The free fatty acids and neutral lipids of the cuticular wax of the Mormon cricket, Anabrus simplex,
Hald
by Joel Mackie Padmore
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY in Chemistry
Montana State University
© Copyright by Joel Mackie Padmore (1964)
Abstract:
The composition of the free fatty acids and the neutral lipids of the Mormon cricket, Anabrus simplex.
Hald., was studied by infrared spectroscopy and gas, column, and thin-layer chromatography. Free
fatty acids were separated on the basis of unsaturation by column chromatography of their methyl
esters on silver nitrate impregnated silicic acid. The presence of normal and branched chain saturated
acids (C8-C36) and unsaturated acids (C14-C24) was demonstrated by gas chromatography on SE-30
silicone arid EGS polyester columns. Structures of the major unsaturated acids, oleic, linoleic; and
linolenic, were determined by infrared spectroscopy and reductive ozonolysis followed by gas
chromatography. Neutral lipids were separated into chemical classes by column chromatography on
various adsorbents. Wax esters, steryl. esters, triglycerides, free sterols, diglycerides, monoglycerides,
and traces of residual fatty acid were found. The fatty acid components were studied by
transesterification followed by gas chromatography. The free sterols and non-saponifiables derived
from the ester fractions were determined by gas chromatography on SE-30 and QF-1 silicone columns. THE FREE FATTY ACIDS AND NEUTRAL LIPIDS OF THE CUTICULAR WAX
OF THE MORMON CRICKET, ANABRUS SIMPLEX. HALD.
by
JOEL MACKIE PADMORE
A t h e s i s su b m itte d to t h e G raduate F a c u lty in p a r t i a l
f u l f i l l m e n t o f t h e re q u ire m e n ts f o r t h e degree
of
DOCTOR OF PHILOSOPHY
in
C hem istry
Approved:
Chairman, Examining Committee
Dean, G raduate D iv is io n
MONTANA STATE COLLEGE
Bozeman, Montana
J u n e , 1964
ACKNOWLEDGEMENT
I wish t o e s p e c i a l l y thank my re s e a r c h a d v i s o r e Dr. Graeme L. Baker,
f o r h i s guidance and encouragement th ro u g h o u t th e c o u rs e o f t h i s work.
I would l i k e t o e x p re ss my th an k s t o th e United S t a t e s Department o f
H e a lth , E d u c a tio n , and W elfare f o r a N a tio n a l Defense E d u catio n Act F e llo w ­
s h ip and t o t h e Montana S t a t e C o lle g e C hem istry Department and Chem istry
S ta tio n fo r fin a n c ia l a ssista n c e .
I wish t o th an k D r s . K. G oering, J . Pepper, R. O lsen , R. O’Connor
f o r t h e i r h e l p f u l s u g g e s tio n s and c r i t i c i s m s .
I a ls o wish t o thank
E lls w o rth H a stin g s and o th e r members o f t h e Department o f Zoology and
Entomology f o r t h e i r c o l l e c t i o n and p r e p a r a t i o n o f th e i n s e c t c u t i c l e s .
T h is t h e s i s i s d e d ic a te d t o my w ife , C a r o l, who has endured much
and g iv en h e r encouragement and moral s u p p o rt d u rin g t h e p a s t fo u r y e a r s .
TABLE OF CONTENTS
PAGE
LIST OF TABLES............................................ ..................... ................................................... .v
LIST OF FIGURES . . . . . . . . . . . . . . .
...................................................
ABSTRACT....................................................
I.
II.
INTRODUCTION...................... .
vi
v iii
. .
EXPERIMENTAL PROCEDURE AND RESULTS.
......................
.1
................................ 3
I s o l a t i o n o f Wax. . . . . . . . . . . . . .
.......................... . . . .3
I n f r a r e d S p e c t r a .......................................
6
Column C h ro m a to g ra p h y ...........................................
.6
T h in -L a y er Chromatography ............................... ........................................... . .7
S o l v e n t s ..........................................................; ........................................... ....
.9
Gas Chromatography. .............................................................
9
P r e p a r a tio n o f Methyl E s te r s o f Ka............................................................. 10
S e p a r a tio n o f P o la r Methyl E s t e r s .......................... . .......................... 10
S e p a ra tio n o f N oh-Polar Methyl E s t e r s .................................................... 14
Gas Chromatography o f S a tu r a te d Methyl E s t e r s , Kam-I.................. . 1 7
Q u a n t i t a t i v e A n a ly sis o f S a tu r a te d Methyl E s t e r s ....................................20
Gas Chromatography o f U n sa tu ra te d Methyl E s te r s ......................
23
S t r u c t u r e D e term in atio n o f t h e U n sa tu rate d A c id s. . ...................... 26
Q u a n t i t a t i v e A n a ly sis o f th e U n sa tu ra te d Methyl E s t e r s ................. 29
I s o l a t i o n o f N e u tra l L ip id s . ......................................................................... 30
T r a n s e s t e r i f i c a t i o n o f E s t e r F r a c t i o n s . . . . ................................... 37
Q u a n t i t a t i v e A n a ly sis o f Methyl E s t e r s o f N e u tra l L i p i d s . . . . 38
A n a ly sis o f S t e r o l s and A lc o h o ls ............................................................. . 38
III.
IV.
DISCUSSION: LIMITATIONS OF RESULTS . . . . . . . . . .
SUMMARY.........................
APPENDIX..........................
LITERATURE CITED
.................. 52
56
57
. . 60
LIST OF TABLES
TABLE
I.
II.
III.
IV.
PAGE
Composition o f Wax K.................................................................. .... ...................... „ 6
Composition o f Methyl E s t e r s , K a m ......................
C om position o f F r a c tio n Kam-I
...................
. „11
„ ..........................
Composition o f th e S a t u r a t e d Methyl E s t e r s , Kam-Is................... „ „ „ „24
V. Methyl E s t e r s o f th e Free D n s a tu r a te d A p id s . . . . . . . . . . .
V I.
V II.
V IIIi
IX.
Composition o f Kno by L ip id C la s s . . . . . . . . .
S a tu r a te d F a t t y Acid Methyl E s t e r s o f
.30
v ............................... .35
th e N e u tra l L i p i d s . ................... 39
D n sa tu ra te d F a t t y Acid Methyl E s t e r s o f th e N e u tra l L i p id s .
. . .
.39
S t e r o l com p o sitio n o f F r a c tio n s Kno-3n and Kno-Ia ................... . . .
.46
X. F a t t y A lco h o ls o f th e Wax E s t e r s ...................................... ......
XI.
„17
R e te n tio n Times o f th e Major Acid Methyl E s te r s on EGS. . . . . . .
.47
„58
LIST OF FIGURES
FIGURE
PAGE
1.
I n f r a r e d spectrum o f Wax Kn ( c a p . f i l m ) ..................................
5
2.
I n f r a r e d spectrum o f Wax Ka ( c a p . f i l m ) ........... ......................................................5
3.
I n f r a r e d spectrum o f methy e s t e r s Kam ( c a p . f i l m ) ..........................
.12
4.
I n f r a r e d spectrum o f methyl e s t e r s -Kam-I(c a p .film )
.12
5.
In frared
spectrum o f methyl e s t e r s Kam-2 (CCI4 s o l . ) ......................................1,3
6.
In frared
spectrum o f methyl e s t e r s Kam-3 (CCI4 s o l . ) ......................................13
7.
Gas chromatogram o f K a m -2 .............................................................................................15
8.
Gas chromatogram o f K a m -3 .............................................................................................16
9.
Gas chromatogram o f K a m - I .............................................................................................18
10.
C hromatographic s e p a r a tio n s o f f r e e f a t t y a c id methyl e s t e r s ....................19
11.
Is o th e rm a l gas chromatogram o f Kam-Is ............................................
12.
Programmed te m p e ra tu re gas chromatogram o f Kam-Is ..........................................22
13.
Gas chromatogram o f K a m - I u .......................................................................................... 25
14.
In frared
spectrum o f Kno-I (c a p .film )
. . . .
15.
In frared
spectrum o f Kno-2 (c a p .film )
. .
16.
In frared
spectrum o f Kno-4 (c a p .film )
............................................
17.
Infrared
spectrum o f Kno-3n (CSg s o l . ) . ......................................
18.
Chromatographic s e p a r a tio n s o f th e n e u t r a l l i p i d s .......................................... 36
19.
Gas chromatogram o f t h e methyl e s t e r s
o f K n o - I a ......................
40
20.
Gas chromatogram o f t h e methyl e s t e r s
o f Kno-Ib ......................
.41
21.
Gas chromatogram o f th e methyl e s t e r s
o f K n o -2 m ............................................. 42
22.
Gas chromatogram o f t h e methyl e s t e r s
o f K n o - 4 a ......................
21
............................................ .32
.................................
32
34
.34
.43
v ii
23.
Gas chromatogram o f t h e methyl e s t e r s o f Kno-4b . . . . . . . . . .
.44
24.
Gas chromatogram o f t h e methyl e s t e r s o f K n o - 4 c .............................................45
25.
Gas chromatogram o f t h e f r e e s t e r o l s , Kno-Sn..................................................... 48
26.
Gas chromatogram o f th e s t e r o l s o f K n o - I a ................................................ . .49
27.
Gas
28.
Gas chromatogram o f t h e n o n -s a p o n .ifia b le s o f K n o - I b ....................................51
chromatogram o f t h e a lc o h o ls o f K n o -Ia......................................................... 50
ABSTRACT
The c o m p o s itio n .o f t h e f r e e f a t t y a c id s and th e n e u t r a l l i p i d s o f th e
Mormon c r i c k e t , Anabrus s im p le x . H a ld ., was s tu d ie d by i n f r a r e d s p e c tro sc o p y
and g a s , column, and t h i n - l a y e r chrom atography. F ree f a t t y a c id s were s e p ­
a r a t e d on th e b a s is o f u n s a tu r a ti o n by column chromatography o f t h e i r methyl
e s t e r s on s i l v e r n i t r a t e im pregnated s i l i c i c a c i d . The p re s e n c e o f normal
and branched c h a in s a t u r a t e d a c id s (CgOgf,) and u n s a tu r a te d a c id s (C14-C24)
was d e m o n strated by gas chromatography on SE-30 s i l i c o n e arid EGS p o l y e s t e r
columns. S t r u c t u r e s o f t h e major u n s a tu r a te d a c i d s , o l e i c , l i n o l e i c ; and
l i n o l e n i c , were d eterm in ed by i n f r a r e d sp ec tro sc o p y and r e d u c t i v e o z o n o ly sis
follow ed by gas ch rom atography. N e u tra l l i p i d s were s e p a r a te d in to chem ical
c l a s s e s by column chromatography on v a rio u s a d s o r b e n t s . Wax e s t e r s , s t e r y l .
e s t e r s , t r i g l y c e r i d e s , f r e e s t e r d l s , d i g l y c e r i d d s , m o n o g ly cerid es, and
t r a c e s o f r e s i d u a l f a t t y a c id were found. The f a t t y a c id components were
s tu d ie d by t r a n s e s t e r i f i c a t i o n follow ed by gas chrom atography. The f r e e
s t e r o l s and n o n - s a p o n if ia b le s d e riv e d from t h e e s t e r f r a c t i o n s were d e t e r ­
mined by gas chromatography on SE-30 and QF-I s i l i c o n e columns.
INTRODUCTION
The c u t i c u l a r wax o f i n s e c t s has been found to be an im p o rtan t f a c t o r
in t h e c o n t r o l o f m o is tu re b a lan c e in i n s e c t s . ^«^0
A brasion o f t h i s la y e r
f r e q u e n t l y r e s u l t s in r a p i d d e s s i c a t ion and d eath o f th e i n s e c t .
Some
i n s e c t s , such as t h e c o c k ro a c h , a re r e s t r i c t e d to moist h a b ita t's whereas
o th e r i n s e c t s , such as t h e Mormon c r i c k e t , have th e a b i l i t y to s u rv iv e in
a r i d r e g io n s .
A stu d y o f th e n a tu re o f t h e c u t i c u l a r l i p i d s would le a d to
a g r e a t e r u n d e rs ta n d in g o f one o f t h e l i m i t i n g f a c t o r s in th e g e o g ra p h ica l
d is trib u tio n of in se c ts.
Beament has p o s t u l a t e d t h a t r e t a r d a t i o n o f w ater
p assage th rough th e c u t i c l e o f t h e cockroach is due t o th e p resen ce o f a
m ono-layer o f f a t t y a lc o h o ls a t th e lower i n t e r f a c e o f th e c u t i c u l a r wax.^
In a r e c e n t stu d y G ilby and Cox*® showed t h a t th e l i p i d s o f th e cockroach
in c lu d e d f a t t y a c i d s , a l i p h a t i c a ld e h y d e s , e s t e r s , and h y drocarbons, but
no f a t t y a l c o h o l s ; th e re b y p a r t i a l l y r e f u t i n g Beament1s p o s t u l a t e d mechanism
o f m o is tu re c o n t r o l .
