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Comparison of results of phasic and chromatographic methods for carotene analysis.

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Comparison of results of phasic and chromatographic methods for carotene analysis.

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Content COMPARISON 0F RESULTS OF PHASIC AND
CHROMATOGRAPHIC METHODS FOR CAROTENE ANALYSIS
A T h e s is
P r e s e n t e d t o
t h e F a c u l t y o f t h e G ra d u a te School
U n i v e r s i t y o f S o u th e rn C a l i f o r n i a
I n P a r t i a l F u l f i l l m e n t
o f t h e R e q u ire m e n ts f o r t h e Degree
M a ste r o f S c ie n c e i n B io c h e m is tr y
by R o b e rt A. M o f f i t t
A ugust 1 1 , 1954
UMI Number: EP41330
All rights reserved
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8 .'o 'SS M te?S
This thesis, written by
...............................
under the guidance of h.X&..Faculty Committee,
and approved by all its members, has been
presented to and accepted by the Faculty of the
Graduate School in partial fulfillment of the
requirements for the degree of
Dean
D ate..
A u g u s t * ! ? ^
Faculty Com m ittee
UxyviiiiiJL
Chairman
i
TABLE OF CONTENTS
PAGE ■
HISTORICAL INTRODUCTION . . . . . . . . . . . 1
S t r u c t u r e o f C a r o te n o id s . . . . . . . . 1
C a ro te n o id s a s a group . . . . . . 1
S t r u c t u r e o f c a r o t e n e s . . . . . . 1
S t a b i l i t y o f Iso m e rs . . . ............................... 5
P r e p a r a t i o n o f P u re E x t r a c t o f a S in g le
Isom er .................................... . 3
E f f e c t o f S t r u c t u r e on S p e c t r a l D ata . . 9
G e n e ra l e f f e c t s o f s t r u c t u r e . . . 9
S t r u c t u r e i d e n t i f i c a t i o n by s p e c t r a l
d a t a ....................................................................10
M ethods o f A n a ly s is o f M ix tu r e s f o r
C a ro te n e . . . . . . .......................... . . . . 11
C a r r - P r i c e m ethod . .....................................11
Phasic, m ethod . . . . . . . . . . . 12
M o d i f i c a t i o n s o f p h a s ic m ethod . . 13
C hrom ato grap hic m ethod . . . . . . 15
Q u a n t i t a t i v e M easurem ent o f C a ro te n e . . 26
C om parison o f M ethods ......................................... 29
STATEMENT OF THE PROBLEM AND PLAN OF ATTACK . 31
MATERIALS AND METHODS ............................................... 32
A cetone . . . 32
ii
l lo o h .o l i c P o ta ssiu m H ydroxide . . . . . . 32
Calcium H ydroxide a s A d so rb en t ..... 32
Hexane ......................... 32
Magnesium Q x id e -S u p e rc e l M ix tu re a s
A d s o r b e n t .................................................................. SB
M e t h a n o l ............................................................................. SS
p -G re s y l M ethyl E th e r ............................... S3
P h a s ic Method f o r D ehy drated A l f a l f a Meal S3
P h a s ic Method f o r F r e s h A l f a l f a . . .. . 34
C hro m ato g rap h ic Method . . . . . . . . . 34
EXPERIMENTAL RESULTS AHD D IS C U S S IO N ..................... 36
Use o f F r a c t i o n G u tte r t o S tu dy P ig m e n ts. 36
P igm ents R em aining on Column A f t e r
Removal o f B e t a - c a r o t e n e ................................... 37
I d e n t i f i c a t i o n o f P igm en ts on Column;
Zone # 4 . . . . . . .................................................... 45
I d e n t i f i c a t i o n o f Zone #3 on Column . . . 49
I d e n t i f i c a t i o n o f Zones # 2 a and 2b on th e
C o l u m n .................................................................................. 52
SUMMARY AND CONCLUSIONS .............................................. 56
BIBLIOGRAPHY................................... 57
iii
LIST OF FIGURES
FIGURE PAGE
1 . Zones on M gO-Supercel Column a f t e r Caro
te n e S o l u t i o n P r e p a r e d "by P h a s ic Method
Was Washed Through w ith 700 c c . 10%
A cetone i n Hexane ........................................38
2. C hrom atography o f P ig m en ts P r e v i o u s l y
P u r i f i e d by P h a s ic Method o r by Chroma
t o g r a p h i c M e t h o d ............................................................39
3 . Zones on M gO -Supercel Column When Caro
t e n e S o l u t i o n P r e p a r e d by P h a s ic Method
Was D eveloped w ith 3fo A ceto ne i n Hexane . 40
4 , S p e c t r a l Curve f o r Zone #4 on MgO-
S u p e rc e l Column . . . . . . . . ..................... 41
5 . S p e c t r a l Curve f o r Zone #3 on MgO-
S u p e rc e l Column .............................................42
6. S p e c t r a l Curve f o r Zone #2b on MgO-
S u p e ro e l Column .............................................................. 43
7. S p e c t r a l Curve f o r Zone # 2 a on MgO-
S u p e rc e l Column .......................................................44
8 . Zones on M gO-Supercel Column f o r X antho-
p h y l l s i n H e x a n e .............................................. ..... . 47
9. S p e c t r a l Curve f o r "Heavy Yellow Band"
o f X a n th o p h y ll Column w ith Curve f o r
Zone #4 P i g m e n t .............................................................. 4 8
1 0 . Zones on M gO-Supercel Column f o r / 3 -
c a r o t e n e i n Hexane from F r e s h A l f a l f a . . 50
11. S p e c t r a l C urves f o r R o s e -c o lo re d Zones
on M gO-Supercel Columns from F re s h and
D eh y d rated A l f a l f a .................................................... 51
12. Zones on M gO-Supercel Column and on C al
cium H ydroxide Column f o r / S ’ - e a r o t e n e
S o l u t i o n i n H e x a n e .......................................................54
LIST OF TABLES
iv
TABLE PAGE-
I . C a ro te n e Iso m ers E x t r a c t e d from A l f a l f a
P r e p a r a t i o n . .......................................................... 6
I I . S p e c t r a l Maxima o f Four Zones Found on
M gO-Supercel Column f o r P h a s ic Method
C a r o t e n e ........................................................................ 45
HISTORICAL IHTRODUCTIOU
S t r u c t u r e o f C a r o te n o id s
C a r o te n o id s a s a g r o u p . The c a r o t e n o i d s a r e n i t r o
g e n - f r e e p ig m e n ts w ith a l o n g a e y c l i e c h a in o f c a rb o n
atoms j o i n e d by c o n ju g a te d d o u b le b o n d s. At each end o f
th e c h a in i s a group w hich may o r may n o t be e y e l i z e d
and w hich h a s is o m e r ic fo rm s. The c a r o t e n o i d s a re
d iv id e d i n t o th e h y d ro c a rb o n s (ly co p e n e and t h e c a r o
te n e s ) , th e c a r o t e n o l s (fo r m e r ly c a l l e d x a n t h o p h y l l s ) ,
and t h e e s t e r s , e t h e r s , k e to n e s , h y d ro x y k e to n e s and
a c i d s . ^ A cco rd in g t o work done by C a ld w e ll and Hughes
th e m ost common c a r o t e n o i d p ig m e n ts a r e a l p h a - c a r o t e n e ,
b e t a - c a r o t e n e , g a m m a-e aro te n e, ly e o p e n e , c r y p t o x a n t h i n e ,
l u t e i n and z e a x a n t h i n . 2
S t r u c t u r e o f c a r o t e n e s . The c a r o t e n e s , e s p e c i a l l y
a s th e y a r e p r e s e n t i n a l f a l f a , a r e t h e p ig m e n ts o f
p a r t i c u l a r i n t e r e s t i n t h i s r e p o r t . The c a r o t e n e f r a c
t i o n o f a l f a l f a i s p r i m a r i l y a m ix tu r e o f a l l - t r a n s -
b e t a - c a r o t e n e and i t s e i s - i s o m e r s .
I n th e b e t a s t r u c t u r e o f c a r o t e n e eac h t e r m i n a l
r i n g h a s a doub le bond i n c o n ju g a ti o n w i t h t h o s e i n th e
l o n g a l i p h a t i c c h a i n . The c a r o t e n o i d s w ith t h e a l p h a
s t r u c t u r e have i n one r i n g a d o u b le bond betw een c a rb o n s
4 ’ and 5 ' r a t h e r t h a n betw een 5 ’ and & }?■ The p h y s i c a l
2
c h a r a c t e r i s t i c s o f t h e s e two g ro u p s a r e q u i t e d i f f e r e n t .
The p r o v i ta m i n A a c t i v i t y i s i n f l u e n c e d a s w e ll by t h e i r
s te r e o c h e m ic a l c o n f i g u r a t i o n . I n th o s e c a r o t e n o i d s
r e f e r r e d t o a s " a l l - t r a n s " , each do u b le bond p o s s e s s e s
t h e t r a n s c o n f i g u r a t i o n . The " a l l - c i s " d e s i g n a t i o n means
t h a t a l l do u b le bonds w h ich a r e s p a t i a l l y u n h in d e re d i n
assu m ing t h e c i s c o n f i g u r a t i o n a r e p r e s e n t i n t h e c i s
form . A ll t h e r e s t a r e t r a n s b e c a u se o f s p a t i a l eon -
f l i c t s . The fo rm u la f o r a l l - t r a n s b e t a c a r o t e n e i s as
f o l lo w s :
H u
C
H , / V f C l U ri H H H V C.Mi
h \ / \ A / / w/c\ // \ j \ / / y / v / \ ^ Y \ u
C m I C C r f C C C . C l *
H C«3 ff C % H h H H H * I
3 5 6 7 ?
The u n h in d e re d double bonds c a p a b le o f e i s - t r a n s r o t a t i o n
a r e num bered.
On t h e b a s i s o f a t h e o r e t i c a l d i s c u s s i o n , a group
o f w o rk e rs r e c e n t l y p o i n t e d o u t t h a t n e o - b e t a - e a r o t e n e O
c o n t a i n s one d o u b le bond i n t h e c i s c o n f i g u r a t i o n and
t h a t t h i s m ust be n e a r one end o f t h e chrom ophoric system.
I t s s t r u c t u r e , th e y p o s t u l a t e , i s a s f o llo w s :
3
I t c o u ld be c a l l e d 3 m o n o - c i s - b e ta - e a r o te n e .®
H e o - a lp h a - c a r o te n e and n e o - b e t a - e a r o t e n e B a r e b o th
d i - c i s - p i g m e n t s , c h a r a c t e r i z e d by one c e n t r a l l y l o c a t e d
and one p e r i p h e r a l l y l o c a t e d c i s d o u b le bond. The bend
in g i n t h e m id d le r e g i o n o f such m o le c u le s seems t o
a c c o u n t f o r t h e i r d e c r e a s e d s t a b i l i t y and t h e i r i n c r e a s e d
te n d e n c y t o r e v e r t to t h e a l l - t r a n s fo rm .^
One o f t h e f i r s t im p o r ta n t s t u d i e s o f t h e isom ers
o f e a r o t e n e was c o n d u c te d by G illa m and E l R i d i i n 1935
and 1936. U s in g p u re b e t a - c a r o t e n e t h e y adsorbed, i t
r e p e a t e d l y on a lu m in a , and o b se rv e d t h a t i t s a b s o r p t i o n
sp e c tru m changed u n t i l i t was t h e same a s t h a t o f a lp h a -
e a r o t e n e . O th e r p r o p e r t i e s , how ever, w ere n o t t h e same
a s th o s e o f a l p h a - e a r o t e n e . T h is iso m er was named by
them " p s e u d o - a l p h a - e a r o t e n e " .$ The s tu d y o f t h e s e men has
been re v ie w e d f r e q u e n t l y by l a t e r w o r k e rs , who have found
i
t h a t " n e o - b e t a - c a r o t e n e B" i s d o u b t l e s s l y th e m ain eon-
4
s t i t u e n t o f ,!p s e u d o - a l p h a - e a r o t e n e n . ^
N e o - b e t a - e a r o t e n e B may be pro d u ced a l s o by b e a t i n g
O
b e t a - c a r o t e n e i n p e tro le u m e t h e r . By t r e a t i n g b e t a -
c a r o te n e i n v a r i o u s w ays, P o l g a r and Z e e h m e is te r were
a b l e t o s e c u r e ab o u t tw e lv e d i f f e r e n t is o m e rs . I n an
e q u i l i b r i u m m ix tu r e p r e p a r e d from c r y s t a l l i n e b e t a -
c a r o t e n e t h e s e same i n v e s t i g a t o r s showed t h a t a p p r o x i
m a te ly 95fo o c c u rs a s t h e a l l - t r a n s form , t h e n e o - b e t a -
e a r o te n e B, and n e o - b e t a - e a r o t e n e U ,^ These same t h r e e
p ig m e n ts a r e t h e ones found i n g r e a t e s t abundance i n
a l f a l f a .
