e
e f f e c t o f th e change in c a l c i f e r o l and e r g o s t e r o l
concentration.
V a r i a t i o n o f Column L e n g t h
Having o b s e rv e d th e p o r t i o n of e l u a t e
there

i n which

is an o v e r la p o f th e e l u t i o n of c a l c i f e r o l and

e r g o s t e r o l t o b e 7 a n d 6 f r a c t i o n s f o r t h e colum ns o f

-19-

7 . 8 and 1 5 . 6

centim eters in length re s p e c tiv e ly ,

s eem s r e a s o n a b l e t o
to see i f

th is

in v estig ate

it

c o lu m n s o f l o n g e r l e n g t h

s i x f r a c t i o n o v e r l a p c a n be r e m o v e d .

The

e l i m i n a t i o n o f a p o r t i o n o f t h e e l u a t e w here c a l c i f e r o l
and e r g o s t e r o l are e lu te d s im u lta n e o u s ly w i l l take p la c e
o n l y when t h e r a t e

o f a d s o r p t i o n - d e s o r p t i o n f o r one o f

t h e c o m p o u n d s . i s more r a p i d t h a n t h e r a t e

of w id e n in g of

t h e r a n g e o v e r w h i c h t h a t compound i s e l u t e d .

The

v a r i o u s column l e n g t h s i n v e s t i g a t e d a r e g i v e n i n Table
and i l l u s t r a t e d g r a p h ic a lly

in F ig u re 7.

o f t h e c o lu m n i s i n c r e a s e d ,

t h e nu m b er o f f r a c t i o n s

c o n t a i n i n g b o t h compounds i s

III

As t h e l e n g t h

slow ly d e c r e a s e d and the

t o t a l nu m ber o f f r a c t i o n s r e q u i r e d t o e l u t e e a c h compound
is

increased.

This l a t t e r p r o p e r t y i s u n f a v o r a b l e f o r

keeping th e i n i t i a l

c a l c i f e r o l and e r g o s t e r o l c o n c e n t r a ­

t i o n s , w hich a r e i n t r o d u c e d o n to columns of l o n g e r
length,
less

co n stan t.

S in c e t h e e l u a t e becomes l e s s and

concentrated per f r a c tio n ,

readings f o r

t h e s e d i l u t e f r a c t i o n s t e n d t o f a l l below

t h e r a n g e o f maximum r e l i a b i l i t y
Ho we ve r, when n e c e s s a r y ,
th is

some o f t h e e x t i n c t i o n

inh erent e rro r.

of th e sp e c tro p h o to m e te r.

the r e s u l t s a r e c o rre c te d f o r

A s e p a r a t i o n o f c a l c i f e r o l fro m

e r g o s t e r o l was a t t a i n e d when t h e c o l u m n l e n g t h was a b o u t
4 4 . 6 c e n t i m e t e r s (25 g r a m s ) .

The c a l c i f e r o l i n i t i a l l y

a p p e a r e d i n f r a c t i o n 66 a n d t e r m i n a t e d i n f r a c t i o n 86

a n d e r g o s t e r o l was e l u t e d i n f r a c t i o n s

87 t h r o u g h 1 1 5 .

The r a n g e i n w h i c h e r g o s t e r o l was e l u t e d b e i n g 8 f r a c t i o n s
more t h a n w e r e r e q u i r e d t o e l u t e

calcifero l.

I t i s n o t s u f f i c i e n t j u s t to have o b ta in e d a
s e p a r a t i o n o f c a l c i f e r o l from e r g o s t e r o l ,

b u t the

c o m p l e t e n e s s o f t h e r e c o v e r y o f e a c h compound s h o u l d be
considered.

F i g u r e 8 shows t h e p e r c e n t o f c a l c i f e r o l

f r e e from e r g o s t e r o l a s

compared t o th e t o t a l p e r c e n t of

c a l c i f e r o l re c o v e re d as the le n g th of th e alum ina
adsorbent is

changed.

The p e r c e n t o f c a l c i f e r o l e l u t e d

f r e e from e r g o s t e r o l i n c r e a s e s q u i t e r a p i d l y up t o and
in c lu d in g the y l . l
lev el o ff.

c e n t i m e t e r column.

I t th e n te n d s to

The t o t a l p e r c e n t o f c a l c i f e r o l r e c o v e r e d

f r o m t h e c o lu m n d e c l i n e s v e r y r a p i d l y a t f i r s t a n d t h e n
becomes a lm o s t l i n e a r betw een th e 7 .8 c e n t i m e t e r and
4 A .6 c e n t i m e t e r c o l u m n s .
free

The p e r c e n t o f e r g o s t e r o l

f ro m c a l c i f e r o l a s c o m p a r e d t o t h e t o t a l p e r c e n t

o f e r g o s t e r o l r e c o v e r e d a s t h e column l e n g t h i s a l t e r e d
is

shown i n F i g u r e 9 .

e rg o ste ro l eluted free

The c u r v e s h o w i n g t h e p e r c e n t o f
fro m c a l c i f e r o l i s q u i t e s i m i l a r

to t h a t f o r th e comparable c a l c i f e r o l c u r v e .

Ho wev er ,

t h e p u r e e r g o s t e r o l r e c o v e r e d f r o m t h e co lum ns o f g r e a t e r
l e n g t h t h a n t h e 15*6 c e n t i m e t e r colum n i n c r e a s e s
c o n s i d e r a b l y more s l o w l y t h a n d i d t h e p u r e c a l c i f e r o l
recovered.

The t o t a l p e r c e n t o f e r g o s t e r o l r e c o v e r e d

-21-

d e c l i n e s more r a p i d l y

t h a n d o e s c a l c i f e r o l f o r co lu m n s

o f s h o r t e r l e n g t h and th e n c o n ti n u e s a l i n e a r d e c l i n a t i o n ,
more r a p i d t h a n c a l c i f e r o l ,
and 44.6 c e n t i m e t e r colum ns.

betw een th e 7 .8 c e n tim e te r
C o n s i d e r i n g t h e co lu m n on

w h i c h t h e two compounds were s e p a r a t e d ,

8 6 . 5^ o f t h e

c a l c i f e r o l a n d 7 6 .2% o f t h e e r g o s t e r o l w e r e r e c o v e r e d .
I t i s q u i t e p o s s i b l e t h a t w ith th e w idening of th e range
of e l u t i o n t h a t i s c o n c u rre n t w ith the le n g th e n in g of
t h e c o lu m n ,

t h e c a l c u l a t e d t o t a l r e c o v e r i e s o f t h e two

compounds a r e somewhat l o w e r t h a n t h e a c t u a l a m o u n t s
recovered.

However , f u r t h e r i n v e s t i g a t i o n shows t h i s

e r r o r t o be l e s s

t h a n tw'o p e r c e n t .

V a r i a t i o n o f S am ple C o n c e n t r a t i o n
The e f f e c t o f v a r y i n g t h e r e l a t i v e a n d t o t a l
c o n c e n t r a t i o n s of c a l c i f e r o l and e r g o s t e r o l to be
i n t r o d u c e d o n t o t h e a l u m i n a was s t u d i e d u s i n g t h e
7 - 8 c e n t i m e t e r ' ( F i g u r e 1 0 ) an d 1 5 .6 c e n t i m e t e r
( F ig u r e 11) colum ns.

R e s u l t s from c o r r e s p o n d in g

i n v e s t i g a t i o n s o f t h e two co l u m n s o f d i f f e r e n t l e n g t h
a r e very com parable.

This i s p a r t i c u l a r l y

t r u e f o r the

t o t a l p e r c e n t r e c o v e r y o f e a c h compound; t h e m a in
c o n s is te n t d iff e r e n c e b e in g t h a t the t o t a l reco v ery f o r
e a c h compound i s s l i g h t l y
column.

g r e a t e r when u s i n g t h e s h o r t e r

The p a r a l l e l i s m b e t w e e n t h e r e s u l t s o b t a i n e d

f r o m t h e two d i f f e r e n t l e n g t h c o l u m n s i s

l e s s f o r the

p e r c e n t s e p a r a t i o n o f c a l c i f e r o l an d e r g o s t e r o l .
However, t h e m ost c o n s i s t e n t d i s s i m i l a r i t y i s t h a t t h e
e l u t i o n o f e a c h compound when f r e e f r o m t h e o t h e r i s
c o n s i d e r a b l y g r e a t e r f o r t h e l o n g e r colum n.
a re fe re n c e the b asic c a l c i f e r o l
m illilite r)

an d e r g o s t e r o l

U s in g as

( 0 . 0 0 0 2 6 0 gram p e r

( 0 . 0 0 0 4 8 8 gram p e r m i l l i l i t e r )

c o n c e n t r a t i o n s and f o r com pariso n an e q u a l c o n c e n t r a t i o n
of c a l c i f e r o l w ith a double q u a n t i t y of e r g o s t e r o l , the
s e p a ra tio n of each is

l e s s e n e d on t h e l o n g e r column b u t

th e s e p a r a tio n of c a l c i f e r o l i s
column.

i n c r e a s e d on t h e

sho rter

D o u b lin g t h e e r g o s t e r o l c o n c e n t r a t i o n and

k eep in g t h a t of c a l c i f e r o l c o n s ta n t i n c r e a s e s th e
s e p a r a t i o n o f c a l c i f e r o l an d d e c r e a s e s t h a t o f e r g o s t e r o l
on b o t h c o l u m n s .

When b o t h compounds a r e d o u b l e d ,

the

c a l c i f e r o l and e r g o s t e r o l s e p a r a t i o n s a r e i n c r e a s e d on
t h e s h o r t e r c o l u m n and d e c r e a s e d o n t h e l o n g e r c o l u m n .
K eeping th e r e f e r e n c e c o n c e n t r a t i o n of c a l c i f e r o l th e
s am e, c h a n g i n g t h a t o f e r g o s t e r o l t o 0 . 0 0 0 9 7 6 gram p e r
m illilite r,

an d c o m p a r i n g t h e r e s u l t s

from t h i s

study

w i t h t h o s e when c a l c i f e r o l i s d o u b l e d and e r g o s t e r o l
i s u n c h a n g e d , t h e s e p a r a t i o n o f e a c h compound i s
i n c r e a s e d on t h e 1 5 . 6 c e n t i m e t e r c o lu m n an d t h a t of
e r g o s t e r o l i s d e c r e a s e d on t h e 7 . 8 c e n t i m e t e r c o l u m n .
U s i n g t h e i m m e d i a t e l y p r e c e d i n g c a l c i f e r o l and e r g o s t e r o l
r e f e r e n c e c o n c e n t r a t i o n s an d m a k i n g a c o m p a r i s o n w i t h

t h e r e s u l t s o b t a i n e d when t h e c a l c i f e r o l c o n c e n t r a t i o n
i s d o u b l e d an d t h a t o f e r g o s t e r o l i s

co n stan t, the

s e p a r a t i o n s o f b o t h co mpounds a r e i n c r e a s e d
colum ns.

on b o t h

When t h e c o n c e n t r a t i o n o f c a l c i f e r o l i s

d o u b l e d and t h a t o f e r g o s t e r o l i s u n c h a n g e d and a
c o m p a r i s o n i s made u s i n g t h e same amount o f c a l c i f e r o l
an d d o u b l i n g t h a t o f e r g o s t e r o l ,
i s decreased w hile th e
on b o t h c o l u m n s .

th e c a lc if e ro l sep aratio n

erg o stero l sep aratio n is increased

In g eneral,

as t h e r a t i o

of the

c o n c e n tra tio n of e r g o s te r o l to c a l c i f e r o l d e c re a se s,

the

p e r c e n t o f c a l c i f e r o l o b t a i n e d f r e e fro m e r g o s t e r o l
increases.

H o w ev er , t h e p r e v i o u s l y s t a t e d f a c t s do n o t

len d th e m se lv e s to any o t h e r g e n e r a l c o n c lu s io n s .
THE USE OF SUPERFILTROL AS AW ADSORBENT
The c o m m e r c i a l p r e p a r a t i o n o f s u p e r f i l t r o l ,
t h e most a c t i v e a d s o r b e n t s ,

one o f

c o n t a i n s i m p u r i t i e s w hich-

a r e s o l u b l e i n t h e o r g a n i c s o l v e n t s u s e d a n d h av e
a b s o r p tio n i n th e u l t r a v i o l e t r e g io n under i n v e s t i g a t i o n .
The m a j o r i t y o f t h i s

i r r e l e v a n t m a t e r i a l i s rem oved fro m

t h e a d s o r b e n t by p r e w a s h i n g t h e c o l u m n w i t h t h e p r o p e r
organic s o lv e n t.