T h ere have been few i n v e s t i g a t i o n s in t o th e chem ical com position o f
c u t i c u l a r waxes.
P a r t i a l a n aly se s have been c a r r i e d out on c u t i c u l a r waxes
from t i c k s a n d silkworm l a r v a e ^
P o s s ib ly th e most com plete study o f
an in s e c t c u t i c u l a r wax p r e s e n t in th e l i t e r a t u r e is t h a t o f Gilby and
C
o x .
18
The n a tu re o f th e h y d ro c a rb o n s, a ld e h y d e s , and f a t t y a c id s were
f a i r l y co m p le tely e l u c i d a t e d .
L i t t l e o r no work was done t o c h a r a c t e r i z e
th e e s t e r s o r t o co n firm t h e t r a c e q u a n t i t i e s o f s t e r o l s r e p o r te d .
The o r i g i n a l i n v e s t i g a t i o n i n to th e l i p i d com position o f th e c u t i c u l a r
wax o f th e Mormon c r i c k e t was i n i t i a t e d by J . H. Pepper and E . J . H astin g s
in th e
m
i d -
1940 ' s
. 2 9
The o b je c t o f t h e stu d y was t o le a r n more about t h e
- 2-
c o n t r o l o f w a ter b a la n c e by s tu d y in g t h e chem ical c o m p o sitio n o f t h e in s e c t
c u tic le .
The Mormon c r i c k e t was s e l e c t e d because o f i t s a v a i l a b i l i t y , la r g e
s i z e , and economic im portance in Montana.
At t h a t tim e i n s e c t waxes were
g e n e r a l l y b e lie v e d t o be r e l a t i v e l y sim ple m ixtures o f long c h a in a l i p h a t i c
a l c o h o l s , long c h a in a l i p h a t i c e s t e r s , and long ch ain a l i p h a t i c a c i d s .
The
e x t r a c t e d c u t i c u l a r wax was s u b je c te d t o s a p o n i f i c a t i o n , r e s u l t i n g in i n ­
t r a c t a b l e e m u lsio n s.
About 1957 t h e p r o j e c t was re v iv e d by Baker^ who u n d e r­
took a s p e c tro p h o to m e tr ic and chrom atographic study o f t h e wax.
This stu d y
showed t h e p re s e n c e o f e s t e r s , f a t t y a c i d s , hyd ro carb o n s, a c i d i c r e s i n s ,
and p o s s ib ly c h o l e s t e r o l .
The a c id s and hydrocarbons were p a r t i a l l y c h a r a c ­
t e r i z e d , b u t no work was done on th e e s t e r s and th e p re s e n c e o f c h o l e s t e r o l
was not co n firm ed .
The o b je c t o f th e p r e s e n t stu d y has been t o c h a r a c t e r i z e as c o m p letely
as p o s s i b l e th e e s t e r s , f r e e s t e r o l s , and f r e e f a t t y a c i d s .
T h is has
in c lu d e d th e d e te r m in a tio n o f double bond p o s i t i o n and c o n f ig u r a tio n o f t h e
m ajor u n s a tu r a te d a c id s and t h e i d e n t i f i c a t i o n and q u a n t i t a t i v e d e te rm in a tio n
o f t h e a c i d s , f a t t y a l c o h o l s , and s t e r o l s o f t h e v a rio u s e s t e r f r a c t i o n s .
Methods were developed a n d /o r m o d ified t o p erm it th e a n a l y s i s o f th e v a rio u s
f a t t y a c i d s , s t e r o l s , and e s t e r s p r e s e n t .
EXPERIMENTAL PROCEDURE AND RESULTS
I s o l a t i o n &£ Max
C r ic k e t s used in t h i s stu d y were c o l l e c t e d d u rin g th e summers o f 1961
and 1962 from v a rio u s l o c a t i o n s in s o u th e a s te r n Montana.
The l i v e c r i c k e t s
were q u ick fro z e n and s t o r e d in a f r e e z e r u n t i l e x c is io n o f t h e c u t i c l e s ,
No s e l e c t i o n was made as t o sex b u t only a d u lts p a s t th e f i f t h moult were
u sed .
The c u t i c l e s were e x c is e d under w a ter and b rushed l i g h t l y t o remove
a d h e rin g c e l l u l a r m a t e r i a l .
The e x c is e d c u t i c l e s were d r ie d and s to r e d
fro z e n u n t i l e x t r a c t e d . .
I n i t i a l s t u d i e s were perform ed on wax i s o l a t e d by s o x h le t e x t r a c t i o n .
The c u t i c l e s were h e ld in a cup o f chloroform -w ashed c h eese c l o t h and
e x t r a c t e d w ith ch lo ro fo rm f o r about tw enty h o u r s .
The s o lv e n t was removed
and th e cru d e wax r e c o v e re d .
F i n a l s t u d i e s were made on m a t e r i a l e x t r a c t e d from 1962 c u t i c l e s a t
room te m p e ra tu re .
C u t i c l e s and ch lo ro fo rm were mixed in a l a r g e s t a i n l e s s
s t e e l b le n d e r and homogenized f o r f i v e m inutes
The homogenate was allow ed
t o s ta n d f o r about t e n m inutes and was th e n f i l t e r e d t o remove t h e ground
c u tic le s.
The ch lo ro fo rm was reduced t o about t e n ml on a r o t a r y e v a p o r a to r
under reduced p r e s s u r e and then tak en t o d ry n ess under a stre am o f n i t r o g e n .
The wax i s o l a t e d by b o th methods had s i m i l a r c h a r a c t e r i s t i c s .
I t was
y e llo w -g re e n in c o l o r , had a c h a r a c t e r i s t i c od o r, and a s o f te n in g range of"
25-50 d e g r e e s .
The cru d e wax o b ta in e d by e i t h e r method was d e s ig n a te d wax K.
Wax K was ta k e n up in hexane and allow ed t o s ta n d f o r about one h o u r.
The hexane s o l u t i o n was f i l t e r e d t o remove t h e a c id i c r e s i n s and o th e r
-4-
hexane i n s o l u b l e s .
The f i l t e r e d hexane s o l u t i o n was imm ediately passed
th rough a K i e s ^ c o u n te r c u r r e n t a p p a ra tu s t o remove th e f r e e f a t t y a c id s .
The t h r e e s ta g e s c o n ta in e d 0.01N KOH, 0.005N KOH1 and w a ter r e s p e c t i v e l y .
T o t a l tim e f o r an average Kies e x t r a c t i o n was about 60 h o u rs .
Ethanol
was added dropwise t o th e f i r s t and second s ta g e s as heeded t o reduce
foaming.
The e x t r a c t e d hexane s o l u t i o n was ta k e n t o dryness and d e s ig n a te d wax
Kn.
The i n f r a r e d spectrum o f Kn i s shown in f i g u r e I .
The i n t e n s i t y o f t h e
bands a t 720 and 730 c m ~ \ t h e r a t i o o f t h e carbon-hydrogen deform ation
bands a t 1380 and 1470 cm~l, and t h e i n t e n s i t y o f t h e carbon-hydrogen
s t r e t c h i n g v i b r a t i o n n e a r 2900 cm~^ i n d i c a t e long hydrocarbon c h a in s .
The
e s t e r carb o n y l a t 174Q cm"I i s c h a r a c t e r i s t i c o f t h a t observed in t r i ­
g l y c e r i d e s and s t e r y l e s t e r s .
The carbon-oxygen s t r e t c h n e a r 1200 cnT^ is
a l s o s i m i l a r in p a t t e r n t o t h a t observed f o r t r i g l y c e r i d e s .
A small amount
o f f a t t y a c id is p ro b a b ly s t i l l p r e s e n t as i n d i c a t e d by t h e s h o u ld e r a t
1705 cm"I , th e c h a r a c t e r i s t i c carb o n y l s t r e t c h o f an a l i p h a t i c a c i d .
Weak
bands n e a r 3300 cm- * s u g g e st th e p o s s i b i l i t y o f hydroxy compounds such as
a l c o h o l s , s t e r o l s , m o n o g ly c e rid e s, and d i g l y c e r i d e s .
The combined aqueous s o l u t i o n s from t h e Kies a p p a ra tu s were a c i d i f i e d
t o pH 3 w ith 1 .0 N s u l f u r i c a c id and e s t r a c t e d t h r e e tim es w ith d ie th y l
e t h e r in 300, 300, and 200 ml p o r t i o n s .
The combines e t h e r e x t r a c t s were
washed w ith 25 ml o f w a ter and d r ie d o v er anhydrous sodium s u l f a t e .
The
e t h e r was removed by e v a p o r a tio n on a r o t a r y e v a p o r a to r under reduced p r e s ­
s u re t o about 10 ml, th en ta k en t o d ry n ess under a stream o f d ry n itr o g e n .
The re c o v e re d a c i d i c m a t e r i a l s were d e s ig n a te d was Ka.
The i n f r a r e d
-5-
W A V ELEN G TH
IN
M IC R O N S
8
W A V EN U M B ER
F ig u re I .
KA Y SER S
I n f r a r e d spectrum o f Wax Kn (cap. f i l m . )
W A V ELEN G TH
F ig u re 2.
IN
12
IN
M IC R O N S
I n f r a r e d spectrum o f Wax Ka (cap. f i l m .)
13
14 15 16
,
- 6-
sp ec tru m , f i g u r e 2 , i s t y p i c a l o f s p e c t r a o b ta in e d from long chairi f a t t y
a c id s.
The weak baijd j u s t below 3000 cm- * on th e s id e o f th e carb o n -
hydrogen s t r e t c h i n g v i b r a t i o n i n d i c a t e s th e p resen ce o f unS a t u r a t io n .
T a b le I shows th e r e l a t i v e amounts o f hexane i n s o l u b l e s , n e u t r a l s , and a c id s
o b ta in e d .
,
TABLE I
Composition o f Wax K
F r a c tio n
Weight P e rc e n t
hexane in s o lu b le s
Wax Ka
Wax Kn
1 .3
* 3 1 .3
6 7 .4
I n f r a r e d S p e c tra
A ll i n f r a r e d s p e c t r a were o b ta in e d on a Beckman IR-4 S p e ctro p h o to m eter
equipped w ith sodium c h l o r i d e o p t i c s w ith p r e s e n t a t i o n l i n e a r in?wavenumbers .
S p e c tra were ta k e n as c a p i l l a r y film s whenever p o s s i b l e .
S p e c tra
o f s o l i d s were o b ta in e d in both carbon d i s u l f i d e and carbon t e t r a c h l o r i d e .
Column Chromatography
A ll chromatography colum ns, r e g a r d l e s s o f a d so rb en t u s e d , were p re p a re d
in an i d e n t i c a l m anner.
The weighed a d so rb e n t was s l u r r i e d w ith hexane and
p la c e d in t o a 1 .2 cm.column.
The column was l i g h t l y ta p p e d t o in s u re u n i ­
form packing o f t h e a d s o rb e n t; th e a d so rb e n t was covered w ith a f i l t e r p a p er
-7-
d i s k ; and th e p re p a re d column washed th o ro u g h ly w ith hexane.
The sample
was in tro d u c e d onto t h e column in a minimal amount o f hexane and washed i n to
th e column w ith 1-2 m„ o f hexane.
Care was tak en a t a l l tim es t o in s u re
t h a t th e column was co v ered w ith s o l v e n t .
In o rd e r to use a b a tc h e l u t i o n te c h n iq u e o f column e l u t i o n , each
column was s ta n d a r d iz e d f o r r e t e n t i o n volumes o f l i p i d c l a s s e s .
The l i p i d
m ix tu res chosen f o r t h e s t a n d a r d i z a t i o n were s e l e c t e d t o r e f l e c t as c l o s e l y
as p o s s i b l e th e l i p i d s to be en co u n tered in th e c u t i c u l a r wax as determ ined
by t h i n - l a y e r chrom atography.
S u f f i c i e n t columns were p re p a re d f o r each
s e p a r a t i o n to f ir m ly e s t a b l i s h r e t e n t i o n volumes.
T h in -L a y er Chromatography
E x te n s iv e use was made o f t h i n - l a y e r chromatography th ro u g h o u t th e
c o u rse o f th e i n v e s t i g a t i o n .
t h a t d e s c r ib e d by Mangold.