Much o f t h e work done w ith t h e c a r o t e n e is o m e rs
employs a Beckman DU S p e c tr o p h o to m e te r. S a t i s f a c t o r y
s l i t w id th s f o r u se w ith such an i n s t r u m e n t were c a l c u
l a t e d by S t i t t , B i e k o f f and a s s o c i a t e s . 10 They a l s o
m easu red t h e a b s o r p t i v i t i e s o f t h e d i f f e r e n t is o m e rs a t
v a r y i n g wave l e n g t h s , em ploying b o th a t u n g s t e n f i l a m e n t
and a m ereu ry a r c a s l i g h t s o u r c e . The v i s u a l maxima o f
n e o - b e t a - e a r o t e n e U were fo und by Z e e h m e iste r and P o lg a r
to d i f f e r by 5 and th o s e o f h e o - b e t a - e a r o t e n e V by
1 3 .5 m/Ufrom t h e p o s i t i o n s o f maxima o f a l l - t r a n s b e t a -
c a r o t e n e . T h is was ta k e n to i n d i c a t e t h a t neo U i s a
m o n o -cis and neo ¥ a d i - c i s compound. N e i th e r c an c o n t a i n
a 6 c i s d o u b le b ond, t h e s e w o rk e rs s a y , b e c a u se o f t h e i r
5
low c i s p e a k s and r e l a t i v e l y h ig h t h e r m o s t a b i l i t y . They
s u g g e s t t h a t n e o - b e t a - e a r o t e n e U i s 3 -m o n o -c is b e t a -
c a r o t e n e and neo V i s 3 , 9 - d i - e i s b e t a - c a r o t e n e , o r p o s s i -
b l y 5 , 7 - d i - c i s b e t a - c a r o t e n e . * U e o - b e t a - e a r o t e n e B,
a c c o r d in g t o Z e e h m e is te r, i s 3 , 6 - d i - e i s b e t a - c a r o t e n e . 4
S t a b i l i t y o f Isom ers
One o f th e im p o r ta n t f a c t o r s i n th e s tu d y o f c a r o
te n e i s t h e s t a b i l i t y o f i t s v a r i o u s is o m e r s . As h as
b een m e n tio n e d , b e t a - c a r o t e n e may r e a d i l y be c o n v e rte d
i n t o a m ix tu r e o f i t s is o m e r ic fo rm s. I t may, how ever,
be k e p t i n a p u re s t a t e . B e a d le and Z s e h e i le k e p t a
s o l u t i o n o f p u re b e t a - c a r o t e n e f o r t h r e e h o u r s a t 30° C.
(one h o u r a t room l i g h t , two h o u rs i n d a rk n e s s ) w ith o u t
any g r e a t change i n th e a b s o r p t i o n s p e c tru m . When t h e y
lo w ered th e te m p e r a tu r e t o 5° th e y found t h e y c o u ld keep
th e s o l u t i o n f o r 60 h o u rs w ith no s i g n i f i c a n t change.
They a l s o t r i e d s h a k in g a p u re hex an e s o l u t i o n o f b e t a -
c a r o te n e f o r one h o u r w ith a l c o h o l i c KOH, and o b se rv ed
no s p e c t r o s c o p i c ch an ge. S i m i la r t r e a tm e n t w i t h o x a li c
a c i d and HC1 showed a 3 - 4 $ lo w e r in g o f th e a b s o r p t i o n
12
c u rv e .
Deuel and h i s a s s o c i a t e s r e p o r t e d on t h e s t a b i l i t y
o f n e o - a lp h a and n e o - b e t a - e a r o t e n e . I n one s tu d y i n 1944
6
th e y i s o l a t e d c r y s t a l s o f n e o - b e t a - c a r o t e n e , w h ic h , t h e y
r e p o r t e d , c o u ld be k e p t s e v e r a l m onths i n t h e a b s e n c e ©f
a i r w ith o u t c h a n g in g t h e i r c o n f i g u r a ti o n .® I n a l a t e r
work t h e s e i n v e s t i g a t o r s found t h a t when n e o - a lp h a or
n e o - b e t a - e a r o t e n e B i n Wesson O il was k e p t c o l d , i t r e
m ained p r a c t i c a l l y unchanged f o r a t l e a s t two w eeks. In
th e o il,* th e y r e p o r t e d , each o f t h e s e compounds had i t s
m ain e x t i n c t i o n maximum a t a wave l e n g t h a b o u t 8 m^
j
s h o r t e r th a n t h a t o f t h e c o rr e s p o n d in g a l l - t r a n s com-
p o u n d s . ‘
The f o l lo w in g t a b l e shows t h e isom er c o m p o s itio n
a f t e r e x t r a c t i n g an a l f a l f a p r e p a r a t i o n w ith p e tro le u m
e t h e r f o r v a r y i n g l e n g t h s o f tim e . E x t r a c t i o n f o r 15
m in u te s to 25 h o u rs p ro d u c e d o nly th e same t h r e e iso m e rs
13
i n any s i g n i f i c a n t am ount.
TABLE I
CAROTENE ISOMERS EXTRACTED FROM ALFALFA PREPARATION
E x t r a c t i o n Neo- Neo- A l l - fo T o ta l
Time b e t a - b e t a - t r a n s C a ro te n e
(h o u rs) B_____ U b e t a e x t r a c t e d
A
4
3 9 ,8 1 7 . 2 4 3 .0 47
1 3 7 . 2 1 6 .8 4 6 .0 52
25 4 1 .5 1 5 ,3 4 3 .2 84
The amount o f c i s is o m e rs found i n d e h y d ra te d a l f a l
f a m eal i s much g r e a t e r t h a n t h a t i n f r e s h t i s s u e s . Re
7
s u i t s o f t e s t s by Thompson, B i o k o f f and t h e i r a s s o c i a t e s
showed t h a t v e r y l i t t l e i s o m e r i z a t i o n to o k p l a c e b e f o r e
e n t r y i n t o th e d e h y d r a t o r , and t h a t even p a r t i a l d r y in g
i n a p r e d r i e r c a u se d no l a r g e amount o f i s o m e r i z a t i o n .
The p r o c e s s o f d e h y d r a tio n i t s e l f , w ith i t s i n c r e a s e d
te m p e r a t u r e , cau sed th e g r e a t e s t amount o f t r a n s - e i s
s h i f t . These w o rk e rs a l s o d e te rm in e d t h a t u se o f a
hammer m i l l gave no s i g n i f i c a n t i n c r e a s e i n th e amount
o f c i s i s o m e r s . ^ I t i s i n t e r e s t i n g t h a t t h e r e i s a p p a r
e n t l y a l a r g e r p r o p o r t i o n o f n e o - b e t a - e a r o t e n e B form ed
d u rin g d e h y d r a t io n o f a l f a l f a t h a n when i t i s t r e a t e d
l />
w ith io d in e a t room te m p e r a t u r e .
Z e e h m e iste r and T uzson, among o t h e r s , a tte m p te d to
d i s c o v e r t o what d e g re e th e a n a l y t i c a l p r o c e s s i t s e l f
( f o r b e t a - c a r o t e n e ) a f f e c t s t h e s u b s ta n c e s w hieh i t i s
d e s ig n e d o n ly t o s e p a r a t e . U sing d i f f e r e n t a d s o r b e n t s
f o r c h ro m a to g ra p h ic d e t e r m i n a t i o n , t h e y found Ca(OH)g
t o be t h e b e s t b o th f o r s h a r p e r s e p a r a t i o n o f p ig m e n ts
and b e c a u se i t p ro d u ce d t h e l e a s t e f f e c t on t h e s e p i g
m e n ts . They r e p o r t e d t h a t "once a sam ple o f b e t a -
c a r o t e n e h a s re a c h e d c h ro m a to g ra p h ic h o m o g en e ity , l i t t l e
o r no pseudo a l p h a - c a r o t e n e a p p e a rs when a d s o r p t i o n i s
s u b s e q u e n tly r e p e a t e d , p ro v id e d t h a t no c o n s i d e r a b le
tim e e l a p s e s betw een each e x p e rim e n t" , and f u r t h e r t h a t
9
t h e e n t i r e p r o c e d u r e he c a r r i e d o u t a t room te m p e r a tu r e
o r b e l o w . ^
S i l k e r , S e h re n e k , and King a l s o lo o k e d i n t o t h i s
m a t t e r and found t h a t m a g n e sia columns c a u se v e r y l i t t l e
l o s s o f c a r o t e n e . T h e i r m ethod, t h e y r e p o r t e d , d id c au se
some i s o m e r i z a t i o n , h u t l e s s t h a n m ethods r e q u i r i n g
r e f l u x i n g . ^ 7
The m ost im p o r ta n t t h i n g s t o a v o id i n o r d e r to p r e
v e n t i s o m e r i z a t i o n d u r in g a n a l y s i s seem t o he t a k i n g to o
lo n g a tim e i n th e p r o c e d u r e and a p p ly in g to o much h e a t .
I t h a s a l s o been r e p o r t e d t h a t t h e e x t r a c t i o n and
chrom atog raph y o f e a r o te n e from a l f a l f a m ust be c a r r i e d
on u n d e r re d u c e d l i g h t , o r t h e n e o -b e ta - B and U iso m ers
1 * 5
w i l l be r e c o n v e r t e d t o th e a l l - t r a n s fo rm .*
P r e p a r a t i o n o f P u re E x t r a c t o f a S in g le Isom er
P r e p a r a t i o n o f p u re e x t r a c t s o f a s i n g l e iso m er i s ■
som etim es d e s i r a b l e . B i c k o f f and a s s o c i a t e s , i n p r e
p a r i n g a l l - t r a n s b e t a - c a r o t e n e , p u t a hexane s o l u t i o n
c o n t a i n i n g com m ercial c a r o t e n e th ro u g h a 28 cm. h ig h
c h ro m a to g ra p h ic column o f MgO and d i c a l c i t e . T h is t h e y
d e v elo p ed w ith a Zfo s o l u t i o n o f a c e to n e i n h e x a n e , t h e n
e x tru d e d th e column and c a rv e d o u t t h e a lp h a and b e t a
b a n d s . The p ig m e n ts w ere e l u t e d w ith a l c o h o l i c h e x a n e ,
9
washed, t o remove a l e o h o l , t h e n d r i e d w ith Ha^SG^. Twice
more t h r o u g h th e column removed a l l a l p h a - q a r o t e n e , t h e n
th e h e t a iso m er was c r y s t a l l i z e d u n d e r n i t r p g e n w ith 5fo
a lc o h o l a t 30° F . 18
E f f e c t o f S t r u c t u r e on S p e c t r a l B ata
G e n e ra l e f f e c t s o f s t r u c t u r e . Almost a l l e a r o t e n o i d
p ig m e n ts have t h r e e s h a r p i y d e f in e d maxima i n th e v i s i b l e
p a r t o f t h e i r sp e c tru m . The p o s i t i o n o f t h e s e maxima i s
d e te rm in e d by th e number o f d o u b le bonds p r e s e n t . Each
new Cl e n t e r i n g i n t o a c o n ju g a te d system o f d ou b le bonds
d i s p l a c e s t h e - a b s o r p t i o n maxima tow ard l o n g e r wave
l e n g t h s by a b o u t £00 t o ££0 A °.1 9 A b s o r p tio n i s s t r o n g e r
f o r e a r o t e n o l s th a n f o r h y d ro c a rb o n c a r o t e n e s , and
i n c r e a s e s w ith t h e number o f h y d ro x y l g ro u p s p r e s e n t . i
F ry e s t a t e s t h a t i n a g iv e n s o l v e n t t h e wave l e n g t h s o f
th e a b s o r p t i o n maxima o f a n u n sy m m e tric a l p o ly e n e a re
a p p ro x im a te ly th e mean v a lu e s o f th e c o rr e s p o n d in g
maxima o f th e two r e l a t e d sy m m etrica l p o l y e n e s . ^ 8
Z e e h m e iste r and P o l g a r , f o l lo w in g a s tu d y co m p lete d
i n 1943, p o i n te d o u t t h a t s p e c t r a l c u rv e s o f c a r o t e n o i d s
i n 'the v i s u a l r e g i o n a r e n o t much a f f e c t e d i n shape by
t r a n s - e i s r e a r r a n g e m e n ts . The s t e r e o c h e m ic a l s i t u a t i o n ,
t h e y s t a t e d , seems t o f i n d a more s p e c i f i c - e x p r e s s io n
10
i n a c e r t a i n r e g i o n ©f th e u l t r a v i o l e t , th e e i s - p e a k
21
a p p e a r in g a t 320 t o 380 mf*. T h is c i s - p e a k a p p e a rs
when th e t r a n s t o c i s re a r ra n g e m e n t "bends t h e m o le c u le .
