Journal of Chromatographic Science, Vol.
29, August 1991
Identification of Minor C Triene and Conjugated Diene 18
Isomers in Hydrogenated Soybean Oil and Margarine
by GC-MI-FT-IR Spectroscopy
Magdi M. Mossoba*
Division of Contaminants Chemistry, Food and Drug Administration, Washington, DC 20204
Richard E. McDonald and David J . A r m s t r o n g
National Center for Food Safety and Technology and Division of Food Chemistry and Technology, Food and Drug
Administration, Summit-Argo, Illinois 60501
Samuel W. Page
Division of Contaminants Chemistry, Food and Drug Administration, Washington, DC 20204
Abstract
Vegetable oils are partially hydrogenated in order to produce
palatable products of suitable plasticity. The constituents of
these new dietary products are complex mixtures of fatty acid
isomers with different nutritional properties. A rapid method is
described for separating a n d identifying fatty acid methyl ester
( F A M E ) i s o m e r s of linolenic (octadecatrienoic, 18:3) acid a n d
of conjugated octadecadienoic (18:2) acid, minor s p e c i e s
found in hydrogenated s o y b e a n oil a n d margarine, by capillary
g a s chromatography-matrix isolation-Fourier transform-
infrared ( G C - M I - F T - I R ) spectroscopy. F A M E s of 18:3 acid
i s o m e r s in margarine, s o y b e a n oil hydrogenated in our
laboratory, a n d isomerized linolenic acid were identified by
this method, a n d M I - F T - I R spectra of F A M E geometric i s o m e r s
of octadecatrienoic a n d conjugated octadecadienoic a c i d s are
reported for the first time. Five major C 1 8 triene G C p e a k s are
found in c h r o m a t o g r a m s of isomerized methyl linolenate,
representing s p e c i e s with tri-cis a n d tri-trans configurations
and three s p e c i e s with cis-trans mixed configurations. F A M E
i s o m e r s with these configurations are a l s o found for a
hydrogenated s o y b e a n oil having a n iodine value of 111.
Methyl linolenate (tri-cis) is no longer found when s o y b e a n oil
is further hydrogenated to an iodine value of 96. IR spectra
characteristic of a tri-trans isomer are obtained for two test
s a m p l e s with iodine v a l u e s of 111 a n d 96. B e s i d e s methyl
linolenate, only i s o m e r s with a mono-trans d i - c i s configuration
are found for the margarine analyzed. Conjugated cis-trans
and trans-trans 18:2 F A M E i s o m e r s are a l s o found in all the
hydrogenated s o y b e a n oil a n d margarine analyzed.
Introduction
S o y b e a n oil is the largest source o f vegetable oil i n the w o r l d
today. It h a s the advantage o f r e m a i n i n g l i q u i d o v e r a relatively
w i d e t e m p e r a t u r e r a n g e a n d it c a n b e u s e d a s a p l a s t i c fat
( s e m i s o l i d ) after partial h y d r o g e n a t i o n . B e c a u s e it is s a v o r y ,
p r o c e s s e d s o y b e a n oil is u s e d i n s h o r t e n i n g s , m a r g a r i n e s , a n d
salad oils. H o w e v e r , because o f its h i g h content o f C H C H C H = 3 2
* Author to whom correspondence should be addressed.
324 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