97
P l a t e s were p re p a red in a manner s i m i l a r t o
The a d so rb e n t was s l u r r i e d w ith w ater in a
m o rtar f o r one m in u te, pouring th e r e s u l t a n t s l u r r y i n t o a Desaga s p r e a d e r ,
and r a p i d l y sp re a d in g i t over t h e p l a t e s .
The p l a t e s were a g i t a t e d f o r a
few seconds w hile s t i l l wet to produce a more uniform l a y e r .
A f te r a i r
d ry in g t h e p l a t e s were a c t i v a t e d in an oven a t IlO0C f o r one h o u r.
These
a c t i v a t e d p l a t e s were s t o r e d in a d e s s i c a t o r u n t i l u sed.
General l i p i d s e p a r a tio n s were perform ed on s i l i c a g e l p l a t e s con­
t a i n i n g 0.04% Bhodamine 6 G i n d i c a t o r .
The Rhodamine c o n ta i n in g p l a t e s
were p re p a re d by adding th e f lo u r e s c e n t dye t o th e w ater used t o p re p a re
th e s lu rry .
The p l a t e s were developed w ith wicks in a 75/25/1 (v /v /v )
h e x a n e - d ie th y l e t h e r - a c e t i c a c id s o lv e n t sy ste rm . This s o lv e n t system gave a
- 8-
s e p a r a t i o n in which s t e r y l e s t e r s „ t r i g l y c e r i d e s , s t e r o l s , d i g l y c e r i d e s , and
m onoglycerides were i d e n t i f i a b l e when c o n ta in e d in th e same m ix tu re .
De­
t e c t i o n was made by viewing th e developed p l a t e s under u l t r a v i o l e t l i g h t
b o th b e f o r e and a f t e r exposure to io d in e vapor followed by s p ra y in g with
50% s u l f u r i c a c id and c h a r r in g in a d ry in g oven.
D e te c tio n l e v e l s f o r most
l i p i d s u sin g t h i s te c h n iq u e were around 0 .1 microgram .^
S t e r y l e s t e r s and wax e s t e r s a re not s e p a ra b le on s i l i c a g e l by th e
u s u a l so lv en ts, b u t may be s e p a r a te d w ith o u t wicks using t r i c h l o r o e t h y l e n e
as a s o l v e n t . 5
e ste rs.
In t h i s s o lv e n t system s t e r y l e s t e r s m ig ra te ahead o f wax
I t was d is c o v e re d t h a t a b e t t e r s e p a r a tio n c o u ld be a f f e c t e d by
chrom atographing s t e r y l and wax e s t e r s on A n a sil p l a t e s
Conn.) developed w ith wicks in a 9 0 /1 0 /1
a c e t i c a c id s o lv e n t system .
ste ry l e s te rs .
(Analabs, Hamden,
(v /v /v ) h e x a n e -d ie th y l e t h e r
In t h i s system wax e s t e r s m ig ra te ahead o f
D e te c tio n was made by sp ra y in g th e A n a sil p l a t e s with
d i c h l o r o f l u o r e s c e i n follow ed by io d in e vapor and s u l f u r i c a c id as used
w ith s i l i c a g e l p l a t e s .
M ixtures o f a lc o h o ls and s t e r o l s were s e p a r a te d by chromatography
on s i l i c a g e l p l a t e s developed w ith o u t wicks in th e s o lv e n t system 90/10
(v/v) b e n z e n e -e th y l a c e t a t e .
p r e v io u s ly d e s c r ib e d .
D e te c tio n methods were s i m i l a r to those
The d e t e c t i o n l e v e l o f a l i p h a t i c a lc o h o ls was
ap p ro x im a te ly I microgram and was in th e neighborhood o f 0.01 microgram
fo r d e lta -5 s te r o ls .
During s u l f u r i c a c id c h a r , s t e r o l s , and s t e r y l e s t e r s
dev elo p an in te n s e p u rp le c o l o r .
S t r a i g h t c h a in a lc o h o ls develop a f a i n t
y e llo w c o l o r and l a n b s t e r o l a re d d is h c o l o r .
-9S o lv e n ts
A ll s o l v e n t s , w hether t e c h n i c a l o r re a g e n t g ra d e , were found to
c o n ta in s e v e r a l m illig ra m s o f im p u r itie s p e r I i t e r ^ th u s n e c e s s i t a t i n g
p u rif ic a tio n p rio r to use.
Hexane (High P u r i t y Normal Hexane, P h i l l i p s
P etroleum C o . ) , r e a g e n t g ra d e m ethanol, and re a g e n t g rad e ch lo ro fo rm were
found t o be s u f f i c i e n t l y p u r i f i e d by a sim ple d i s t i l l a t i o n .
Benzene was
p u r i f i e d by s t i r r i n g w ith c o n c e n tr a te d s u l f u r i c a c id (5/1 v /v ) f o r a t l e a s t
8 hours p r i o r t o d i s t i l l a t i o n .
D ie th y l e t h e r was p u r i f i e d by d i s t i l l a t i o n
from f r e s h l y c u t sodium m e t a l .
P u r i t y o f s o lv e n ts was checked by concen­
t r a t i n g 200 ml t o l e s s th a n I nil and chrom atographing 50 micro I i t e r s o f t h e
r e s i d u e on s i l i c a g e l t h i n - l a y e r p l a t e s .
Gas Chromatography
P r e lim in a r y gas chromatograms wejre o b ta in e d using a Wilkens A90-G
Aerograph equipped with; a fo u r f ila m e n t th erm al c o n d u c t i v i t y d e t e c t o r .
F i n a l gas chromatograms were o b ta in e d u s in g an FGM Model 400 Biomedical
Gas Chromatograph equipped w ith a flame i o n i z a t i o n d e t e c t o r .
used as a c a r r i e r gas a t a s ta n d a rd flow o f 65 ml p e r m in u te .
c o n s tr u c te d o f 6 mm O.D
pyrex g l a s s t u b i n g .
Helium was
Columns were
I n j e c t i o n was made d i r e c t l y
on column t o circum vent t h e p o s s i b l e decom position o f sample in a ho t m etal
i n j e c t i o n b lo c k .
Columns c o n ta in in g l e s s th a n 3.0% l i q u i d phase were packed
on s i l a n i z e d 60 /8 0 mesh Chromasorb W (John M an sv ille I n c . ) and packed in t o
s ila n iz e d g la ss u -tu b e s.
H e x a m e th y ld isila z a n e was i n j e c t e d in t o th e column
! ■
p r i o r t o i n i t i a l use in o rd e r t o reduce s i t e s which cause peak t a i l i n g and .
r
'
i r r e v e r s i b l e a d s o r p tio n o f sample. The 3.8% SE-30 column used in t h i s stu d y
-10-
was packed on 80/100 mesh D ia to p o rt S (FOl S c i e n t i f i c C o r p . ) .
A ll o th e r
columns were packed on 6 0 /8 0 mesh Gas Chrom P (Applied S c ie n ce L a b s . ) .
P r e p a r a t i o n o f Methyl E s t e r s o f Ka
In o rd e r t o f a c i l i t a t e t h e i r h a n d lin g in subsequent column and gas
chromatograms th e f r e e f a t t y a c id s were c o n v e rte d to t h e i r methyl e s t e r
d e riv a tiv e s.
, I :
The f a t t y a c id s were r e f lu x e d w ith boron t r i c h l o r i d e - m e t h a n o l
o r boron t r i f l o u r i d e -m ethanol re a g e n t (2 ml p e r 100 mg o f a cid ) f o r f i v e
m in u te s .
I d e n t i c a l r e s u l t s were ach ie v e d w ith e i t h e r r e a g e n t .
A f te r r e f l u x
t h e r e a c t i o n m ix tu re was poured i n to 25 ml o f w a ter and e x t r a c t e d t h r e e tim es
w ith 25 ml p o r ti o n s o f hexane.
The hexane e x t r a c t s were combines, washed
w ith 25 ml o f w a te r , th e n d r i e d o v er anhydrous sodium s u l f a t e . The hexane
•i. ; .
s o l u t i o n was e v a p o ra te d t o y i e l d t h e f a t t y a c id methyl e s t e r s , wax Kam.
The i n f r a r e d spectrum o f t h e methyl e s t e r s is shown in f i g u r e 3.
Hydroxy methyl e s t e r s a r e in d ic a te d by t h e hydroxyl bands a t 3350 and
3425 c m -J .
B n s a tu r a tio n i s i n d i c a t e d by t h e sm all band a t 2980
T h i n - l a y e r chromatography on s i l i c a g e l p l a t e s a l s o i n d i c a t e d th e p re se n c e
o f hydroxy diethyl e s t e r s as w e ll a s more p o l a r e s t e r s .
The n o n -p o la r
e s t e r s app eared t o be u n s a tu r a te d as i n d i c a t e d by th e q u a n t i t y o f io d in e
vapor ad so rb ed on t h e above t h i n l a y e r p l a t e . 33
Sepa r a t i o n o f P o la r Methyl E s te r s
The methyl e s t e r s o f t h e f r e e f a t t y a c id s were chromatographed on a
20 g column o f F l o r i s i l (Research S p e c i a l t i e s , Richmond, C a l i f . ) in o rd e r
t o s e p a r a t e t h e n o n -p o la r methyl e s t e r s from t h e p o l a r methyl e s t e r s .
The
- 11-
non- p o l a r methyl e s t e r s were e l u t e d from t h e column w ith 100 ml o f 10%
d i e t h y l e t h e r in hexane.
The hydroxy a c id methyl e s t e r s a n d /o r methyl
e s t e r s o f s i m i l a r p o l a r i t y were e l u t e d from t h e column w ith 100 ml o f 5G%
d i e t h y l e t h e r in hexane.
The more p o l a r methyl e s t e r s were e l u t e d with
TOO ml o f 10% methanol in d i e t h y l e t h e r .
A y e llo w pigment and o th e r m ate­
r i a l s were not e l u t e d from th e column and no t i n v e s t i g a t e d .
The th r e e
e l u a n t s from t h e f l o r i s i l column were d e s ig n a te d Kara-I5 Kara-2, and Kam-3,
re sp e c tiv e ly .
The i n f r a r e d s p e c t r a o f t h e t h r e e methyl e s t e r f r a c t i o n s
i s shown in f i g u r e s 4 t o 6 ,
T a b le I I shows t h e r e l a t i v e amounts o f th e
in d iv id u a l methyl e s t e r f r a c t i o n d e riv e d from a c id s o b ta in e d from both
1961 and 1962 c u t i c l e s .
TABLE I l
C om position o f Methyl E s t e r s , Kara
Year
1961
1962
% Kam-I
8 3 .9
9 0 .8
% Kam-2
% Kam-3
% not e lu te d
3 .9
5 .0
0 .6
1 .3
11.7
2 .9
The f r a c t i o n s from th e F l o r i s i l Column were s u b je c te d t o t h i n - l a y e r
chromatography on s i l i c a g e l p l a t e s developed in a 75/25/1 (v /v /v ) hexane
d i e t h y l e t h e r - a c e t i c a c id s o lv e n t sy stem .
F r a c tio n Kara-1 showed only a
s i n g l e sp o t c h a r a c t e r i s t i c o f n o n -p o la r methyl e s t e r s such as methyl
{■stearate o r l i n o l e n a t e .
F r a c tio n Kam-2 showed a spot which m ig rate d l i k e
methyl I 2 - h y d r o x y s t e a r a t e „ p lu s o t h e r s p o ts o f a more p o l a r n a t u r e .
Kam-3
12
W A V ELEN G TH
F ig u re 3.
IN
M IC R O N S
I n f r a r e d spectrum o f Methyl e s t e r s Kam (cap. film)
W A V ELEN G TH
IN
M IC R O N S
12
W A V EN U M B ER
F ig u re 4 .
IN
13
14 15 16
KA Y SER S
I n f r a r e d s p e c t r u m o f M e th y l e s t e r s Kam-I
(cap . film )
-13-
W A V ELEN G TH
)
1600
1500
W A V EN U M B ER
F ig u re 5 .
F ig u re 6 .
IN
M IC R O N S
1400
1300
IN KAYSEI
I n f r a r e d spectrum o f methyl e s t e r s Kam-2 (CCI4 s o l . )
W A V ELEN G TH
IN
M IC R O N S
W A V EN U M B ER
IN
KA Y SER S
I n f r a r e d s p e c t r u m o f m e t h y l e s t e r s Kam-3 (CCI 4 s o l . )
-14-
s bowed l i t t l e m a te r ia l which m ig rate d from th e o r i g i n i n d i c a t i n g m a te r ia ls
o f a high degree o f p o l a r i t y .