H o w ev er , t h e s e i m p u r i t i e s a r e s e e m i n g l y

n e v e r c o m p l e t e l y removed from t h e

s u p e r f i l t r o l and,

b e y o n d a c e r t a i n a m ount o f p r e w a s h i n g , t e n d t o a p p r o a c h
a rep etitio u s

c o n c e n t r a t i o n an d a b s o r p t i o n s p e c t r u m w h i c h

are ap p a re n t in the a n a ly s is of su cc e ssiv e e q u iv a le n t

-2 4 -

fra c tio n s of elu ate.
spectrum of t h i s

The n e a r l y l i n e a r a b s o r p t i o n

ir r e le v a n t substance lends i t s e l f w ell

to th e m athem atical tre a tm e n t,
an aly sis,

o r a m o d i f i e d form o f t h e
13
p r e s e n t e d by M o r t o n a n d S t u b b s
. T heir

c o r r e c t i o n f o r an i n t e r f e r i n g s u b s t a n c e ,

which

a c c o m p a n i e s a compound w h o s e a b s o r p t i o n s p e c t r u m i n t h e
pure s ta t e

i s known, i s

e n tire ly

g e n e r a l and r e l a t i v e

to any l i n e a r a b s o rb in g m a t e r i a l .
A few s u r v e y i n v e s t i g a t i o n s ,

u s i n g c a l c i f e r o l an d

e r g o s t e r o l i n c o m b i n a t i o n on s u p e r f i l t r o l c o l u m n s o f
various len g th s,

i n d i c a t e d v ery p o s i t i v e l y t h a t an

i r r e l e v a n t m a t e r i a l , whose u l t r a v i o l e t a b s o r p t i o n s p e c ­
t r u m was n o t a t a l l l i n e a r ,

was a p p e a r i n g i n t h e e l u a t e .

H o w ev er , b e c a u s e o f s m a l l e r t o t a l and r e l a t i v e c o n c e n ­
t r a t i o n s o f t h e two compounds an d t h e p r a c t i c e o f
t e r m i n a t i n g t h e developm ent of t h e chromatogram a t t h e
p o i n t w h e r e o n l y c a l c i f e r o l and s e e m i n g l y n o t h i n g e l s e
had b e e n e l u t e d ,

th is nonlinear in te rfe rin g

h a s n o t p r e v i o u s l y b e e n o f much c o n c e r n .

substance

Thus, th e

M o r t o n - S t u b b s c o r r e c t i o n a l o n e i s no l o n g e r a p p l i c a b l e ;
and b e c a u s e e q u i l i b r i u m b e t w e e n s o l u t i o n an d a d s o r b e n t
i s n o t i n s t a n t a n e o u s an d from o b s e r v a t i o n t h e e f f e c t o f
d i f f u s i o n ( n o n s h e r p b e n d in g ) i s f a r from n e g l i g i b l e ,
t h e o r y o f c h r o m a t o g r a p h i c a n a l y s i s g i v e n by W i l s o n ^ offers l i t t l e

in fo rm a tio n r e l a t i v e to th e a n a l y s is of

the

the c o n te n t of th e e l u a t e .

The more r e c e n t k i n e t i c

t h e o r y o f c h r o m a t o g r a p h y p r o p o s e d by T h o m a s ^ j_s Qf
no a i d i n t h e s o l u t i o n o f t h e c o m p o s i t i o n o f t h e e l u a t e .
b e c a u s e o f t h e b i n a r y compound a n d s o l v e n t s y s t e m u s e d
f o r t h i s work.

H e n c e , a d i f f e r e n t m etho d o f a n a l y s i s

m u st b e f o u n d f o r t h e s e p a r a t i o n o f c a l c i f e r o l f r o m
e r g o s t e r o l when u s i n g s u p e r f i l t r o l a s t h e a d s o r b e n t .
The e l u a t e f r o m a 4 . 6 c e n t i m e t e r ( b y w e i g h t ,

2 grams)

s u p e r f i l t r o l co lum n on w h i c h 0 . 0 0 0 2 6 0 g ram o f c a l c i f e r o l
had i n i t i a l l y

b e e n p l a c e d was c a r e f u l l y a n a l y z e d .

f r a c t i o n was c h e c k e d f o r p u r i t y by a r a t i o

Each

t e s t a t no

l e s s t h a n t e n d i f f e r e n t wave l e n g t h s o v e r t h e r a n g e o f
230 m i l l i m i c r o n s t o 3 0 0 m i l l i m i c r o n s .

H aving d e te rm in e d

each f r a c t i o n to c o n ta in only pure c a l c i f e r o l ,

the

p e r c e n t o f c a l c i f e r o l r e c o v e r e d f r o m t h e a d s o r b e n t was
co m p u te d a n d f o u n d t o be 9 7 ,6% .

T herefore,

the non­

l i n e a r i m p u r i t y f o u n d i n t h e e l u a t e when c a l c i f e r o l a n d
e r g o s t e r o l a r e p l a c e d on t h e column t o g e t h e r i s n o t
lik ely

t o be due t o a s t e r i c o r c h e m i c a l ch a n g e i n t h e

c a l c i f e r o l c a u s e d by i t s

c o n ta c t w ith the

E r g o s t e r o l was t h e n i n i t i a l l y

p l a c e d on a c olum n

of s u p e r f i l t r o l and most of t h e e l u a t e ,
co llected fractionw ise,

su p e rfiltro l.

w h i c h was

d id n o t have an a b s o r p t i o n

spectrum re se m b lin g t h a t of e r g o s t e r o l a t a l l .

V arying

t h e e r g o s t e r o l c o n c e n t r a t i o n f r o m 0 . 0 0 0 2 4 4 gram p e r

m i l l i l i t e r t o 0 . 0 0 1 9 5 2 gram p e r m i l l i l i t e r by m u l t i p l e s
o f two and u s i n g a 4 . 6 c e n t i m e t e r a d s o r b e n t colum n, a
v e r y c o n s i s t e n t p a t t e r n o f a b s o r p t i o n c u r v e s was n o t e d
a s b e i n g e l u t e d from t h e colum n i n e a c h c a s e .

F u rth er

i n v e s t i g a t i o n s , u s i n g t h e s e same c o n c e n t r a t i o n s o f
e r g o s t e r o l and a 2 . 3 c e n t i m e t e r s u p e r f i l t r o l colum n,
gave com p arab le p a t t e r n s f o r th e a b s o r p t i o n s p e c t r a o f
t h e column e l u a t e .

The c o n t e n t o f t h e e l u a t e c o u ld

t h e n be c l a s s i f i e d i n t o t h r e e d i s t i n c t co m p on en ts;
( 1 ) a s u b s t a n c e w i t h a maximum a t 282 m i l l i m i c r o n s ,
( 2 ) a s u b s t a n c e w i t h a v e r y d i s t i n c t maximum a t 250 m i l l i ­
m ic r o n s , and ( 3 ) t h e pure e r g o s t e r o l u n a l t e r e d .
a r a tio

test,

A p p ly in g

s im ila r to t h a t used p r e v io u sly to

d e t e r m in e t h e p u r i t y o f f r a c t i o n s

c o n ta in in g c a l c i f e r o l ,

t o s e v e r a l s u c c e s s i v e e l u a t e f r a c t i o n s w h ich a p p e a r e d
to c o n t a i n o n l y t h e s u b s t a n c e w i t h a maximum a t 282
m i l l i m i c r o n s o r th e s u b s t a n c e w i t h a maximum a t 250
m i l l i m i c r o n s , a v e r y c o n s i s t e n t a b s o r p t i o n s p e c tr u m ,
w h ich d i f f e r e d o n l y i n m a gn itu d e from f r a c t i o n t o
f r a c t i o n , was fo u n d f o r e a c h o f t h e two s u b s t a n c e s
( s e e F ig u r e 1 2 ) .

H e n cefo rth , th e m a te r ia l w ith a

maximum a t 282 m i l l i m i c r o n s s h a l l b e c a l l e d s u b s t a n c e 282
and t h a t w i t h a maximum a t 250 m i l l i m i c r o n s s h a l l be
c a lle d su b sta n ce 2 5 0 .

Thus, t h e n o n l i n e a r i r r e l e v a n t

m a t e r i a l i n t h e e l u a t e from a column o f s u p e r f i l t r o l

in itia lly

c o n t a i n i n g o n l y p u r e c a l c i f e r o l an d e r g o s t e r o l

i s v e r y l i k e l y d u e t o a c o n v e r s i o n o f some o f t h e
ergosterol,

c a u s e d by i t s

c o n ta c t w ith the ad so rb en t i n

th e presence of th e s o lv e n t,

i n t o a t l e a s t two d i f f e r e n t

substances.
A s u b s t a n c e w i t h a maximum a t 250 m i l l i m i c r o n s was
r e p o r t e d by K i m b a l l
of i r r a d i a t e d

, who, i n s e p a r a t i n g t h e p r o d u c t s

e r g o s t e r o l c h r o m a to g ra p h ic a lly , noted i t s

appearance j u s t p r i o r t o the e l u t i o n of th e rem aining
non irrad iated e rg o ste ro l.
th o u g h t t o be t o x i s t e r o l ,

At f i r s t ,

the

s u b s t a n c e was

b u t upon c h ro m a to g ra p h in g

e r g o s t e r o l i n t h e a b s e n c e o f c a l c i f e r o l an d a n y o t h e r
p r o d u c t s o f i r r a d i a t i o n an d s t i l l

o b t a i n i n g a maximum

o f some s u b s t a n c e a t 250 m i l l i m i c r o n s ,

t h i s i d e a was

e lim in ated because t o x i s t e r o l presupposes the presence
of c a l c i f e r o l - ^ .

S e v e r a l i n v e s t i g a t i o n s were c a r r i e d

out r e l a t i v e to w hether o r not th e i n t e r f e r i n g m a te r ia l
c o u l d b e a p r o d u c t f ro m t h e i r r a d i a t i o n p r o c e s s , a
r e a c tio n of the e lu e n t w ith the a d so rb e n t,
of th e e lu e n t w ith the e r g o s t e r o l ,
in the e rg o s te ro l,
r e a c t i o n of the
itself.

a reactio n

an im p u rity p re se n t

o r a r e s u l t f r o m some d e c o m p o s i t i o n

e r g o s t e r o l o n t h e s u p e r f i l t r o l c olum n

K im ball concluded t h a t th e s u b sta n c e w ith a

maximum a t 250 m i l l i m i c r o n s c o u l d o n l y be f r o m some
r e a c t i o n b e t w e e n t h e e r g o s t e r o l ' and t h e a d s o r b e n t .

Com putations
H aving i s o l a t e d r e p r e s e n t a t i v e a b s o r p t i o n s p e c t r a
f o r two s u b s t a n c e s , w h i c h a r e a p p a r e n t l y o b t a i n e d fro m
a d e c o m p o s i t i o n o f p u r e e r g o s t e r o l w h i c h i s c a u s e d by
c o n tact w ith s u p e r f i l t r o l ,

the a d d itiv ity

of t h e

e x t i n c t i o n c u r v e s , w h i c h was d i s c u s s e d i n d e t a i l f o r
two compounds w h e n u s i n g a n a l u m i n a a d s o r b e n t ,
be a b l e

should

t o be e x t e n d e d t o f o u r s u b s t a n c e s i n v o l v i n g

th e u s e of a sy ste m of f o u r s im u l t a n e o u s l i n e a r
e q u a tio n s i n f o u r unknowns,

If

d e v i a t i o n s from t h i s

t h e o r y a r e s i g n i f i c a n t when t h e l e n g t h o f t h e s u p e r f i l t r o l
c olum n i s v a r i e d ,

t h e t o t a l an d r e l a t i v e

of c a l c i f e r o l and e r g o s t e r o l i n i t i a l l y

co ncentrations

p l a c e d on t h e

co lum n a r e v a r i e d ,

or t h e r e l a t i v e c o m p o sitio n of the

eluent i s a lte r e d ,

then e ith e r

fo r th e pro d u cts obtained

the ab so rp tio n sp ectra

fro m t h e d e c o m p o s i t i o n of

e r g o s te r o l are i n c o r r e c t or e r g o s t e r o l i n com bination
w ith c a l c i f e r o l i s n o t changed i n t o the

form o b s e r v e d

when e r g o s t e r o l was c h r o m a t o g r a p h e d a l o n e .
The f o u r u n k n o w n s , w h i c h r e p r e s e n t

each o f th e

f o u r c o n s t a n t s i n th e system of f o u r s im u lta n e o u s
lin e a r equations,

a r e some f o r m of a. c o n c e n t r a t i o n

c o n s t a n t , w h i c h d e p e n d s on t h e
the e q u a tio n s.

other u n its in use in

L e t w, x , y , a n d z b e t h e unknown

concentration co n stan ts fo r c a l c i f e r o l,

s u b s t a n c e 282^

su b stan ce 250, and e r g o s t e r o l r e s p e c t i v e l y

in a so lu tio n

o f one c e n t i m e t e r t h i c k n e s s .

and d be

Let a, b,

c,

th e ir respective

e x t i n c t i o n c o e f f i c i e n t s a t a wave

l e n g t h X and a ' ,

b ',

c ',

and d '

th e ir resp ectiv e

e x t i n c t i o n c o e f f i c i e n t s a t a s e c o n d wave l e n g t h X* .
Then l e t a " , b " ,

c " , a n d d" b e t h e i r r e s p e c t i v e

e x tin ctio n co efficien ts
a ’", b'",

c"* a n d d H'

a t a t h i r d wave l e n g t h x " a n d

th e ir respective ex tin ctio n

c o e f f i c i e n t s a t a f o u r t h wave l e n g t h A"'.
a n d D1" a r e t h e r e s p e c t i v e d e n s i t i e s ,

I f D, E)1 , D",

lo g ( I 0/ l ) ,

o f the

m i x t u r e o f t h e f o u r s u b s t a n c e s a t e a c h o f t h e f o u r wave
l e n g t h s , then
at A
at

a

a
'

a t X"
and a t x " 1

w + b

a 'w

x + c

+ b 'x

y + d

+ c 'y

+ d'

z = D
z = D'

a " w + b" x + c" y + d" z = D"
a ' " w + b'" x + c IMy + d™ z = D1"

The c r i t i c a l wave l e n g t h s s e l e c t e d f o r u s e i n t h e comp­
u t a t i o n o f w, x , y ,
m illim icrons.

a n d z w er e 2 5 0 , 2 o 5 , 2 6 1 , a n d 293

T heoretically,

e x t i n c t i o n valu es observed

a t an y f o u r d i f f e r e n t wave l e n g t h s i n t h e r a n g e o f
i n v e s t i g a t i o n c o u l d b e u s e d , b u t t h e maximum a c c u r a c y
I n t h e r e s u l t s w i l l be o b t a i n e d by u s i n g wave l e n g t h s
w here a c r i t i c a l maximum ( o r minimum) e x t i n c t i o n o f
e a c h c o m p o n e n t i s m o s t r e a d i l y a l t e r e d by a f o r e i g n

-3 0 -

substance.