C H C H C H = C H C H C H = C H ( C H ) C O O H , linolenic (cis-9,cis-
2 2 2 7
12,cw-15-octadecatrienoic [ c 9 , c l 2 , c l 5 - 1 8 : 3 ] ) acid ( 6 - 8 % ) , s o y
bean oil is partially hydrogenated to prevent flavor reversion (1).
D u r i n g h y d r o g e n a t i o n , the levels o f linolenic a n d linoleic (cis-
9, c i s - 1 2 - o c t a d e c a d i e n o i c ) acids are l o w e r e d , w h i l e the h i g h e r
melting cis- a n d t r a n s - m o n o e n e components are increased. I n a d
dition to the partial saturation o f double b o n d s , isomerization re
actions also o c c u r i n polyunsaturated fatty acids. S u c h reactions
lead to the f o r m a t i o n o f a n u m b e r o f positional a n d geometric
i s o m e r s ( 2 ) . I s o m e r s o f linolenic acid ( 1 8 : 3 ) , w h i c h is a n e s s e n
tial fatty a c i d ( 3 ) , h a v e b e e n f o u n d i n c o m m e r c i a l salad oil ( 4 ) ,
margarine (5,6), a n d f r y i n g oil (7), but the nature o f their metabo
lites a n d their p h y s i o l o g i c a l effects are unclear ( 8 ) . O n the other
h a n d , conjugated i s o m e r s o f octadecadienoic a c i d ( 1 8 : 2 ) that
were f o u n d i n butter a n d m a r g a r i n e (9) are n e w l y reported anti-
c a r c i n o g e n i c fatty acids ( 1 0 ) .
P e r k i n s a n d S m i c k (4) studied octadecatrienoic fatty a c i d i s o
mers o f a partially hydrogenated s o y b e a n oil, u s i n g a protocol i n
v o l v i n g fractional crystallization, r e v e r s e d - p h a s e h i g h - p e r f o r
m a n c e l i q u i d c h r o m a t o g r a p h y ( R P - H P L C ) , a n d capillary g a s
c h r o m a t o g r a p h y . L a b o r - i n t e n s i v e separation t e c h n i q u e s w e r e
also u s e d for margarine analysis b y R a t n a y a k e a n d B e a r e - R o g e r s
(5). W e recently characterized m o n o e n e a n d diene fatty a c i d
m e t h y l ester ( F A M E ) i s o m e r s derived f r o m m a r g a r i n e a n d h y
drogenated s o y b e a n oil ( 1 1 , 1 2 ) b y g a s c h r o m a t o g r a p h y - m a t r i x
isolation-Fourier transform-infrared ( G C - M I - F T - I R ) spec
t r o s c o p y ( 1 3 , 1 4 ) . I n the present w o r k , w e are e x t e n d i n g o u r
study to include triene a n d conjugated diene F A M E i s o m e r s .
T h i s paper reports a r a p i d m e t h o d for d e t e r m i n i n g the c o n f i g u
rations o f 1 8 : 3 F A M E i s o m e r s obtained f r o m h y d r o g e n a t e d s o y
b e a n oil, m a r g a r i n e , a n d i s o m e r i z e d linolenic a c i d m e t h y l ester,
as w e l l as those o f conjugated C 1 8 diene F A M E s .
Experimental
Materials. R e f i n e d s o y b e a n oil w a s p u r c h a s e d f r o m a local
distributor. A sulfur-containing n i c k e l catalyst ( 1 % S , 2 0 % N i )
was obtained f r o m a c o m m e r c i a l source. A c o m m e r c i a l m a r
garine c o n t a i n i n g s o y b e a n o i l o n l y w a s a c q u i r e d f r o m a l o c a l
f o o d retailer.
Standard and test sample preparation and analysis. All solu-
 Journal of Chromatographic Science, Vol. 29, August 1991