F r a c tio n Kam-I was a ls o chromatographed on
s i l i c a g e l p l a t e s c o n ta i n in g 5% s i l v e r n i t r a t e . ^8
The p l a t e s were developed
in a 50/50 (v/v) h e x a n e -d ie th y l e t h e r s o lv e n t system .
t e c t e d by s u l f u r i c a c id c h a r .
The s p o ts were de­
S a t u r a t e d , monoenoic, d i e n p i c , and t r i e n o i c
methyl e s t e r s were i n d i c a t e d .
F r a c tio n s Kam-2 and Kam-3 were an aly zed by gas chromatography on a
4 f o o t 3.8% SE-30 s i l i c o n e column a t 195°C.
The major peaks in th e chrom ato-
gram o f f r a c t i o n Kam-2 appeared t o be s a t u r a t e d and an u n s a tu r a te d
h y d ro x y o c ta d e c a n o a te .
B romination o f th e sample follow ed by r e i n j e c t i o n
in t o t h e chromatograph b rought about th e d isap p e a ra n ce o f t h e peak th o u g h t
t o be th e u n s a tu r a te d hydroxy methyl e s t e r .
The assignm ents o f c h ain le n g th
f o r th e two methyl e s t e r s were based oh th e o b s e r v a tio n t h a t th e l a t e r major
peak in t h e chromatogram had a r e t e n t i o n time i d e n t i c a l to t h a t o f methyl
12-hydroxy s t e a r a t e .
^
The peak immediately p reced in g i t in th e chromatogram,
,',f ■; u'! '' I".
‘
which d is a p p e a rs upon b ro m in a tio n , was a l s o a ss ig n e d th e 18 carbon s t r u c t u r e
as u n s a ttira te d e s t e r s a re e l u t e d from SE-30 columns b e fo re t h e i r s a t u r a t e d
a n a l o g s . v -No compounds were i d e n t i f i e d in f r a c t i o n Kam-3.
R e p r e s e n ta tiv e
chromatograms o f th e s e two f r a c t i o n s d e r iv e d from 1961 a c id s a re shown in
f i g u r e s 7 rand 8 .
C orresponding f r a c t i o n s from 1962 a c id s e x h ib ite d minor
q u a n tita tiv e d iffe re n c e s .
S e p a r a t ion o f N on-Polar Methyl E s te r s
F r a c tio n Kam-I was chromatographed on a 4 fo o t 15% e th y le n e g ly c o l
s u c c in a te p o l y e s t e r (EGS) column a t ITO0C .
The chromatogram f o r th e 1962
15
F ig u re 7.
Gas c h ro m a to g ra m o f Kam-2
F ig u re 8 . Gas chromatogram o f Kam-3
-17r
methyl e s t e r s i s shown in f i g u r e 9.
Due to th e com p lex ity o f th e chrom ate-
\
gram o b ta in e d from th e t o t a l n o n -p o la r e s t e r f r a c t i o n , i t was decided t o
s e p a r a t e f r a c t i o n Kam-I in t o u n s a tu r a te d and s a t u r a t e d methyl e s t e r f r a c ­
tion's .
The methyl e s t e r s were a c c o rd in g ly chromatographed on a 25% s i l v e r
n i t r a t e - A d s o r b o s i l column (Applied S cien ce L ab s, S t a t e C o lle g e , P a . ) . 14
S a t u r a t e d methyl e s t e r s were e l u t e d from th e column w ith 40 ml o f 18%
benzene in hexane.
U n sa tu ra te d methyl e s t e r s were i s o l a t e d as a group by
e l u t i o n w ith 150 ml o f 100% d i e t h y l e t h e r .
d e s ig n a te d Kam-Is and Kam-Iu.
The r e s u l t a n t f r a c t i o n s were
The r e l a t i v e q u a n t i t i e s o f s a t u r a t e d and
u n s a tu r a te d f a t t y a c id s from 1961 and 1962 a re shown in T a b le I I I .
A flow
diagram f o r t h e s e p a r a t i o n o f methyl e s t e r s is shown in f i g u r e 10.
TABLE I I I
C om position o f F r a c tio n Kam-iI
Year
% Kara-Is
% Kam-Iu
1961
1962
3 6.2
2 4 .0
6 3 .8
7 6 .0
Gas Chromatography p f S a tu r a te d Methyl E s t e r s » Kam-I
In o r d e r t o d eterm in e th e f a t t y a c id methyl e s t e r s p r e s e n t in Kam-Is,
th e y were chromatographed on EGS a t ITO0C .
The r e s u l t a n t chromatograms
r
in d i c a t e d th e p re se n c e o f s a t u r a t e d e s t e r s from m y r i s ta te t o a r i c h i d a t e .
No
t r a c e s o f u n s a tu r a te d methyl e s t e r s c o u ld be d e te c te d by t h e d isap p e a ra n ce o r
-16-
—t
r “ T*'
F ig u re 9.
Gas c h ro m a to g ra m o f Kam-I
Ka m
FLOR ISlL
T
Kam-I
K am-2
K am-3
25% AgNO3 SILICIC ACID
I
h—«
01
T
Kam-Is
Kam-Iu
25% Ag NO3 SILICIC ACID
Karn-Iul
F i g u r e 10 .
K am-lu2
K nm-lu3
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 s o f f r e e f a t t y a c i d m e th y l e s t e r s
- 20 -
s h i f t o f a peak when chromatographed subsequent t o b ro m in atio n o f th e sam ple.
A r e p r e s e n t a t i v e chromatogram o f 1962 methyl e s t e r s on EGS is shown in
f i g u r e 11.
F r a c tio n Kam-Is was a ls o chromatographed on SE-30.
A programmed
te m p e ra tu re gas chromatogram from 100 deg rees to above 300 degrees shows th e
p re se n c e o f s a t u r a t e d normal and branched c h a in methyl e s t e r s from l a u r a t e
t o t r i c o n t a n a t e and above.
A p o r ti o n o f t h e chromatogram from 1962 methyl
e s t e r s is shown in f ig u r e 12.
Is o th e rm a l chromatography on SE-30 a t 80°C
showed th e p re sen c e o f t r a c e amounts o f a c id s from Cg t o
.
Programming
on SE-30 from 200° i n d i c a t e d th e p re sen c e o f a c id e s t e r s up t o C36 and
beyond.
Q u a n t i t a t i v e A n a ly sis o f th e S a tu r a te d Methyl E s te r s
F r a c tio n Kam-Is was q u a n t i t a t i v e l y analy zed by a com bination o f gas
ch rom atographic t e c h n i q u e s .
The normal a c id s from Cg to C24 were analyzed
by gas chromatography on EGS a t 120» 170, and 195°C.
between chromatograms by means o f common p e a k s .
C o r r e l a t i o n was made
Methyl l a u r a t e (C^g) was
used t o r e l a t e th e chromatograms p re p a re d a t 120 and ITO0C ; methyl a r a c h i d a t e (Cgg) was used to r e l a t e th e chromatograms p re p a red a t 170 to I 950C,
P e rc e n tag e com p o sitio n was determ ined through th e use o f a Disk i n t e g r a t o r
a ttach m en t on a I mv Brown r e c o r d e r .
l i c a t e and th e v alu es av erag ed .
Chromatograms were p re p a re d in t r i p ^
Data on branched c h a in methyl e s t e r s and
normal e s t e r s o f carbon numbers h ig h e r th a n C24 were o b ta in e d from th e p ro ­
grammed te m p e ra tu re runs on SE-30.
shown in f i g u r e 11.
A p o r ti o n o f a t y p i c a l chromatogram i s
Peak h e ig h ts were ta k e n t o be p r o p o r tio n a l to th e amount
o f component p r e s e n t in a sample as peak w idths remained n e a r ly c o n s ta n t
21
F ig u re 11.
Iso th e rm a l gas chromatogram o f Kam-Is
Kam-Is
F i g u r e 12 .
Programmed t e m p e r a t u r e g a s c h ro m a to g ra m o f Kam-Is
—2 3 —
th ro u g h o u t t h e r u n s .
The peaks o f methyl m yris t a t e CC14) and methyl
a r a c h i d a t e (Cgg) were used t o c o r r e l a t e d a ta from th e programmed te m p e ra tu re
chromatograms on SE-30 w ith th e is o th e rm a l chromatograms on EGS.
Normal
s h o r t c h a in methyl e s t e r s from Cg to C ^ were p re s e n t in t r a c e q u a n t i t i e s
f o r both 1961 and 1962 a c i d s .
were not d e t e c t e d .
C orresponding branched c h a in methyl e s t e r s
Both normal and branched c h ain a c id s from Cgg up t o a t
l e a s t C35 were a ls o p r e s e n t in t r a c e q u a n t i t i e s .
The co m p o sitio n o f th e
normal and b ranched c h a in s a t u r a t e d methyl e s t e r s from Cjg up t o Cgg is
shown in T a b le IV.
Gas Chromatography o f (J n sa tu ra te d Methyl E s te r s
To determ in e th e u n s a tu r a te d a c id s p r e s e n t methyl e s t e r f r a c t i o n s
Kam-Iu from 1961 and 1962 were chromatographed on SE-30 a t I 950C .
u r a t e d a c id s from C jg t o Cg4 were found.
O n sat-
As SE-30 s i l i c o n e does not
r e s o lv e t h e v a rio u s u n s a tu r a te d a c id s o f th e same carbon number b u t con­
t a i n i n g d i f f e r e n t numbers o f double b o n d s„ f r a c t i o n Kam-Iu was chromato­
graphed on EGS a t both 170 and 195°C.
a c id s i s shown in f i g u r e 13.
A chromatogram o f Kam-Iu from 1962
The mnemonic sh o rth an d o f S t o f f e l 1^S and
F a rq u h a rlS w i l l be used t o d e s c r ib e u n s a t u r a t e d f a t t y a c id methyl e s t e r s .
In t h i s co n v en tio n two numbers s e p a r a te d by a colon a re used; th e f i r s t
number i s t h e number o f carbon atoms in t h e molecule and t h e second is th e
number o f double bonds in th e m o le c u le .
The major, components as determ ined
by peak s i z e s on SE-30 appeared t o be C jg a c i d s .
The p o s i t i o n s o f th e peaks
on EGS i n d i c a t e d th e s e methyl e s t e r s t o be 1 8:1, 1 8 :2 , and 1 8 :3 .
The
r e t e n t i o n tim es o f t h e t ^ r e e methyl e s t e r s were i d e n t i c a l t o th o s e o b ta in e d
-2 4 -
TABLE IV
Composition o f S a tu r a te d Methyl E s t e r s , Kam-I
Carbon *
% 1961
% 1962
12
13br
13
14b r
14
15br
15br
15
I6 b r
16br
16
ITbr
ITbr
IT
18br
18br
18
19br
19br
19
20b r
20b r
20
21br
21b r
21
22b r
22b r
22
0 .2
T
T
T
4 .4
0.1
0 .3
I.T
0.1
0 .2
5 3 .0
0 .5
0 .7
2 .8
0 .5
1 .0
31.1
0 .3
0 .2
0 .6
T
0 .2
0 .9
T
T
0.1
T
T
0 .2
0 .3
T
T
T
4 .4
0 .2
0 .4
1.4
0.1
0 .1
5 1 .0
0 .4
O.T
3 .2
0.1
1 .4
3 1.4
0 .2
0 .3
O.T
T
T
1 .9
T
T
0.1
T
T
0 .3
T denotes l e s s than 0,1% o f f r a c t i o n
b r d en o tes branched s t r u c t u r e s
-25-
F ig u re 13.
Gas chromatogram o f Kam-Iu
-26-
f o r o l e a t e , l i n o l e a t e and l i n o l e n a t e methyl e s t e r s .
O v erlap o f carbon numbers occur when chrom atographing u n s a tu r a te d
a c id s on EGS0 th u s making peak assignm ents d i f f i c u l t .
To make unequivocal
assig n m en ts a p o r ti o n o f Kam-Iu from 1961 was chromatographed on 25%
s i l v e r n i t r a t e - s i l i c i c a c id column.
Monoenoic a c id s were e l u t e d with
100 ml o f 3% d i e t h y l e t h e r in h e x an e„ d ie n o ic acid s w ith 100 ml 15%
d i e t h y l e t h e r in h e x an e, and t r i e n o i c a c id s w ith 200 ml 10(% d i e t h y l e t h e r .
The f r a c t i o n s so o b ta in e d d e s ig n a te d K am -Iul0 Kam-I u 2 0 and Kam-IuS.
Gas
chromatography on th e in d iv id u a l f r a c t i o n s on EGS showed t h a t monoenoic
e s t e r s p r e s e n t ranged from C14 t o C24, d ie n o ic e s t e r s from C14 t o C22, and
t r i e n o i c a c id s from
t o Cg#.