W h enever p o s s i b l e ,

t h e wave l e n g t h s s h o u l d

be sp a c e d a t r e a s o n a b l e i n t e r v a l s and s e l e c t e d where th e
e x t i n c t i o n v a l u e s h a v e b een r e a d from t h e m ost a c c u r a t e
settin g s

of the sp e c tro p h o to m e te r.

S o l u t i o n s f o r t h e f o u r s im u l t a n e o u s e q u a t i o n s were
fo u n d by u s i n g a method of a d d i t i o n an d s u b t r a c t i o n
w hich i s

c o m p i l e d by Dwyer^0*.

For n eq u atio n s in n

u n k n o w n s , whose m a t r i x o f c o e f f i c i e n t s

i s nonsym m etric,

t h i s method o f s o l u t i o n r e q u i r e s o n ly

ro w s a n d

has th e a d d i t i o n a l advantages of having le s s
n u m erical e r r o r ,
calcu late,

le ss recording,

more a c c u r a t e ,

less

chance f o r

and b e i n g e a s i e r t o

t i m e c o n s u m i n g , a n d more

compact t h a n o t h e r methods o f s o l u t i o n .
s u b s t a n c e s were p r e s e n t in th e e l u a t e ,

When o n l y t h r e e
th e system of

e q u a t i o n s was r e d u c e d t o t h r e e a n d t h e f o u r t h e q u a t i o n ,
i n v o l v i n g t h e o b s e r v e d d e n s i t y f o r t h e m i x t u r e a t 293
m illim icron s,

was o m i t t e d .

appeared in the e lu a te ,

When o n l y two s u b s t a n c e s

the e q u a tio n in v o lv in g the

d e n s i t y o b s e r v e d a t t h e wave l e n g t h s u s p e c t e d t o b e t h e
l e a s t c r i t i c a l was e l i m i n a t e d .
F o r 0 . 0 0 0 2 6 0 gram o f c a l c i f e r o l a n d 0 . 0 0 0 4 8 8 gram
o f e r g o s t e r o l p l a c e d o n a 4 . 6 c e n t i m e t e r c olum n o f
su p erfiltro l,

t h e s e v e n t h f r a c t i o n o f e l u a t e was s e l e c t e d

as an i l l u s t r a t i o n o f th e agreem ent betw een th e ob serv ed
a b s o r p t i o n s p e c tru m and t h a t c a l c u l a t e d from t h e

a s s u m p t io n o f t h e p r e s e n c e o f pu re c a l c i f e r o l ,
s u b s t a n c e 2 8 2 , and s u b s t a n c e 25 0 ( s e e T ab le I V ) .

The

c o n c e n t r a t i o n c o n s t a n t s w ere fo u n d t o b e 1 . 0 5 1 0 f o r
c a lc ife r o l,

0 . 1 3 7 3 f o r s u b s t a n c e 2 8 2 , and 0 . 0 0 4 6 f o r

su b stan ce 2 5 0 .

The com puted e x t i n c t i o n c u r v e h a s b e e n

b r o k e n down i n t o t h e a b s o r p t i o n due t o c a l c i f e r o l and
t h a t due t o s u b s t a n c e 28 2 and s u b s t a n c e 2 5 0 .

The g r a p h i c

r e p r e s e n t a t i o n o f t h i s d a t a and a n a l y s i s i s g i v e n i n
F ig u re 1 3 .

The g r a p h s o f t h e o b s e r v e d and com puted

e x t in c t io n curves are very n ea rly c o in c id e n t.

The

v a r i a t i o n s b e tw e e n t h e s e two c u r v e s a r e w i t h i n t h e l i m i t s
o f e x p e r i m e n t a l e r r o r and a r e n o t s i g n i f i c a n t l y d i f f e r e n t .
The r e l a t i v e

c o m p o s i t io n o f t h i s f r a c t i o n a t 28 l m i l l i ­

m ic r o n s i s a b o u t 8 9 . 5 $ c a l c i f e r o l ,
and 0 . 2 $ s u b s t a n c e 2 5 0 .

1 0 .4 $ su b sta n ce 282,

The c a l c i f e r o l i n t h i s f r a c t i o n

i s 2 8 . 3$ o f t h e t o t a l amount o f c a l c i f e r o l i n i t i a l l y
p l a c e d on t h e colu m n .
A ssu m in g t h e p r e s e n c e o f p u re c a l c i f e r o l ,

su b stan ce

2 8 2 , s u b s t a n c e 2 5 0 , and pure e r g o s t e r o l i n t h e f o u r t h
f r a c t i o n o f t h e e l u a t e from a 1 . 1 + c e n t i m e t e r s u p e r ­
f i l t r o l colu m n , a h exam p le o f t h e marked s i m i l a r i t y
b e t w e e n t h e o b s e r v e d and computed a b s o r p t i o n s p e c t r a i s
g i v e n In T a b le V.

The q u a n t i t i e s o f c a l c i f e r o l and

e r g o s t e r o l o r i g i n a l l y p l a c e d on t h e column were t h e same
as b e fo r e .

The c o n c e n t r a t i o n c o n s t a n t s w ere fo u n d to be

-32

0 .9^29 f o r c a l c i f e r o l ,
f o r s u b s ta n c e 250,

0 .2 3 2 3 f o r s u b s ta n c e 282, 0.1346

and 0 .6 4 4 9 f o r e r g o s t e r o l .

The

computed e x t i n c t i o n v a l u e s h a v e b e e n g i v e n s e p a r a t e l y
f o r t h e a b s o r p t i o n due t o c a l c i f e r o l ,
erg o stero l,
250.

t h a t due t o

an d t h a t due t o s u b s t a n c e 282 and s u b s t a n c e

In fa c t,

t h e a b s o r p t i o n s p e c t r u m d u e t o o n e o r any

c o m b i n a t i o n o f t h e s e f o u r s u b s t a n c e s c a n v e r y e a s i l y be
found.

F i g u r e 14 shows t h e g r a p h s o f t h e

o b s e rv e d and

co m p u t e d e x t i n c t i o n c u r v e s a r e a g a i n v e r y n e a r l y
co in cid en t.

As b e f o r e , t h e d e v i a t i o n s o f t h e c o m p u t e d

v a l u e s from t h e o b s e r v e d v a l u e s a r e n o t s u f f i c i e n t l y
l a r g e t o make t h e tw o c u r v e s s i g n i f i c a n t l y
It

d ifferen t.

i s i n t e r e s t i n g t o n o te t h e d i f f e r e n c e betw een t h e

e x t i n c t i o n c u rv e due t o t h e p r e s e n c e o f only p u re
c a l c i f e r o l an d e r g o s t e r o l ,

C u r v e D, an d t h e o b s e r v e d

e x t i n c t i o n c u r v e f o r t h e f o u r co m p o n e n ts, Curve F .
There i s n ' t

any p o s s i b l e c o m b i n a t i o n o f c a l c i f e r o l and

e r g o s t e r o l alo n e t h a t could g iv e a r e s u l t a n t a b s o r p t i o n
s p e c t r u m w i t h a maximum a t 262 m i l l i m i c r o n s .
e l u a t e f r a c t i o n m ust be c o n t a m i n a t e d ,

Thus,

th is

and t h e i n t r o d u c t i o n

o f t h e a b s o r p t i o n s p e c t r u m d u e t o s u b s t a n c e 282 and
s u b s t a n c e 2 50 seem s t o a c c o u n t f o r t h i s maximum, a n d
o th e r v a r i a t i o n s , - very w ell.
of t h i s

The r e l a t i v e c o m p o s i t i o n

f r a c t i o n a t 281 m i l l i m i c r o n s i s

c alcifero l,

X

a b o u t 46.5/5

1 0 . 2 $ s u b s t a n c e 2 8 2 , 3 . 2 $ s u b s t a n c e 2 5 0 , and

4-0.0$ e r g o s t e r o l .

Th e a m o u n t s o f c a l c i f e r o l an d

erg o stero l,

"based u p o n t h e i n i t i a l q u a n t i t i e s p l a c e d on

t h e colum n,

in th is

f r a c t i o n a r e 2 5 . 3$ and 1 2 . 9 $

resp ectiv ely .
A l l o f t h e o b s e r v e d an d c o m p u t e d a b s o r p t i o n c u r v e s
a r e i n a t l e a s t a s c l o s e a g r e e m e n t a s t h e two p r e c e d i n g
e x a m p l e s f o r t h e e n t i r e i n v e s t i g a t i o n and f o r a l l
fractio n s.
S in ce a l l of th e c o n d itio n s f o r each group of
i n v e s t i g a t i o n s a r e k e p t c o n s t a n t e x c e p t t h e one f o r w h i c h
a v a r ia tio n i s being stu d ied ,
conditions is

an e r r o r i n h e r e n t to t h e s e

t h o u g h t t o r e s u l t f r o m t h e f a c t t h a t a few

( f r o m one t o t h r e e )

o f th e i n i t i a l and f i n a l

may b e c a r r y i n g v e r y

sm all q u a n t i t i e s

fractio n s

of c a l c i f e r o l

a n d / o r e r g o s t e r o l a n d a r e d i s c a r d e d b e c a u s e o f v e r y low
e x ti n c t i o n re a d in g s a t the c r i t i c a l p o in ts .
f r a c t i o n concerned,

F o r -each

t h i s ' e r r o r h a s b e e n found t o be l e s s

t h a n 0 . 5 $ o f t h e i n i t i a l q u a n t i t y o f e i t h e r compound
p l a c e d on t h e colum n.
V a r i a t i o n o f Column P r e w a s h
The c o n t e m i n e n t s i n t h e c o m m e r c i a l p r e p a r a t i o n o f
s u p e r f i l t r o l a r e r e m o v e d by p r e w a s h i n g t h e p a c k e d c o l u m n
w ith th e most p o la r s o lv e n t,

developer,

used d u rin g th e i n v e s t i g a t i o n .
hexane, in th e r a t io

o r e l u e n t t o be

The q u a n t i t y o f e t h e r -

o f 1 : 5 p a r t s by v o l u m e , pre'wash

-3 4 -

s o lu tio n re q u ire d to reduce th e se i m p u r i t i e s 't o a
minimum an d t h a t n e c e s s a r y t o i n d i c a t e p a r t i a l
t i o n of the ad so rb en t are q u ite d i f f e r e n t .
4 m illilite rs

of t h i s

deactiv a­

H aving found

e t h e r - hexane s o l v e n t to be th e

minimum v olu m e o f p r e w a s h s o l u t i o n t h a t w o u ld s a t i s f a c t o ­
rily

f r e e a 4 . 6 c e n t i m e t e r s u p e r f i l t r o l column from

im p u rities,

p r e w a s h s o l u t i o n s o f 16 m i l l i l i t e r s

m illiliters

of t h i s

were i n v e s t i g a t e d .

a n d 30

same e t h e r - h e x a n e c o n c e n t r a t i o n
I n e a c h c a s e ., t h e i n i t i a l am ount

o f c a l c i f e r o l p l a c e d on t h e a d s o r b e n t was 0 . 0 0 0 2 6 0 gram
a n d t h a t o f e r g o s t e r o l was 0 . 0 0 0 4 8 8 g r a m .
from F i g u r e 15, t h e r e i s e s s e n t i a l l y
betw een th e r e s u l t s

As i s

evident

no d i f f e r e n c e

o b t a i n e d from p r e w a s h i n g t h e

ad so rb en t w ith 4 m i l l i l i t e r s

of s o lv e n t and th o se

o b t a i n e d f r o m h a v i n g p r e w a s h e d t h e c o l u m n w i t h 16
m illilite rs

of s o l v e n t .

H o w e v e r , when t h e c o lu m n was

p r e w a s h e d w i t h 30 m i l l i l i t e r s

of s o l v e n t ,

o f c a l c i f e r o l r e c o v e r e d was v e r y s l i g h t l y

t h e amount
increased

w h i l e t h e am oun t o f e r g o s t e r o l e l u t e d f r o m t h e c o l u m n
was q u i t e d e c i d e d l y i n c r e a s e d t o 3»5%.