tions were prepared i n isooctane. A l l F A M E standards were pur v o l u m e ) to the G C carrier g a s ( h e l i u m ) a n d trapping the effluent
c h a s e d f r o m A l l t e c h A s s o c i a t e s . T h e test s o l u t i o n s were p r e onto the outer rim o f a s l o w l y rotating (at about 3 m m / m i n )
p a r e d f r o m transesterified m a r g a r i n e , transesterified s o y b e a n g o l d d i s k h e l d at c r y o g e n i c temperatures. D u r i n g a r u n , h e l i u m
o i l , or h y d r o g e n a t e d , transesterified s o y b e a n oil. w a s r e m o v e d b y the v a c u u m p u m p s , a n d the analyte m o l e c u l e s
Hydrogenation, esterification, and isomerization. S o y b e a n oil s u r r o u n d e d b y a cluster o f a r g o n a t o m s were f r o z e n into a s o l i d
w a s h y d r o g e n a t e d i n a 1-gal p r e s s u r e v e s s e l reactor ( A u t o c l a v e matrix. T h e analytes isolated i n the I R - t r a n s p a r e n t a r g o n matrix
E n g i n e e r s ) at 1 0 0 0 r p m a n d 138 k P a H pressure. T h e sulfur-con
2 were subsequently a n a l y z e d b y I R spectroscopy. T h e p o s i t i o n o f
taining n i c k e l catalyst w a s u s e d at a level o f 0 . 2 5 % ( 2 . 5 g : 1 0 0 0 each analyte p e a k o n the C r y o l e c t collection d i s k w a s i n d e x e d b y
g/catalyst:oil) for h y d r o g e n a t i o n , w h i c h w a s carried out at 2 0 0
( 1 0 ) C for 10 a n d 2 5 m i n (test s a m p l e s 2 4 B a n d 2 4 D , respec
tively) to obtain iodine values o f 1 2 3 a n d 1 1 1 , respectively, as de
termined b y the official procedure o f the A s s o c i a t i o n o f O f f i c i a l
A n a l y t i c a l C h e m i s t s ( A O A C ) ( 1 5 : sec. 9 2 0 . 1 5 9 ) . A n o t h e r test
s a m p l e ( 7 H ) w a s h y d r o g e n a t e d to a n i o d i n e v a l u e o f 9 6 ( 0 . 1 %
catalyst, 1 7 5 ( + 5 ) C , 4 h ) . F A M E s o f the s o y b e a n oil a n d o f the
fat i n a c o m m e r c i a l m a r g a r i n e w e r e prepared b y transesterifica-
tion w i t h b o r o n t r i f l u o r i d e - m e t h a n o l a c c o r d i n g to the official
A O A C m e t h o d ( 1 5 : sec. 9 6 9 . 3 3 ) . L i n o l e n i c a c i d m e t h y l ester
( N u c h e k Prep) w a s isomerized with p-toluenesulfinic acid
( P T S A ) ( E a s t m a n K o d a k ) a c c o r d i n g to the procedure d e s c r i b e d
b y M c D o n a l d et al. ( 1 6 ) .
Instrumentation. G a s c h r o m a t o g r a p h i c separations were per
f o r m e d o n a H e w l e t t - P a c k a r d M o d e l 5 8 9 0 instrument e q u i p p e d
w i t h a f l a m e i o n i z a t i o n detector ( F I D ) a n d a n H P M o d e l 3 3 9 2 A
integrator. A 5 0 - m 5 0 . 2 2 - m m (i.d.) C P - S i l - 8 8 capillary c o l u m n
( C h r o m p a c k ) w i t h a 0 . 1 9 - m stationary phase film w a s u s e d . H e
l i u m c o n t a i n i n g 1 . 5 % a r g o n ( M a t h e s o n G a s P r o d u c t s ) at a p
p r o x i m a t e l y 2 7 c m / s linear velocity w a s u s e d as the carrier g a s
a n d h e l i u m ( 9 9 . 9 9 5 % ) at 3 0 m L / m i n w a s u s e d as the m a k e u p g a s
to the detector. T h e injector a n d detector temperatures were 2 5 0
a n d 3 0 0 C , respectively. T h e carrier g a s mixture w a s purified b y
u s i n g a H y d r o - P u r g e I I filter ( A l l t e c h A s s o c i a t e s ) a n d a heated
g a s purifier filter ( S u p e l c o ) . T h e injection m o d e w a s splitless a n d
a 10-L H a m i l t o n 7 0 I N s y r i n g e w a s u s e d . Injections o f about
1 L were m a d e 6 s after the start o f a r u n , a n d the total injection
time w a s 15 s . T h e injector w a s p u r g e d 1 m i n after the start o f a
r u n . T h e initial c o l u m n o v e n temperature w a s 7 5 C w i t h a 2 - m i n
h o l d , f o l l o w e d b y a 2 0 / m i n increase to 1 8 5 C , a n d the o v e n w a s
h e l d at this temperature until the a n a l y s i s w a s complete.
A c o l u m n effluent split ratio o f 4 : 1 , I R : G C , w a s calculated
f r o m the F I D r e s p o n s e factors (area c o u n t s / n a n o g r a m injected)
f o u n d w i t h the c o l u m n directly connected to the detector, a n d
subsequently to the G C - M I interface.
A M a t t s o n I n s t r u m e n t s M o d e l S i r i u s 1 0 0 F T - I R spectrometer
e q u i p p e d w i t h a n M I C r y o l e c t interface ( F i g u r e 1) operating at
12 under v a c u u m w a s u s e d . T h i s s y s t e m h a s b e e n described i n
detail ( 1 3 , 1 4 ) . T h e M I m e t h o d i n v o l v e d a d d i n g a r g o n ( 1 . 5 % b y