The major methyl e s t e r in each case was th e
C10 and accounted f o r a t l e a s t 85% o f th e weight o f t h e in d iv id u a l f r a c t i o n s .
Gas chromatography o f th e u n s a tu r a te d e s t e r fr a c tio n s , i n d i c a t e d t h a t th e
s e p a r a t i o n s were about 95% co m plete.
S t r u c t u r e D e term in atio n o f th e U n sa tu ra te d Acids
O zo n o ly sis was used t o determ ine th e p o s i t i o n s o f th e double bonds in
t h e u n s a tu r a te d a c i d s .
O zonolysis has been e x te n s iv e ly re v ie w as a
te c h n iq u e f o r th e d e te r m in a tio n o f u n s a t u r a t i o n in f a t t y a c id methyl
e ste rs .4
Both o x i d a t i v e * *^
and r e d u c t i v e
^
te c h n iq u e s have been used
t o c le a v e t h e ozonides formed from u n s a tu r a te d f a t t y a c id methyl e s t e r s .
The r e d u c t i v e method a f f o r d e d th e b e s t r e s u l t s when working w ith m illig ra m
q u a n t i t i e s o f u n s a tu r a te d e s t e r s because th e aldehydes and aldehyde e s t e r s
produced may be d i r e c t l y a n aly zed by gas chrom atography.
o x id a tio n p ro d u c ts a re c o m p le tely ^ b s e n t . ^
In a d d itio n o v e r­
-2?-
The method used in t h i s s tu d y was based on th e method o f S t e i n and
N i c o l a i d e s .24
The f a t t y a c id methyl e s t e r 1-2- mg, was d is s o lv e d in e i t h e r
I ml o f d ich lorom ethane a t -70 o r I ml o f methyl c a p r a te a t -20°C.
Ozone
was bubbled i n to t h e s o l u t i o n through a c a p i l l a r y u n t i l t h e s o lu tio n
tu r n e d b l u e , i n d i c a t i n g i t was s a t u r a t e d w ith ozone.
The ex cess ozone was
removed by b u b b lin g dry n itr o g e n th rough t h e r e a c t i o n m ix tu re f o r approx­
im a te ly I m in u te.
A 2 -3 molar ex cess o f t r i p h e n y l phosphine was added
d i r e c t l y to t h e r e a c t i o n m ix tu re t o e f f e c t th e r e d u c tio n o f t h e ozonides
formed.
A f t e r about 30 seconds th e r e a c t i o n m ix tu re was removed from th e
c o ld b a th , allow ed t o warm t o room te m p e r a tu r e , and a sample i n j e c t e d i n to
th e gas chrom atograph.
The c h o ic e o f s o lv e n t used was d i c t a t e d by t h e n a tu r e o f t h e f a t t y
a c id t o be o z o n ize d .
Aldehyde p ro d u c ts w ith a r e t e n t i o n tim e s h o r t e r th a n
t h a t o f cap ro ald eh y d e a r e b u r ie d in t h e s o lv e n t peak o f 'd ich lo ro m eth an e and
o t h e r v o l a t i l e , ozone i n e r t s o l v e n t s .
Thus p o ly u n s a tu r a te d f a t t y acid s
o f te n r e q u i r e o z o n o ly sis in both s o l v e n t s .
The y i e l d o f aldehydes o b ta in e d
by o z o n o ly sis in methyl c a p r a t e i s c o n s id e r a b ly lower th a n t h a t o b ta in e d
in dich lo ro m eth an e due to th e h ig h e r r e a c t i o n te m p e ratu res and th e d e crea se d
s o l u b i l i t y o f th e t r i p h e n y l ph o sp h in e.
Each o f th e t h r e e u n s a tu r a te d e s t e r f r a c t i o n s gave t h e same aldehyde
e s t e r f r a c t i o n upon o z o n o ly sis in dich lo ro m eth an e as d eterm in ed by gas
chromatography on EGS a t I 70°C.
As t h i s fragment was t h e same as t h a t
o b ta in e d from known samples o f o l e a t e , l i n o l e a t e , and l i n o l e n a t e , each o f
t h e unknown C^g e s t e r s was a ss ig n e d a 9 ,1 0 double bond.
Thus th e p o s i t i o n
o f t h e double bond o f t h e 18:1 methyl e s t g r was th e same as methyl o l e a t e .
“28-
Chromatography o f th e r e a c t i o n m ix tu res on EGS a t 120°C showed the presence
o f p elarg o n ald e h y d e in th e r e a c t i o n m ix tu re o f Kam-Iul9 tlius co nfirm ing t h e
s t r u c t u r e as a 9 ,1 0 double bond.
No o t h e r s h o r t c h a in aldehyde was p r e s e n t
in th e m ix tu re in d e t e c t a b l e q u a n t i t y .
The only a l i p h a t i c aldehyde ob­
t a i n e d from th e r e a c t i o n m ix tu re o f Kam-lu2 on EGS a t 120°C was c a p ro a ld eh y d e , th u s co n firm in g i t s s t r u c t u r e t o be a 9, 1 2 -o c ta d ie n o ic methyl
e ste r.
No s h o r t c h a in e s t e r fragm ents were d is c e r n a b le f o r Kam-IuS by
chromatography on EGS.
For t h i s reaso n th e t r i e n o i c a c id f r a c t i o n was a l s o
ozonized in methyl c a p r a t e and th e r e a c t i o n m ix tu re an aly z e d by gas chrom­
ato g ra p h y on a 20% XF-1150 s i l i c o n e a t 25°C.
The only aldehyde found was
p ro p io n a ld e h y d e , th u s c o n firm in g th e s t r u c t u r e as 9, 12, 1 5 - o c t a t r i e n o i c
methyl e s t e r .
The i n f r a r e d s p e c t r a o f t h e t h r e e u n s a tu r a te d methyl e s t e r f r a c t i o n s
c o rre sp o n d almost e x a c t l y to th o s e o b ta in e d from methyl o l e a t e , I i n o l e a t e 9
and l i n o l e n a t e .
The com plete absence o f a t r a n s band a t 960 cm- ^ in th e
i n f r a r e d s p e c t r a o f th e f r a c t i o n s Kam-Iul and Kam-I u2 i n d i c a t e s t h a t th e
double bonds must be in th e c i s c o n f i g u r a t i o n .
A weak band n e a r 960cm~l in
t h e spectrum o f Kam-IuS i s not n e a r ly as s tro n g as t h a t observed in th e
spectrum o f methyl l i n o l e n a t e , so th e m a jo r ity o f th e double bonds in Kam-IuS
a r e p ro b a b ly in th e c i s c o n f i g u r a t i o n .
S u b s t a n t i a t i n g th e c i s c o n f ig u r a tio n
o f th e 18:1 i s th e occurence o f only one s p o t c o rre sp o n d in g t o methyl o l e a t e
when t h e f r a c t i o n is chromatographed on s i l i c a g e l p l a t e s c o n ta in in g 5%
silv e r n itra te .
A ccording to Morris^® a m ix tu re o f o l e a t e (c is c o n f ig u r a tio n )
and e l a i d a t e ( tr a n s c o n f ig u r a tio n ) would show two d i s t i n c t s p o t s « Thus th e
t h r e e m ajor u n s a tu r a te d a c id s a re ta k e n t o be i d e n t i c a l t o o l e i c , l i n o l e i c ,
-2 9 -
and l i n o l e n i c a c i d s .
Q u a n t i t a t i v e A n a ly sis o f th e U n sa tu ra te d Methyl E s te r s
Q u a n t i t a t i v e a n a l y s i s o f t h e methyl e s t e r s o f th e f r e e u n s a tu r a te d
a c id s was perform ed on a 4 fo o t 15% EGS column a t both 170 and 1950C.. A ll
chromatograms were run in t r i p l i c a t e and t h e v alu es av erag e d .
C o rrelatio n
was made between th e two s e t s o f chromatograms by means o f th e common methyl
l i n o l e n a t e peak.
T a b le V shows t h e com position o f th e methyl e s t e r s d e­
r i v e d from t h e u n s a tu r a te d a c i d s .
cent lis te d .
Peaks not re s o lv e d have a combined p e r ­
The m ajor component in th e u n re so lv ed peaks i s u n d e rlin e d .
In t h e c a s e o f th e 18:1 and 18:3 p e a k s , t h e Cjq a c id s make up more than
95% o f t h e t o t a l peak as i n d i c a t e d by gas chromatograph o f th e monoenoic
and t r i e n o i c methyl e s t e r f r a c t i o n s .
-30-
TABLE V
Methyl E s t e r s o f t h e F ree U n sa tu ra te d A c id s , Kam-Iu
% Kam-Iu
1961
-
% Kam-Iu
1962
1
T
T
T
3 .0
0 .9
T
5 4 .3
29.1
9 .8
0 .3
1 .0
1 .3
T
14:1
14:2
15:1
16:1
16:2
17:1
* 1 8 ;1 -1 6 :3
18:2
* 1 8 ;3 -2 0 :I
20:2
* 2 2 :I -2 0 ;3
22:2
* 2 2 ;3 - 2 4 :I
T
T
T
2 .4 .
0 .3
T
3 9 .9
4 4 .8
1 1 .3
0 .3
0 .7
T
T
T d e n o tes l e s s th a n 0.1% o f f r a c t i o n
* not r e s o lv e d under c o n d itio n s u sed, major component u n d e rlin e d
I s o l a t i o n o f N e u tra l L ip id s
C la s s s e p a r a t i o n s o f l i p i d s a r e n e c e s s a ry t o e f f e c t i v e l y study t h e i r
c o m p o sitio n by gas chrom atography.
A g r e a t d e al o f r e s e a r c h has been
devoted t o t h e development o f column chrom atographic te c h n iq u e s f o r th e
sep aratio n of l i p i d s .
s e p a r a tio n s by one
Most o f th e s e methods have been b a se d on complete
c o l u m n . ® 6*
®
,21
Due t o th e c o m p lex ity o f th e l i p i d s
o b ta in e d from t h e c u t i c u l a r wax o f t h e Mormon c r i c k e t , a more e f f e c t i v e
method has proved t o be a com bination o f s e v e r a l columns in c o n ju n c tio n
w ith p r e p a r a t i v e t h i n - l a y e r chrom atography.
-31 -
The n e u t r a l l i p i d s and hydrocarbons o f wax Kn were chromatographed on
a 4 .5 g column o f U n i s i l (Clarkson C h em ical, W illia m s p o rt, P a . ) , an a c t i ­
v a te d s i l i c i c a c i d .
f o r 75% o f wax Kn.
Hydrocarbons were e l u t e d from t h e column and accounted
The n e u t r a l l i p i d s were e l u t e d from t h e column in f o u r
f r a c t i o n s by th e s u c c e s s iv e use o f 18% benzene in hexane, 100 ml o f 60%
benzene in h ex an e, 75 ml o f 100% b enzene, and 100 ml 100% methanol
The fo u r f r a c t i o n s were, d e s ig n a te d K n o -I, Kno-2, Kno-3, and Kno-4 , r e s p e c ­
tiv e ly .
T h i n - l a y e r chromatography was used to an aly ze each o f t h e f r a c t i o n s
from t h e U n i s i l column.
Kno-I gave one s p o t c o rre sp o n d in g to ,a m ixture of.
wax and s t e r y l e s t e r s p lu s a second sp o t which had m ig r a tio n c h a r a c t e r i s t i c s
s i m i l a r t o th o s e o f methyl o r C th y l1 e s t e r s , o r a c e t a t e e s t e r s o f long c h ain
a lc o h o ls.
The i n f r a r e d spectrum o f K n o -I, f i g u r e 14, i s s i m i l a r t o t h a t -
o b ta in e d from known c h o l e s t e r y l e s te r s ,.
The in te n s e p u rp le c o l o r developed
by t h e !spot on t h i n - l a y e r a f t e r b eing -sprayed with s u l f u r i c a c id and c h a r r e d
i s i n d i c a t i v e o f d e l t a s ? s t e r y l e s t e r s . , i Kno-2 was found, b y . t h i n - l a y e r
chrom atography; t o be t r i g l y c e r i d e s .
The i n f r a r e d .spectrum , f i g u r e 15, is
s i m i l a r t o t h a t o f known t r i g l y c e r i d e m i x t u r e s .
Kno-3 was found to be com­
posed o f s t e r o l s and h ig h ly u n s a tu r a te d t r i g l y c e r i d e s .
Kno-4 appeared t o . b e
a m i x t u r e ; o f rm o n o g ly cerid es, d i g l y c e r i d e s , and f r e e f a t t y a c i d s .
The i n f r a r e d
spectrum , f i g u r e 16, i s s i m i l a r t o t h a t o f m ono-olein.
