[2] Separation of methyl esters of fatty acids by gas chromatography on capillary columns, including the separation of deuterated from nondeuterated fatty acids

8

[2]

GENERAL ANALYTICAL METHODS

[2] S e p a r a t i o n

of Methyl

Chromatography Separation

By

Esters of Fatty Acids by Gas

on Capillary Columns,

of Deuterated

Including the

from Nondeuterated

Fatty Acids

GEORGE M. PATTON, STEFANIE CANN, HENRI BRUNENGRABER, and JOHN M. LOWENSTEIN

Analyses of the composition of mixtures of fatty acid methyl esters by gas c h r o m a t o g r a p h y have been improved greatly by the introduction of capillary columns. The inner wall of such columns is coated with a thin film of stationary phase. They possess a much greater resolving power than columns packed with an inert granular material coated with the stationary phase. For a general introduction to the gas c h r o m a t o g r a p h y of methyl esters of fatty acids, the reader is referred to the article by Ackman.1 For a detailed treatment o f gas c h r o m a t o g r a p h y with glass capillary columns, the reader is referred to the book by Jennings. la Drawing of Capillaries. Soda glass tubing, 2-2.5 mm i.d., 7 mm o.d. × 120 cm (VWR Scientific) is washed with chromic acid, rinsed with distilled water, and dried, z The tube is then drawn into a capillary using a Shimadzu GDM-1 glass drawing machine. The drawing ratio is usually 64 : 1, and the oven temperature is 620-640 °.3 The temperature o f the tube coiling heater is adjusted to the highest temperature that will give a smooth coil. 4 Allowing for wastage o f the ends of the tubing, a 120-cmlong glass tube yields a 70-m capillary with an internal diameter of about 0.25 mm. Etching of Capillaries. Before coating the capillary with stationary phase, it is necessary to etch its inside. This facilitates the application of an even coat of the stationary phase and increases the amount of stationary

1 R. G. Ackman, this series, Vol. 14, [49]. la W. Jennings "'Gas Chromatographywith Glass Capillary Columns," Second edition, New York, Academic Press, 1980. 2 Soda glass (soft glass) is used in preference to Pyrex because soda glass can be etched more conveniently. 3 If the oven temperature is too hot, the internal diameter of the resulting capillary may vary, and this adversely affects column performance. If the oven temperature is too low, the capillary is brittle and breaks in the coiling tube of the machine. 4 If the temperature of the heater for coiling the capillary is too low, the finished capillary is very brittle; too high a temperature results in an irregular coil. Neither condition affects the performance of the resulting column, but either condition makes the column difficult to handle.

METHODS IN ENZYMOLOGY, VOL. 72

Copyright © 1981 by Academic Press, lnc, All rights of reproduction in any form reserved. ISBN 0-12-181972-~

[2]