I t i s a new .marked maximum, and o c c u r s , i n t h e e a s e o f
b e t u - c a r o t e n e i n h e x a n e , a t 3 3 9 .5 mji.
T hese same w o rk e rs , i n an e a r l i e r r e p o r t , r e a c h e d
t h e c o n c lu s io n t h a t t h e g r e a t e s t i n t e n s i t y o f l i g h t ab
s o r p t i o n i s shown by a l l - t r a n s c a r o t e n o i d s , w h ile c i s
bonds d e c r e a s e i n t e n s i t y , each one s h i f t i n g t h e maxima
f o u r to s i x mjU to w a rd t h e v i o l e t . They s t a t e d t h a t n e o -
b e t a - e a r o t e n e U seems t o have one e i s b o n d .21
S t r u c t u r e i d e n t i f i c a t i o n by s p e c t r a l d a t a . Employ
in g a c h ro m a to g ra p h ic column f o r th e i d e n t i f i c a t i o n o f
is o m e rs i s a c o m p a r a tiv e ly sim p le p r o c e d u r e . A s o l u t i o n
c o n t a i n i n g an unknown iso m er i s m ixed w ith t h a t o f a
known p u re p ig m e n t. I f a s i n g l e zone a p p e a r s i n t h e
a d s o r p t i o n colum n, t h e two iso m e rs a r e a l i k e . I f t h e r e
a r e two z o n es t h e y a r e d i f f e r e n t . 1
As an exam ple o f such i d e n t i f i c a t i o n , Kemmerer and
F r a p s s e p a r a t e d two iso m e r p r e p a r a t i o n s from a l f a l f a
l e a f m e a l. One was m ixed w ith a s o l u t i o n o f n e o - b e t a -
e a r o te n e U p r e p a r e d from c r y s t a l l i n e e a r o t e n e . When t h e
m ix tu r e was c h ro m a to g ra p h e d , no s e p a r a t i o n i n t o bands
11
a p p e a re d ; so t h i s " c a r o t e n o i d IV was i d e n t i f i e d , The
o t h e r f r a c t i o n , i n a s i m i l a r m anner, was shown t o be
Q
n e o - b e t a - e a r o t e n e B,
A lso w o rk in g w ith a l f a l f a , B i c k o f f and Thompson to o k
ab o u t 10 gm. o f e i t h e r s u n - c u r e d or d e h y d ra te d m eal and
soak ed i t i n 25 m l. o f p e tr o le u m e t h e r i n t h e d a r k f o r
15 m in u te s . Then th e y f i l t e r e d t h e m a t e r i a l and th e
e x t r a c t was re a d y f o r a s p e c t r o s c o p i c stu d y o f t h e
s t e r e o i s o m e r s . B i e k o f f found t h a t good q u a l i t y d e h y d r a t
ed m ea ls c o n t a i n a b o u t 40 m ierog ram s o f c a r o t e n e p e r
m i l l i l i t e r o f e x t r a c t . 1 ®
U sin g th e a d s o r p t i o n colum n, S t r a i n h a s b e e n a b l e
t o s e p a r a t e a s l i t t l e a s 0 .001 5 mg. o f c a r o te n e i n t o
a lp h a and b e t a i s o m e r s . 1 The r e s o l u t i o n i s im proved by
w o rk in g a t re d u c e d t e m p e r a t u r e s , i n w hich c a s e t h e a d
s o r p t i o n bands on th e eolumn become n a rr o w e r.
M ethods o f A n a ly s is o f M ix tu re s f o r C a ro te n e
C a r r - P r i c e m ethod. S e v e r a l d i f f e r e n t m ethods have,
been u se d f o r m e a su rin g th e amount o f c a r o t e n e i n a
g iv e n s o l u t i o n . One o f th e f i r s t was th e C a r r - P r i c e
m ethod. The C a r r - P r i c e r e a g e n t was d e v e lo p e d i n 1926.
I t i s a s a t u r a t e d s o l u t i o n o f antim ony t r i c h l o r i d e i n
c h lo ro f o rm . A b lu e c o l o r d e v e lo p s when t h i s compound
12
r e a c t s w ith v i t a m i n A o r one o f t h e c a r o t e n o i d s . The
C a r r - P r i c e d e te r m i n a ti o n i s b a se d on m e a s u rin g t h e i n t e n
s i t y o f t h e b lu e c o lo r and a l s o th e sp e ed w ith w hich
maximum i n t e n s i t y i s r e a c h e d . T h is method s t i l l o c c u p ie s
"a p o s i t i o n o f f i r s t im p o rta n c e i n m ost l a b o r a t o r i e s
p
where v i t a m i n A i s e s t im a t e d by c h e m ic a l m eans". I t i s ,
how ever, n o t to o r e l i a b l e f o r c a r o t e n e . I t s non-
s p e c i f i c i t y and t h e t r a n s i e n t n a t u r e o f th e b lu e c o lo r
s ta n d a g a i n s t i t , and t h e a c c u ra c y o f e s t i m a t i n g th e b lu e
c o l o r due t o c a r o te n e i s p o o r. V a lu es f o r th e b lu e
e q u i v a l e n t o f c a r o te n e ra n g e d from 3 . 5 to 1 3 , c a l c u l a t e d
a s meg. o f a p p a r e n t v i ta m in A p e r 100 gm. o f c a r o t e n e . 22
P h a s ic m eth o d . P i o n e e r i n g work done a s e a r l y a s
1918 by W i l l s t a t t e r and S t o e l l r e s u l t e d i n what i s com
monly r e f e r r e d t o a s t h e p h a s i c m e th o d .2® I n t h i s p r o
c e d u re t h e c a r o t e n o i d s a r e s e p a r a t e d from i n t e r f e r i n g
p ig m e n ts by v i r t u e o f th e d i f f e r e n c e s i n t h e i r d i s t r i b u
t i o n c o e f f i c i e n t s i n im m isc ib le s o l v e n t s . A m ix tu re o f
p e tro le u m e t h e r , i n w hich c a r o t e n e s a r e s o l u b l e , and
aqueous m eth y l a l c o h o l , w hich i s a s o l v e n t f o r t h e c a r o -
t e n o l s (one o f th e c h i e f i n t e r f e r i n g p ig m e n ts i n fe e d
i n g r e d i e n t s } , i s em ployed. When t h e p ig m e n ts a r e th u s
s e p a r a t e d t h e b e t a - c a r o t e n e can be m easured d i r e c t l y by
c o l o r i m e t r i c m eans. F o r many y e a r s t h i s method w ith i t s
13
m o d i f i c a t i o n s was c o n s i d e r e d th e m ost im p o r t a n t .
M o d i f i c a t i o n s o f p h a s i c m ethod. One o f t h e m o d if i
c a t i o n s o f th e p h a s ic m ethod was worked o u t by G -uilbert*
He d i g e s t e d h i s m a t e r i a l f o r 30 m in u te s w ith KOH i n
e t h y l a l c o h o l , t h e n added e t h y l e t h e r t o t h e m ix t u r e .
The c h l o r o p h y l l was rem oved by w ashing w i t h w ater* The
re m a in in g s o l u t i o n c o n t a i n i n g c a r o te n e and x a n th o p h y ll
\
was e v a p o r a te d to remove th e e t h e r . The r e s i d u e was
e x t r a c t e d w ith p e tro le u m e t h e r and x a n th o p h y l l s w ere r e
moved w i t h 90io m e th a n o l.
B a k e r, B rooks and T y l e r ’ s m o d i f i c a t i o n o f t h e
o r i g i n a l p h a s ic m ethod i s v e ry much l i k e t h e o f f i c i a l
p h a s i c method o f th e AOAC. Two grams o f m a t e r i a l a r e
p l a c e d i n a 100 c c . f l a s k . 15 c c . o f 10fo KOH i n e t h y l
a lc o h o l a r e add ed . T h is i s r e f l u x e d 30 m in u t e s , c o o le d ,
and p la c e d i n a s e p a r a t o r y f u n n e l . The c a r o t e n o i d s a r e
e x t r a c t e d by a d d in g 15 c c . o f p e tro le u m e t h e r , s h a k in g
th e f u n n e l , and d r a i n i n g o f f th e bottom a l c o h o l - w a t e r
l a y e r . T h is s t e p i s r e p e a t e d s i x tim e s , t h e n w a te r i s
added to th e p e tro le u m e t h e r s o l u t i o n , and t h e m ix tu r e
a llo w e d t o s t a n d . The w a te r l a y e r i s d r a in e d o f f , and
20 c c . p o r t i o n s o f 89fo m e th a n o l a r e added r e p e a t e d l y a s
b e f o r e , w ith s h a k in g , i n o r d e r t o remove t h e x a n t h o p h y l l s .
The p e tro le u m e t h e r - c a r o t e n e s o l u t i o n i s f i l t e r e d th ro u g h
14
an h y d ro u s HagSO^ i n t o a 100 e c . v o l u m e t r i c f l a s k and made
24
up t o volume f o r r e a d i n g .
W ild er and B ethke p l a c e d t h e i r sam ple i n a c e to n e and
s t o r e d i t 48 h o u rs u n d er r e f r i g e r a t i o n i n a s to p p e r e d
f l a s k . The s o l u t i o n was th e n d e c a n te d i n t o a s e p a r a t o r y
f u n n e l and th e r e s i d u e r e - e x t r a c t e d w ith a c e to n e and
l o w - b o i l i n g p e tro le u m e t h e r by g r i n d i n g i n a m o r ta r .
E x t r a c t i o n s were c o n tin u e d u n t i l no more p igm ent c o u ld
be rem oved from t h e r e s i d u e . W ater was t h e n added t o
th e a c e to n e - p e tr o le u m e t h e r m ix tu r e t o remove th e a c e to n e .
P o ta s s iu m h y d ro x id e i n a lc o h o l was added to s a p o n if y t h e
p e tr o le u m e t h e r l a y e r . 89$ m eth an o l was u se d w ith s h a k in g
to remove th e x a n t h o p h y l l s , t h e n t h e p e tro le u m e t h e r was
washed a g a i n w ith w a te r to remove th e a l k a l i and m eth -
25
a n o l .
A n o t h e r - m o d i f ic a t i o n o f th e p h a s ic method employs
85$ o r th o - p h o s p h o r i c a c i d i n s t e a d o f m ethanol to s e p a r a t e
t h e x a n th o p h y l l s from t h e c a r o t e n e s i n s o l u t i o n . T h is
m ethod i s more r a p i d th a n t h e o f f i c i a l m e th a n o l-p e tro le u m
e t h e r m eth od, and i s c la im e d t o be more a c c u r a t e f o r
m a t e r i a l w ith low o r o n ly a v e r a g e c a r o t e n e c o n t e n t . I t
w i l l n o t remove ly c o p e n e n o r s e p a r a t e a lp h a and beta-*
e a r o t e n e . £6
15
C h ro m atographic m ethod . The c h ro m a to g ra p h ic m ethod
i s a c o m p a r a tiv e ly r e c e n t dev elo p m en t. Column ch rom ato
grap h y i s employed t o s e p a r a t e i n t e r f e r i n g s u b s ta n c e s
f r o m .th e b e t a - c a r o t e n e . T h is i s p o s s i b l e b e c a u se each
o f t h e s e s u b s ta n c e s h a s a d i f f e r e n t s p e c i f i c a b s o r b t i v i t y
on t h e a d s o r b e n t i n th e colum n. The r e s u l t i n g s o l u t i o n
may th e n be m easured d i r e c t l y f o r b e t a - c a r o t e n e by c o l o r
i m e t r i c m eans. I n t h i s method a s o l u t i o n c o n t a i n i n g
c a r o t e n o i d p ig m e n ts i s f i l t e r e d th ro u g h a g l a s s tu b e con
t a i n i n g one or a n o th e r a d s o r b e n t . T here a r e s e v e r a l
s i g n i f i c a n t v a r i a b l e s i n t h i s m ethod, w h ich , a s B io k o f f
and h i s c o -w o rk e rs w arn, sh o u ld be c a r e f u l l y c o n t r o l l e d .