Th e p r e s e n c e of

t h i s q u a n t i t y o f e r g o s t e r o l i n t h e e l u a t e may be
i n c i d e n t a l b u t i s m or e l i k e l y due t o a s l i g h t d e a c t i v a ­
t i o n o f t h e s u p e r f i l t r o l w h i c h was c a u s e d by t h e l a r g e
volum e o f e t h e r - h e x a n e w h i c h came i n c o n t a c t w i t h i t .
T h e re i s n ' t any d i f f e r e n c e i n th e p o s i t i o n o f th e

-3 5 -

in itia l

ap p earan ce of c a l c i f e r o l ,

s u b s t a n c e 250 i n t h e
prewash s o l u t i o n s .

su bstance 282,

an d

elu ate fo r th e th re e d if f e r e n t
H o w ev er ,

th e f r a c t i o n s i n w hich

t h e s e c o m p o n e n t s t e r m i n a t e v a r y ’w i t h i n one o r two
m illilite rs

(see F ig u re 16).

E r g o s te r o l i s found i n

more f r a c t i o n s a n d t e r m i n a t e s l a t e r f r o m t h e c o l u m n
w h i c h was p r e w a s h e d w i t h 30 m i l l i l i t e r s

of e t h e r -

hexane.
As w ould b e e x p e c t e d ,

the r e la tiv e

com position of

th e t o t a l e x tin c tio n f o r e l l

fractio n s

gation d if f e r s

w i t h r e s p e c t t o e a c h com­

only s l i g h t l y

p o n e n t a t 281 m i l l i m i c r o n s

f o r each i n v e s t i ­

(see F igure 17).

h a s t h e most v a r i a b l e p o s i t i o n s due t o

its

E rg o stero l
appearance,

d i s a p p e a r a n c e , an d r e a p p e a r a n c e f r o m t h e c o l u m n s w h i c h
were p rew ash ed w i t h 4, 16,

a n d 30 m i l l i l i t e r s

of s o lv e n t

resp ectiv ely .
V a r i a t i o n of E l u e n t P o l a r i t y
F o r the s t u d i e s r e l a t i v e t o t h e v a r i a t i o n of the
co m p o sitio n of th e e lu e n t,

0 . 0 0 0 2 6 0 gram o f c a l c i f e r o l

a n d 0 . 0 0 0 4 8 8 gram o f e r g o s t e r o l w e r e i n t r o d u c e d o n t o a
4 . 6 c e n t i m e t e r s u p e r f i l t r o l column.

Three d i f f e r e n t

e t h e r - hexane c o m b i n a t i o n s were c o n s i d e r e d .
in the r a t i o s

of 1:1,

h e x a n e by v o l u m e .
decreased,

1:3,

These were

and 1 : 5 p e r t s o f e t h e r t o

As t h e r a t i o

of e th e r to hexane i s

t h e t o t a l amoun t o f c a l c i f e r o l r e c o v e r e d f r o m

t h e c o l u m n v a r i e s "between 99.6% arid 9 8 . 7 % .
o usly,

Sim ultane­

t h e r e c o v e r y o f e r g o s t e r o l from t h e column

d e c r e a s e s f r o m 80.1% t o a b o u t 1 . 3 % .

F i g u r e 18 shows t h e

c o n s i s t e n t l y h i g h r e c o v e r y of c a l c i f e r o l ,

w h i c h seem s t o

be i n d e p e n d e n t o f t h e e t h e r c o n t e n t o f t h e e l u e n t ,

and

th e ra p id d e c lin e of the e r g o s te r o l reco v ery as th e
p o larity

of th e e lu e n t i s decreased .

100% h e x a n e was u s e d a s t h e e l u e n t ,

Eowever,

when

n e i t h e r compound

was d e s o r b e d o r r e c o v e r e d i n t h e c o l l e c t i o n o f over60 f r a c t i o n s .
A l t h o u g h t h e r e c o v e r y o f e a c h compound i s
tan t,

im por­

t h e s e p a r a t i o n o f t h e two compounds i s a l s o v e r y

im portant.

The i n i t i a l and t e r m i n a l f r a c t i o n s i n w h i c h

e a c h o f t h e f o u r c o m p o n e n t s a r e f o u n d a r e shown i n
F i g u r e 19 f o r e a c h e l u e n t v a r i a t i o n .

When t h e e l u e n t i s

co mposed o f e t h e r an d h e x a n e i n e q u a l p a r t s by v o l u m e ,
t h e e l u t i o n o f a l l f o u r s u b s t a n c e s b e g i n s and e n d s q u i t e
quickly,

b u t t h e r e a r e no f r a c t i o n s i n w h ic h any one

substance i s
is

found a l o n e .

As t h e p o l a r i t y

of th e e l u e n t

d e c r e a s e d , t h e v o lu m e i n w h i c h t h e e l u t i o n o f a l l

components t a k e s p l a c e i s
hexane e lu e n t r a t i o

is

increased.

When t h e e t h e r -

1:3, th e f i r s t th re e fra c tio n s

a re f r e e of s u b s ta n c e 282.

Very l i t t l e

ergosterol is

e l u t e d f r o m t h e a d s o r b e n t when e t h e r - h e x a n e i n t h e
ra tio

of 1:5 i s used as the e l u e n t ,

-3 7 -

a s was m e n t i o n e d

p reviously.

T h is sm a ll p e rc e n ta g e of e r g o s t e r o l i s

f o u n d o n l y i n t h e l a s t tw o f r a c t i o n s ;

and h e n c e ,

a ll pre­

c e d in g f r a c t i o n s c o n t a i n c a l c i f e r o l f r e e from e r g o s t e r o l .
U n f o r t u n a t e l y , th e d e co m p o sitio n p r o d u c ts of e r g o s t e r o l
are found i n each f r a c t i o n .
When t h e e l u e n t i s
little

1:1,

relativ e ly

of th e e x t i n c t i o n v a lu e f o r th e t o t a l

elu ate fo r

each i n v e s t i g a t i o n i s

e th e r - hexane,

d e v o t e d t o s u b s t a n c e 282 and

s u b s t a n c e 250 a t 281 m i l l i m i c r o n s ,
p o larity

of t h e e l u e n t i s

especially
in the

however,

decreased,

as the

s u b s t a n c e 2 5 0 an d

s u b s t a n c e 282 o b t a i n more a n d more p r o m i n e n c e

com position of th e e lu a te .

A t t h e same t i m e ,

p o s i t i o n of e r g o s t e r o l d e c r e a s e s to a lm o st z e r o ,
be e x p e c t e d ,

since i t s

desorption i s

th a t the r e la tiv e

a s w o u ld

tending to zero,

the g a in in p o s i t i o n of c a l c i f e r o l i s
expense of e r g o s t e r o l (F ig u re 2 0 ).

the

It

and

probably a t the
is q u ite

evident

e t h e r - hexane c o n te n t of th e e lu e n t

h a s a d i r e c t i n f l u e n c e on t h e c o n v e r s i o n o f e r g o s t e r o l
in to

s u b s t a n c e 282 a n d s u b s t a n c e 2 5 0 .

This c o n v e r s io n .

m u st a l s o r e s u l t i n p a r t f r o m t h e v o l u m e o f e l u e n t
necessary to e lu te

the

compounds f r o m t h e c o l u m n a n d / o r

th e l e n g t h of tim e and sub seq u en t a d s o r b e n t - a d s o r b a t e elu en t c o n tact.
V a r i a t i o n o f Column L e n g t h
By c h a n g i n g t h e amount o f s u p e r f i l t r o l p l a c e d i n a

-5 8 -

c h r o m a to g r a p h ic tu b e o f c o n s t a n t d i a m e t e r ,

the h e ig h t to

w h ich t h e a d s o r b e n t w i l l r i s e i s v a r i a b l e .
a l l p r a c t ic a l purposes,

In f a c t ,

for

the v a r i a t io n in h e ig h t i s a

l i n e a r f u n c t io n o f the w e ig h t.

S i n c e p r e v i o u s work h as

shown an a l m o s t c o m p le te s e p a r a t i o n o f c a l c i f e r o l from
e r g o s t e r o l u s i n g a 4 . 6 c e n t i m e t e r column,

columns o f

b o t h s h o r t e r and l o n g e r l e n g t h s w ere s t u d i e d .

The e l u e n t

c h o s e n f o r t h i s work was e t h e r - h exan e i n th e r a t i o o f
1 : 5 p a r t s b y v o lu m e , and t h e am ounts o f c a l c i f e r o l and
e r g o s t e r o l i n i t i a l l y p l a c e d o n to e a c h colum n o f s u p e r ­
f i l t r o l w ere 0 . 0 0 0 2 6 0 gram and 0 . 0 0 0 4 8 8 gram r e s p e c t i v e l y .
The p a t t e r n s r e s u l t i n g from t h i s work a r e v e r y c l e a r l y
shown i n F i g u r e s 2 1 - 3 0 .

Each f i g u r e i s

com posed so a s

to p r e s e n t the a b s o r p tio n s p e c tr a o f s e v e r a l r e s i d u e s ..
Each r e s i d u e i s from a f r a c t i o n o f th e same number and
from a column o f d i f f e r e n t l e n g t h .
colum ns v a r y from 1 . 1 + c e n t i m e t e r s

The l e n g t h o f t h e s e
( 1 / 2 gram) t o 7 . 0

cen tim eters

(3 gram s) i n i n t e r v a l s o f a b o u t 1 . 2 c e n t i ­

m e te r s ( l / 2

g r a m ).

In a l l c a s e s ,

th e e x t i n c t i o n v a l u e s

a r e t h o s e f o r w h ic h e a c h f r a c t i o n r e s i d u e was v o lu m e t r ic a lly

t a k e n up i n 4 m i l l i l i t e r s

of hexane.

The f i r s t

f r a c t i o n f o r e a c h run i s n o t g i v e n b e c a u s e none o f t h e
s u b s t a n c e s w ere e l u t e d i n any i n v e s t i g a t i o n .
colum ns o f s h o r t e r l e n g t h s ,
ce n tim e te r s,

For t h e

1 . 1 + c e n t i m e t e r s and 2 . 3

the c a l c i f e r o l i s e lu t e d q u it e q u ick ly

in th e f i r s t
calcifero l,

few f r a c t i o n s .
w hich i s f r e e

In f a c t,

most of th e

from e r g o s t e r o l ,

is

eluted

b e f o r e th e e l u t i o n o f t h e c a l c i f e r o l from t h e

longer

columns i s e v e n s t a r t e d

As t h e

(see F ig u res 21 -2 3 ).

e l u t i o n o f t h e s h o r t e r co lu m n s t e n d s t o c o m p l e t i o n ,
d i s t o r t i o n of the c a l c i f e r o l curve to t h a t
and s u b s t a n c e 250 i s

very ev id e n t

the

of e r g o s t e r o l

(F igures 2 4 -2 7 ).

S u b s t a n c e 250 seems t o g a i n i n p r o m i n e n c e and t h e n t a p e r
back t o an a b s o r p t i o n s p e c tru m s i m i l a r t o t h a t o f p u r e
ergosterol,

a s can be seen i n F i g u r e s 2 5 - 2 9 .

F o r the

l o n g e r c o l u m n s , 5 . 8 c e n t i m e t e r s an d 7 . 0 c e n t i m e t e r s ,
there is l i t t l e

v i s u a l e v i d e n c e o f an y v e r y p r o m i n e n t

q u a n t i t y of c a l c i f e r o l i n th e e lu a te b e f o re th e s i x t h
f r a c t i o n , F ig u re 25.

P r io r to th is f r a c t i o n the

m a j o r i t y o f t h e e l u a t e a p p e a r s t o b e c o m p o se d o f
substance 282.

C alcifero l is

e l u t e d v e ry h e a v i l y from

t h e s e l o n g e r columns i n f r a c t i o n s 7 - 9 .

L e s s e r amounts

o f c a l c i f e r o l end t h e b e g i n n i n g o f a s l i g h t d i s t o r t i o n
of t h e c a l c i f e r o l c u r v e a r e e v i d e n t i n F i g u r e 29.
a b s o rp tio n s p e c tra f o r th e rem aining f r a c t i o n s

The

elu ted

f r o m t h e 7 . 0 c e n t i m e t e r c o l u m n a r e shown i n F i g u r e 3 0 .
A l a r g e r e x tin c tio n s c a le i s used f o r t h i s

graph i n

o r d e r t o show t h e d i s t o r t i o n more c l e a r l y .
the c o n tr ib u tio n ,

i n term s of c o n c e n tr a tio n ,

A lthough
of substance

250 t o t h e r e s u l t a n t a b s o r p t i o n s p e c t r a may be s m a l l , i t s

-4 0 -

presence Is very e v id e n t.
250 ten d s to c o m p le tio n ,

As t h e e l u t i o n o f s u b s t a n c e
th e s p e c t r a l p a t t e r n s , w h ich

may be p a r t i a l l y due t o some i m p u t i t i e s

s t i l l b ein g

r e l e a s e d from t h e a d s o r b e n t , h a v e a t e n d e n c y t o r e t u r n
to n e a r l y l i n e a r s p e c t r a .