SEPARATION OF METHYL ESTERS BY GAS CHROMATOGRAPHY

9

phase that will adhere to the glass. ~ The capillary is etched by passing through it a stream of HCI gas (30-40 ml/min) for 3 hr while heating the tubing in an oven at 300°. A suitable pressure of HCI gas is obtained by cooling a lecture bottle or small cylinder of HCI gas in dry ice2 After etching, the capillary column is flushed with N~ gas and washed with 10 ml of dry acetone. Since the etching procedure results in the deposition of a layer of NaCI on the wall of the capillary, it is imperative that the acetone be dry and that moisture be avoided at all stages after etching, otherwise column performance will be adversely affected. 7 Coating of Capillaries. The interior wall of the column is coated by passing a solution of the stationary phase through the column. EGSS-X and Silar stationary phases are used as 5% solutions in chloroform (w/v); BDS (butanediol succinate) is used as a 1% solution in chloroform. An amount of coating solution equivalent to-between a quarter and a third of the column volume is forced through the column with nitrogen gas at a velocity of 0.6 rn/min. This is normally accomplished with a gas pressure of 1 kg/cm z. The column is then dried with a stream of N2 for about 4 hr. The velocity at which the solution passes through the column increases when it is near the end of the column, which tends to cause uneven coating at the end of the column. This effect can be reduced by attaching a 20- to 30-m-long capillary to the end of the column. The rate of nitrogen flow should not be too high during the drying period, otherwise the stationary phase is mechanically disturbed and uneven coating results. If a nitrogen pressure much greater than 1 kg/cm 2 is required to force the coating solution through the column, the pressure must be reduced once the coating solution has left the column and the drying phase is started. During the drying there is a tendency for the solution of stationary phase to puddle at the bottom of the column. In order to minimize this tendency, the column should be packed with the plane of the coils horizontal, and it should be turned over every 5-10 min while the column is drying. After the drying has been completed, 5-10 m are broken off each end of the column and discarded. The resulting column is approximately 50 m long. The column is conditioned for 12-24 hr at the maximum operating temperature recommended by the manufacturer for the coating material, using N2 as purging gas. Polyester and other highly polar stationary Capillary c o l u m n s are easily overloaded. This problem is ameliorated by increasing the a m o u n t of stationary phase. However, column efficiency decreases s o m e w h a t with increasing a m o u n t s of stationary phase. '~ Low pressure, HCI-resistant connections can be made with standard M-inch stainless steel Swagelok fittings by wrapping Teflon tape between the front and back ferrules. r A t t e m p t s to etch c o l u m n s with a m m o n i u m bifluoride resulted in uneven etching even when the column was coated with an even layer of a m m o n i u m bifluoride. Etching can also be accomplished with HF, but this is technically more difficult. Etching ether (2-chlorol,l,2-trifluoroethylmethyl ether, obtainable from Alltech Associates, Deerfield, Illinois 60015) provides an alternative method that we have not tested.

10

GENERAL ANALYTICAL METHODS

[2]

phases are self-deactivating and generally require no special treatment to deactivate the column before coating. Operation of Columns. The capillary coil is installed in the gas chromatography apparatus by wrapping the ends with Teflon tape and using standard Swagelok or similar fittings over the taped ends. Machines specially manufactured to take capillary columns are available. The work described here was performed using a Shimadzu GC Mini 1 gas chromatography apparatus equipped with flame ionization detectors. Once installed, a capillary column can perform consistently for a year or more if care is taken to keep moisture out and if injection of impurities of low volatility is avoided. Water rapidly degrades the performance of the column both by hydrolyzing the polyester stationary phase and by destroying the NaC1 substrate. It is recommended to reverse the direction of flow through the column every few months. A clean and dry petroleum ether solution (1-5 ~1) of methyl esters of fatty acids is injected into a stream splitter (Shimadzu) which provides a split ratio between 50:1 and 100:1 depending on the resistance of the column. Nitrogen is used as carrier gas. Figures 1 and 2 show chromatograms of fatty acid methyl esters from rat liver on Silar-5 and EGSS-X columns, respectively. Table I provides the equivalent chain lengths 8 calculated from these and other chromatograms. Both columns readily separate all major components. The separation between saturated and unsaturated fatty acid methyl esters with the same carbon number is greater on the more polar column (EGSS-X), but is adequate on the less polar column (Silar-5). For a given temperature and flow rate a complete chromatogram is obtained more quickly on Silar-5 columns, because the polyunsaturated fatty acids are eluted sooner on these than on EGSS-X columns. The resolution of positional isomers of unsaturated fatty acids is about equally good on both columns. Many more minor components are evident at greater sample loads. The EGSS-X and Silar-5 columns show significant differences in the separation of minor components. Neither column separates all minor components completely and the choice of coating material will depend on the components to be separated. For example, the Silar-5 column resolves all the common fatty acid methyl esters except those of 20 : 3to9 and 20 : 2o~6, which run together with 21 : 0. On the EGSS-X column, isosaturated fatty acid methyl esters run very close to the monounsaturated esters with one s Equivalent chain length = [log R R T x - log RRTn]/[log RRT (n + 1) - log RRT n], where RRT is the relative retention time, n and (n + 1) are the saturated fatty acids that emerge immediately before and after unknown x, respectively.

l

to tO 3 0

,!