These a re th e a d s o r b e n t , t h e d e v e lo p e r , th e a d s o r b a t e ,
27
t h e s i z e o f column a n d . t h e m ethod o f p a c k in g i t .
When an a d s o r b e n t i s b e in g ehosen f o r t h e r o u t i n e
a n a l y s i s o f c a r o t e n e , t h e r e a r e s e v e r a l r e q u ir e m e n ts
w hich s h o u ld be k e p t i n m ind. I t sh o u ld a d s o rb a l l
i n t e r f e r i n g p ig m e n ts , b u t n o t b e t a - c a r o t e n e . i n c e r t a i n
s t u d i e s o t h e r th a n r o u t i n e a n a l y s i s , i t may be d e s i r a b l e
to choose an a d s o r b e n t w hich would r e t a i n t h e b e t a -
c a r o te n e on th e colum n. I t sh o u ld be i n e x p e n s i v e ,
r e a d i l y o b ta in e d co m m ercially ,, e a s i l y packed i n t o th e
column, s h o u ld n o t c o n t r a c t from th e column w a l l s i f
drawn dry o f s o l v e n t , and sh o u ld a llo w a r a p i d flow o f
16
I s o l v e n t th ro u g h t h e eolumn and a r a p i d developm ent o f
I th e chrom atogram.^®
Soda a s h i s a l o w - c o s t a d s o r b e n t and r e q u i r e s no
m ix in g or p r e l i m i n a r y t r e a t m e n t . When p e tro le u m e t h e r
I o r a s i m i l a r s o l v e n t i s u se d , th e s o l u t i o n o f p ig m e n ts
!
i may be p o u re d o n to a d ry colum n. S in c e soda a s h h a s
: a lm o st no a d s o r p t i v e power f o r c a r o t e n e s , th e y may be
washed on th ro u g h t h e colum n, r e q u i r i n g no s e p a r a t e s o l -
( v e n t t o e l u t e t h e c a r o t e n e s from t h e n o n - c a r o t e n e s . The
; d i s a d v a n ta g e s o f soda a s h a s an a d s o r b e n t a r e : i t does
n o t s e p a r a t e n o n - c a r o t e n e p ig m e n ts i n t o s h a r p l y d e f i n e d
' b a n d s; i t m ust be p r o t e c t e d from a tm o s p h e ric m o is tu r e ;
! and o n ly one t e c h n i c a l g r a d e h a s th e p r o p e r a d s o r p t i v e
p r o p e r t i e s (W yandotte l i g h t soda ash).**®
I
! J . B. DeW itt o f t h e Food and Drug A d m i n i s t r a t i o n ,
I
j a f t e r s t u d y i n g d i f f e r e n t a d s o r b e n t s , d e c id e d t h a t MgO
j was b e t t e r t h a n CaHPO^, Ca(OH)g , o r A lg02 i n s e p a r a t i n g
j th e c a r o t e n e s and x a n th o p h y lls .^ ®
i
! B io k o f f and co w orkers found s e v e r a l h y d r a te d lim e s
I
! w ere s u i t a b l e . One o f t h e s e was M a l li n e k r o d t c a lc iu m
i
i
h y d ro x id e USP. They c o n s id e r e d t h a t t h e m o is tu r e c o n te n t
; o f h y d r a t e d lim e i s an im p o r ta n t f a c t o r i n f l u e n c i n g i t s
1 07
; a d s o r p t i v e p o w e r s .&
17
M oore, i n h i s w ork, found d ic a lc iu m p h o s p h a te to be
th e b e s t a d s o r b e n t . I t removed a l l p ig m e n ts e x c e p t
e a r o t e n e and no s p e c i a l p r e c a u t i o n s were n e e e s s a r y d u r in g
t h e p r o c e d u r e . MgO and MgCOg, he fo u n d , te n d e d to change
some o f th e c a r o t e n e s d u r in g f i l t r a t i o n , g i v i n g to o low
a v a l u e . D i e a l p i u m p h o s p h a te , how ever, does n eed to be
a c t i v a t e d . F or t h i s p ro c e d u re Moore w eighed 100 gm. o f
i n a c t i v e d ic a lc iu m p h o sp h a te i n t o a 2 l i t e r E rle n m e y er
f l a s k . He p r e f e r r e d M erck’ s p r o d u c t b e c a u se i t s c o a r s e r
g r i n d gave a f a s t e r f i l t r a t i o n r a t e , and he r e p o r t e d t h a t
b o i l i n g more th a n 100 gm. u s u a l l y c a u se d c o n s i d e r a b l e
bum ping. 5 grams o f KOH were added i n one l i t e r o f w a te r .
The m ix tu r e was b o i l e d 1 5 -3 0 m in u te s , t r a n s f e r r e d t o a
B uchner f u n n e l , and washed w ith 0 .5 to 1 l i t e r o f w a te r .
I t was th e n p la c e d i n an e v a p o r a t i n g d i s h , d r i e d f o r 24
h o u r s a t 100° G . , b ro k e n up w ith a m o r t a r , and u s e d .
Moore s u g g e s te d a com m ercial d e x tr o s e such a s ^dyno” o f
31
Corn P r o d u c ts R e f in i n g a s a f i l t e r a i d .
O th e r a d s o r b e n t s a r e powdered s u g a r , CaCO^ and
i n u l i n . Of t h e s e th e pow dered su g ar i s b e s t , l a c k i n g
28
on ly i n h ig h flow r a t e .
H yflo Super C e l , C e l i t e 501, C e l i t e 535, and C e l i t e
545 a r e a l l s a t i s f a c t o r y f o r c h ro m a to g ra p h ic w ork. The
C e l i t e 501 i s e s p e c i a l l y good f o r th e s e p a r a t i o n o f
1
18
x a n t h o p h y l l s , g i v i n g c l e a r l y d e fin e d band s and a h ig h
f i l t r a t i o n r a t e . H yflo Super Gel i s t h e s t r o n g e s t o f
t h e s e a d s o r b e n t s . I t s e p a r a t e s e a r o te n e from e r y p t o -
x a n t h i n , x a n th o p h y ll e s t e r s , and th e a c i d d e c o m p o s itio n
p r o d u c t s o f x a n t h o p h y l l s , none o f w hich a r e e x t r a c t e d
from t h e p e tr o le u m e t h e r s o l u t i o n by 90$ m eth an o l i n t h e
p h a s i c m eth o d . I t does n o t , how ever, s e p a r a t e ly c o p e n e
from th e c a r o t e n e s . Among th e o t h e r a d v a n ta g e s o f H yflo
Super Gel a r e t h a t i t i s in e x p e n s i v e , u n ifo rm i n p r o p e r
t i e s , h a s r a p i d flow r a t e , and i s e x c e p t i o n a l l y s t a b l e .
Open s a c k s o f H yflo Super Gel have b e en s t o r e d f i v e y e a r s
and rem a in e d a s a c t i v e a s when new.
Some w o rk e rs have chosen a m ix tu r e o f H y flo Super
Gel and m ie ro n -b ra n d magnesium o x id e . One r e a s o n i s t h a t
t h e h e a t - t r e a t e d s i l i c e o u s e a r t h s a r e c o m p a r a tiv e ly weak
a d s o r b e n t s , and a r e more s t r o n g l y a f f e c t e d by t r a c e s o f
p o l a r s o l v e n t s such a s a c e to n e o r m ethanol th a n i s a
m ix t u r e . She o f f i c i a l AOAG m ethod employs a 1 to 1 m ix
t u r e o f t h e s e two compounds. The c h i e f d is a d v a n ta g e i s
t h a t th e m a t e r i a l s need to be m ixed b e fo r e u s e , and m ust
be m o is te n e d i n th e column w ith p e tro le u m e t h e r b e f o r e
f i l t e r i n g . When t h e s o l u t i o n o f p ig m e n ts h a s been added
and a d s o rb e d , i t m ust be e l u t e d w ith a c e to n e i n p e tr o le u m
e t h e r . 28
19
F o r iso m er s t u d i e s i t i s d e s i r a b l e t h a t t h e c a r o t e n e s
re m a in i n th e column and t h a t each iso m er form a s e p a r a t e
c l e a r l y - d e f i n e d b an d . Some a d s o r b e n t s a r e p a r t i c u l a r l y
good f o r t h i s p u r p o s e . S e v e ra l w o rk e rs h av e t r i e d
CaHPO^. E n t w i s t l e and H u n te r found i t d id n o t p ro d u ce
s a t i s f a c t o r y s e p a r a t i o n o f p ig m e n ts , an d f i l t r a t i o n was
slow and t e d i o u s . B la c k had d i f f i c u l t y p r e p a r i n g a dry
column w ith CaHPO^ and MgCQg. He u s e d them i n a 1 t o 1
m ix tu r e w ith H yflo Super C e l. H a t h e r e l l a n d Lamb found
t h a t t h e CaHPO^ o b t a i n a b l e f o r t h e i r work was u n s a t i s
f a c t o r y f o r p u r i f i c a t i o n o f c a r o t e n e e x t r a c t s . ^ 9
B e ad le and Z s c h e i l e u se d an alu m in a column f o r r e
s o l v i n g b e t a - c a r o t e n e is o m e r s .
I n o r d e r to s e p a r a t e a lp h a and b e t a - c a r o t e n e , B ic k -
o f f and h i s a s s o c i a t e s u se d a 1 to 1 m ix tu r e o f MgO and
D i c a l e i t e (a d iato m ac eo u s e a r t h ) . F o r s t u d i e s o f th e
iso m e rs o f b e t a - c a r o t e n e th e y u sed S h e l l b ra n d l im e ,
c h em ic al h y d r a t e , 325 Mesh, a s recommended by P o l g a r and
Z e c h m e i s t e r D i a t o m a c e o u s e a r t h , th e y fo u n d , speeded
up developm ent o f t h e chrom atogram , b u t b e c a u s e o f
d i f f u s e n e s s and r e s u l t i n g low c o l o r i n t e n s i t i e s i t was
po
c o n s id e r e d to be u n s a t i s f a c t o r y . They fo u n d a ls o t h a t
sp eed was p a r t i c u l a r l y i m p o r t a n t , b o th from t h e s ta n d -
£0
p o i n t o f economy o f tim e and to m in im iz e t h e p o s s i b i l i t y
o f r e i s o m e r i z a t i o n o f any band d u rin g th e s e p a r a t i o n . ® 3
I n o r d e r to a c h ie v e t h i s d e s i r e d speed th e y fou nd t h a t
i
t h e i r c h ro m a to g ra p h ic columns c o u ld b e s t be packed u n i - I
i
fo rm ly u n d e r vacuum w ith a minimum o f t a m p i n g . ^ i
K ernohan recommended t h a t f o r p a c k in g a column when
t o t a l c a r o te n e i s to be d e te r m in e d , a p lu g o f c o t t o n o r
g l a s s wool be p la c e d i n th e bottom o f t h e tu b e , and t h e
tu b e t h e n be fo r c e d down i n t o a c o n t a i n e r o f th e a d s o r
b e n t . When enough a d s o r b e n t (he u se d H yflo Super Gel)
i
i s i n t h e t u b e , i t s h o u ld be p r e s s e d down f i r m l y w ith a j
p l u n g e r . I f i t i s d e s i r e d t h a t some band be rem oved,
more u n ifo rm p a c k in g may be o b ta in e d by a d d in g a d s o r b e n t
28
a l i t t l e a t a tim e and p r e s s i n g i t i n t o p la c e f i r m l y .
Choice o f s o l v e n t s i s a l s o im p o r ta n t f o r t h e s u c c e s s
o f t h e c h ro m a to g ra p h ic m ethod. Two, o r som etim es t h r e e ,
a r e u s e d . The f i r s t i s f o r e x t r a c t i n g th e c a r o t e n o i d
p ig m e n ts from th e m a t e r i a l b e in g s t u d i e d . T hen, a f t e r
t h i s s o l u t i o n h as b een f i l t e r e d th ro u g h th e colum n, a
second d e v e lo p in g s o l v e n t i s a d d ed , and som etim es a t h i r d
s o l v e n t f o r t h e p u rp o se o f e l u t i n g one o r more s p e c i f i c
p ig m e n ts from th e column. I n t h e AOAC m ethod th e d e v e l-
i
op in g and e l u t i n g s o l v e n t s a r e n o t added s e p a r a t e l y . !