The e l u a t e f r a c t i o n s from t h e

colum ns o f i n t e r m e d i a t e l e n g t h s ,
4 .6 c e n tim e te r s ,

3 . 4 + c e n t i m e t e r s and

f o l l o w p a t t e r n s b e tw e e n t h o s e f o r t h e

s h o r t e r and l o n g e r colu m n l e n g t h s .
A c o m p ila tio n o f th e t o t a l reco v ery o f c a lc ir fe r o l
and e r g o s t e r o l from e a c h column o f d i f f e r e n t l e n g t h i s
shown i n F i g u r e 31*
recovery,

F or e a c h colu m n , t h e c a l c i f e r o l

s e e m i n g l y in d e p e n d e n t o f t h e colum n l e n g t h ,

c o n s is t e n t ly above 98^ .

is

On t h e o t h e r h an d , t h e e r g o s t e r o l

r e c o v e r y d e c l i n e s s t e a d i l y from 2 8 . 7 ^ from t h e 1 . 1 + c e n t i ­
m eter column t o 0 . 0 % from t h e 5 . 8 c e n t i m e t e r colu m n .
E r g o s t e r o l i s a b s e n t from t h e e l u a t e from t h e 7 . 0 c e n t i ­
m eter column a l s o .
R e g a r d le ss o f th e f a c t t h a t s e v e r a l - o f th e curves
i n t h e f i g u r e s f o r t h e s u r v e y o f t h e v a r i o u s column
l e n g t h s a p p ea r t o c o n t a i n o n l y p u re c a l c i f e r o l , f u r t h e r
m a th e m a tic a l i n v e s t i g a t i o n show6 a l m o s t a l l o f t h e s e
f r a c t i o n s to b e c o n t a m in a t e d w i t h v a r y i n g p o r t i o n s o f
s u b s t a n c e 282 and s u b s t a n c e 2 5 0 .

F ig u r e 32 p r e s e n t s i n

a com pact form t h e i n i t i a l and t e r m i n a l f r a c t i o n s i n
w h ich e a c h o f t h e com ponents a p p e a r s f o r e a c h r u n .

-4 1 -

As

th e l e n g th o f the

column i s

increased ,

f r a c t i o n s r e q u ir e d to f r e e the
co ntents i s in creased .
great,

however,

adsorbent.
filtro l

t h e number of

s u p e r f i l t r o l of i t s

T his in c r e a s e i s

not n e a r ly as

a s i t w as when a l u m i n a was u s e d a s t h e

For an in c re a s e in the le n g th of th e super­

column,

e a c h component i s d e la y e d i n i t s

a p p e a r a n c e and t e r m i n a t e s

in itia l

two o r t h r e e f r a c t i o n s

later

t h a n f o r t h e im m e d ia te ly p r e c e d i n g s h o r t e r colum n.
S u b s t a n c e 282 a n d s u b s t a n c e 250 acco m pa ny o r p r e c e d e t h e
e l u t i o n of c a l c i f e r o l .

As t h e

column i s l e n g t h e n e d ,

t h e s e two i n t e r f e r i n g s u b s t a n c e s p r e c e d e t h e c a l c i f e r o l
by a n e v e r i n c r e a s i n g n u m b e r o f f r a c t i o n s .

There are

no f r a c t i o n s w h i c h a r e f r e e f r o m e i t h e r c a l c i f e r o l o r
e r g o s t e r o l t h a t a r e a l s o f r e e f r o m b o t h s u b s t a n c e 282
and s u b s t a n c e 2 5 0 .
S u b s t a n c e 282 and s u b s t a n c e 2 5 0 c a n n o t be a n a l y z e d
on a t o t a l p e r c e n t r e c o v e r y b a s i s s u c h a s t h a t u s e d
p r e v i o u s l y f o r c a l c i f e r o l and e r g o s t e r o l

(see F ig u re 51).

T h i s w ould r e q u i r e a k n o w l e d g e o f t h e amoun t o f e r g o s t e r o l
r e t a i n e d o n t h e c o l u m n , i n t h e c o n v e r t e d and u n c o n v e r t e d
form s,

a s . w e l l a s some a d d i t i o n a l i n f o r m a t i o n a b o u t t h e

two p r o d u c t s o f c o n v e r s i o n .

However, the r e l a t i v e

co m p o sitio n of each com plete run,
f o r a n y d e f i n e d wave l e n g t h ,
be c o m p u t e d

(F ig u re 33)•

or any f r a c t i o n t h e r e o f ,

s a y 281 m i l l i m i c r o n s ,

S in ce the

-4 2 -

can

amount of e r g o s t e r o l

e l u t e d f r o m t h e c o l u m n ae t h e
to zero,

the r e l a t i v e

length is

i n c r e a s e d goes

p o s it i o n of e r g o s t e r o l in the

c o m p o s i t i o n o f t h e e l u a t e w i l l go t o z e r o .
an i n c r e a s e i n column l e n g t h ,

calcifero l,

Hence,

w ith

s u b s t a n c e 282,

a n d s u b s t a n c e 2 5 0 s h o u l d be p r e s e n t i n t h e r e l a t i v e
c o m p o s itio n of th e e l u a t e

to a g r e a t e r d eg ree.

e x t i n c t i o n v alu es f o r the e lu a te
slig h tly

The

f o r each run d ecrease

and the c o n c e n t r a t i o n c o n s ta n ts f o r s u b sta n c e

2o2 a n d s u b s t a n c e 2 p 0 i n c r e a s e
t h e c o lu m n i s i n c r e a s e d .
e rg o s te ro l into th ese

s l i g h t l y as tn e l e n g t h of

Hence,

two c o m p o n e n t s a p p e a r s t o be a

f u n c t i o n of t h e column l e n g t h .
le n g th g r e a t e r th an 4.6

H owev er , f o r c o l u m n s o f

centim eters,

e r g o s t e r o l i s no l o n g e r e l u t e d ,
and c o n c e n t r a t i o n c o n s ta n ts
Therefore,

th e c o n v e r s i o n of

from w hich

the e x t i n c t i o n v a lu e s

t e n d t o become c o n s t a n t .

t h e c o n v e r s i o n o f e r g o s t e r o l m u st be a

f u n c t i o n o f t h e l e n g t h o f t h e colu mn a n d t n e a m o u n t o f
pure e r g o s t e r o l a v a i l a b l e f o r d e s o r p tio n a n d /o r e l u t i o n
from th e colum n,.
V a r i a t i o n o f Sam ple C o n c e n t r a t i o n
To s t u d y t h e e f f e c t o f t h e c h a n g e o f ' c o n c e n t r a t i o n
upon th e s e p a r a t i o n o f c a l c i f e r o l from e r g o s t e r o l ,

two

d i f f e r e n t l e n g t h columns of s u p e r f i l t r o l c o n t a i n i n g
various q u a n titie s
The c o lu m n o f 2 . 3

o f e a c h compound w e r e i n v e s t i g a t e d ..
centim eters

-4p-

i n l e n g t h was s e l e c t e d f o r

f u r t h e r s t u d y "because t h e r e h a d d e f i n i t e l y b e e n p r o v e n
(by a p r e v i o u s i n v e s t i g a t i o n )

t h a t some u n c o n v e r t e d

e r g o s t e r o l had b e e n e l u t e d from i t .

The e f f e c t o f

h a l v i n g and d o u b lin g th e i n i t i a l q u a n t i t y of e r g o s t e r o l
( 0 . 0 0 0 4 5 5 gr am ) a n d k e e p i n g t h a t o f c a l c i f e r o l
g r a m ) c o n s t a n t was s t u d i e d ( s e e F i g u r e 3 4 ) .

(0.0 0 0 2 6 0

The t o t a l

c a l c i f e r o l r e c o v e r y r e m a i n e d a b o v e 9 9 % u n t i l t h e r u n in.
w h i c h t h e am o u n t o f e r g o s t e r o l was d o u b l e d .
c a l c i f e r o l r e c o v e r y d r o p p e d t o a b o u t 9~5%.
d ecline

Here th e
This sudden

i n r e c o v e r y c a n p r o b a b l y be a t t r i b u t e d

t o some

m o le c u la r en ta n g le m e n t betw een th e c a l c i f e r o l and
e r g o s t e r o l a t th e s u r f a c e of th e s u p e r f i l t r o l p a r t i c l e s
s i n c e t h e e r g o s t e r o l was i n i t i a l l y
l a r g e am ount.

p resen t in a r e la tiv e ly

Trie t o t a l r e c o v e r y ( i n p e r c e n t )

e r g o s t e r o l i s s o m ew h at more v a r i a b l e .

of

However,

the

a c t u a l g a i n i n amount of pure e r g o s t e r o l e l u t e d from
t h e co lum n i n c r e a s e s s t e a d i l y f r o m 0 . 0 0 0 0 5 5 g r a n t o
0 . 0 0 0 0 7 5 gram t o 0 . 0 0 0 1 5 9 gram f o r q u a n t i t i e s
p l a c e d on t h e c o lu m n o f 0 . 0 0 0 2 4 4 gram,

in itia lly

0 . 0 0 0 4 5 8 gram,

a n d 0 . 0 0 0 9 7 6 gram r e s p e c t i v e l y .
Ae

the e rg o ste ro l co ncen tratio n is

increased,

fo u r c o n s titu e n ts appear in the e lu a te a l i t t l e
and term in ate s l i g h t l y

later

(Figure 3 5 ).

T hus ,

v o lu m e o f e l u e n t n e c e s s a r y f o r t h e i r e l u t i o n i s
The r e l a t i v e

the

earlier
the
g reater.

c o m p o s itio n ( F ig u r e 3o) of th e e l u a t e

-4 4 -

f o r e a c h r u n a t 281 m i l l i m i c r o n s

is

in agreem ent w ith

the f a c t t h a t th e q u a n tity of e l u te d e r g o s t e r o l in c r e a s e s
as the r a t i o

of e r g o s te r o l to

Sim ultaneously,

c a lc if e r o l is

in creased .

t h e c o n t r i b u t i o n of s u b s t a n c e 282 and

s u b s t a n c e 250 to t h e t o t a l e x t i n c t i o n v a lu e i s g r e a t e r .
This i s

t o be e x p e c t e d s i n c e

t h e s e two s u b s t a n c e s a r e a

r e s u l t o f a c o n v e r s i o n o f some o f t h e e r g o s t e r o l .

The

c o n t r i b u t i o n of c a l c i f e r o l to th e t o t a l a b s o r p tio n
spectrum w i l l n a t u r a l l y d e c l i n e .
As a c o m p a r i s o n f o r t h e a b o v e w o r k , a s i m i l a r s u r v e y
was made on a co lu m n o f 4 . 6 c e n t i m e t e r s

in le n g th .

This

l e n g t h o f co lu m n was s e l e c t e d b e c a u s e t h e r e was v e r y
little

e r g o s t e r o l e l u t e d f r o m i t olS G.e t e r m i n e d f r o m a

p r e v i o u s s tu d y i n which th e i n i t i a l am ounts of c a l c i f e r o l
a n d e r g o s t e r o l w e r e 0 . 0 0 0 2 6 0 gr am a n d 0 . 0 0 0 4 8 8 gr am
resp ectiv ely .
was h a l v e d ,

For t h i s study th e q u a n t i t y of e r g o s t e r o l

doubled,

and q u ad ru p led w hile t h a t of

c a l c i f e r o l was h e l d c o n s t a n t .

As t h e o r i g i n a l q u a n t i t y

o f e r g o s t e r o l on t h e co lu m n i s

i n c r e a s e d from 0 .0 0 0 2 4 4

gram t o 0 . 0 0 1 9 5 2 g r a m ,

the t o t a l c a l c i f e r o l re c o v e ry

d e c r e a s e s from 100^ t o 93^ ( s e e F i g u r e 3 7 ) .
decrease i s alm ost li n e a r ,
graphing.

In com parison,

i n c r e a s e s f r o m 0% t o 1 3 $ .

This

d e p e n d i n g on t h e m e t h o d o f
the e r g o s t e r o l recovery
T h i s i s s om ew hat more s i g n i f ­

i c a n t t h a n t h e p e r c e n t a g e s seem t o i n d i c a t e s i n c e t h e

amount o f e r g o s t e r o l r e c o v e r e d i n c r e a s e s from 0 .0 0 0 0 0 0
gram t o 0 . 0 0 0 0 0 6 gram t o 0 . 0 u 0 0 2 0 g r a m t o 0 . 0 0 0 2 6 3 gram
f o r i n i t i a l q u a n t i t i e s o f e r g o s t e r o l o f 0 . 0 0 0 2 4 4 g r am ,
0 . 0 0 0 4 8 8 g r am , 0 . 0 0 0 9 7 6 g r a m , en d 0 . 0 0 1 9 5 6 gr am r e s p e c ­
tiv ely .
The p o s i t i o n o f t h e i n i t i a l a p p e a r a n c e o f t h e f o u r
components i n t h e

eluate is

th e 2 . 3 c e n t i m e t e r colum n.
the

very com parable to t h a t f o r
However,

e l u t i o n of e a c h component i s

(see F ig u re 3 8 ).
column,

the t e r m i n a t i o n of

somew hat more v a r i a b l e

As c o m p a r e d t o t h e 2 . 3 c e n t i m e t e r

th e volume of e l u e n t r e q u i r e d t o f r e e

th e column

o f t h e c o n s t i t u e n t s w i l l be g r e a t e r b e c a u se of the
i n c r e a s e i n column l e n g t h .
tio n of the c o n s titu e n ts

P erhaps the

e a r l i e r term ina­

f ro m t h e c o l u m n c o n t a i n i n g

0 . 0 0 0 4 8 8 gram o f e r g o s t e r o l a s c o m p a r e d t o t h e

c o lu m n

c o n t a i n i n g 0 . 0 0 0 2 4 4 gram o f e r g o s t e r o l c a n p a r t i a l l y

be

a t t r i b u t e d t o t h e e a r l i e r e l u t i o n o f s u b s t a n c e 282 end
su b sta n c e 250.