F r a c t i o n Kno-I was chromatographed on s i l i c a , g e l p l a t e s developed in
t r i c h l o r o e t h y l e n e and on A n a sil p l a t e s developed in 9 0 /1 0 /1 (v /v /v ) hexaned i e t h y l e t h e r - a c e t i c a c id in o rd e r t o d eterm in e i f wax e s t e r s were p r e s e n t .
A sp o t was o bserved which m ig rate d ahead o f th e s t e r y l e s t e r s on th e A n a s il
p l a t e s b u t behind t h e s t e r y l e s t e r s on s i l i c a g e l , th u s i n d i c a t i n g th e
-32-
W A V ELEN G TH
2000
F ig u re 14.
1900
1800
)
1600
1500
W A V EN U M B ER
IN
M IC R O N S
1400
1300
I
IN KA Y SER S
I n f r a r e d spectrum o f Kno-I (cap. film )
W A V ELEN G TH
IN
M IC R O N S
13
1900
F ig u re 15.
1800
17W
1600
1500
W A V EN U M B ER
1400
1300
1200
IN KA Y SER S
1100
1000
900
I n f r a r e d spectrum o f Kno-2 (cap. film)
800
14 15 16 .
700
-3 3 -
p re s e n c e o f wax e s t e r s .
F r a c tio n Kno-I was s e p a r a te d i n t o two s u b f r a c t io n s by means o f p re p ­
a r a t i v e t h i n - l a y e r chromatography on A d s o r b is i l -2 (no b in d e r) p l a t e s .
The
Kno-I was s p o tte d in a co n tin u o u s s t r i p 2 cm above th e bottom o f th e p l a t e .
The p l a t e was developed in a 9 0 /1 0 /1 (v /v /v ) h e x a n e -d ie th y l e t h e r - a c e t i c
a c i d s o lv e n t system u n t i l t h e s o lv e n t f r o n t had t r a v e l e d 15 cm.
The
m a t e r i a l s were lo c a t e d by chrom atographing s ta n d a rd s along th e edges o f t h e
p l a t e , s p ra y in g t h e s ta n d a rd s w ith Bhodamine 6 G in e t h a n o l , and viewing
un d er u l t r a v i o l e t l i g h t .
The p o r ti o n s o f th e a d so rb en t c o n ta in in g th e un­
knowns were scrap e d from th e p l a t e u sin g t h e d is ta n c e s t r a v e l e d by th e
s ta n d a r d s as a g u id e .
The ad so rb en t removed from t h e p l a t e was s l u r r i e d
w ith d i e t h y l e t h e r and f i l t e r e d th ro u g h a f r i t t e d g l a s s fu n n el o f very f i n e
p o ro sity .
The a d so rb e n t was washed t h r e e tim es w ith 5 ml p o r ti o n s o f
d ie th y l e th er.
The s o lv e n t was ta k e n t o n e a r dryness on a r o t a r y evap­
o r a t o r , th en tak en t o d ry n ess under a stre am o f n i t r o g e n .
The wax and
s t e r y l e s t e r f r a c t i o n was d e s ig n a te d Kno-Ia and t h e second f r a c t i o n , s i m i l a r
t o a c e t a t e s o r methyl e s t e r s , was d e s ig n a te d K no-Ib.
g e l was found in t h e i s o l a t e d f r a c t i o n s .
No i n d i c a t i o n o f s i l i c a -
A ll m a t e r i a l s in b o th f r a c t i o n s
were s o lu b le in hexane, le a v in g no u n d is s o lv e d r e s i d u e .
A b la n k p l a t e run
as a c o n t r o l f a i l e d t o show t h e p re se n c e o f e i t h e r s i l i c a g e l o r e x tra n eo u s
lip id s a fte r e lu tio n .
The u n s a tu r a te d t r i g l y c e r i d e s were removed from f r a c t i o n Kno-3 by
chromatography on an alumina column ( F is c h e r , F a irla w n , N . J . ) .
The
t r i g l y c e r i d e s were e l u t e d w ith 50 ml benzene and th e s t e r o l s w ith 50 ml
d ie th y l e th e r.
The t r i g l y c e r i d e s re c o v e re d were added t o f r a c t i o n Kno-2.
34
W A V ELEN G TH
IN
MICRON!
12
)
1600
1500
W A V EN U M B ER
F ig u re 16.
1400
1300
1200
IN KA Y SER S
1100
1000
I n f r a r e d spectrum o f Kno-4 (cap. film )
W A V ELEN G TH
IN
M IC R O N S
Kn o- 3n
4500
2000
F ig u re 17.
1800
1700
1600
1500
W A V EN U M B ER
1400
1300
I
IN KA Y SER S
I n f r a r e d spectrum o f Kno-Sn (CSg s o l . )
13
14
-3 5 -
T h is f r a c t i o n was th e n r e d e s ig n a te d Kno-2m.
The s t e r o l s contained very
minor t r a c e s o f d i g l y c e r i d e , i n d i c a t i n g some h y d r o ly s is o f t h e t r i g l y c e r i d e s
on t h e alumina column.
column.
The f a t t y a c id s r e l e a s e d were not e l u t e d from th e
The q u a n t i t y o f d i g l y c e r i d e p r e s e n t was j u s t s u f f i c i e n t to g iv e a
f a i n t sp o t when 75 micrograms o f t h e s t e r o l f r a c t i o n was examined by t h i n l a y e r chrom atography.
shown in f i g u r e 17.
The i n f r a r e d spectrum o f t h e s t e r o l s , Kno-Sn, i s
The t r a c e o f d i g l y c e r i d e i s i n d i c a t e d by th e small
e s t e r c a rb o n y l band n e a r 1740 cm"^.
The rem ainder o f th e spectrum i s id e n ­
t i c a l t o t h a t o b ta in e d f o r c h o l e s t e r o l .
F r a c tio n Kno-4 was chromatographed on a 5 g F l o r i s i l column to s e p a r a t e
t h e m o n o g ly c e rid e s, th e d i g l y c e r i d e s , and th e f a t t y a c i d s .
D ig ly c e rid e s
were e l u t e d w ith 100 ml 5(% d i e t h y l e t h e r in hexane, m onoglycerides by 100 ml
10% methanol in d i e t h y l e t h e r , and f a t t y a c id by 50 ml 2% a c e t i c a c id in
d ie th y l e th e r.
Kno-4c.
The r e s p e c t i v e f r a c t i o n s were d e s ig n a te d Kno-42, Kno-4 b , and
The com p o sitio n o f Kno by l i p i d c l a s s is shown in T a b le VI.
F ig u re 18 shows t h e s e p a r a t i o n scheme f o r th e n e u t r a l l i p i d s .
TABLE VI
Composition o f Kno by L ip id C la ss
F rac tio n
Kno-Ia
Kno-Ib
Kno-2m
Kno-Sn
Kno-4a
Kno-4b
Kno-4c
Weight P e rc e n t
22.6
9 .1
36.5
9.1
4 .9
12,3
'' 5 .5
Kn o -2
K no-2 m
THIN- LAYER
Kno-Io
Kno-Ib
F ig u re 18.
Kno-3
Kno- 3 n
FLORISIL
K n o -4 a
Kn o- 4b
Chromatographic s e p a r a tio n s o f th e n e u tr a l l i p i d s
Kno- 4o
-37-
T r a n s e s t e x i f i c a t Ion o f E s t e r F r a c tio n s
The v a rio u s e s t e r f r a c t i o n s were t r a n s e s t e r i f ie d by th e method o f
Luddy e t a l .
26
by r e f l u x i n g in sodium m ethoxide-m ethanol s o l u t i o n s .
The
sodium methoxide was f r e s h l y p re p a re d imm ediately p r i o r t o use by d is s o lv i n g
sodium b a l l s in r e d i s t i l l e d m ethanol.
S t e r y l and wax e s t e r s were t r a n s -
e s t e r i f i e d w ith 10 ml o f 0.4N s o l u t i o n f o r I h our.
The otfyer e s t e r f r a c t i o n s
were t r a n s e s t e r i f i e d w ith 0.1N methoxide f o r f i f t e e n m in u te s .
The r e a c t i o n
m ix tu re s were a c i d i f i e d w ith 0.5N s u l f u r i c a c id in methanol a f t e r r e f l u x
was co m p leted .
The a c i d i f i e d r e a c t i o n m ix tu res were poured i n to 25 ml o f
w a te r and e x t r a c t e d f i v e tim es w ith 25, 25, 15, 15, and 15 ml p o r ti o n s o f
hexane.
The h e x a n e . e x t r a c t s were combined, washed w ith 10 ml o f w a te r, and
d r i e d o v er anhydrous sodium s u l f a t e .
e v a p o r a to r t o about 2 ml.
The hexane was removed on a r o t a r y
F r a c tio n s Kno-2m, Kno-4a, Kno-4b were ta k en t o
d ry n e ss w ith a stre am o f n i t r o g e n .
The hexane s o l u t i o n s o f Kno-Ia and
Kno-Ib were chromatographed on F l o r i s i l t o s e p a r a te t h e n o n - s a p o n if ia b le s
from t h e methyl e s t e r s .
The methyl e s t e r s were e l u t e d w ith 100 ml 15%
d i e t h y l e t h e r in hexane and t h e n o n - s a p o n if ia b le s were removed w ith 100 ml
10% methanol in d i e t h y l e t h e r .
The methyl e s t e r s so removed were d e s ig n a te d
Kno-Iam and Kno-Ibm; t h e n o n s a p o n if ia b le s were d e s ig n a te d Kno-Ian and
K n o -Ib n .
F r a c t i o n Kno-4c was c o n v e rte d t o i t s methyl e s t e r s w ith boron
t r i c h l o r i d e methanol re a g e n t in a manner i d e n t i c a l t o t h a t used f o r th e f r e e
a cid s.
T h i n - l a y e r chromatography was used t o m onitor each o f th e t r a n s ­
e s t e r i f i c a t i o n m ix tu re s f o r co m pleteness o f r e a c t i o n .
A ll r e a c t i o n s went
t o a p p a re n t c o m p letio n ex cep t f o r t h e wax and s t e r y l e s t e r s .
Small
q u a n t i t i e s o f s t e r y l e s t e r c o u ld be d e te c te d in th e r e a c t i o n m ixture a f t e r
-38-
one hour o f r e f l u x .
Q u a n t i t a t i v e A n a ly sis o f Methyl E s te r s o f N eu tral L ip id s
The. methyl e s t e r s from th e t r a n s e s t e r i f i c a t i o n were chromatographed
on EGS a t ITO0C .
Chromatograms o b ta in e d from th e methyl e s t e r s a re shown
in f i g u r e s 19 t o 24.
To s im p lif y q u a n t i t a t i v e a n a ly s is t h e methyl e s t e r s
were chrom atographed on columns o f 25% s i l v e r n itr a t e - A d s o r b o s i I to
s e p a r a t e th e s a t u r a t e d a c id s from t h e u n s a tu r a te d a c i d s .
The f a t t y a c id s
were th e n gas chromatographed on EGS a t b o th 170 and 195°C.
were computed th rough th e use o f a Disk i n t e g r a t o r .
P ercen tag es
C o r r e l a t i o n was made
between chromatograms, p re p a re d in t r i p l i c a t e , o b ta in e d a t th e two tem p er­
a t u r e s by t h e comparison o f common p e a k s .
T a b le V II shows t h e p a t t e r n s o f
t h e s a t u r a t e d f a t t y a c id s o f th e n e u t r a l l i p i d s .
p a t t e r n s o f th e u n s a tu r a te d a c i d s .
Table V III shows th e
Only c r i c k e t s c o l l e c t e d du rin g th e
summer o f 1962 were used f o r th e stu d y o f t h e n e u t r a l l i p i d s .
A n a ly sis o f S t e r o l s and A lcohols
F re e s t e r o l s and s t e r t i l s from I h e 1S t e r y l eister f r a c t i o n s ' Aere
a n aly z e d by gas chromatography on SE-30 and QF-I s i l i c o n e gas chromato­
graphy colum ns.
Due t o t h e peak o v e rla p in t h e s t e r o l s , p e rc e n ta g e com­
p o s i t i o n was determ ined by t r i a n g u l a t i o n o f th e p e a k s .
F ig u re s 25 and
26 show th e chromatograms o f th e f r e e and e s t e r i f i e d s t e r o l s .
The gas
chromatograms were o b ta in e d on a 4 fo o t 3.E% SE-30 column a t 250°C.