I~, ,~ iI :3 c',. 3 ; ,

oo ~

-~o i~_~ -

S'

i i;

~'i

i

:1

~o

0

o ~

t',,l

3

--

¢q

~o

co

¢b

_3 o

:22 o

oo

co 0

H

H~

~

H

FIG. I. Chromatogram of total fatty acids from the liver of a rat fed laboratory chow (Charles River Breeding Laboratories, Wilmington, Massachusetts 01887). The methyl esters were run on a glass capillary column (50 m long and 0.25 mm internal diameter) coated with Silar-5. The column temperature was 175°C and the linear flow velocity of the N 2 carrier gas was 8 cm/min. One microliter of sample was injected and the split ratio was 25: 1. The retention time for heptadecanoate was 22.0 minutes. Two separate runs are shown. In run X, the concentration o f the mixture injected was adjusted to bring all major peaks on scale; in run Y, the concentration of the mixture injected was increased about 5-fold in order to visualize minor constituents. (A) Chromatograms run through 24: lco l l . (B) Enlargement o f the same chromatograms from the solvent peak through 18: 3os6. Peaks in the interval marked by an asterisk (*) include the following methyl esters, listed with increasing retention times: trailing shoulder, 18: los5; first distinct peak, 18: los3; small bump, 18 : 2m?; second distinct peak, 18 : 2os9.

3

~D 3 ~1"

I%1

0

0¢

-c2~

t ~

.

/,

0 oo Od C~l Od

X

y

~ o

,~

o

~

~o ~

o

..

N

~I"

~

o,1

L__

-\

3 --

23

o o

t'M

/

~,.fi~

Ll-.

0

O0

~

0

H

H,~

__H

~

&

FIG. 2. C h r o m a t o g r a m of the s a m e mixture of fatty acid methyl esters s h o w n in Fig. 1 on a glass capillary c o l u m n (50 m long a n d 0.25 m m internal diameter) coated with EGSS-X. T h e c o l u m n t e m p e r a t u r e w a s 160°C and the linear flow velocity of the N 2 carrier gas was 10 cm/min. O n e microliter of sample was injected and the split ratio w a s 25 : I. T h e retention time for heptadecanoate w a s 22.7 minutes. Two separate r u n s are s h o w n . In run X, the concentration o f the mixture injected w a s adjusted to bring all major peaks on scale; in r u n Y, the concentration o f the mixture injected was increased about 5-fold in order to visualize minor constituents. (A) C h r o m a t o g r a m s run t h r o u g h 22 : 6to3. (B) E n l a r g e m e n t o f the s a m e c h r o m a t o g r a m s from the solvent peak t h r o u g h 18:3to6. Peaks in the interval m a r k e d by an asterisk (*) include the following methyl esters, listed with increasing retention times: first distinct peak, 18 : Ira5 a n d isol9 : 0; small b u m p following first peak, 18 : 2to?; twin peak, anteisol9: 0, 18: ltu3, 18: 2co9.

[2]