The e a r l i e r a p p e a r a n c e of e r g o s t e r o l

fr o m t h e c o l u m n c o n t a i n i n g 0 . 0 0 0 9 7 6 gram o f e r g o s t e r o l
i s q u i t e i n l i n e w ith th e p a r a l l e l
2 . 3 c e n t i m e t e r column.

s itu a tio n fo r th e

The r e t u r n o f e r g o s t e r o l t o a

l a t e r i n i t i a l a p p e a r a n c e from t h e column w hich c o n t a i n e d
0 . 0 0 1 9 5 2 gram o f e r g o s t e r o l i s q u i t e
large quantity

likely

due t o t h e

of e r g o s t e r o l which h a s f o r c e d t h e

conversion products to appear s t i l l

-4 6 -

e a r l i e r an d r e t a i n e d

ca lcifero l for a la te r

fractio n .

i s u n d o u b t e d l y due t o t h e i n c r e a s e

The i n c r e a s e d b a n d i n g
in e r g o s te r o l concen­

tra tio n .
The r e l a t i v e

com position of th e t o t a l i t y

r u n a t 261 m i l l i m i c r o n s i s

shown i n F i g u r e 3 9 .

of each
The

p a t t e r n f o r each component i s a l m o s t i d e n t i c a l to t h a t
f o r t h e c o m p a r a b l e c o n d i t i o n s on t h e s h o r t e r c o l u m n .
Hence,

as the r a t i o

calcifero l

is

o f th e amount o f e r g o s t e r o l to

increased ,

ergo sterol,

s u b s t a n c e 2&2, a n d

s u b s t a n c e 250 i n c r e a s e w h i l e c a l c i f e r o l d e c r e a s e s
prom inence i n th e r e l a t i v e

in

com position of the e l u a t e .

SUMMARY
The s e p a r a t i o n o f c a l c i f e r o l f r o m e r g o s t e r o l u s i n g
an e t h e r or e t h e r - hexane s o lv e n t and an alum ina or
s u p e r f i l t r o l ad so rb en t has been p re se n te d .

E t h e r was

fo u n d to be t h e b e s t s o l v e n t and e l u e n t f o r th e accom­
p l i s h m e n t o f t h e s e p a r a t i o n by means o f a n a l u m i n a
ad sorbent.

When s u p e r f i l t r o l was u s e d a s t h e a d s o r b e n t ,

e t h e r - hexane in the r a t i o

o f 1 : 5 p a r t s by v olum e was

f o u n d t o be a v e r y s a t i s f a c t o r y s o l v e n t f o r t h e s e p a r a ­
tio n .
I n o b t a i n i n g a c o m p l e t e s e p a r a t i o n o f t h e two
co m p o u n d s ,

th e w eig h t of th e a d s o r b e n t and th e h e i g h t

t o w h i c h i t was p a c k e d i n t h e c h r o m a t o g r a p h i c t u b e w e r e
very im p o rta n t.

A l u m i n a g a v e a v e r y good s e p a r a t i o n o f

t h e two c o m p o u n d s,

th e r e s u l t a n t f o r n of each b e in g pure

and f r e e from any o t h e r m a t e r i a l .

H owev er ,

the le n g th ­

e n i n g o f t h e c olu m n a n d t h e r e s u l t i n g b r o a d e r b a n d i n g
o f e a c h compound w i t h i n t h e co lum n w e r e f o u n d t o be
u n d esirab le.

S u p e r f i l t r o l gave a v e r y s h a r p s e p a r a t i o n

much more q u i c k l y a n d on a v e r y much s h o r t e r c o l u m n .
H o w ev er , a c o n v e r s i o n o f t h e e r g o s t e r o l i n t o two
i r r e l e v a n t s u b s t a n c e s t o o k p l a c e on t h e a d s o r b e n t a n d ,
w h i l e c a l c i f e r o l a n d e r g o s t e r o l were o b t a i n e d f r e e f r o m
each o th e r,

n e i t h e r compound was f o u n d i n t h e e l u a t e

c o m p l e t e l y f r e e f r o m one o r b o t h o f t h e
m aterials.

irrelev an t

T h i s o b s e r v a t i o n l e a d to a p a t t e r n a n a l y s i s

of th e c o n v ersio n pro d u cts and as a r e s u l t ,
fractio n s

c o u ld be a n a l y z e d by a system o f s i m u l t a n e o u s

lin e a r equations.

A lso,

th e c o n c e n t r a t i o n of c a l c i f e r o l

or e r g o s t e r o l in th e t o t a l e l u a t e ,
th ereo f,

the e l u a t e

or any f r a c t i o n

c o u l d be d e t e r m i n e d e x a c t l y .

The r e l a t i v e a n d t o t a l
and e r g o s t e r o l i n i t i a l l y
very l i t t l e

c o n c e n tr a tio n s of c a l c i f e r o l

p l a c e d on e i t h e r a d s o r b e n t h a d

e f f e c t on th e t o t a l

c a lc ife ro l recovery.

Howe ver , b o t h t h e s e p a r a t i o n o f t h e two compounds a n d
th e t o t a l e r g o s t e r o l r e c o v e r y were i n f l u e n c e d c o n s i d e r a b l y
by t h i s

property.

TABLE I
F ig .

Alum ina
( c m .)
7 .8

i
vr
0
1
15.6

E lu e n t
E th e r Hexane
( p a r t s by v o l . )

C a l. fr e e
from E r g .

R ecovery
T otal
Erg. fr e e
C a l.
from C a l .

T otal
Erg.

{ %)

( %)

{ %)

{ %)

^0

50

34.0

31+.2

9 0 .3

81.5

70

50

3 4 -5

3 7 .6

9 3 .4

85.2

80

20

3 3 .8

38.8

9 4 .7

8 8 .0

100

0

3 2 . i|

3 3 .1

96.2

90.1

70

30

48.1+

1+5.7

90.1

82.9

80

20

1+7 . 1

1+4.7

92.8

85.2

1 00

0

51.6

4 9 .0

9 4 .3

87.2

I n i t i a l c a l c i f e r o l c o n c e n tr a tio n :
I n i t i a l e r g o s te r o l co n c e n tr a tio n :

0 .0 0 0 2 6 0 g . / m l .
0 .0 0 0 4 8 8 g . / m l .

TABLE I I
'ig .

Alum ina

(cm .)
7 .8

6

I 5.6

E lu e n t
E th e r Hexane

In itia l
F r a ctio n
C o n t a in in g
C a lc ife r o l

( p a r t s by v o l .)

E lu a te
I n itia l
F in a l
F r a c tio n
F r a ctio n
C o n t a i n in g C o n t a i n i n g
E rgosterol C a lc ife r o l
(num ber)

F in a l
F r a c tio n
C o n t a in i n g
E rgosterol

F r a c tio n s
C o n ta in in g
C a l . E r g . Cal
Only Only and
Erg
(num ber)

50

50

37

46

6o

72

9

12

15

70

30

24

30

4o

50

6

10

11

8o

20

19

25

32

4i

6

9

8

100

0

15

20

26

35

5

9

7

70

30

4o

51

6l

78

li

17

ll

8o

20

34

43

51

65

9

14

9

100

0

29

37

42

56

8

14

6

I n i t i a l c a l c i f e r o l c o n c e n tr a tio n :
I n i t i a l e r g o s te r o l co n c e n tr a tio n :

0 .0 0 0 2 6 0 g . / m l .
0.000[j.88 g . / m l .

TABLE I I I
F ig .

A lu m in a

In itia l
F r a ctio n
C o n t a in i n g
C a lc ife r o l
(g.)

( c m .)

E lu a te
In itia l
F in a l
F r a ctio n
F r a ctio n
C o n t a i n in g
C o n ta in in g
E rgosterol C a lc ife r o l
(number)

F in a l
F r a c tio n
C o n t a in in g
E rgosterol

F r a c tio n s
C o n t a in i n g
C a l. E rg. C a l.
Only Only and
E rg.
(number)

2

5-9

9

12

19

24

3

5

8

1+

7 .8

15

20

26

55

5

9

7

8

15.6

29

37

42

56

8

11+

6

16

51.1

49

63

65

86

14

21

3

22

42.7

64

84

84

110

20

26

1

23

4 4 .6

66

87

86

115

21

29

0

E lu e n t: D ie t h y l e th e r
I n i t i a l c a l c i f e r o l c o n cen tra tio n :
I n i t i a l e r g o s t e r o l co n cen tra tio n :

0 .0 0 0 2 6 0 g . / m l .
0 .0 0 0 4 8 8 g . / m l .

TABLE IV

S u p e r f i l t r o l : 4.6 c e n tim e te rs
I n itia l c a lc ife ro l co n cen tratio n :
I n itia l, e rg o ste ro l co ncentration:
F ractio n :
#7

0.000260 g . / m l .
0.000488 g . / m l .

C orrection

230

0 .0 3 8

0.462

0 .5 0 0

0 .5 2 0

240

0.038

0.592

0 .6 3 0

0.632

250-

0.040

0.736

0.776

0.776

260

0.043

0.845

0.888

0.893

262

0.045

CO
LH
CO
«
O

E xtinction
C alcifero l
Computed.

Wave L e n g t h
(m mu)

0.903

0.906

26 4

0.046

0 .8 6 2

0.908

0.909

2o 5

0.047

0.861

0.908

0.908

266

0.048

0.854

0.902

0.904

270

0.054

0.812

0.866

0.866

271

0.055

0.795

0 .8 5 0

0.852

272

0.055

0.777

0 .8 3 2

0.835

276

0.057

0.688

0.745

0.741

280

0.063

0.577

0.640

0.636

281

0 .0 6 5

0.545

0.610

O .o lO

282

0 .0 6 5

0.516

0.581

0.580

289

0 .0 5 3

0.324

0.377

0.380

293

0.049

0.230

• 0.280

0.282

300

0.033

0.111

0.145

0 .1 5 0

y = 0.0046

w = 1.0510

x = 0 .1 3 7 3

-5 3 -

Observed.

TABLE V

S u p erfiltro l:
1 .1 + centim eters
I n it i a l c a lc ife ro l concen tratio n :
I n it i a l e rg o ste ro l co ncentration:
F ractio n :
//4

0.000260 g ./in l.
0.000463 g . /m l .

7/ave L e n g t h
E x tin ctio n
(m mu)
C o r r e c t i o n C a l c i f e r o l E r g o s t e r o l Computed O b s e r v e d
230

0.217

0 .414

0.076

0.707

0.730

240

0.263

0.531

0.081

0.876

0.872

250

0 .315

0.661

0.144

1.120

1.120

260

0.262

0.758

0.272

1.292

1.294

262

0 .2 5 0

0.770

0.283

1.303

1.300

264

0.229

0.773

0 .2 8 6

1.288

1.288

265

0.214

0.772

0.296

1.282

1.282

266

0.200

0.766

0 .310

1.276

1.278

270

0 .153

0.729

0.397

1.279

1.278

271

0.146

0.714

0.398

1.258

1.260

272

0.139

0.697

0.3o9

1.225

1.224

276

0.130

0.618

0.339

1.086

1.086

280

0.140

0.518

0.410

1.066

1.068

281

0.142

0.489

0.421

1 .0 5 2

1.052

282

0.142

0.463

0.416

1.021

1.018

289

0.113

0.290

0.221

0.623

0.620

293

0.106

0.206

0.244

0.556

0.556

300

0.072

0.100

0 .0 5 0

0.221

O.23O

w = 0.9429

y = 0 . 13^6

x = 0.2323

z = 0 .6449

1.0

0.8

0.6

0 .4

0.2
-o —

0.0
230

240

250
W ove

F ig u re 2 .

270
260
L e n g t h (m illim ic ro n s )

A b s o r p tio n s p e c t r a o f c a lc ife r o l a n d
A - C a l c i f e r o l ( 0 . 0 0 0 0 1 7 5 g ./m l.)
B -E rg o s te ro l
( 0 0 0 0 0 2 4 4 g ./m l.)

e rg o s te ro l

280

in

290

hexane.

300

c
_____ —

<

— ---------------

M—

D

-----------------------------------------

A

0 5 0 E th e r
5 0 H exone

60
40

70
30
E lu e n t (p e rc e n t by

F igure 3. R ecovery o f co lc ife ro l a n d
A - P u r e c a lc ife r o l
£3- P u r e e rg o s te ro l
C - T o t a l c a lc ife ro l
D - T o t a l e rg o s te ro l

80
20
volume)

e rg o ste ro l from

a

7 .8

90
10
c e n tim e te r

100
0

a lu m in a

colum n.

100

80

5 0 E th e r
5 0 Hexone

60
40

F igure 4 , R ecovery o f c a lc ife ro l
A - P u r e co lc ife ro l
B —P u re erg o stero l
C —T o ta l c a lc ife r o l
D - T o to l e rg o s te ro l

70
30
E lu e n t (p e rc e n t

by

on d

fro m

e rg o s te ro l

00
20
volume)
a

100

90

0

10

15.6 c e n tim e te r

alumina

colum n.