The
s t e r o l f r a c t i o n s were a l s o chromatographed on a 4 fo o t 0.75% QF-I f l u o r i n a t e d s i l i c o n e column a t 202°C t o s u b s t a n t i a t e th e i d e n t i f i c a t i o n o f th e
-39TABLE VII
S a t u r a t e d F a t t y Acid Methyl E s te r s o f th e N e u tra l L ip id s
Carbon No.
Kno-Ia
Kno-Ib
Kno-2m
Kno-4a
Kno-4b
8 t o 12
13
14
15
16
17
18
19
20
T
T
5 .1
6.1
5 6 .3
7 .2
23.5
0 .4
7 .7
T
T
6 .2
1 .3
54.6
2 .7
2 7 .0
T
T
T
0 .1
5 .6
1 .2
5 5 .7
2 .5
2 1 .2
0 .7
2 .6
T
T
10.6
1 .9
56.5
5 .7
20.5
T
4 .8
T•
T
6 .1
2 .3
4 9 .8
5 .3
2 2 .7
1 .6
12.1
Kno-4c
T
T
4 e0
1.5
39.2
3 .8
44.1
1.4
6.1
T denotes l e s s th a n 0.1% o f f r a c t i o n
TABLE V III
'
D n s a tu r a te d F a t t y A cid Methyl E s t e r s o f th e N e u tra l L ip id s
Acid
Kno-Ia
14:1
14:2
15:1
16:1
16:2
17:1
* 1 8 :1 -1 6 :3
18:2
* 1 8 :3 -2 0 :1
20:2
* 2 2 :1 -2 0 :3
22:2
* 2 2 :3 -2 4 :1
0 .7
0 .4
0 .3
7.1
1 .0
T
4 1 .5
4 0 .3
7 .6
0 .2
0 .3
0 .6
T
Kno-Ib
T
T
T
1,8
0 .4
T
39,2
4 6 .2
2.1
0 .2
0 ,5
, 9 .4
T
% com position
Kno-2m
Kno-4a
T
T
T
2 .5
0 .3
T
2 6 .7
4 6.6
11.6
0 .2
1 .2
0 .7
T
T
T
T
4 .3
T
T
4 6 .8
40.8
6 .4
1.6
T
T
T
Kno-4b
IKno-4c
T
T
T
4 .9
T
T
5 9 .4
29 .4
5 .6
T
T
T
T
T
T
T
2.4
T
2 .6
73.2
10,6
3 .3
5 .1
T
T
T
T den o tes l e s s th a n 0.1% o f f r a c t i o n
* not r e s o l v a b l e under c o n d itio n s u sed . major e s t e r u n d e rlin e s
-40-
F i g u r e 19.
Gas c h ro m a to g ra m o f t h e m e th y l e s t e r s o f K n o-Ia
-41-
F ig u re 20.
Gas c h ro m a to g ra m o f t h e m e th y l e s t e r s o f K no-Ib
42
F ig u re 21.
Gas c h ro m a to g ra m o f t h e m e th y l e s t e r s o f Kno-2m
-43-
F i g u r e 22
Gas c h ro m a to g ra m o f t h e m e th y l e s t e r s o f Kno-4a
-4 4 -
f \ •-
F ig u re 23.
Gas c h ro m a to g ra m o f t h e m e th y l e s t e r s o f Kno-4b
-4 5 -
"4—t4-
F ig u re 2 4 .
Gas c h ro m a to g ra m o f t h e m e th y l e s t e r s o f Kno-4c
-4 6 -
ste ro ls.
T a b le IX shows t h e s t e r o l c o m p o sitio n o f Kno-Sn and K no-la.
U n id e n t i f i e d s t r u c t u r e s a re i n d i c a t e d by l e t t e r .
Known samples o f c h o l e s ­
t e r o l , c a m p e s te r o l, and b e t a - s i t o s t e r o l have r e t e n t i o n tim es on both columns
i d e n t i c a l t o th o s e l i s t e d f o r th e c u t i c u l a r s t e r o l s which were i d e n t i f i e d .
TABLE IX
S t e r o l Composition o f F r a c tio n s Kno-Sn-and Kno-Ia
Rt
OF-I
y
Rt
SE- 30
' 2 .8
3 .1
3 .6
4 .1
5 .0
6 .0
1.6
1 .8
2 .0
2 .3
2 .9
3 .2
X
S te ro l
% Kno-Sn
A
C h o le s te r o l
B
C am pesterol
{ 3 - s ito s te r o l
C
0 .7
9 2 .2
0 .9
2 .5
3 .7
T
% of s te ro ls
in Kno-la
,
1 .3
8 6 .2
2 .7
3 .5
5 .5
0 .8
T d en o tes l e s s th a n 0.1% o f f r a c t i o n
x r e t e n t i o n tim es r e l a t i v e t o c h o l e s t a n e , t r 3 .2 m inutes
y r e t e n t i o n tim e s r e l a t i v e t o c h o l e s t a n e , t = 6 .2 m inutes
The f a t t y a lc o h o ls o f t h e n o n - s a p o n if ia b le s were chromatographed on
SE-SO a t 2150C f o r tw enty m inutes fo llo w ed by te m p e ra tu re programming t o
SOO0C.
F ig u re 27 shows a p o r ti o n o f th e gas chromatogram o f t h e a l c o h o l s .
T a b le X shows t h e c o m p o sitio n o f t h e f a t t y a lc o h o ls d e r iv e d from th e wax
e s t e r s o f K no-la.
chrom atography.
U n s a tu r a tio n was d eterm in ed by b ro m in atio n followed by r e ­
Those peaks which d is a p p e a re d o r were s h i f t e d in p o s i t i o n
were ta k e n t o be u n s a t u r a t e d .
The n o n - s a p o n if ia b le s o f Kno-Ib were chromatographed on SE-SO.
chromatogram i s shown in f i g u r e 28.
The
C h o le s te r o l was p r e s e n t in t h e sample
-47a s shown by t h e peak a t 5 .6 m in u te s, B t= I .8 .
by chromatography on Q F -I.
T h is assignm ent was confirmed
The peak a t 9 .1 m inutes in f i g u r e 27, R t=2.9,
appeared t o be e i t h e r b e t a - s i t o s t e r o l o r Cgy a l c o h o l .
Chromatography on
Q F -I, however, showed t h a t t h i s peak was n e i t h e r b e t a - s i t o s t e r o l o r Cgy
I
a lc o h o l as i t had an Bt o f 5 , 2 . The peak a t 9 .8 m inutes in f i g u r e 27 had
t h e same r e t e n t i o n tim e as s t e r o l C in T a b le IX.
T h i n - l a y e r chromatography
on s i l i c a g e l su g g e s te d t h a t t h i s f r a c t i o n was mainly composed o f m a te r ia ls
sim ila r to la n o s te ro l.
Gas chromatography on SE-30, however showed t h a t
none o f t h e m a t e r i a l s had t h e same r e t e n t i o n tim e as l a n o s t e r o l .
None o f
t h e m a t e r i a l s o f t h i s f r a c t i o n , w ith t h e e x c e p tio n o f c h o l e s t e r o l , were
id e n tifie d .
TABLE X
F a t t y A lcohols o f t h e Wax E s te r s
Carbon No.
% o f A lcohols
16
17
18
19
20u
20
21
22u
22
23?
24
• 25 .
26
T
T
1.6
T
0 .8
23.6
2 .0
13.6
17.4
27.1
5 .5
4 .1
4 .3
T den o tes l e s s th a n 0.1% o f f r a c t i o n
u den o tes uns a t u r a t e d s t r u c t u r e s
? i n d i c a t e s t h e peak does not f a l l on a log p lo t o f e i t h e r th e
s a t u r a t e d o r u n s a tu r a te d a lc o h o ls
Kno-3n
■
at
CD
F i g u r e 25 .
Gas c h ro m a to g ra m o f t h e f r e e s t e r o l s , Kno-Sn
F ig u re 26.
Gas chromatogram o f th e s t e r o l s o f Kno-Ia
Kno-Ia
I
0
1
in
F ig u re 27.
Gas c h ro m a to g ra m o f t h e a l c o h o l s o f K n o -Ia
Kno-Ib
F ig u re 28.
Gas c h ro m a to g ra m o f t h e n o n - s a p o n i f i a b l e s o f Kno-Ib
•DISCUSSION:
LIMITATIONS OF RESULTS
Some o f t h e r e s u l t s o f t h i s stu d y were ambiguous and o th e r s were based
on assum ptions not i n d i c a t e d in th e s e c t i o n on e x p erim en tal p r o c e d u re .
In
an a tte m p t t o i n d i c a t e t h e n a tu re o f some o f th e s e assum ptions and ambig­
u i t i e s th e fo llo w in g d is c u s s io n i s in c lu d e d .
P e rc e n ta g e c o m p o sitio n s f o r t h e v a rio u s methyl e s t e r f r a c t i o n s a re
b a se d on th e o b s e r v a tio n t h a t th e re sp o n se o f a flame d e t e c t o r i s p ro ­
p o r t i o n a l t o th e number o f carbon atoms in a m o lecu le.
The p r o p o r t i o n a l i t y
c o n s ta n t i s t h e same f o r a l l members o f a homologous s e r i e s , th u s th e a re a
p e r c e n t computed from a r e c o r d e r t r a c e i s d i r e c t l y c o n v e r t i b l e t o weight
p e rc en t.
E t t r e and K a b o t have shown t h a t t h i s same r e l a t i o n s h i p holds
f o r a l l ty p e s o f n o q -p o la r methyl e s t e r s so t h a t f a t t y a c id methyl e s t e r s
may be compared d i r e c t l y f o r weight p e rc e n t from th e d e t e c t o r re sp o n se .
T a b le s IV and V l i s t t h e r e l a t i v e p e rc e n ta g e s o f th e f r e e s a t u r a t e d
a c id s d eterm in ed as methyl e s t e r s .
Small d i f f e r e n c e s may b e noted between
t h e s a t u r a t e d f r e e f a t t y a c id s o f 1961 and 1962.
In alm ost a l l case s t h e s e
d e v i a t i o n s between th e p e rc e n ta g e s computed f o r th e two y e a rs f a l l w ith in
t h e s ta n d a r d d e v ia t io n s computed f o r th e p a r t i c u l a r a c id s t u d i e d .
In t h e
c a s e o f t h e u n s a tu r a te d a c id s th e s ta n d a r d d e v ia tio n s do n o t account f o r
t h e wide v a r ia n c e o f p e rc e n ta g e c o m p o sitio n .
f e r e n c e s in sample t r e a t m e n t .
These might r e p r e s e n t d i f ­
A second p o s s i b i l i t y i s v a r i a t i o n between
t h e two y e a rs due t o en v iro n m en tal s e l e c t i o n .
O b se rv a tio n s on th e b i g ­
headed g ra s sh o p p e r have shown d i f f e r e n c e s in c h a r a c t e r i s t i c s between i s o ­
l a t e d p o p u la tio n s d u rin g th e same y e a r . ^
Svoboda^b has found d i f f e r e n c e s
-53-
i n l i p i d s between g ra s sh o p p e r eggs c o l l e c t e d one m ile a p a r t on th e same day.
Thus t h e observed d i f f e r e n c e s may be due t o any one o f s e v e r a l b i o l o g i c a l
fa c to rs.
During th e i n v e s t i g a t i o n in t o th e n a tu r e o f t h e f r e e f a t t y a c id s i t
was n oted t h a t t h e r e were m a t e r i a l s not e l u t e d from t h e F l o r i s i l column
used to s e p a r a t e t h e methyl e s t e r s i n t o p o l a r groups (Table T I ) .
m a t e r i a l no t e l u t e d c o u ld r e p r e s e n t n o n - e s t e r i f i e d f a t t y a c i d s .
The
T his i s
u n l i k e l y as th e i n f r a r e d spectrum , f i g u r e 3, o f t h e t o t a l methyl e s t e r s
shows no ev id en c e o f an a c id carb o n y l a t 1705 c m . I n
a d d itio n V o r b e c .
has shown t h e m e th y la tio n p ro ced u re used t o be q u a n t i t a t i v e .
A more l i k e l y
p o s s i b i l i t y i s t h a t th e s e m a t e r i a l s a r e p h o s p h a tid ic a c i d s .
Such m a t e r i a l s
would not be c o n v e rte d t o methyl e s t e r s b u t would be r e t a i n e d on th e column
as magnesium s a l t s .
A t h i r d p o s s i b i l i t y i s t h a t th e s e m a t e r i a l s , as well as
t h e p o l a r methyl e s t e r s Kam-2 and Kam-S0 r e p r e s e n t o x id a tio n p ro d u cts o f t h e
uns a t u r a t e d a c i d s .