F o r th e e x t r a c t i o n s t e p K ernohan u se d p e tro le u m J
21
e t h e r . W ilkes r e p o r t s t h a t a m ix tu r e o f p e tr o le u m e t h e r
and a c e to n e i s b e t t e r * He sa y s t h a t an e x t r a c t i o n tim e
o f 16 to 18 h o u rs i s s a t i s f a c t o r y , and t h a t even 70 h o u rs
o f e x t r a c t i o n c au se no l o s s o f c a r o t e n e when p r e c a u t i o n s
have been ta k e n to a s s u r e th e p u r i t y o f th e a c e to n e . I f
t h e a c e to n e i s n o t p u r e , t h e r e may be l o s s by o x i d a t i o n . 2^1
i
Gooley and Koehn e x t r a c t e d w ith e q u a l p a r t s o f
t o l u e n e , e t h y l a l c o h o l , and e t h y l a e e t a t e , th e n removed
t h o s e s o l v e n t s w i t h s u c t i o n and w a te r b a th and d i s s o l v e d
t h e r e s i d u e i n an a e e to n e - p e tr o le u m e t h e r s o l u t i o n .
I
S t r a i n and Manning recommend th e a d d i t i o n o f 0 .5 $
r e d i s t i l l e d d im e th y l a n i l i n e to th e e x t r a c t i n g s o l v e n t
when p l a n t m a t e r i a l s a r e b e in g s t u d i e d . The p u rp o se o f j
t h i s i s to n e u t r a l i z e th e p l a n t a c i d s d u r in g e x t r a c t i o n s ,
t o c o u n te r a c t a c i d i t y w hich m ight o th e r w is e d ev elo p on j
2Q !
t h e colum ns, and to m inim ize pigm ent o x i d a t i o n .
F o r t h e d e v e lo p in g and e l u t i n g s o l v e n t s , two f a c t o r s
a r e in v o lv e d : f i r s t th e e l u t i n g s t r e n g t h o f each s o l v e n t
r e l a t i v e to th e o t h e r s ; and se co n d , t h e e f f i c i e n c y o f
th e s o l v e n t s f o r s e p a r a t i n g th e com ponents o f th e pigm ent
m ix t u r e . T rappe h a s d e m o n s tra te d t h a t a group o f s o l
v e n ts can be a rr a n g e d i n a d e f i n i t e o r d e r o f d im in is h in g
e l u t i n g p o w e rs, in d e p e n d e n tly o f t h e n a t u r e o f th e a d
s o r b e n t or o f th e s u b s ta n c e s a d s o r b e d . 9
£2
B ic k o f f p o i n t s o u t t h a t "when e t h e r a lo n e was u se d
a s th e d e v e lo p e r , a l l th e han ds w ere wide and d i f f u s e
and t h e r e was no v i s i b l e s e p a r a t i o n betw een t h e two lo w e r
z o n e s , a lth o u g h t h e r e was a s l i g h t s e p a r a t i o n o f th e
u p p e r (n e o -b e ta -U ) zone from t h e o t h e r two. A lc o h o ls
were m ost e f f e c t i v e i n i n c r e a s i n g th e s h a r p n e s s o f t h e
zones by n a rro w in g t h e i r width-* However, a l c o h o l s a l s o
made t h e bands a p p ro a c h one a n o th e r so c l o s e l y t h a t t h e r e
was no s e p a r a t i o n . A c e to n e , e th y l e t h e r , and benzene
a l l i n c r e a s e d th e i n t e r z o n a l d i s t a n c e b e tw ee n a l l - t r a n s
b e t a - c a r o t e n e and n e o - b e t a - e a r o t e n e U, b u t had no e f f e e t
on t h e n e o - b e t a - c a r o t e n e B zo n e. A ll t h e e s t e r s s t u d i e d
cau se d a s l i g h t s e p a r a t i o n o f t h i s n e o -b e ta -B zo ne.
Among t h e b e s t was p - c r e s y l m eth y l e t h e r . I t was t e s t e d
w ith c a r o t e n e e x t r a c t s from d e h y d ra te d a l f a l f a a s w e ll
a s c r y s t a l l i n e i o d i n e - i s o m e r i z e d m i x t u r e s . I n b o th c a s e s
Q
a good s e p a r a t i o n was o b t a i n e d . I n a n o th e r stu d y B ic k
o f f and h i s eo w orkers u se d a 1 . 5 ^ s o l u t i o n o f th e p - c r e s y l
m e th y l e t h e r i n p e tr o le u m e t h e r , and a ls o i n i s o - o c t a n e .
They found t h e fo rm er gave c l e a n e r s e p a r a t i o n w h ile t h e
l a t t e r had th e a d v a n ta g e o f u n i f o r m i t y from b a tc h to
b a t c h .
At a n o th e r tim e B i c k o f f and h i s a s s o c i a t e s u se d a
d e v e lo p in g s o l u t i o n o f Zfo a c e to n e i n h e x a n e — t h i s f o r a
23
M g Q - d ie a lc ite colum n. And f o r h y d r a te d lim e columns
th e y u se d e i t h e r by volume o f a c e to n e i n hexane o r a
1 2
l^fo s o l u t i o n o f p - c r e s y l m eth y l e t h e r i n h e x an e .
The p r e s e n t o f f i c i a l AOAC c h ro m a to g ra p h ic m ethod i s
a s f o l lo w s : A one to two gram sam ple i s r e f l u x e d one
h o u r w ith 30 m l. o f a m ix tu re o f 3 p a r t s a c e to n e and 7
p a r t s S k e l l y s o l v e B. I t i s th e n t r a n s f e r r e d q u a n t i t a
t i v e l y to a 100 m l. v o lu m e tr ic f l a s k , and th e e n t i r e
sam ple i s ch ro m a to g ra p h ed on a 10 cm. column made up w ith
a 1 t o 1 m ix tu r e o f m agnesia Bo. 2642 and H yflo Super Q el.
The c a r o t e n e i s e l u t e d w ith 70 m l. o f a m ix tu r e o f one
p a r t a c e to n e and n i n e p a r t s S k e l ly s o lv e B. I t i s c o l
l e c t e d i n a 100 m l. f l a s k and t h e d e n s i t y d e te rm in e d a t
440 a i/^ in a c o l o r i m e t e r o r s p e c tr o p h o to m e te r .
An a l t e r n a t i v e AOAC m ethod i s t o e x t r a c t t h e sam ple
o v e r n ig h t i n th e d a rk a t room te m p e r a t u r e . In t h i s e a s e ,
a 5 m l. a l i q u o t o f th e e x t r a c t e d pigm ent s o l u t i o n (made
up t o 100 m l. w ith S k e l ly s o lv e B) i s ch ro m ato g rap h ed on
a column 12 mm. i n d ia m e te r packed to 40 mm. i n h e i g h t .
S o l v e n ts , a d s o r b e n t and f i n a l m e a su rin g a r e t h e same a s
a b o v e .3 ®
A recommended change i n o r d e r to s h o r t e n t h e AOAC
method i s t o p la c e a 2 gm. sam ple on Ko. 1 Whatman f i l t e r
p a p er and wash w ith h o t w a te r (500 m l. a t 50° 0 . ) . A
24
vacuum i s u se d to move th e w a te r th ro u g h t h e m e a l. The
volume o f w a te r a b s o r b e d by th e f i l t e r p a p e r and t h e r e
h y d r a te d m eal i s a s c e r t a i n e d . The f i l t e r p a p e r c o n t a i n
i n g t h e sam ple i s t r a n s f e r r e d t o an e l e c t r i c a l b le n d o r
and d i s i n t e g r a t e d a t h ig h sp e e d f o r one m in u te i n th e
p re s e n c e o f 100 m l. o f a c e t o n e . A 5 m l. a l i q u o t o f th e
a c e to n e e x t r a c t i s t a k e n and added to 5 m l. o f hexane
and 3 m l. o f w a te r i n a 50 m l. s e p a r a t o r y f u n n e l . The
fu n n e l i s s w i r l e d g e n t l y f o r a m in u te and m ost o f th e
a c e to n e t r a n s f e r r e d i n t o t h e lo w e r w a te r l a y e r , w hich i s
t h e n w ithdraw n and d i s c a r d e d . The e n t i r e h y p e rp h a se i s
chrom ato g rap h ed on a column 12 mm. i n d ia m e te r packed
t o 70 mm. i n h e i g h t w ith t h e o f f i c i a l l y ap p ro v e d a d
s o r b e n t m en tio n ed b e f o r e . The c a r o te n e i s e l u t e d w ith
a m ix tu r e o f one p a r t a c e to n e and n in e p a r t s hexane
u n t i l 25 m l. i s c o l l e c t e d i n a v o l u m e tr ic f la s k * The
a b so rb a n c e o f t h e s o l u t i o n i s r e a d a s ab o v e, and a llo w
ance i s made i n t h e c a l c u l a t i o n s f o r th e w a te r a b s o rb e d
rz f.
by t h e o r i g i n a l sam ple.
The AOAC m ethod h a s come a s a r e s u l t o f th e work o f
many i n v e s t i g a t o r s * Some whose m ethods a r e v e ry s i m i l a r
t o i t a r e : Wall and K e l l e y , 37 K e rn o h a n ,3 ® S i l k e r , j
S c h re n k and King.,'1 ' 7 and Q uaekenbush*39 The f i r s t o f j
t h o s e m en tio n ed h e r e e x t r a c t e d by r e f l u x i n g , th e se co n d J
25
two s e t s o f w o rk e rs f a v o re d o v e r n ig h t e x t r a c t i o n i n c o ld
s o l u t i o n s , w h ile Quackenbush t r i e d b o th .
Z s e h e i l e and Whitmore d e v e lo p e d a v a r i a t i o n o f th e
W all and K e lle y m ethod. They p u t t h e i r sam ple b etw een
two l a y e r s o f c o t t o n i n a Goors p o r c e l a i n e x t r a c t i o n
th im b le su sp en d e d i n an A. S. T. M. e x t r a c t i o n a p p a r a t u s .
The s o l v e n t u s e d was 30$ a c e to n e i n h e x a n e , w ith w hich
e x t r a c t i o n was c a r r i e d on f o r t h r e e h o u r s on a w a te r
b a t h . They made th e e x t r a c t up to 100 m l. w ith h e x a n e ,
and to o k a 15 m l. a l i q u o t t o c h ro m a to g ra p h . T h e ir a d -
s o r b e n t was 1 to 1 MgO 2.642 and H yflo Super G e l .3
Some w o rk e rs u se d a m o d i f i c a t i o n o f t h e p h a s i c
m ethod d e s c r i b e d h e r e f o r th e e x t r a c t i o n s t e p , and t h e n
'
!
employed a c h ro m a to g ra p h ic column i n o r d e r t o p u r i f y t h e j
c a r o te n e s o l u t i o n b e fo r e f i n a l r e a d i n g w ith a s p e c t r o
p h o to m e te r. Among th e s e w ere M oore, 40 Kemmerer and
P r a p s , 4 1 » 42 and Gharkey and W ilg u s .43
One o f th e v a r i a b l e s i n t h e a n a l y t i c a l m ethods men
t i o n e d i s th e amount o f c a r o te n e s o l u t i o n w hich i s drawn
th ro u g h t h e c h ro m a to g ra p h ic colum n. B ic k o f f and h i s co
w o r k e r s , who have c a r r i e d on e x t e n s i v e stu d y o f is o m e r s ,
found t h a t f o r t h e i r f i e l d t h e t o t a l amount o f e a r o t e n e j
ad ded t o a eolumn i s more im p o r ta n t t h a n i t s e o n c e n tr a - j
26
t i o n i n d e te r m in in g th e d e g re e o f s e p a r a t i o n o f is o m e rs .
However, t h e y f e l t i t i s p r e f e r a b l e to add t h e d e s i r e d
q u a n t i t y i n a volume o f 1 m l. o r l e s s t o p ro d u c e com pact,
w e l l - d e f i n e d h a n d s . T h e i r s t u d i e s showed t h a t i n c r e a s i n g
th e amount o f c a r o t e n e on th e column from 20 t o 1000
m icrogram s g i v e s s u c c e s s i v e l y p o o r e r s e p a r a t i o n . . Too
sm all an am ount, how ever, i s n o t v i s i b l e on th e column.
About 4:0 g o f t o t a l c a r o t e n e was foun d b e s t f o r l i q u i d
27
c h ro m a to g ra p h ic p r o c e d u r e .