Figure 2. Truncated GC-FID chromatograms for FAMEs derived from (a)

isomerized linolenic acid methyl ester, (b) soybean oil partially hydro
genated to iodine values of 123, (c) 111, and (d) 96. Retention times and
normalized area % are given in Table I. Triene isomer configuration: A, ttt,
B, ttc; C, ttc and tcc; D, tcc; E, ccc. IS = internal standard. Asterisk (*) de
notes GC peak (r 15.90 min) for two coeluting FAMEs: 20:0 and cis,cis
t

18:2 species. Peaks F, G, and are due to conjugated FAMEs. 22:0 FAME
Figure 1. Diagram of GC-MI interface. gave rise to peak I.

325
 Journal of Chromatographic Science, Vol. 29, August 1991

its o b s e r v e d G C retention time. P r o c e d u r e s were detailed p r e v i m o r e coeluting positional i s o m e r s w i t h the s a m e c o n f i g u r a t i o n .

o u s l y ( 1 7 ) for reproducibly locating a p e a k m a x i m u m o n the c o l
lection d i s k a n d for o p t i m i z i n g the p e r f o r m a n c e o f the s y s t e m .
T h e s e latter p r o c e d u r e s , w h i c h include optical a l i g n m e n t , c a n
m i n i m i z e the extent o f p o s t c o l u m n p e a k b r o a d e n i n g . W i t h the
C r y o l e c t interface, detection limits for s o m e c o m p o u n d s are i n
the p i c o g r a m (parts per trillion) r a n g e ( 1 7 ) . T h e y are l o w e r than
those obtained w i t h d y n a m i c s a m p l i n g light-pipe interfaces b e
cause w i t h the M I interface, I R spectra are measured after the G C
separation is f i n i s h e d ( p o s t - G C r u n ) , a n d e a c h c o m p o n e n t i s
trapped o n t o a c o m p a c t spot ( 0 . 5 - m m diameter) i n the s o l i d
a r g o n matrix.
GC-MI-FT-IR analysis. T h r e e h u n d r e d analyte interferograms
were c o a d d e d ( 2 m i n 4 3 s at 4 c m - 1
resolution), a n d the b a c k
g r o u n d ( 3 0 0 scans) w a s u s u a l l y collected just before or after
the analyte peak.
Results and Discussion
T h e c h r o m a t o g r a m obtained for linolenic a c i d m e t h y l ester
( c 9 , c l 2 , c l 5 - 1 8 : 3 ) a n d its i s o m e r i z a t i o n products exhibited five
p e a k s labeled A , B , C , D , a n d ( F i g u r e 2 a ) , f o u r o f w h i c h
( B - E ) h a d the same retention times as those f o u n d for the various
h y d r o g e n a t e d s o y b e a n o i l s ( F i g u r e s 2 b - 2 d ) a n d the m a r g a r i n e
a n a l y z e d . T h e c h r o m a t o g r a m s i n F i g u r e 2 s h o w that p e a k D e x
hibits a shoulder, is broader than the other triene p e a k s , a n d thus
is p r o b a b l y the result o f m o r e than o n e analyte. A listing o f re
tention times o b s e r v e d at 1 8 5 C for these f i v e F A M E p e a k s is
g i v e n i n Table I. T h e o b s e r v e d retention time for peak matched
that o f the m e t h y l linolenate standard. T h e i s o m e r i z a t i o n o f cis
fatty a c i d s w i t h P T S A d o e s not alter the p o s i t i o n o f d o u b l e
b o n d s ( 1 8 ) . H o w e v e r , the catalytic hydrogenation o f oils converts
the c o n f i g u r a t i o n o f s o m e o f the d o u b l e b o n d s f r o m cis to trans
a n d leads to the formation o f positional i s o m e r s i n w h i c h the