SEPARATION OF METHYL ESTERS BY GAS CHROMATOGRAPHY

13

carbon atom less. Monounsaturated esters of the co7, co6, and co5 families do not resolve from the isosaturated esters. However, monounsaturated esters of the to8 and higher families are resolved. Two different approaches are available for complete resolution of all components. One is to use two columns of different polarity. The other is to make use of a peculiarity of the EGSS-X column, namely, that on this column the equivalent chain lengths of the polyunsaturated fatty acid methyl esters change significantly with temperature (Table I). Thus resolution of different components can be achieved on the same column at different temperatures. The Silar-5 column does not manifest this property. Quantitation. Capillary columns may not have the same response correction factor for all fatty acid methyl esters. With the columns described here, the response correction factor increases with increasing carbon number (Table 2, top). The number of double bonds, on the other hand, does not affect the response correction factor appreciably (Table 2, bottom). The response correction factor diminishes somewhat at higher temperatures and at slower flow rates, but these effects are marginal. For any particular column there is a 100- to 1000-fold range of concentrations over which the response correction factors are reproducible and almost insensitive to concentration. Outside of this range quantitation becomes unreliable. The high efficiency of capillary columns, which results in narrow peaks, generally makes it impractical to determine areas of peaks on a complex chromatogram by triangulation or peak height measurements. Areas of peaks are best determined with a mechanical or digital integrator. Separation of Deaterated from Nondettterated Fatty Acids. A specialized application of capillary gas chromatography of fatty acid methyl esters is the measurement of the rates of fatty acid biosynthesis by incorporation of deuterium from D20. 9'1° Silar-5, Silar-10, EGSS-X, and BDS coated capillary columns all separate deuterated fatty acid methyl esters from the corresponding nondeuterated species. At low temperatures, fatty acid methyl esters with as few as three deuteriums can be resolved from the corresponding nondeuterated compounds. Figures 3-6 show chromatograms o f total fatty acids from livers that had been perfused with D20 for 2 hr. J. M. Lowenstein, H. Brunengraber, and M. Wadke, this series, Vol. 35 [34]. ~0 G. M. Patton and J. M. Lowenstein, Biochemistry 18, 3186 (1979). 11 The following notation is used: to7, to9, etc., position of first double bond counted from highest n u m b e r e d carbon atom; I, iso; A, anteiso; t, trans; and D, deuterated. Retention times are quoted from the time of injection to the front tangent of the peak.

A

el)

O

~2

Y

~

o~ 3 ~

0

~\ ~ o N

OJ

~_~2

, I

on

,i Y OO

O

O0

~0

Q,,~

H

I...4~

~_..H

O~

FIG. 3. Silar-5 c o l u m n c h r o m a t o g r a m s of total fatty acids from a rat liver that had been perfused with 70% D20 for 2 hr '° (liver 839). The donor o f the liver was fed laboratory chow. T h e methyl esters were run u n d e r the conditions described in the legend to Fig. 1. Two separate r u n s are shown. In run X, the a m o u n t of mixture injected was adjusted to bring all major peaks on scale; in run Y, the a m o u n t o f mixture injected was increased 5.5-fold in order to visualize minor constituents. (A) C h r o m a t o g r a m s through 22 : 6to3 (retention time 147.8 rain); (B) an enlargement o f the s a m e c h r o m a t o g r a m s from the solvent peak to 18 : 3(o3 (retention time 37.8 min). '1 Peaks in the interval m a r k e d by an asterisk (*) include the following methyl esters, listed with increasing retention times: 18: lto5, 18: lto3, 18: 2to?, 18 : 2to9, 18 : 2to7. Internal s t a n d a r d s of 15 : 0 and 17 : 0 were added at different stages of the isolation procedure.

[2]

SEPARATION

OF METHYL

~

ESTERS

BY GAS CHROMATOGRAPHY

15

00~

3

P~ ~

0

J :3

.3

3

~

oOo

Od

6

^

~_

3

3

t%l

0

0

~

I°

0

~"

0

~--

×

.

_

Y

i

II

I c~ d5

db -

.-

~ 0~

- - - -

m ~

FIG. 4. Silar-5 c o l u m n chromatograms of total fatty acids from a rat liver that had been perfused with 70% DzO for 2 hr. The donor of the liver was fed a fat-free, high-carbohydrate diet for 2 w e e k s 1° (liver 608). In run X, the amount injected was adjusted to bring all major peaks on scale; in run Y, the amount injected was increased 7.5-fold in order to visualize minor constituents. For other details see legend to Fig. 3. The peak marked with an asterisk (*) contains D I 8 : R o 9 and trans-18: 1~o7.H An internal standard of 15:0 was added during the isolation procedure.