Q u a n t i t a t i v e M easurem ent o f C a ro te n e
When th e c a r o t e n e h a s b een e x t r a c t e d and p u r i f i e d
by any o f th e m ethods d e s c r i b e d , a q u a n t i t a t i v e m e a su re
ment i s o b ta in e d by d e te r m in in g t h e amount o f c o l o r or
d e g re e o f a b so rb a n c y o f th e c a r o t e n e s o l u t i o n .
E a r l y work was done w i t h v i s u a l ty p e c o l o r i m e t e r s
i n w hich t h e unknown s o l u t i o n was compared w ith s t a n d a r d s
(p o ta ssiu m d ic h ro m a te was som etim es u se d ) and c o n c e n tr a
t i o n was d e te rm in e d from c a l i b r a t i o n c u r v e s . T h is was a
r a p i d and c o m p a r a tiv e ly sim p le m ethod, b u t had s e v e r e
l i m i t a t i o n s .
More r e c e n t l y two much im proved m ethods have b een
d e v e lo p e d . The p h o t o - e l e c t r i c f i l t e r p h o to m e te r can g iv e
v e ry s a t i s f a c t o r y r e s u l t s where m ix t u r e s a r e n o t to o
27
com plex and need n o t be r e s o l v e d . Iso m e rs o f b e t a -
c a r o t e n e can n o t be d e te rm in e d , b u t a lp h a o r b e t a - e a r o -
t e n e can , w ith an a c c u ra c y o f 1 fo. The p h o t o - e l e c t r i c
s p e c tr o p h o to m e te r was f i r s t u se d w i t h c a r o t e n o i d p ig m e n ts
by M i l l e r i n 1934. I t h a s , a s Z s e h e i le s a y s , na g r e a t e r
number o f a d v a n ta g e s t h a n any o t h e r m eth o d ". The tim e
r e q u i r e d f o r eaeh d e t e r m i n a t i o n i s o n ly m in u te s , t h e
amount o f m a t e r i a l r e q u i r e d i s o n ly .0 3 mg. i n 10 c c . ,
and s e n s i t i v i t y i s h i g h . The d i s a d v a n t a g e s o f th e
s p e c tr o p h o to m e te r a r e t h a t t h e o p t i c a l a p p a r a t u s i s v e r y
e x p e n s iv e , fu n d a m e n ta l a b s o r p t i o n d a ta m ust be o b t a i n e d
from p u re sam ples o f e a c h pigm ent t o be i n c lu d e d i n t h e
a n a l y t i c a l sy ste m , an d s p e c t r a o f a l l p ig m en ts p r e s e n t
m ust be known b e f o r e a n a l y t i c a l d a t a upon unknown sam ples
can be i n t e r p r e t e d a c c u r a t e l y . ^
W orkers i n t h e U n ite d S t a t e s a r e now i n f a i r a g r e e
ment a s t o t h e a b s o l u t e v a l u e s o f a b s o r b t i v i t i e s i n t h e
v i s i b l e r e g i o n a s w e ll a s t o wave l e n g t h s o f maximum
a b s o r p t i o n o f t h e more common c a r o t e n o i d s o l u t i o n s . T h is
i s e s p e c i a l l y t r u e o f l a b o r a t o r i e s w ith s p e e t r o p h o t o -
e l e c t r i c m eth ods. T here a r e r a t h e r l a r g e d i f f e r e n c e s
betw een v a l u e s g e n e r a l l y a c c e p te d i n t h i s c o u n tr y and
th o s e i n t h e E uropean l i t e r a t u r e , p a r t i c u l a r l y Kuhn and
S m aku la.1
28
I n o r d e r t o a c h ie v e maximum p r e c i s i o n i n iso m er
a n a l y s i s , wave l e n g t h s m ust he cho sen f o r a b so rb a n c e
m easurem ents su c h t h a t a t e a c h wave l e n g t h : f i r s t , a j
i
p a r t i c u l a r component a b s o r b s s t r o n g l y com pared to o t h e r !
com ponents; and se co n d , t h e a b s o r p t i o n c u rv e o f t h e I
i
s t r o n g l y a b s o r b in g component i s n o t s t e e p I n a n a -
i
l y z i n g f o r b e t a - c a r o t e n e a w a v e - le n g th o f 436 m ^(or 440 m y ,
on an E v ely n i n s t r u m e n t ) i s u s e d . I f a Beckman s p e c t r o
p h o to m e te r i s u se d t h e s p e c i f i c a b s o r b t i v i t y i s 19 6, and :
39
t h e e q u a t io n i s :
c a r o t e n e ( m g . / l b . ) s l o g ( I n/ l ) x V x 454
“ x " L x W :
V r f i n a l volum e, I s l e n g t h o f c e l l (cm.), Wswt. o f sam ple
S t i t t , B i c k o f f and c o w o rk ers found t h a t e m p i r i c a l l y a d - .
I
j u s t e d a b s o r p t i v i t y v a l u e s were n e c e s s a r y t o g e t c o r r e c t
r e s u l t s i n d e te r m in in g b e t a - c a r o t e n e is o m e rs i n a l f a l f a
w i t h th e s p e c t r o p h o t o m e t e r . They f e l t t h a t w h ile l e s s ,
r e l i a b l e , th e t u n g s t e n f i l a m e n t s o u rc e wave l e n g t h s w ere j
e a s i e r t o u s e . They a l s o worked w ith a m erc u ry a r e a s !
t h e l i g h t s o u r c e . S in c e a l f a l f a c o n t a i n s c h i e f l y t h r e e
c a r o t e n e p ig m e n ts , t h e s e w o rk e rs p o i n t e d o u t t h a t th e
s p e c t r o p h o t o m e t r ie a n a l y s i s o f t h e i r s o l u t i o n " r e q u i r e s
a b so rb a n e y m easu rem ents a t t h r e e d i f f e r e n t wave l e n g t h s ;
i
and a knowledge o f t h e a b s o r p t i v i t i e s o f t h e t h r e e p u re
£9
com ponents a t each, o f t h e wave l e n g t h s . M a th e m a tic a lly ,
f o r m easurem ents re d u c e d t o t h e same p a th l e n g t h (1 cm.)
and d i l u t i o n , we may w r i t e t h r e e s im u lta n e o u s e q u a tio n s
e x p r e s s in g th e f a c t t h a t t h e a b so rb a n c y a t eac h wave
l e n g t h i s th e sum o f th e a b s o r b a n e i e s o f t h e i n d i v i d u a l
c om ponents. " ^ ®
I n com paring t h e m ethods o f m e a su rin g p ig m e n t,
T a y lo r s a i d , " I t i s so u n d er te c h n iq u e to a s s a y t h e m a t
e r i a l s p e e t r o p h o t o m e t r i e a l l y by m e a su rin g th e a b s o r p t i o n
(
o f l i g h t a t th e wave l e n g t h o f maximum a d s o r p tio n * The
m e a su re i s , t h e n o f t h e p rim a ry phenomenon ( s p e c i f i c a d
s o r p t i o n ) and n o t o f t h e s e c o n d a ry phenomenon ( c o l o u r ) ,
w hich i s e a s i l y c o n ta m in a te d , i s t o some e x te n t s u b j e c t -
44
i v e , and i s n o t i n t r i n s i c . "
C om parison o f M ethods
S e v e r a l w ork ers have compared t h e p h a s ic and c h r o
m a to g ra p h ic m eth ods. R e s u l t s o f t h e i r work seem t o a g re e
t h a t t h e p h a s i c m ethod i s n o t a s good b e e a u se c e r t a i n
n o n - c a r o t e n e chromogens rem a in i n th e p e tro le u m e t h e r
p h a se and a r e m easured a s c a r o t e n e , when a c t u a l l y , by
c h ro m a to g ra p h ic or b i o l o g i c a l means th e y may be shown to
be n o n - c a r o t e n e s p o s s e s s i n g no v i ta m in A a c t i v i t y . * 5®
Z s c h e i l e p o i n te d o u t t h a t t h e l a c k o f p r e c i s e s e p a r a t i o n
30
by w ash in g i s e s p e c i a l l y t r u e o f b i o l o g i c a l m ix tu r e s
b e c a u se i m p u r i t i e s c a u se changes i n t h e s o l u b i l i t y o f
p ig m e n ts .-
A lth o u g h i t i s g e n e r a l l y c o n s i d e r e d b e t t e r , th e
ch ro m a to g ra p h ic m ethod h a s i t s l i m i t a t i o n s . S o u rc e s o f
e r r o r a r e : r e - i s o m e r i z a t i o n , p a r t i a l d e s t r u c t i o n o f
c a r o t e n e s , in c o m p le te rem oval o f c a r o t e n e d u r in g e x t r a c
t i o n , in c o m p le te e l u t i o n o f th e colum n, and l o s s o f a c e -
35
to n e i n r e f l u x i n g . The d i f f i c u l t y i n o b t a i n i n g a d s o r b -
p g
e n ts o f u n ifo rm p erfo rm an c e i s a l s o a l i m i t i n g f a c t o r .
B i c k o f f o b se rv e d t h a t iso m e r s e p a r a t i o n would be
much s i m p l i f i e d i f i t were p o s s i b l e t o c o l l e c t t h e i s o
m ers s e p a r a t e l y by c o n t i n u i n g t h e d evelopm ent u n t i l each
zone p a s s e s c o m p le te ly th ro u g h t h e colum n.^
C r i t i c i s m s o f th e AOAC c h ro m a to g ra p h ic m ethod i n
p a r t i c u l a r a r e on t h e b a s i s t h a t f i r s t , i t does n o t ex
t r a c t a l l o f t h e n e o - b e t a - c a r o t e n e IT from t h e sam ple,
and s e c o n d , t h a t r e c o v e r y o f c a r o t e n e s on t h e column i s
in c o m p le te . T h is l a t t e r i s th o u g h t to be due to th e f a c t
i
t h a t i n s u f f i c i e n t e l u e n t i s p r e s c r i b e d and t h a t a change j
may o c c u r i n r e t e n t i v i t y o f c a r o te n e by t h e a d s o r b e n t .
I t i s p o s t u l a t e d t h a t t h e r e may be enough i s o m e r i z a t i o n
o f n e o - b e t a t o a l l - t r a n s c a r o t e n e t o com pensate f o r
K
low column r e c o v e r y . °
'________________________________________________________________________________:_____________________ :__________ _i
STATEMENT OF THE PROBLEM AND PLAN OF ATTACK
The problem a s u n d e rta k e n i s to i d e n t i f y th e p i g
m ents o r segm ents t h a t a r e h e ld i n t h e e p i p h a s i e (hexane)
l a y e r a lo n g w ith h e t a - c a r o t e n e i n th e d e t e r m i n a t i o n o f
c a r o t e n e hy t h e p h a s ic method* These p ig m e n ts g iv e a t
1
i
f a l s e l y h ig h q u a n t i t a t i v e v a lu e f o r c a r o t e n e i n dehydrated?
i
a l f a l f a a s compared t o t h e v a lu e o b ta in e d by t h e chrom a- |
\
\
t o g r a p h i c m ethod. j
The a p p ro a c h u sed i n t h i s s tu d y was f i r s t t o ta k e I
1
a s o l u t i o n o f c a r o t e n e i n h e x a n e , p u r i f i e d by t h e p h a s ic j
|
m etho d, and p u t i t th r o u g h a column o f M gO -supereel such j
t
a s i s u se d f o r p u r i f i c a t i o n i n th e c h ro m a to g ra p h ic method.I
i f
i
From t h i s we w ish ed t o l e a r n how many p ig m e n ts were i
I
washed th r o u g h th e 'c o lu m n and how many zon es o f p ig m e n ts j
rem ain ed on th e colum n. The n e x t s t e p was to i d e n t i f y j
i n so f a r a s p o s s i b l e th o s e re m a in in g z o n es o f pigm ent
by u se o f s p e c t r a l d a t a . j
MATERIALS AMD METHODS
A cetone
The a c e to n e u se d was a n a l y t i c a l r e a g e n t g r a d e , n o t
p u r i f i e d f o r t h i s s t u d y , a s i t was d e s i r e d t h a t a l l
s o l u t i o n s sh o u ld he th e same a s th o s e used i n th e r o u t i n e
p h a s ie and c h ro m a to g ra p h ic m ethods f o r c a r o t e n e d e t e r
m in a t io n s by com m ercial l a b o r a t o r i e s .
A lc o h o lic P o ta s s iu m H ydroxide
T h is s o l u t i o n was p r e p a r e d by a d d in g 1 5 .5 gms.
p o ta s s iu m h y d ro x id e p e l l e t s , a n a l y t i c a l r e a g e n t , t o 80
c c . o f fo rm u la #3A e th y l a l c o h o l , h e a t i n g u n t i l d i s s o l v e d .