d o u b l e b o n d p o s i t i o n s m a y n o l o n g e r be the s a m e a s i n the
starting materials. H e n c e , e a c h G C p e a k for h y d r o g e n a t e d s o y
b e a n oil ( F i g u r e 2 ) or m a r g a r i n e m a y p o s s i b l y be due to t w o or
It is also p o s s i b l e that t w o geometric i s o m e r s m a y coelute.
U p to four triene F A M E G C p e a k s (labeled B , C , D , a n d E )
were obtained for the different s o y b e a n oils ( F i g u r e 2 , Table I )
a n d the margarine a n a l y z e d . M e t h y l linolenate (peak E ) w a s o b
tained for s o y b e a n o i l s that were h y d r o g e n a t e d to i o d i n e v a l u e s
o f 1 2 3 a n d 111 ( F i g u r e s 2 b a n d 2 c , respectively) a n d for m a r
garine. It w a s n o l o n g e r f o u n d ( F i g u r e 2 d ) i n a m o r e h i g h l y h y
drogenated s o y b e a n oil (iodine v a l u e 9 6 ) . P e a k w a s absent
f r o m the c h r o m a t o g r a m o f the m a r g a r i n e . O f the F A M E triene
p e a k s , o n l y B , C , a n d D were c o m m o n to the c h r o m a t o g r a m s o f
all four test s a m p l e s s h o w n i n F i g u r e 2 .
I R spectra obtained for G C p e a k s A - E ( F i g u r e 3 , Table I I )
were consistent w i t h triene F A M E structures. Inspection o f these
spectra revealed g r a d u a l variations i n spectral features between
adjacent pairs. Specifically, the relative intensity o f the cis = C H
out-of-plane deformation vibrations increased f r o m spectra A to
E , w h e r e a s that o f the trans = C H out-of-plane deformation v i
brations decreased. T h e s e trends are consistent with a progressive
increase i n cis character a n d decrease i n trans character, a n d
they s u g g e s t that p e a k s A a n d are due to tri-trans (ttt) a n d tri
cis (ccc) 1 8 : 3 F A M E i s o m e r s , respectively. T h e intermediate
peaks B , C , a n d D are consistent w i t h triene species h a v i n g both
cis a n d trans configurations. T h e I R spectrum for peak
m a t c h e d that o f the m e t h y l linolenate standard. T h e s e results
demonstrate that G C - M I - F T - I R c a n discriminate between g e o
metric i s o m e r s .
G C p e a k s a n d D were attributed to F A M E s w i t h di-trans
mono-cis (ttc) a n d mono-trans di-cis (tcc) F A M E s , respectively,
b a s e d o n the intensity o f the = C H out-of-plane deformation v i
bration for a trans g r o u p , relative to that o f the c a r b o n y l b a n d
(Table I I ) . It i s noted that the m e t h y l ester o f e i c o s e n o i c a c i d
( 2 0 : 1 ) , a m i n o r constituent o f s o y b e a n oil ( 1 9 ) , coeluted w i t h the
p e a k D species u n d e r o u r experimental c o n d i t i o n s . T h i s w a s
c o n f i r m e d b y coinjecting the 2 0 : 1 standard w i t h the test samples.
T h u s , for margarine the I R spectrum for peak D w a s obtained b y
spectrally subtracting the contribution ( 2 0 % ) o f the 2 0 : 1 F A M E
standard f r o m the spectrum observed at the retention time o f peak
D . T h e difference spectrum matched that o f p e a k D obtained for

Table I. Retention Time and Normalized Area % for C Triene and Conjugated Diene FAME GC Peaks 1 8