16

GENERAL

ANALYTICAL

TABLE

METHODS

[2]

I

E Q U I V A L E N T C H A I N L E N G T H S OF U N S A T U R A T E D A N D S O M E ISO A N D A N T E I s o F A T T Y A C I D M E T H Y L ESTERS ON C A P I L L A R Y C O L U M N S C O A T E D W I T H S I L A R - S C P AND EGSS-X a Fatty acid methyl

ester

Silar-SCP 200 °

EGSS-X 160 °

EGSS-X 185 °

14:lco9

14.33

--

--

14 : lro5

14.52

14.55

14.48

14:lto3 I 15 : 0 A 15 : 0

14.63

--

--

14.63

14.55

14.48 14.72

14.78

14.73

15.48

--

--

15.58

15.55

15.47

16: Itoll

--

16.30

--

16 : lto9 16 : lto7

16.33 16.40

16.47 16.50

16.50 16.57

16 : lto3

16.61

16.73

16.86

16:2o>5

--

17.14

17.18

I 17:0 A 17 : 0 17:loJ9

--

16.64

--

16.76

16.73

16.72

17.33

17.38

--

17:1(o8

17.41

17.44

17.50

17:lto5

17.51

--

--

17.60

17.57

17.50

18.33

18.40

18.40

--

18.27

--

18 : loJ7

18.42

18.48

18.45

18:loJ5

--

18.60

18.66

1 8 : Ito?

--

18.67

--

18 : 1(o3 18 : 2to9

18.56 18.66

18.74 18.91

18.90 --

18 : 2(o?

18.70

18.83

--

18 : 2to7

18.80

19.00

19.08

18 : 2t06

18.85

19.06

19.17

18 : 2to5

--

19.15

19.25

18 : 3to7 18 : 3to6

18.95 19.18

19.37 19.56

-19.73

19.44

19.91

20.08

--

18.60

18.52

18.78

18.74

18.74

19.27

19.32

19.39

19.37

19.40

19.41

19.87

20.25

--

20.31 20.33

20.28 20.33

20.36 20.40

20.42 20.50 20.60

20.43 ---

20.49 ---

20.65 20.75

---

---

20.83

20.96

21.12

15 : 1co5 I 16 : 0

I 18:0 18 : I ~ 9 t 18:1co7

18 : 3to3 I 19:0 A 19:0 19 : l t o l 0 19 : 1co8 19 : 3(o8 20:1o~10 20 : lto9 20 : lto7 20 : 2 w ? 20 : 2to? 20 : 2oJ9 2 0 : 2o>7 20 : 2o~6

[2]

SEPARATION OF METHYL ESTERS BY GAS CHROMATOGRAPHY

17

TABLE I (continued) Fatty acid methyl ester 20 : 3609 20: 3w? 20 : 360? 20 : 3(o6 20 : 4
Silar-5CP 200° 20.76 20.83 20.99 21.08 21.20 21.28 21.73 21.87 23.30 23.40 23.88 24.03 24.22