100

80

E 60

20

5 0 E th e r
5 0 Hexone

70
30
E lu en t (p e rc e n t by vo!
volume)

60
40

Figure 5 . E lu tio n o f c o lc ife ro l a n a e rg o s te r o l fro m
A - I n it ia l f ra c tio n containing c a lc ife ro l
B - Initial fro c tio n containing e rg o s te ro l
C - F i n a l f ro c tio n c o n ta in in g c o lc ife ro l
D - Final f ra c tio n c o n ta in in g e rg o s te ro l

a

7 .8

100

90

c e n tim e te r

a lu m in a

colum n.

100

80

= 60

40

20

5 0 E th e r
5 0 Hexane

70
30
E lu en t ( p e r c e n t

60
40

F ig u re 6. E lu tio n o f
In itia l
B —In itia l
C -F in a l
D -F in a l

c o lc ife ro l
f r a c tio n
f ra c tio n
f r a c ti o n
f ro c tio n

80
20
by volume)

a n d e rg o s te r o l from
c o n ta in in g c a lc i f e r o l
co n ta in in g e r g o s t e r o l
c o n ta in in g c a lc ife r o l
co n tain in g e r g o s t e r o l

o

90

1 5 .6 c e n tim e te r

100

a lu m in a colum n.

tejasji

Eluate

Fraction

(number)

120

Ol0

10

I

20
Column L en g th

I■

30
(ce n tim e te rs)

Figure 7. E lu tio n o f colciferol a n d e rg o s te ro l u sin g
A — I n itia l f r a c ti o n c o n ta in in g calcifero l
0 - In itia l f ra c tio n co n ta in in g e rg o s te ro l
C —Final fro c tio n c o n ta in in g calcifero l
D —Final fro c tio n co n to in in g e rg o ste ro l

a

pure e th e r

I

40

elu en t.

50

100

00

£40

20

20
30
Column Length (ce n tim e te rs)
F igure 8. R e co v ery of c a lc ife ro l using
A - P u r e c a lc ife ro l
B - T o t a l c a lc ife ro l

a

p u re

40

50

e th e r eluent.

100

80

*60

£ 40

20

20
Column
C
olum n
F igure 9. R ecovery o f e rg o s te ro l
A - P u r e e rg o s te ro l
B - T o t a l e rg o s te ro l

u sing

30
L e n g th (ce n tim e te rs)
a

pure

e th e r eluent.

40

50

100

80

60

£40

20

C alcifero l
E rg o ste ro l

2 .6 0
4 .8 8

2 .6 0
9 .7 6

2 .6 0 5 . 2 0 2 .6 0 5 .2 0 2 .6 0 5 . 2 0 2 .6 0 5 . 2 0
4 .8 8 4 .8 8 4 .8 8 9 .7 6 9 .7 6 4 .8 8 9 .7 6 9 .7 6
C o n c e n tra tio n X I0 “ 4 (g ram s p er m illilite r)

F igure 10. R ecovery of c a lc ife ro l ond
A —Pure colciferol
B - P u r e erg o stero l
C - Total colciferol
D - T o to l ergosterol

e rg o s te ro l

fro m

a

7 .8

5 .2 0
4 .8 8

c e n tim e te r

5 .2 0
9 .7 6

a lu m in a

column.

100

80

£ 60

20

Calciferol
E rg o stero l

2 .6 0
4 .8 8

2 .6 0
9 .7 6

2 .6 0 5 . 2 0 . 2 .6 0 5 .2 0 2 .6 0 5 .2 0 2 . 6 0 5 .2 0
4 .8 8 4 .8 8 4 .8 8 9 . 7 6 9 .7 6 4 .8 8 9 .7 6 9 .7 6
C oncen tratio n X I 0 " 4 (grom s p er m illiliter)

Figure 11. Recovery of colciferol
A—Pure c a lc if e r o l
B - P u r e erg o stero l
C - T o to l calciferol
D - T o to l erg o stero l

ond erg o stero l

from

5 .2 0
4 .8 8

5 .2 0
9 .7 6

o 15.6 cen tim eter olumino

colum n.

230

240

250
W ave

F igure

260
L e n g th

A b s o rp tio n s p e c t r a o f s u b s ta n c e
A -S u b s ta n c e 2 8 2
B -S u b s to n c e 2 5 0

270
(m illim icro n s)
282

and

280

s u b s to n c e

290

250

300

in hexane.

0.8

0.6
c
o
o
_e
x

LU

0 .4

0.2

0.0

230

240

250

260
270
Wave Length (m illim
n s)
imicro
icrons)

Hi

280

290

300

F ig u re 13. S ev en th fra c tio n o f. e lu a te from a 4 . 6 centim eter s u p e rfiltro l column.
A -C o m p u ted ab sorption sp e c tra due t o su b stan ce 2 0 2 a n d su b stan ce 2 5 0
B -C o m p u ted absorption sp e ctra due to pure calciferol
C - R e s u lta n t computed obsorption s p e c t r a - curve A + c u rv e B ( o )
D -O b s e rv e d a b so rp tio n sp e ctra in h exane (•)

0 .9
c
o

o

.c

X
U
J

0.6

0 .3

ao
230

240

250
Wave

260
Length

270
(m illim icrons)

290

300

Figure 14. Fourth fractio n of eluate from a U + c en tim eter s u p e rf iltro l column.
A -C om puted absorption sp e ctra due to substance 2 6 2 a n d sub stan ce
250
B -C o m p u ted ab sorption sp e c tra due to pure calciferol
C -C o m p u ted absorption sp e ctra due to pure ergosterol
D -C o m p u ted absorption s p e c tra due to pure calciferol a n d pure ergosterol —
curve B + curve C
E -R e s u lta n t com puted absorption s p e c t r a - c u r v e A + curve B + curve C (O)
F -O b s e rv e d obsorption s p e c tra in h e x a n e (•)

100
96
96

10
15
20
E th e r - h e x a n e , K 5 , p re w a s h so lu tio n
F ig u re 15. R eco v ery o f c a lc ife ro l a n d
s u p e rf iltro l colum n.
A - T o ta l c a lc ife r o l
B - T o t a l e rg o s te ro l

e rg o s te ro l from a

25
(m illilite rs)

30

4 . 6 c e n tim e te r

yz

•w y
•w x y z
•Z

•X

•w

•W

•X Y

wZ
X

W

XY

•X Y

30

10
15
20
25
E th e r-h e x a n e , 1:5, prew ash solution (m illiliters)
Figure 16. E lution o f c a lc ife ro l, s u b s ta n c e 2 8 2 , s u b s ta n c e 2 5 0 , and
a 4 . 6 c e n tim e te r su p e rfiltro l column.
W -lnitial fra c tio n containing calcifero l
X -In itia l fro ctio n containing su b s ta n c e 2 9 2
Y -in itia l fractio n containing su b sto n c e 2 5 0
Z -ln itio l fra c tio n containing e rg o ste ro l
w -F in a! fra c tio n containing c alcifero l
x -F in a l fractio n containing su b sta n c e 2 8 2
y —Final fractio n containing s u b sta n c e 2 5 0
z -F in a l fractio n containing ergosterol

erg o stero l

from

ea>
Jf 7 5
0a)
c
o

°0

5

10
15
20
25
E th er-h ex o n e, 1:5, p rew ash solution ( m illilite r s )

F igure 17 R elative com position of to ta l e lu a te
column a t 281 m illim ic ro n s.
A -C o lcifero l
B - S u b s ta n c e 2 8 2
C - S u b s ta n c e 2 5 0
D - E rg o stero l

from a 4 .6 c e n tim e te r

30

su p e rfiltro l

35

100
75

50

25

E th e r
Hexane

3
E lu en t ( p a r ts by volume)

F ig u re 18. R ecovery o f c a lc ife r o l and
su p e rfiltro l column.
A—Total c alcifero l
8 - T o t a l e rg o ste ro l

e r g o s te r o l

fro m

5

a

4 .6

c e n tim e te r

• w xyz
•Z

10
•z
•Z
• WXY

•w

•W X Y

•X Y

E th e r
Hexone
E lu e n t ( p o r t s by volum e)

F ig u re 19. E lu tio n o f c a lc ife r o l, su b s ta n c e 2 8 2 , s u b s ta n c e 2 5 0 , a n d
from a 4 .6 c e n tim e te r s u p e rf iltro l column.
W—Initial fra c tio n co n ta in in g c o lc ife ro l
X -ln itio l fra c tio n co n tain in g s u b s ta n c e 2 8 2
Y -ln itia l fra c tio n co ntaining s u b s ta n c e 2 5 0
Z - ln itia l fra c tio n co ntaining e rg o ste ro l
w -F in o l f ra c tio n co ntaining c a lc ife ro l
x - F in a l f ra c tio n co n tain in g s u b s ta n c e 2 8 2
y - F in a l fro c tio n containing s u b s ta n c e 2 5 0
z - F i n a l fra c tio n containing e rg o ste ro l

e rg o s te ro l

75

50

25

E ther
H exane

5

3
E lu en t ( p a r ts by volum e)

F ig u re 2 0 . R e la tiv e c o m p o s itio n of to to l e lu a te fro m
s u p e rf iltro l c o lu m n a t 281 m illim icro n s.
A - C olciferol
B -S u b s ta n c e 2 8 2
C -S u b s ta n c e 2 5 0
D - E r g o s te r o l

a

4 .6 c e n tim e te r

0.8

0.6
c
o

o

_c

K
L
l)
0 .4

0.2

0.0
230

240

250
W ove

F ig u r e 21.

o
oe
c

260
L en g th

270
(m illim icr o n s)

A b s o r p tio n s p e c t r a o f s e c o n d f r a c t i o n
A - 1 .1 * c e n t im e t e r s u p e r f ilt r o l co lu m n
B - 2 . 3 c e n t im e t e r s u p e r f i l t r o l colu m n

of

260

e lu a te

in

290

300

h exon e.

0 .9

x
UJ

0.6

0 .3

240

250
W ave

260
L e n g th

270
(m illim ic r o n s )

F ig u r e 2 2 . A b s o r p tio n s p e c t r a o f th ird f r a c t i o n o f
A - U + c e n t im e t e r s u p e r f ilt r o l co lu m n
B - 2 . 3 c e n t im e t e r s u p e r filtr o l colu m n
C - 3 . 4 * c e n t im e t e r s u p e r f ilt r o l co lu m n
D - 4 . 6 c e n t im e t e r s u p e r f i l t r o l colu m n

280

e lu a te

in

290

h exon e.

300

E x tin c tio n

0 .9

0.6

0 .3

0.0
230

240

250
W ave

260
L en gth (m illim icr o n s)

F ig u r e 2 3 . A b so rp tio n s p e c t r a o f fo u r th f r a c t i o n
A - (.(^ c e n tim e te r su p e r filtr o l co lu m n
6 -2 .3
c e n t im e t e r s u p e rfiltro l c o lu m n
C - 3 . 4 + c e n t im e t e r
su p e r filtr o l co lu m n
0 —4 .6
c e n t im e te r s u p e r filtr o l co lu m n
E —5 . 8
c e n t im e te r s u p e rfiitro l co lu m n
F —7 .0
c e n t im e t e r su p e r filtr o l c o lu m n

280

of

e lu a te

290

in

300

h exan e.

Extinction

0 .9

0 .3

0.0
230

240

250
W ave

F ig u r e 2 4 . A b s o r p tio n
sp ectra
A - l . I 4" c e n t i m e t e r
B - 2 3 c e n t im e te r
C - 3 . 4 + c e n t im e te r
0 - 4 . 6 c e n t im e t e r
E - 5 . 8 c e n t im e t e r
F - 7 . 0 c e n t im e t e r

260
L en gth

270
(m illim icr o n s)

o f f i f t h fr a c t io n
s u p e r filtr o l c o lu m n
s u p e r filtr o l co lu m n
s u p e r filtr o l co lu m n
su p e rfiltro l co lu m n
su p erfiltrol
c o lu m n
su p e rfiltro l co lu m n

of

290

260

elu a te

in

h exan e.

300

1.2

E x tin c tio n

0 .9

0.6

0 .3

0.0
23 0

240

^250
W ave

F i g u r e 2 5 . A b s o r p tio n
sp ectra
A - 1 .1 + c e n t im e t e r
8 - 2 3 c e n t im e t e r
C - 3 .4 + c e n t i m e t e r
D - 4 . 6 c e n t im e t e r
E - 5 . 8 c e n t im e t e r
F - 7 . 0 c e n t im e t e r

260
L e n g th

270
(m illim ic r o n s )

of
s ix th fr a c t io n
s u p e r f ilt r o l colu m n
s u p e r f i l t r o l co lu m n
s u p e r filtr o l colu m n
s u p e r filtr o l co lu m n
s u p e r filtr o l colu m n
s u p e r filtr o l colum n

of

280

e lu a te

290

in

300

hexon e.

1.0

3 .8

E x tin ctio n

0.6

0 .4

0.2

0.0
2 3 0

240

250
W ave

260
L e n g th

F i g u r e 2 6 . A bsorp tion s p e c t r a o f s e v e n t h
A - l.l + c e n t im e t e r s u p e r filtr o l
B - 2 . 3 c e n t im e te r s u p e r filtr o l
C - 3 . 4 * c e n t im e t e r s u p e r filtr o l
D - 4 . 6 c e n t im e t e r s u p e r filtr o l
E - 5 .8
c e n t im e t e r s u p e r filtr o l
F - 7 . 0 c e n t im e t e r s u p e r filtr o l

270

280

290

(m illim icr o n s)
fr a c tio n
colum n
colum n
colu m n
colu m n
colu m n
colum n

of

e l u a t e in

h exan e.