I t m ust be p r e p a r e d f r e s h each tim e i t i s t o be u s e d .
C alcium H ydroxide a s A d so rb en t
The c a lc iu m h y d ro x id e u sed was M a l li n c k r o d t ’ s
a n a l y t i c a l r e a g e n t g r a d e .
Hexane
The hexane u se d was S k e l l y s o l v e B, a s p u rc h a s e d w ith
no p u r i f i c a t i o n .
Magnesium O x id e - s u p e r c e l M ix tu re a s A d so rb en t
T h is was a m ix tu r e o f e q u a l p a r t s by w e ig h t o f w e st”
vaco $2641 magnesium o x id e and Jo h n s M a n v ille s u p e r e e l .
33
M ethanol
The m eth an o l u se d was r e f i n e d , h u t n o t r e p u r i f i e d
f o r t h i s study* A 90fo s o l u t i o n was p r e p a r e d by a d d in g
900 g o . o f m eth an o l to 100 e c . o f w a te r and m ix in g
th o r o u g h ly .
p - C r e s y l M ethyl E th e r
The p - c r e s y l m eth y l e t h e r u se d was E a stm a n ’ s
D i s t i l l a t i o n P r o d u c ts r e a g e n t .
P h a s ic Method f o r D e hy drated A l f a l f a Meal
A 4 gm. sam ple o f d e h y d ra te d a l f a l f a m eal i s .
r e f l u x e d f o r 30 m in u te s w ith 80 e c . o f a l c o h o l i c p o t a s
sium h y d ro x id e . A f t e r c o o l i n g , th e s o l u t i o n i s d e c a n te d j
i n t o a s e p a r a t o r y f u n n e l . Hexane i s added t o th e r e s i
d u e, and th e m ix tu r e i s sh a k e n . Then t h e hexane f r a c
t i o n i s d e c a n te d i n t o t h e s e p a r a t o r y f u n n e l . T h is ex
t r a c t i o n o f p ig m e n ts from t h e r e s i d u e i s c o n tin u e d u n t i l
th e hexane p o r t i o n s a r e c o l o r l e s s . The s o l u t i o n o f
a l c o h o l i c p o ta s s iu m h y d ro x id e i n th e s e p a r a t o r y fu n n e l
i s washed w ith w a te r u n t i l i t i s f r e e o f a l k a l i (as
shown by t e s t i n g w i t h p h e n o l p h t h a l e i n ) . The hexane
s o l u t i o n i n t h e s e p a r a t o r y fu n n e l i s t h e n washed w ith
90$ m eth an o l i n w a te r u n t i l th e m eth an ol l a y e r i s c o l o r
l e s s . The m eth an o l f r a c t i o n i s d i s c a r d e d , and t h e hexane
34
f r a c t i o n i s f i l t e r e d th r o u g h anhy d ro u s sodium s u l f a t e .
The c a r o t e n e s a r e now i n th e h exane s o l u t i o n , and th e
x a n th o p h y l l s a r e i n t h e 90$ m e th a n o l s o l u t i o n .
P h a s ic Method f o r F r e s h A l f a l f a
A 40 gm. p o r t i o n o f f r e s h a l f a l f a i s c u t w ith
s c i s s o r s i n t o sm a ll p i e c e s and t h e n p l a c e d i n a W aring
h le n d o r w ith '80 c c . o f a l c o h o l i c p o ta s s iu m h y d ro x id e .
A few m in u te s o f m ix in g i s employed t o p ro d u ce a homo
gen eo u s h le n d . T h is m ix tu r e i s t h e n r e f l u x e d f o r 30
m in u t e s , and th e re m a in d e r o f t h e s e p a r a t i o n p ro c e d u re
i s t h e same as t h a t d e s c r i b e d above when d e h y d ra te d
m a t e r i a l was u se d .
C hro m ato g rap h ic Method
A £ gm. p o r t i o n o f d e h y d ra te d a l f a l f a m eal i s r e
f l u x e d f o r one h o u r w ith a s o l u t i o n o f 30$ a c e to n e i n
h e x a n e . The m ix tu re i s c o o le d i n a w a te r b a th to room
t e m p e r a t u r e , and t h e s o l u t i o n d e e a n te d i n t o a 100 m l.
v o lu m e tr ic f l a s k , u s i n g a s m a ll p lu g o f c o t t o n a s a
f i l t e r . The r e s i d u e i s washed w ith s e v e r a l sm a ll p o r
t i o n s o f h exane i n o r d e r to e x t r a c t any r e m a in in g p i g
m en t. T hese hexane f r a c t i o n s a r e a l s o d e c a n te d i n t o
th e v o l u m e t r i c f l a s k , and a f t e r t h e l a s t one (which
s h o u ld be c o l o r l e s s ) h a s been a d d ed , th e t o t a l volume
35
i s made up t o 100 m l. w i t h h e x a n e . The hexane s o l u t i o n ,
w hich now sh o u ld c o n t a i n a l l th e c a r o t e n e i n t h e o r i g i n a l
sam ple, i s shak en t h o r o u g h ly , t h e n p a s s e d th r o u g h a
c h ro m a to g ra p h ic column i n w hich a m ix tu re o f e q u a l p a r t s
o f magnesium o x id e f Z 641 and s u p e r e e l i s employed a s
a d s o r b e n t . The column u se d had th e d im e n sio n s 20 mm.
x 100 mm. I t had a g rou nd g l a s s ^ o i n t a t th e botto m so
t h a t th e a d s o r b e n t m ight be e x tru d e d from t h e u p p e r end
o f th e column by a p p ly in g p r e s s u r e a t t h e b o tto m .
EXPERMENTAL RESULTS AND DISCUSSION
36
Use o f F r a c t i o n C u t t e r to S tu d y P ig m en ts
T hree sam p les o f d e h y d ra te d a l f a l f a m eal were ob
t a i n e d from I m p e r i a l V a lle y f o r t h i s w ork. The h exane
f r a c t i o n from t h e p h a s ic m ethod f o r c a r o te n e e x t r a c t i o n ,
r e a d y f o r s p e c t r o p h o t o m e t r ie r e a d i n g , was d e p o s i t e d on a
column o f MgO #2641 and s u p e r e e l and w ashed th ro u g h t h e
column w ith 700 e c . o f 13^> a c e to n e i n h ex an e. U sin g a
f r a c t i o n e u t t e r , s u c c e s s iv e 5 c c . p o r t i o n s o f t h e e l u e n t
w ere c o l l e c t e d i n t e s t tu b e s f o r s p e c t r o p h o t o m e t r ie r e a d
i n g . R e ad in g s w ere made a t 436 m^ 4 on a Beckman DU
s p e c t r o p h o t o m e t e r , u s in g a t u n g s t e n lam p.
A n o th e r p o r t i o n o f th e d e h y d ra te d a l f a l f a m eal was
e x t r a c t e d by t h e c h ro m a to g ra p h ic m ethod u s i n g 700 c c . o f
13$ a c e to n e i n hexane to wash i t th r o u g h th e colum n. A
f r a c t i o n c u t t e r was a g a i n u se d t o g a t h e r t h e e lu e n t i n
s u c c e s s iv e 5 c c . p o r t i o n s a s i t came th ro u g h t h e colum n.
B oth t h e p h a s i c and c h ro m a to g ra p h ic r u n s gave m a jo r
i n t e n s i t y p e a k s i n t h e f i r s t f i f t e e n tu b e s c o ll e c t e d *
The c h ro m a to g ra p h ic r u n s gave i n t e n s i t y p e a k s a g a i n a f t e r
a p p ro x im a te ly 50 to 70 t u b e s . The p h a s i c r u n s gave no
m a jo r p e a k s i n t h e 50 t o 100 tu b e r e g i o n . The i n t e n s i t y
p e a k s i n t h i s u p p e r r e g i o n o f t h e c h ro m a to g ra p h ic r u n s
37
were assumed t o be th e x a n th o p h y l l s b e in g washed th r o u g h
t h e column.
I n t h e column a f t e r p h a s io ru n s t h e r e a p p e a re d th e
zones shown i n F ig u r e 1 . F ig u r e 2 shows a p l o t o f den
s i t y v e r s u s tu b e number f o r b o th c h ro m a to g ra p h ic and
p h a s i c m ethods a f t e r g o in g th ro u g h a M gO -supereel column
w ith 700 c c . o f 13$ a c e to n e i n hexane a s d e v e lo p e r .
M easurem ents w ere made a t 436 m p-w ith a Beckman DU
s p e c tr o p h o to m e te r .
P ig m en ts R em aining on Column A f t e r Removal o f B e ta -
c a r o t e n e
A h exane s o l u t i o n o f b e t a - c a r o t e n e p u r i f i e d by th e
p h a s ic method was e v a p o r a te d u n d e r vacuum to remove any
s o l v e n t s w ith b o i l i n g p o i n t s u n d e r 60° 0. t h a t m ig h t be
p r e s e n t . The s o l u t i o n was t h e n ch ro m ato graphed th r o u g h
a M gO -supercel colum n. The ch ro m a to g ra p h was d e v e lo p e d
w ith 3 fo a c e to n e i n hexane u n t i l a l l b e t a - c a r o t e n e had
been washed th ro u g h and t h e e l u e n t coming t h r o u g h th e
column was c o l o r l e s s . F i g u r e 3 shows t h e a p p e a ra n c e o f
th e column a f t e r a l l t h e b e t a - c a r o t e n e had b e e n e l u t e d .
F i g u r e s 4 th r o u g h 7 show t h e s p e c t r a l c u rv e s o f eac h o f
t h e s e zon es a s d e te rm in e d on a Beckman DU s p e c t r o p h o t o
m e te r u s i n g a t u n g s t e n lam p. P l o t s show a b s o rb a n c e
38
e
V
$
m / / / m yellow
v / m m
k /
pink
yellow
Figure 1 . Zones on MgO-Supsreel Column a fter
Carotene Solution Prepared by Phasic Method W as
Washed Through w ith 700 cc. o f 10$ Acetone in
Hexane
■ H -f
N o . 6 3 1 1 , U n i v e r s i t y B o o k s t o r e , L o s A n g e l e s
-1 0
d k
yellow - zone
rose zone
yellow - zone #2b
yellow - zone #2a
v
Figure 3* Zones on MgO-Supereel Column W han
Carotene Solution Prepared by Phasic Method
Was Developed w ith 3p Acetone in Hexane
N o . 6 3 1 1 , U n i v e r s i t y B o o k s t o r e , L o s A n g e l e s
: :: : : :: : : ::
N o . 6 3 1 1 , U n i v e r s i t y B o o k s t o r e , L o s A n g e l e s
&
i
^ 1
N o . 6 3 1 1 , U n i v e r s i t y B o o k s t o r e , L o s A n g e l e s
N o . 6 3 1 1 , U n i v e r s i t y B o o k s t o r e , L o s A n g e l e s
45
v e r s u s wave l e n g t h . The zones w ere e u t out o f t h e column
and r e d i s s o l v e d i n hexane i n one e a se and i n e a rb o n d i
s u l f i d e i n t h e o t h e r .
T ab le I I shows t h e maxima o f t h e s e f o u r z o n es i n
b o th hexane and c a rb o n d i s u l f i d e .
TABLE I I
SPECTRAL MAXIMA OF FOUR ZONES FOUND OR MgO-SUPERCEL
COLUMN FOR CAROTENES PURIFIED BY PHASIC METHOD
Maxima (mju) Maxima (mfJ) i n
i n Hexane Carbon D i s u l f i d e
Zone #4 422, 4 45, 473 4 5 1 , 4 73, 504
Zone #3 445 481
Zone #2b 42 5, 4 4 5 , 472 4 5 0 , 47 3, 503
Zone # 2a 42 2, 443, 470 445, 472£, 502^
Beta-? c a r o t e n e * 425, 4 4 9 , 476 481, 505
* B e ta - c a r o te n e was o b ta in e d from th e a c e to n e - h e x a n e
s o l u t i o n by e v a p o r a t i o n .