EGSS-X 160°

EGSS-X 185°

21.12 -21.24 21.40 -21.77 -22.60 23.67 24.05 24.50 24.90 24.25

21.31 -21.46 21.60 -22.00 -22.91 23.94 24.43 24.89 25.33 24.20

The following notation is used: 097,609,etc., position of first double bond counted from highest numbered carbon atom; I, iso; A, anteiso: t, trans. T h e m e t h y l e s t e r s of d e u t e r a t e d 1 4 : 0 , 1 6 : 0 , a n d 1 8 : 0 all s e p a r a t e readily from the c o r r e s p o n d i n g n o n d e u t e r a t e d species. R e s o l u t i o n of d e u t e r a t e d 1 6 : k o 7 f r o m the n o n d e u t e r a t e d 1 6 : 0 p r e s e n t s some difficulties, b e c a u s e highly d e u t e r a t e d 16:loJ7 r u n s into the p r e c e d i n g , n o n d e u t e r a t e d 16 : 0 on m o d e r a t e l y p o l a r c o l u m n s , such as Silar-5 a n d B D S (see Figs. 4B a n d 6B). H o w e v e r , these species s e p a r a t e c l e a n l y from all o t h e r p e a k s on v e r y p o l a r c o l u m n s , such as Silar-10 a n d E G S S - X . At m o d e r a t e d e u t e r i u m c o n t e n t s , d e u t e r a t e d 16:loJ7 does not s e p a r a t e f r o m 16: l o 9 , a n d d e u t e r a t e d 1 8 : k o 9 is partially o b s c u r e d b y a small trans-18: loJ9. At low d e u t e r i u m c o n t e n t s , d e u t e r a t e d 16 : loJ7 falls b e t w e e n 16 : ko9 a n d ~o7 a n d c a n be q u a n t i t a t e d . This is o b s e r v e d m o s t clearly w h e n the c o l u m n t e m p e r a t u r e is l o w e r e d to 160°, w h e n significantly i m p r o v e d r e s o l u t i o n o f the m a j o r d e u t e r a t e d c o m p o n e n t s is a c h i e v e d . H o w e v e r , o n e o f the c o n s e q u e n c e s of l o w e r i n g the t e m p e r a t u r e is that the p e a k s b r o a d e n greatly a n d m i n o r c o m p o n e n t s fade into the b a s e l i n e . O n highly efficient c o l u m n s d e u t e r a t e d 18 : ko9 resolves f r o m n o n d e u t e r a t e d 18 : ko9. (See Silar-5 colu m n , Fig. 4B. N o t e that trans- 18 : 1oJ7, w h i c h r u n s with d e u t e r a t e d 18 : 1oJ9, is n o r m a l l y a v e r y m i n o r c o m p o n e n t . ) At the l e a d i n g edge of 18 : 1609, t h e r e is a small p e a k o f 18:!o~10 that o v e r l a p s with the b a c k of d e u t e r a t e d 18 : 1o)9. Acknowledgments We wish to thank Dr. R. G. Ackman for many helpful comments. This work was supported by a grant from the National Institutes of Health (AG00120).

18

GENERAL ANALYTICAL METHODS

[9.]

t,D

0IM /

~

~

~

F)

~®__

0~ ID

~_

.°

x

B

rl ~_ o ~b o

3- 3

~

H

aa

:¢:*:

_a

FIG. 5. E G S S - X c o l u m n c h r o m a t o g r a m s o f total fatty acids from a rat liver that had been perfused with 70% D20 for 2 hr (liver 839). The donor of the liver was fed laboratory chow. T h e methyl esters were run u n d e r conditions described in the legend to Fig. 2. Two separate runs are shown. In run X, the a m o u n t o f mixture injected was adjusted to bring all major peaks on scale; in r u n Y, the a m o u n t of mixture injected was increased 7-fold in order to visualize minor constituents. (A) C h r o m a t o g r a m s through 2 2 : 6 ~ 3 (retention time 195.2 min); (B) an e n l a r g e m e n t o f the s a m e c h r o m a t o g r a m s from the solvent peak to 18:3to6 (retention time 40.4 min).;; Peaks in the intervals m a r k e d with asterisks include the following

[2]

19

SEPARATION OF M E T H Y L ESTERS BY GAS C H R O M A T O G R A P H Y

T A B L E II RESPONSE CORRECTION FACTORS IN RELATION TO NUMBER OF CARBON ATOMS AND DOUBLE BONDS a'b

Number of carbons

RCF" EGSS-X

Silar-5CP

14:0 16:0 18:0 20:0 22:0

0.860 0.920 1.000 1.200 1.350

0.785 0.860 1.000 1.200 1.400

Number of double bonds 20:0 20:1w9 20:2~6 20:3~3

RCF EGSS-X 1.000 0.966 0.967 1.057

Silar-5CP 1.000 0.964 0.956 1.014

The detector response for each c o m p o n e n t on the c h r o m a t o g r a m is corrected relative to a reference c o m p o u n d which is set to have a response correction factor (RCF) o f 1.000. A calibration run is conducted with known a m o u n t s of c o m p o u n d s a, b, c, etc., and reJ~ the reference c o m p o u n d . a m o u n t of a area o f r e f RCF~ × area of a amount ofref The a m o u n t of a relative to r~f is given by A m o u n t of a