300

E x tin c tio n

0 .9

0 .3

0.0
230

240

250
W ove

F ig u re 27. A b s o rp tio n s p e c t r a
B - 2 . 3 c e n tim e te r
C - 3 . 4 + c e n tim e te r
D —4 .6 c e n tim e te r
E - 5 . 8 c e n tim e te r
F - 7 . 0 c e n tim e te r

260
L e n g th

o f e ig h th
s u p e rfiltro l
s u p e rf iltro l
s u p e rf iltro l
s u p e rfiltro l
s u p e rf iltro l

270
(m illim icro n s)
f r a c tio n
colum n
colum n
colum n
colum n
colum n

of

290

280

e lu a te

in

300

h ex an e.

0.8

Extinction

0.6

0 .4

0.2

0.0
230

240

250

260
W ave L e n g th

Figure 2 6 . A b so rp tio n s p e c t r a
B - 2 .3
c e n tim e te r
C - 3 . 4 + c e n tim e te r
D -4 .6
c e n tim e te r
E - 5 .8
c e n tim e te r
F - 7 .0
c e n tim e te r

270
(m illim icrons)

o f n in th f ra c tio n o f
s u p e rf iltro l colum n
su p e rfiltro l column
s u p e rfiltro l colum n
s u p e rfiltro l colum n
s u p e rfiltro l colum n

280

e lu a te

in

290

hexone.

300

0 .3

E x tin c tio n

0.2

0.1

OjO
230

240

250
W ave

270
(m illim ic r o n s )

o f te n th f r a c t i o n
s u p e r filtr o l co lu m n
s u p e r filtr o l co lu m n
s u p e r filtr o l colu m n
s u p e r filtr o l colu m n

of

280

e lu a te

290

In

300

h exan e.

_

■

F ig u r e 2 9 . A b s o r p tio n s p e c t r a
C - 3 . 4 + c e n t im e te r
D - 4 . 6 c e n t im e te r
E - 5 . 8 c e n t im e te r
F - 7 . 0 c e n t im e t e r

260
L en g th

E xtin ction

12

0.1

0.0
230

240

250
W ave

260
L e n g th

F ig u r e 3 0 . A b s o r p tio n
sp ectra
o f e iu a t e
s u p e r f i l t r o l co lu m n .
A - E l e v e n t h fr a c tio n
B - T w e l f t h fr a c t io n
C -T h ir te e n th
fr o c tio n
0 - F o u r te e n th
fr a c tio n
E - F ifte e n th
fr a c tio n
F -S ix te e n th
fr a c t io n
6 -S e v e n te e n th
fr a c tio n
H - E ig h t e e n t h
fr a c tio n

270
( m illim ic r o n s )
in

h exane

280

fr o m

a

290

7 .0

c e n t im e t e r

300

1001
971
30

C o lu m n
F ig u r e 31.

L e n g th

R e c o v e r y o f c a l c if e r o l on d
A - T o t a l c a l c if e r o l
B - T o t a l e r g o s te r o l

(c e n tim e t e r e )

er g o ste r o l

u s in g

an

e t h e r - h e x a n e , 1 :5 , e lu e n t.

wy
•

wxy

•wX

X

*Z

• w x yz
•Z
• w x y 2
•Z
•Z

^

"

0

■l -

-

1

-

■■

•
• X Y
I

2
C olu m n

•w

w

• W
•X Y

• WX Y

. . . .

3
L en g th

I

•X Y

•X Y

XY

1

I

I

4
( c e n t im e t e r s )

5

F ig u r e 3 2 . E lu tio n o f c a l c i f e r o l , s u b s t a n c e 2 8 2 , s u b s t o n c e
u s in g a n e t h e r - h e x a n e , 1 : 5 , e lu e n t.
W - I n i t i a l f r o c t i o n c o n ta in in g c a lc if e r o l
X -In itia l
f r a c t i o n c o n ta in in g
su b sta n c e 2 8 2
Y - I n itia l
f r a c t i o n c o n ta in in g
su b sta n c e 2 5 0
Z — I n itia l f r a c t i o n c o n ta in in g e r g o s t e r o l
w - F in a l
f r a c t i o n c o n ta in in g c a l c if e r o l
x -F in a l
f r o c t i o n c o n ta in in g
su b sta n c e 2 8 2
y -F in a l
f r a c t i o n c o n ta in in g
su b sta n ce 2 5 0
z - Final
fr a c t io n co n ta in in g e r g o s te r o l

250,

6

and

7

e r g o s te r o l

100

75

" 50

25

-Ok

Colum n

L e n g th

F ig u r e 3 3 . R e la t iv e c o m p o s itio n o f to to l
a t 2 8 1 m illim ic r o n s .
A - C a lc if e r o l
B -S u b sta n c e 2 8 2
C - S u b sta n c e 2 5 0
D -E r g o ste r o l

( c e n t im e t e r s )

e lu a te

u s in g

an

e t h e r - h e x a n e , 1 :5 ,

e lu e n t

100
90
S 30

'20

TBT

Z&O
2 .4 4

4 .8 8
C o n c e n t r a tio n

X I0~ 4

F ig u r e 3 4 . R e c o v e r y o f c a l c if e r o l o n d
s u p e r f i l t r o l c o lu m n .
A - T o t a l c a lc if e r o l
B - T o t o l e r g o ste r o l

(g r a m s p e r m illilite r )

er g o ster o l

fr o m

a

2 .3

c e n t im e t e r

20

|

15

S
|,0

--------------------------------------------------------------------------------------------------------------------------------------------------- e - y Z -

• yz
• wx

• wx y z

*Z

-Z

•WXY

-W
•XY

-------------------------------------------- Z ~

C a lc ife r o l
E rg o ste ro l

2 .6 0
2 .4 4

•

2 .6 0
4 .8 8
C o n c e n tr a tio n

F i g u r e 3 5 . E lu tio n o f
fr o m
W -I n itia l
X -I n itia l
Y — I n itia l
Z — I n it ia l
w - 'F i n a l
x -F in a l
y - F in a l
z - F in a l

X

I 0 ~ 4 (g r a m s

w xy

2 .6 0
9 .7 6
p e r m illilite r )

c a l c if e r o l , s u b s t a n c e 2 8 2 , s u b s t a n c e 2 5 0 , a n d
a 2 . 3 c e n t im e t e r s u p e r f i l t r o l co lu m n .
f r a c t i o n c o n ta in in g c a l c i f e r o l
f r a c t i o n c o n t a in in g s u b s t a n c e 2 8 2
f r a c t i o n c o n ta in in g s u b s t a n c e 2 5 0
f r o c t i o n c o n ta in in g e r g o s t e r o l
f r a c t i o n c o n ta in in g c o l c if e r o l
f r o c t i o n c o n ta in in g s u b s t a n c e 2 8 2
f r a c t i o n c o n t a in in g s u b s t a n c e 2 5 0
f r a c t i o n c o n t a in in g e r g o s t e r o l

e r g o ste r o l

100

• 75

8 50

£ 25

2 .6 0
2 .4 4

2 .6 0
4 .8 8
C o n c e n t r a tio n

F i g u r e 3 6 . R e la t iv e c o m p o s it io n o f
s u p e r f i l t r o l c o lu m n
A - C a lc ife r o l
B - S u b sta n c e 2 8 2
C - S u b sta n c e 2 5 0
D -E r g o ste r o l

X

2 .6 0
9 .7 6
I 0 “ 4 (g r a m s

p er

t o t a l e l u a t e fr o m 0
a t 2 6 1 m illim ic r o n s .

m illilite r )
2 .3

c e n t im e t e r

I

100
95

c 90

C a lc ife r o l
E r g o s te r o l

2 .6 0
2 .4 4

2 .6 0
4 .8 8

2 .6 0
9 .7 6

C o n c e n t r a t io n

X

I0~ 4

F ig u r e 3 7 . R e c o v e r y o f c o l c if e r o ) a n d
s u p e r filtr o l co lu m n .
A - T o t a l c a l c if e r o l
B - T o t a l e r g o s te r o l

2 .6 0
1 9 .5 2
(g r a m s

p er

e r g o ste r o l

m illilite r )

fr o m

a

4 .6

c e n t im e t e r

20
• y z
• y z
• w x

*w x y

'
♦ x

• w x y 2
•2

5 io
*W X Y

*w Z

• W
•X Y

-w —

• W
• X Y

•X Y
C a lcife ro l
E r g o s te r o l

2 .6 0
2 .4 4

2 .6 0
4 .8 8

2 .6 0
9 .7 6

C o n c e n t r a t io n
F ig u r e 3 8 . E lu tio n o f
from
W—Initial
X -In itia l
Y —In itial
Z -ln itia l
w - F in a l
x -F in a l
y -F in a l
z —F in a l

X

I 0 '4

2 .6 0
1 9 .5 2

(g r a m s p e r

m illilite r )

c a l c if e r o l , s u b s t a n c e 2 8 2 , s u b s t a n c e 2 5 0 ,
a 4 . 6 c e n t im e t e r s u p e r f ilt r o l c o lu m n .
fr a c tio n co n ta in in g c a l c if e r o l
fr a c tio n c o n ta in in g s u b s t a n c e 2 8 2
fr a c tio n c o n ta in in g s u b s t a n c e 2 5 0
fr a c tio n c o n ta in in g e r g o s te r o l
fr a c t io n c o n ta in in g c a lc if e r o l
fr a c t io n c o n ta in in g s u b s t a n c e 2 8 2
f r a c t i o n c o n ta in in g s u b s t a n c e 2 5 0
f r a c t i o n c o n ta in in g e r g o s t e r o l

and

e r g o ste r o l

100

75

2 25

C alciferol
E r g o s te r o l

2 .6 0
2 .4 4

2 .6 0
4 .8 8
C o n ce n tra tio n

2 .6 0
9 .7 6
X I0“ 4

F igure 3 9 . R e la t iv e c o m p o s itio n o f
s u p e rfiltro l co lu m n
A - C a lcife ro l
B - S u b sta n ce 2 8 2
C — S u b s ta n c e 2 5 0
D - E r g o s te r o l

(g r a m s

2 .6 0
1 9 .5 2
per

to ta l e lu a t e from a
a t 28 1 m illim ic r o n s .

m illilite r )
4 .6

c e n t im e t e r

BIBLIOGRAPHY
C a r l s o n , C. W . , P h . D. T h e s i s , M i c h i g a n
C o lle g e , 1946,
pp. 11-13.
B a k e r , D. H . , M. S. T h e s i s ,
1944, pp. 1 5 -2 4 .

S tate

M ichigan S t a t e C o lle g e ,

B u l l a r d , L. J . , M. S . T h e s i s , M i c h i g a n S t a t e
C o lle g e , 1945, pp. 1 5 -2 1 .
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C o lle g e , 1948,
pp. 11-13,
26-28.
Chen, F . , M. S . T h e s i s ,
1950, p . 2 1 .

M ichigan S t a t e C o lle g e ,

C a r y , H. H . , a n d Bec km an, A. 0 . ,
2 1 , 682 ( 1 9 4 1 ) .

J.

O p t i c a l S o c . Am

R o s e n b e r g , H. R . , C h e m i s t r y a n d P h y s i o l o g y o f t h e
V itam ins, I n te r s c ie n c e P u b lis h e r s , I n c .,
New Y o r k , 1 9 4 2 , p p . 3 4 1 - 4 3 2 .
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C h e m i c a l P u b l i s h i n g Company, I n c . , New Y o r k ,
1947, p . 18.
S t r a i n , H. H . , C h r o m a t o g r a p h i c A d s o r p t i o n A n a l y s i s ,
I n t e r s c i e n c e P u b l i s h e r s , I n c . , New Y o r k ,
1945, pp. 47-69.
H o g n e s s , T. R . , S i d w e l l , A. S . , a n d Z s c h e i l e ,
J . B i o l . Chem. 1 2 0 , 2 3 9 ( 1 9 3 7 ) .
H u b e r , W., E w in g , G. W . , a n d K r i g e r ,
Chem. S o c . 6J , 6 0 9 ( 1 9 4 5 ) .

J.,

F. P.

J . Am.

H a r r i s , R . S . , a n d T h i m a n n , K. V . , V i t a m i n s a n d
Hormones, Academ ic P r e s s I n c . P u b l i s h e r s ,
New Y o r k , Volume I , 1 9 4 3 , p p . 1 2 4 - 1 2 6 .
M o r t o n , R. A . , a n d S t u b b s , A. L . , A n a l y s t 7 1 .
348 ( 1 9 4 6 ) .
W ilson,

J.

N .,

J . Am. Chem. S o c . 6 2 ,

1583 ( 1 9 4 0 ) .

15.

Th omas, H. C . , A n n . N. Y. A c a d . S c l .

16.

K i m b a l l , L. B . , M. S . T h e s i s , M i c h i g a n S t a t e
C o lle g e , 1950, pp. 35-36.

17.

R e e r i n k , E. H . , a n d v a n W i j k , A . , B i o c h e m . J .
1294 ( 1 9 2 9 ).

18.

Dwyer, P . S . ,

P sychom etrika 6,

-5 6 -

49,

16 1 ( 1 9 4 8 ) .

101 ( 1 9 4 1 ) .

23.