Normalized area %

Hydr ogenated Soybean Oil

FAME Retention Margarine

GC peak configuration time*
I.V. 91 I.V. 123 I.V. 111 I.V. 96

A ttt 15.95 ** **

B
C
ttc
ttc + tcc
16.39
16.61
0.3
0.7
0.5
0.5
0.2
D tcc 17.03 0.2 0.9 0.6
ccc 17.26 1.5 3.2 0.4
F conj c-t 17.62 0.2 0.4 0.4
G conj c-t 17.95 0.2 0.3 0.3
conj t,t 18.78 0.2 0.4 1.1 0.3

* 0 . 0 1 min at 185C.
Average of triplicate runs; average C V w a s 1 2 % .
Normalized area % for methyl linolenate in refined s o y b e a n oil (I.V. 131) w a s 6 . 1 ,
w h e r e a s that of 22:0 (GC peak I) w a s 0.3 in all the test samples analzyed.
I.V. = iodine value.
* * O b s e r v e d by IR; see text,
Less than 0 . 1 .
tcc only.

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 Journal of Chromatographic Science, Vol. 29, August 1991

1
Table II. Postion (cm ) and Relative Intensity* of
Characteristic MI-FT-IR Bands for 18:3 FAME
-1
Isomers at 4 c m Resolution

FAME =CH out-of- plane def.

GC peak =CH str. CH str. C=0 str. trans cis

A 3035t 2937 1754 972

(0.12) (0.59) (1.00) (0.58) ()

CD

3020 2938 1753 972 730
(0.16) (0.63) (1.00) (0.44) (0.15)
C 3020 2938 1753 971 729
(0.25) (0.67) (1.00) (0.26) (0.18)
D 3019 2938 1753 970 728
(0.27) (0.69) (1.00) (0.19) (0.20)
3019 2938 1752 721
(0.44) (0.74) (1.00) () (0.24)

* In parentheses.
- 1
A shoulder at 3 0 0 5 c m w a s also observed.
1
Broad because of shoulders at 3 0 3 5 a n d 3 0 0 5 c m .

Figure 4. IR spectra (a) for soybean oil test sample hydrogenated to an io

1
dine value of 96, obtained at the retention time of peak A by spectral sub
Figure 3. IR spectra at 4 cm resolution acquired by coadding 300 scans
traction (see text), and (b) for the ttt species of GC peak A obtained for iso-
(2 min 43 s) for 18:3 FAME isomers that gave rise to GC peaks A - E . Po
merized methyl linolenate.
sition and relative intensity for selected bands are given in Table II.

i s o m e r i z e d m e t h y l linolenate standard. T h e fact that spectral
subtraction w a s not required for the h y d r o g e n a t e d s o y b e a n oils
investigated implies that the level o f 2 0 : 1 w a s negligible i n these
materials.
T h e intensity o f the trans b a n d at 9 7 1 c m - 1
for the G C p e a k C
species i n i s o m e r i z e d m e t h y l linolenate a n d i n the h y d r o g e n a t e d
s o y b e a n o i l s fell w i t h i n the r a n g e o f v a l u e s f o u n d for species
a n d D , s u g g e s t i n g the presence o f a m i x t u r e o f triene i s o m e r s
h a v i n g ttc a n d tec configurations. T h e coelution o f these t w o g e
ometric i s o m e r s is consistent w i t h the results o f G r a n d g i r a r d et
al. ( 2 0 ) a n d R a t n a y a k e a n d B e a r e - R o g e r s ( 5 ) , w h o i s o m e r i z e d
linolenic acid a n d characterized the reaction products. It is noted
that for the m a r g a r i n e a n a l y z e d , the I R e v i d e n c e o b s e r v e d for
p e a k C is consistent w i t h the presence o f o n l y tcc species.
B e c a u s e the i s o m e r i z a t i o n reaction o f c 9 , c l 2 , c l 5 - 1 8 : 3 p r o
duces triene i s o m e r s w i t h double b o n d s at carbon positions 9 , 1 2 ,
a n d 15 ( 1 8 , 2 0 ) , the fact that triene F A M E retention times o b
tained for h y d r o g e n a t e d s o y b e a n oil a n d m a r g a r i n e were the
s a m e as those f o u n d for i s o m e r i z e d m e t h y l linolenate p r o b a b l y
indicates that the m o s t abundant triene h y d r o g e n a t i o n products
f o u n d i n these materials are a l s o unsaturated at C , C 9 1 2 , and C 1 5 .
P e r k i n s a n d S m i c k (4) tentatively identified three triene F A M E

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Figure 6. IR spectra found for GC (a) peaks F and G (conjugated cis-trans

18:2), and (b) peak (conjugated trans,trans 18:2).

Figure 5. Expanded IR spectral range showing the CH stretch bands ob

served for a hydrogenated soybean oil test sample (iodine value 111).
From top to bottom, the spectra are attributed to the following configu
-1
rations, respectively: ccc (3019 c m , GC peak E), tcc (GC peak D), ttc(GC
-1 1
peak B), and ttt (3035 cm and a shoulder at 3005 cm- ). The bottom
spectrum, observed for the ttt species of GC peak A obtained for isomer
ized methyl linolenate, is shown for comparison. Figure 7. Expanded CH stretch region observed for conjugated (a)
cis-trans, and (b) trans,trans 18:2 FAMEs.

i s o m e r s o f c 9 , c l 2 , c l 5 - 1 8 : 3 for a partially h y d r o g e n a t e d s o y
b e a n oil f r o m c o m m e r c i a l s a l a d oil as t9,tl2,cl5-18:3, G C p e a k A ( F i g u r e 2 a ) for i s o m e r i z e d m e t h y l linolenate,
c 9 , c l 2 , t l 5 - 1 8 : 3 , a n d t 9 , c l 2 , c l 5 - 1 8 : 3 . P r o b a b l e triene structures w h i c h exhibited a n I R s p e c t r u m attributed to a ttt-l8:3 FAME,
w i t h di-trans mono-cis a n d mono-trans di-cis configurations w a s not f o u n d for the hydrogenated s o y b e a n oils ( F i g u r e s 2 b - 2 d )
were reported for s o m e o f the f o u r margarine types a n a l y z e d b y or the m a r g a r i n e a n a l y z e d . U n d e r o u r experimental c o n d i t i o n s ,
R a t n a y a k e a n d B e a r e - R o g e r s (5). T h e s e configurations are c o n the retention time o f peak A ( 1 5 . 9 5 m i n ) differed b y 3 s f r o m that
sistent w i t h o u r f i n d i n g s . o f another p e a k ( 1 5 . 9 0 m i n , labeled w i t h asterisk i n F i g u r e 2 ) o b -