RCF x area a

The absolute a m o u n t of each peak can be calculated if the reference c o m p o u n d that is added as an internal standard (is) is a s u b s t a n c e not present in the mixture. Then a m o u n t of is area of is T h e c o l u m n s used were glass capillaries (50 m long × 0.25 m m i.d.) coated with Silar-5 or EGSS-X. In the case of the Silar-5 column, the temperature was 190°: the injector a n d detector temperature was 250 °. In the case of the E G S S - X column, the temperature was 185°; the injector and detector temperature was 250 °. Nitrogen was used as carrier gas at a linear flow velocity of 0.5 cm/sec. The injector split ratio was 67: I. Commercially available standard mixtures of methyl esters of fatty acids were used (Nu-Check-Prep, Elysian, Minnesota). Standard mixture A contained c o m m o n saturated fatty acids; standard mixture B contained the C20 acids s h o w n above. Four different dilutions were used, which contained 10, 1.0, 0.1, and 0.03 mg/ml of each component. Each concentration was run in triplicate. Peak areas were m e a s u r e d with a Spectra-Physics Model I computing integrator. c Occasionally we have had RCFs very close to one for all fatty acid methyl esters between 14 : 0 and 22 : 0: however, the values s h o w n are more representative. The frequency of determining R C F s is dictated by experience. Typically, R C F s m a y be determined every 3 days. Amount ofa = RCF x areaa ×

methyl esters, listed with increasing retention times: *, D I 6 : 1co7, 16: ko9, 16: ko7, iso17:1, 16:ko5; **, 16:1oJ3, anteiso-17:0, 16:2¢09?; ***, iso-19:0 and 18:ko5, 18:2oJ?; ****, anteiso-19 : 0, 18 : 1o93, 18 : 2co9 (all in the biggest of these peaks), 18 : 2xo?, and 18 : 2o~?. Internal s t a n d a r d s o f 15:0 and 17:0 were added at different stages of the isolation procedure.

A

0 --0~

if),

i,l, 00

_¢

tO

3333 ~ ?

0

0

0

' !

..

i

.. 0

3 3 ro~'~ 0

o

/

j

N

__.A_

X

i ! I

J

_Jl

il i

Y

X Y I I

I

O

O

3 tO

tO

tO

~

eJ t%l

& 0d

~

....

: aD --

b3

O

3 O

Oo

°T

J

x

)

_

:*:

:g

:g

~t

•

~:*:

_

....

H

)g F1G. 6. E G S S - X column chromatograms of total fatty acids from a rat liver perfused with 70% D=(/for 2 hr (liver 608). The donor of the liver was fed a fat-free, high carbohydrate diet for 2 w e e k s prior to the perfusion experiment. In run X, the amount injected was adjusted to bring all major peaks on scale; in run Y, the amount injected was increased 5-fold in order to visualize minor constituents. For other details see legend to Fig. 5. H Peaks in the intervals marked with asterisks include the following methyl esters listed with increasing retention times: (A) *, D18 : lto9 and trans-18 : lto7. (B) *, iso-15 : 0, 14 : lto5, 14 : lto?, anteiso-15 : 0; **, anteiso 17:0, 16:2to9; ***, 17:1c09, 17:1to8, 17:1oJ6, iso-18:0; ****, i s o - 1 9 : 0 and 18 : 1o~5, 18 : 2to?; *****, anteiso-19 : 0 and 18 : 1c03 and 18 : 2t09. An internal standard of 15 : 0 was added during the isolation procedure.