T h is i s q u i t e l i k e l y as th e a c id s from 1961, which showed
a high p e rc e n ta g e o f m a t e r i a l s not e l u t e d from F l o r i s i l , were c o l l e c t e d from
a number o f Kies e x t r a c t i o n s and S to red in w ater s o l u t i o n as sodium s a l t s
.
f o r s e v e r a l weeks.
The f a t t y a c id s from 1962, which were c o n v e rte d to t h e i r
methyl e s t e r s imm ediately upon i s o l a t i o n , showed, a: much low er p e rc en ta g e o f
m a t e r i a l s not e l u t e d . In a r e c e n t s tu d y A st^ has found t h a t m e th y le n e :i n te r r u p te d p o ly ­
u n s a t u r a t e d f a t t y a c id s may be i n a d v e r t a n t l y co n v erted t o t h e co n ju g ated
isomers d u rin g methyl e s t e r p r e p a r a t i o n .
No evidence f o r such is o m e r iz a tio n
was found in t h i s stu d y as c o n ju g a te d s tr u c tu r e s ,w o u ld have g iven e i t h e r
lo n g e r aldehydes o r aldehyde e s t e r s th a n th e ones a c t u a l l y found.
\
-54No d i r e c t e v id en ce was o b ta in e d f o r t h e assignm ents o f b ra n ch in g to
some o f t h e s a t u r a t e d a c i d s .
These assig n m en ts were based on th e g e n e r a l ­
i z a t i o n t h a t branched c h a in s t r u c t u r e s a r e e l u t e d b e fo re t h e i r , s t r a i g h t
c h a in c o u n t e r p a r t s d u rin g gas chromatography on .1SE-SO s i l i c o n e .
The gas
c h ro m ato g ram s;o f t h e s a t u r a t e d a c id s show two s e r i e s ^ f branched c h ain com­
pounds e x i s t .
These two ,s e r ie s might.! r e p r e s e n t is o ,and a n t e i s o s t r u c t u r e s
as Leibrand^S showed th e p re se n c e o f ; s t r u c t u r e s : o f t h i s ty p e in th e hydro­
carbons o f t h e Mormon c r i c k e t .
I t i s a l s o h ig h ly p o s s i b l e t h a t th e o th e r
h ig h ly b ranched a c id s e x i s t but a re masked by th e peaks o f t h e s t r a i g h t
c h a in a c i d s . H
C onversion o f th e methyl e s t e r s t o hydrocarbons followed by
m o le c u la r s ie v e s t u d i e s co u ld be used t o co n firm th e p re se n c e o f branched
c h a in a c i d s .
Mass s p e c t r a l d a ta on th e branched c h a in methyl e s t e r s c o u ld
be used t o e l u c i d a t e t h e b ra n c h in g i f enough m a t e r i a l s co u ld be c o l l e c t e d by
p r e p a r a t i v e gas chrom atography.
Wax and s t e r y l e s t e r s co u ld have been s e p a r a te d f o r independent stu d y
b y p r e p a r a t i v e t h i n - l a y e r chromatography on A n a sil p l a t e s .
This was not
done in t h e p r e s e n t stu d y d u e .to th e sm all amount o f wax e s t e r p r e s e n t ,
a p p ro x im a te ly 1/6 o f th e w eight o f f r a c t i o n Kno-Ia as e s tim a te d f o r t h i n s,
l a y e r chrom atography.
The s t e r o l s i d e n t i f i e d as c h o l e s t e r o l , c a m p e s te ro l, and b e t a - s i t o s t e r o l
c o u ld be o th e r e p im e rs.
V a r i a t i o n in t h e c o n f ig u r a tio n about one carbon
atom would not be l i k e l y t o change t h e r e l a t i v e r e t e n t i o n tim es o f th e
s te ro ls.
The s t e r o l r e p o r te d as cam p e stero l in t h i s s tu d y is u s u a lly
r e p o r te d as g a m m a - s i to s te r o l.
Thompson e t a l.* ^ „ however, have r e c e n t l y
shown t h a t t h e supposed g a m m a -sito ste ro l t o be c a m p e s te ro l.
The cam p e stero l
-55~ .
s t a n d a r d used in t h i s s tu d y was t h e im p u rity in b e t a - s i t o s t e r o l shown by
Thompson t o be c a m p e s te r o l.
SUMMARY
The c u t i c u l a r l i p i d s o f t h e Mormon c r i c k e t were i s o l a t e d by chloroform
e x t r a c t i o n follow ed by hexane p r e c i p i t a t i o n .
The crude l i p i d s were s e p a ­
r a t e d i n t o n e u t r a l l i p i d s and f a t t y a c id s by a Kies c o u n te r c u r r e n t ex ­
tra c tio n .
The f a t t y a c id s were c o n v e rte d t o t h e i r methyl e s t e r s and chromato­
graphed on F l o r i s i l t o e f f e c t a s e p a r a t i o n in to p o la r g ro u p s .
The n o n -p o la r
methyl e s t e r s were f u r t h e r f r a c t i o n a t e d on s i l v e r n i t r a t e - s i l i c i c a c id
columns in t o s a t u r a t e d methyl e s t e r s and uns a t u r a t e d methyl e s t e r s .
Quan­
t i t a t i v e a n a l y s i s showed t h a t t h e s a t u r a t e d a c id s ranged from Cg t o a t
le ast
and in c lu d e d both normal and branched c h a in s t r u c t u r e s .
a c id s were found t o be p a l m i t i c and s t e a r i c .
The major
The u n s a tu r a te d a c id s were
found t o range from C ig t o Cg^ w ith th e m ajor a c id s bein g i d e n t i c a l with
o l e i c „ l i n o l e i c , and l i n o l e n i c a c i d s .
The s t r u c t u r e s o f th e m ajor u n s a t­
u r a t e d a c id s were determ in ed by th e use o f r e d u c tiv e o z o n o ly sis and i n f r a r e d
s p e c tro s c o p y .
The n e u t r a l l i p i d s were f r a c t i o n a t e d i n t o chem ical c l a s s e s by a com­
b i n a t i o n o f column chrom atographic m ethods.
The n e u t r a l l i p i d s included
wax e s t e r s „ s t e r y l e s t e r s , t r i g l y c e r i d e s , f r e e s t e r o l s , d i g l y c e r i d e s , and
m o n o g ly c e rid e s .
The f r e e s t e r o l s were d e term in e d , and t h r e e o f them were
i d e n t i f i e d as b e in g c h o l e s t e r o l , c a m p e s te r o l, and b e t a - s i t o s t e r o l .
The
e s t e r f r a c t i o n s were t r a n s e s t e r i f i e d and t h e a c id s and n o n -s a p o n if ia b le s
d eterm in ed by gas chrom atography.
The methyl e s t e r s o f t h e n e u t r a l l i p i d s
were found t o be s i m i l a r t o th o s e found in th e f r e e a c id s ^
APPENDIX
X
X
-5 8 -
TABLE XI
R e t e n t i o n Times*of t h e Major Acid Methyl E s t e r s on EGS**
*
**
F ig u r e No.
16:0
18:0
9
11
13
19
20
21.
22
23
24
3.3
3 .2
6.3
6 .2
— — —
— — —
3 .2
3.3
3.0
3 .0
3.0
3.0
6 .2
6 .2
5 .5
5.6
5.6
5.6
Methyl E s t e r
18:1
18:2
18:3
7.3
9 .5
13.2
— — —
— — —
7.0
7 .0
7.1
6.4
6.5
6.5
6 .5
9.1
9.1
9 .2
8.3
8.5
8 .5
8 .5
Time in minutes from t h e s o l v e n t peak
Column t e m p e r a t u r e — ITO0C
I
1 2 .6 \
12.6
12.7
11.4
11.7
11.7
11.7
LITERATURE CITED
1.
Ackraant R . G . , R e t s o n , M.E., G a l l a y , L . R . , and Vandenheuvel, F . A . ,
Can. J . Chem. .22, 1956 (1961)
2.
Amin, E . S . ,
3.
A s t , H . J . , A nal. Chem. .25, 1539 (1963)
4.
B a i l e y , P . S . , Chem. Rev. 5S, 925 (1958)
5.
Baker, G .L ., P r i v a t e Communication
6.
Baker, G . L . , Peppe r, J . H . , John son , L.H ., and H a s t i n g s , E . J , ,
J . I n s . P h y s i o l . 2 , 47 (1960)
7.
Beament, J .W . L . , B i o l , Rev. .26 , 281 (1961)
8.
Bergstrom, B . , Acta P h y s i o l . Sc a nd . 25, H l
9.
Burton-Browne, L . B . , 1964 Annual Review o f Entomology v. 9
J . Chem. S o c . 1410 (1960)
(1952)
10.
C a r r o l l , K.K.,
J . L i p i d R e s . 2 , 135 (1961)
11.
Cason, J . , and T a v s , P . ,
12.
Cason, J . , Lange, G . L . , M i l l e r , W.T., and Weiss, A ., T e t r a h e d r o n 20.
91 (1964)
13.
DeVries, B . , Chera. and I n d . 1049 (1962)
14.
D eV ri es, B . ,
15.
E t t r e , L . S . , and Kabot, F . J . ,
16.
F a r q u h a r , J . W ., I s s u l l , W., S t o f f e l , W., Rosen, P . , and Ahrens, E .11.,
N u t r . Rev. 1 7, s u p p l. I (1959)
17.
G i l b y , A . R . , Arch. Biochera. Biophy. &7 , 320 (1957)
18.
G i l b y , A . R . , and Cox, M.E.,
19.
H a s t i n g s , E . J . , and Pepper, J . H . , Ann. Entomol. S o c . Amer. 22« 216
(1964)
20.
H i r s c h , J . , and Ahrens, E.H ., J B i o l . Chem. 233. 311 (1958)
21.
Horning, M.G., W i l l i a m s , E .A ., and Horning, E .C .,
482 (1960)
J B i o l . Chem. 234. 1401 (1959)
J . Am. O i l Chem. S o c . 4 0 . 184 (1963)
J . Chromatog. JL1, 114 (1963)
J . Ins P h y s i o l . 9 , 671 (1963)
J . L i p i d Res. JL,
-60
2 2.
K i e s , M.W., and D a vis , P . L . ,
J . B i o l . Chem. Ifi9, 637 (1951)
23.
L e ib ra nd , R . J . , Unpublished Masters T h e s i s , Montana S t a t e C o l l e g e
(1962)
24.
L i n d g r e n , F . T . , N i c h o l s , A.V., Freeman, N .K ., and W i l l s , R . D . ,
J . L i p i d Res. 3 , 390 (1962)
25.
Lough, A . K . , F e l i n s k i , L . , and C ar to n, G.A.,
(1962)
26.
Luddy, F .E . , B a r o f r d , R.A ., and R i e m en s c hn ei de r, R.W.,
Soc . £ L , 447 (1960)
27.
Mangold, H.K.,
28.
M o rr is , L . J . , Chem. and I n d . 1238 (1963)
29'.
P e p p e r, J . H . , P r i v a t e Communication
30.
P r i v e t t f O . S . , and N i c k e l l , E . C . , J . Am. O i l Chem. S o c . .32» I (1962)
31.
P r i v e t t , O . S . , and N i c k e l l , E . C . , J . L i p i d Res.
32.
S h i k a t a , M.,
3 3.
Sims, R . P .A ., and L a r o s e , J . A . G . , J . Am. O il Chem. S o c . 52, 232
34.
S t e i n , R.A. , and N i c o l a i d e s , N., J . L i p i d Res. 5» 476 (1962)
35.
S t o f f e l , W., I s s u l l , W., Rosen, P . , and A hre ns , E .H ., P r o c . S o c . Exp.
B i o l . , N.Y. 2 2 , 238 (1958)
36.
Svoboda, J . A . , P r i v a t e Communication
37.
Thompson, M.L., Robbins, W.E., and Baker, G .L ., S t e r o i d s Z , 505 (1963)
38.
Vorbeck, M.L., M a t t i c k , L . R . , Lee, F .A ., and Pederson, C . S . , Anal.
Chem. 5 3 , 1512 (1961)
39.
Vroman, H . E . , and Bmker, G .L ., Manuscript in P r e p a r a t i o n
40.
Wi gglesworth, V . B . , 1957 Annual Review o f Entomology V. 2
J . Lopid Res. 5 , 468
J . Am. O il Chem.
J . Am O i l Chem. S o c . 3 8 . 708 (1961)
208 (1963)
J . A p p l . E n t . Z o o l . A , 187 (1960)
(1962)
MONTANA CTAr e
— _____
3 1762 10011135 8
D378
P136
cop.2
Padmorej J. M.
The free fatty acids and
neutral lipids..._________
n a MA
An o AODw Kaa
■p-3'78
r~ c £ > .