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Journal of Chromatographic Science, Vol. 29, August 1991
Figure 8. Expanded IR spectral range showing the out-of-plane deforma
tion bands for conjugated (a) cis-trans, and (b)trans,trans18:2dienes.
Spectra for methylene-interrupted (c) cis-trans, and (d) trans,trans 18:2
dienes are shown for comparison.
s e r v e d f o r all the test s a m p l e s a n a l y z e d . T h i s G C p e a k w a s due
to t w o c o e l u t i n g s p e c i e s : a r a c h i d i c a c i d ( 2 0 : 0 ) m e t h y l ester,
w h i c h i s a m i n o r c o m p o n e n t o f s o y b e a n o i l ( 1 9 ) , a n d a n unsat
urated c,c-18:2 F A M E . A n I R search o f the a r g o n matrix at the
k n o w n retention time o f p e a k A ( 1 5 . 9 5 m i n ) revealed the p r e s
ence o f trans species f o r t w o o f the h y d r o g e n a t e d s o y b e a n oils
( w i t h i o d i n e v a l u e s o f 111 a n d 9 6 ) . Spectral subtraction (factor
0.4) o f the spectrum obtained at 1 5 . 9 0 m i n f r o m the one obtained
at the retention time o f p e a k A y i e l d e d the difference s p e c t r u m
s h o w n i n F i g u r e 4 a . T h e difference s p e c t r u m w a s characteristic
- 1
o f a ttt-18:3 species (e.g., unique b a n d at 3 0 3 5 c m ) . T h e spectra
presented i n F i g u r e 5 are further evidence that GC-MI-FT-IR
c a n p r o v i d e structural specificity f o r geometric i s o m e r s i n c o m
plex mixtures.
F o u r additional G C peaks ( F - I ) were observed i n chro
matograms o f hydrogenated soybean oil (Figures 2 b - 2 d ) a n d
m a r g a r i n e (Table I ) . T h e c o m p o u n d s that g a v e rise to the first
three o f these G C peaks exhibited I R spectra ( F i g u r e s 6 - 8 ) char
acteristic o f conjugated structures. S u b t l e differences i n the p o
sitions a n d relative intensities o f the w e a k = C H stretching v i
bration b a n d s i n conjugated F A M E s are s h o w n i n F i g u r e 7. T h e
- 1 - 1
bands at 9 8 6 and 9 5 0 c m (trans) and 7 3 1 c m (cis) (Figure 8a)
were attributed to conjugated cis-trans 1 8 : 2 ( G C peaks F a n d G ) ,
- 1
a n d the b a n d at 9 9 0 c m ( F i g u r e 8 b ) w a s d u e to conjugated
trans,trans 1 8 : 2 ( G C p e a k H ) fatty a c i d species ( 2 1 ) . C o n j u
gated fatty acids i n margarine a n d butter were determined b y u l
traviolet s p e c t r o s c o p y ( 9 ) . T h e c o m p o u n d that eluted last ( G C
p e a k I ) w a s determined to be 2 2 : 0 F A M E because its o b s e r v e d
s p e c t r u m exactly m a t c h e d that o f a standard.
P o r k fat ( 2 2 ) , fried g r o u n d beef, a n d dairy products ( 2 3 , 2 4 )
also contain c o n j u g a t e d octadecadienoic a c i d i s o m e r s . T h e s e
species accumulate i n the depot fats a n d cardiac triglycerides o f
h u m a n s a n d other primates ( 2 2 ) , a n d h a v e b e e n isolated f r o m
h u m a n s e r u m , b i l e , a n d d u o d e n a l j u i c e s ( 2 5 ) . O t h e r dietary
sources s h o u l d be investigated f o r conjugated octadecadienoic
acid i s o m e r s , because these c o m p o u n d s h a v e been reported to be
anticarcinogens a n d antioxidants ( 1 0 , 2 6 ) .
Conclusions
Capillary G C - M I - F T - I R p r o v e d to b e a p o w e r f u l h y p h e n
ated technique f o r rapidly identifying m i n o r C 1 8 triene a n d c o n
jugated diene fatty a c i d m e t h y l esters i n h y d r o g e n a t e d s o y b e a n
oil a n d margarine. C o m p u t e r i z e d spectral subtraction, or prefer
ably the u s e o f t w o - d i m e n s i o n a l G C , c a n further enhance the c a
pability o f this technique. W h e n standards are available,
G C - M I - F T - I R c a n be u s e d to quantitate the levels o f F A M E s i n
c o m p l e x mixtures ( 1 1 ) .
The C 1 8 triene F A M E constituents o f partially h y d r o g e n a t e d
s o y b e a n o i l were dependent o n h y d r o g e n a t i o n conditions. A t an
iodine value o f 1 1 1 , methyl linolenate and its tri-trans, di-trans
mono-cis, and mono-trans di-cis geometric isomers were identi
fied. A t a higher iodine value o f 1 2 3 , the tri-trans i s o m e r w a s not
o b s e r v e d , whereas at a l o w e r iodine value o f 9 6 , tri-cis m e t h y l
linolenate was no longer found. -cis and mono-trans di-cis con
figurations were f o u n d f o r the margarine a n a l y z e d . D o u b l e b o n d
positions i n 1 8 : 3 i s o m e r s are probably the same as i n c 9 , c l 2 , c l 5 -
18:3. T h e identities o f conjugated cis-trans and trans,trans C 1 8
diene F A M E s a n d 2 2 : 0 F A M E were c o n f i r m e d i n the margarine
and h y d r o g e n a t e d s o y b e a n oils a n a l y z e d .
Acknowledgment
T h e authors thank D r . D . F i r e s t o n e f o r m a n y helpful c o m
ments a n d K . F a r r e n f o r technical assistance.
329

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