I d e n t i f i c a t i o n o f P ig m en ts on Column; Zone
A 4 gm. p o r t i o n o f d e h y d ra te d a l f a l f a was r e f l u x e d
i n 80 e c . o f a l c o h o l i c p o ta s s iu m h y d ro x id e . The hexane
l a y e r was washed f r e e o f a l k a l i , and x a n t h o p h y l l s were
removed w ith 90$> m e th a n o l. The hexane f r a c t i o n a f t e r
x a n th o p h y ll rem oval was ch ro m a to g ra p h ed on a column o f
) - s u p e r e e l and zone #4 was removed and t a k e n up in
46
a e e to n e and h e x a n e . The a e e to n e was e v a p o r a t e d , l e a v i n g
t h e zone #4 pigm ent i n hexane* Hext th e x a n t h o p h y l l s i n
t h e 90 % m e th a n o l f r a c t i o n w ere p u t i n t o a s e p a r a t o r y
f u n n e l . Hexane was a d d e d , f o llo w e d by a l a r g e p o r t i o n
o f w a t e r . The d i l u t e d m e th a n o l f r a c t i o n was e x t r a c t e d
w ith h exane u n t i l th e h e x an e l a y e r was c o l o r l e s s . The
h e x an e f r a c t i o n s w ere c o l l e c t e d i n a s e p a r a t o r y f u n n e l .
The x a n t h o p h y l l s now i n h ex ane w ere washed s e v e r a l tim e s
w i t h w a t e r and d r i e d o v e r an h y d ro u s sodium s u l f a t e . The
h ex an e s o l u t i o n c o n t a i n i n g t h e zone #4 p igm en t and t h e
s o l u t i o n o f x a n t h o p h y l l s i n hexane w ere m ixed and p u t
th r o u g h a M gO -supercel colum n. R e s u l t s were compared
w i t h t h o s e o b ta in e d on a column u s i n g x a n t h o p h y l l s i n
h ex an e o n ly . The z o n es on e a c h w ere i d e n t i c a l , show ing
t h a t zone #4 c o in c id e d w i t h one o f t h e x a n th o p h y l l z o n e s .
F i g u r e 8 shows t h e M gO -supercel colum n a f t e r p a s s i n g
th r o u g h i t t h e hexane s o l u t i o n o f x a n th o p h y l l and d e v e l
o p in g i t w i t h 3 f o a c e to n e i n h e x a n e .
A m ix tu r e i n hexane o f th e pig m en t from th e "heavy
y e llo w zone'1 on t h e x a n th o p h y l l column and o f o u r "zone
#4 p ig m e n t" o b t a i n e d a s b e f o r e w ere p a s s e d th r o u g h a
M gO -supercel colum n. A s i n g l e band o f c o l o r was form ed.
The s p e c t r a l c u rv e o f t h i s "h eav y y e llo w band" o f
th e x a n t h o p h y l l s i s shown i n F ig u r e 9. W ith i t i s t h e
s
I
yellow
pinki sb-orange
heavy yellow
Hguxe 8* Zone s on M g O — Supercel Column
for Xanthophylls in Hexane
N o . 6 3 1 1 , U n i v e r s i t y B o o k s t o r e , L o s A n g e l e s
49
s p e c t r a l c u rv e o f th e zone #4 p ig m e n t.
I d e n t i f i c a t i o n o f Zone #3 on Column
A 45 gm. p o r t i o n o f f r e s h a l f a l f a was t r e a t e d by t h e
p h a s i c m ethod f o r f r e s h a l f a l f a . (We u se d f r e s h m a t e r i a l
i n t h i s p h a se o f th e s tu d y i n o r d e r to d e te rm in e w h e th e r
th e zone we c a l l e d #3 r e p r e s e n t e d a p ig m en t found i n
f r e s h a s w e ll a s d e h y d ra te d a l f a l f a o r w h e th e r i t m ig h t
be c a u s e d by some p ig m en t form ed d u r in g th e p r o c e s s o f
d e h y d r a t i o n . ) The b e t a - c a r o t e n e i n h exane t h u s p u r i f i e d
was washed f r e e o f m e th a n o l, d r i e d o v e r an hyd ro us sodium
s u l f a t e , and c h ro m a to g ra p h e d on a M gO -supercel colum n.
Two zonCs a p p e a re d a s shown i n F ig u r e 1 0 . The r o s e -
c o l o r e d zone was n o t i n a s heavy a c o n c e n t r a t i o n a s i n
th e d e h y d ra te d a l f a l f a , b u t i t was p r e s e n t . Zones c o r
r e s p o n d in g t o t h o s e numbered 2a and 2b i n our work w ith
d e h y d ra te d a l f a l f a d i d n o t a p p e a r on t h i s column. The
s p e c t r a l c u r v e s f o r th e r o s e z o n es from f r e s h a l f a l f a
and d e h y d ra te d a l f a l f a a r e shown i n F i g u r e 1 1 .
A s e a r c h o f th e l i t e r a t u r e d id n o t shed to o much
l i g h t on w hat t h i s r o s e band m ig h t b e . A p e r s o n a l l e t t e r
to 1 . Z e c h m e is te r b r o u g h t a n answ er w hich was h e l p f u l
b u t a l s o o f a g e n e r a l n a t u r e . However a r e c e n t stu d y
j u s t r e p o r t e d by B i c k o f f and a s s o c i a t e s gave q u i t e de-
Figure
for ft -
77/////////,
'////////M i
U
a
-to-
n > t y >
4 m m * yellow
4 m m . rose
10. Zone 8 on MgO-Super e el Column
•Carotene In Hexane from Fresh A lfa lfa
1st
m
N o . 6 3 1 1 , U n i v e r s i t y B o o k s t o r e , L o s A n g e l e s
^
t a i l e d i n f o r m a tio n on c h ro m a to g ra p h ic s e p a r a t i o n o f c a ro -
t e n o i d s o f f r e s h and d e h y d ra te d a l f a l f a . 4 5 T h is p a p e r
c o r r o b o r a t e s th e e x i s t e n c e and a p p e a ra n c e o f our r o s e -
c o lo r e d zone u s in g c a lc iu m h y d r o x id e and magnesium o xide
colum ns f o r b o th f r e s h and d e h y d ra te d a l f a l f a . T hese
w o rk e rs , u s in g a s u c r o s e colum n, f u r t h e r r e s o l v e d t h i s
r o s e zone i n t o f i v e b an ds w hich th e y s t a t e a r e z e a x a n t h in
and z e a x a n t h i n ^ l i k e p ig m e n ts . The g e n e r a l s p e c t r a l curve
t h e y o b ta in e d f o r t h e f i v e p u r i f i e d p ig m e n ts i s a l s o
shown i n F ig u r e 11 .
I d e n t i f i c a t i o n o f Zones # 3 a and 2b on t h e Column
A b e t a - c a r o t e n e s o l u t i o n i n h exane a s p u r i f i e d by
t h e p h a s ic method f o r d e h y d ra te d a l f a l f a was p u t on a
c a lc iu m h y d ro x id e column and dev elo p ed w i t h 1 .5 $ p - c r e s y l
m e th y l e t h e r i n h e x a n e . A sm a ll y e llo w band p re c e d e d
t h e l a r g e b e t a - c a r o t e n e band down th e eolum n. A dding
15 m l. o f dfo a c e to n e i n hexane e l u t e d a t h i r d y e llo w band
a f t e r th e b e t a - c a r o t e n e band was o u t o f t h e colum n.
A second b e t a - c a r o t e n e s o l u t i o n i n hexane a s p u r i
f i e d by t h e p h a s ic m ethod f o r d e h y d ra te d a l f a l f a was
d iv id e d i n h a l f . One h a l f was chrom ato g rap h ed th ro u g h
a M gO -supercel eolumn w ith 3$ a c e to n e i n h e x a n e . The
o t h e r h a l f was c h ro m a to g ra p h ed on a c a lc iu m h y d ro x id e
53
column u s i n g 1 .5 $ p - c r e s y l m eth y l e t h e r i n hexane a s a
d e v e lo p e r . F ig u r e 12 shows r e s u l t s o b t a i n e d on t h e MgO-
s u p e r c e l column and th e c a lc iu m h y d ro x id e column.
A hex an e s o l u t i o n o f t h e pigm ent i n zone (1) on t h e
c a lc iu m h y d ro x id e eolumn was m ixed w i t h a s i m i l a r s o l u
t i o n c o n t a i n i n g p igm ent from zone # 2 a o f t h e M gO -supercel
column and t h e n ch ro m ato g rap h ed on a M gO -supercel eolumn
u s in g 3 $ a e e to n e i n hexane a s d e v e lo p e r . One band r e
s u l t e d on t h e colum n. S in c e zone (1) i s neo b e t a - c a r o -
45
te n e B, a c c o r d in g t o B i c k o f f and c o w o rk e rs , th e n zone
#2a i s a l s o neo b e t a - c a r o t e n e B.
A m ix tu r e s i m i l a r to t h e a b o v e , made up o f pigm ent
from zone (3) from t h e c a lc iu m h y d ro x id e column and zone
#2b from t h e M gO -supercel eolumn was ch ro m ato grap hed on
a M gO -supercel column u s i n g 3$ a c e to n e i n h e x a n e . One
band r e s u l t e d . S in c e zone (3) i s neo b e t a - c a r o t e n e U
a c c o rd in g t o B i c k o f f , 45 th e n zone #2b i s a ls o neo b e t a -
c a r o t e n e U.
A m ix tu r e o f zone (2) o f th e c a lc iu m h y d ro x id e c o l
umn and b e t a - c a r o t e n e e l u t e d from th e M gO -supercel column
i n hexane was ch ro m ato grap hed on a M gO -supercel column
u s i n g 3fo a c e to n e i n hexane a s d e v e lo p e r . Only one n arrow
band p a s s e d th r o u g h t h e eolum n, i n d i c a t i n g t h a t our
54
't/n//nut
2 0 — '
ZZZZZZZZZ2Z
ntn •///// 77
' / / / / / / / / / /
pink (5)
yellow-
orange (^)
yellow (3)
yellow (2)
(heavy
hand)
light
yellow (l)
T ? 0
m m
n'>‘ n ttjT
w m ,
///Vff/z/j
yellow — zone #U
pink - zone
yellow •* zone #2b
light yellow
zone #2a
Calcium Hydroxide
MgO-Supercel
Figure 12. Zones on MgO-Supercel Column and on Calcium
Hydroxide Column for p — carotene Solution in Hexane
55
zone (2) was a c t u a l l y b e t a - c a r o t e n e , and our work t h e r e
f o r e w ith th e c a lc iu m h y d ro x id e column was n o t i n e r r o r
6
and m ight be compared w ith t h a t r e p o r t e d upon by B i e k o f f .
The s p e c t r a l s h i f t s and t h e h i g h e r a b s o r p t i o n o f zone
#2a a t 340 ( s u g g e s t i n g a h i g h e r c i s peak) a r e c o n s i s t e n t
w ith t h e c o n c l u s i o n t h a t zone # 2 a i s neo b e t a - c a r o t e n e B
and zone #2b i s neo b e t a - c a r o t e n e U.
56
SUMMARY AID C0KCLUSI01S
A s o l u t i o n o f b e t a - c a r o t e n e i n hexane p u r i f i e d by
t h e 'p h a s i c method and th e n ch ro m ato g rap h ed on a MgO-
s u p e r c e l column u s in g Zfo a c e to n e i n hexane a s d e v e lo p e r
l e f t f o u r zones on th e colum n, The e l u a t e d id n o t con
t a i n any p ig m e n ts t h a t w ere n o t found i n a b e t a - c a r o t e n e
s o l u t i o n p u r i f i e d by t h e c h ro m a to g ra p h ic method* These
f a c t s i n d i c a t e t h a t t h e g r e a t e r a p p a r e n t c o n c e n t r a t i o n
found by t h e p h a s ic m ethod a s compared to t h e chro m ato
g r a p h ic m ethod f o r b e t a - c a r o t e n e i s due to th e p ig m e n ts
o f t h e s e f o u r z o n e s. These zo nes were i d e n t i f i e d a s
f o l l o w s :
zone # 2 a : neo b e t a - c a r o t e n e B
zone #2b: neo b e t a - c a r o t e n e U
zone #3: z e a x a n t h in and z e a x a n t h i n ^ l i k e
p ig m e n ts
zone #4: u n i d e n t i f i e d x a n th o p h y ll member
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Asset Metadata

Creator Moffitt, Robert A. (author)

Core Title Comparison of results of phasic and chromatographic methods for carotene analysis.

Degree Master of Science

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, analytical,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Mehl, John W. (committee chair), [illegible] (committee member), Deuel, H.J. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-778030

Unique identifier UC11348817

Identifier EP41330.pdf (filename),usctheses-c17-778030 (legacy record id)

Legacy Identifier EP41330.pdf

Dmrecord 778030

Document Type Thesis

Rights Moffitt, Robert A.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA