SHORT COMMUNICATION

Gas Chromatographic Separation of Wax Esters Based on the Degree of Unsaturation

ABSTRACT

The wax esters of sperm whale head oil have been characterized by gas-liquid chromatography on an APOLAR 10C column according to their carbon number and number of double bonds. The novel technique permits the direct quantitative analysis of saturated and unsaturated wax esters.

INTRODUCTION

Introduction of the new thermally stable cyanosiloxane liquid phase has permitted gas- hquid chromatography (GLC) of lipid corn-

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FIG. 1. Gas-liquid chromatographic resolution of wax esters in sperm whale head oil on APOLAR 10C. (A) Total wax esters. (B) Saturated wax esters. (C) Monoenoic wax esters. (D) Dienoic wax esters. The fractions B, C, and D were obtained by AgNO3-thin layer chromatography of the original wax esters.

ponents of higher mol wt based on their degree of unsaturation as indicated in the analysis of diacylglycerols (1). This report describes an application of this technique to the analysis of wax esters.

Usually, GLC of wax esters has hitherto been performed on a nonpolar column, and the results have been presented in the compositions based on the carbon number only (2-6). The analysis of wax esters based on their carbon number and degree of unsaturation has been reported in a few papers: open-tubular GLC of short chain wax esters of carbon numbers < 18 in the jaw fat of the Atlantic bottle-nosed dolphin (7); GLC with a nonpolar column on saturated, monoenoic, and dienoic fractions obtained by AgNO3-silicic acid column chroma- tography of wax esters in sperm whale head oil (8); and GLC of wax esters in some lantern fish lipids on a diethylene glycol succinate polyester column (9).

This paper presents a simple GLC technique for the resolution of saturated and unsaturated wax esters having carbon numbers up to 40 on a thermally stable polar column.

EXPERIMENTAL PROCEDURES

Wax esters were obtained by silicic acid column chromatography of the head oil of the sperm whale captured at the Antarctic Ocean. The GLC packing material containing 5% APOLAR 10C on Gas Chrom Q (100-120 mesh) was obtained from Applied Science Laboratories (State College, PA). GLC was carried out with a Yanagimoto GC-80 instru- m e n t e q u i p p e d with dual glass columns (150 cm length, 4 mm inside diameter) and a flame ionization detector. The columns were conditioned at 260 C for 10 hr. Detector and injection heaters were operated at 280 C. The colunm temperature was programmed from 200 to 260 C at the rate of 2 C/min. Difficulties in determining peak areas due to the abnormal shapes of the peaks were overcome by the use of a digital integrator (Shimadzu ITG-4AX). Thin layer chromatographic (TLC) separation of wax esters based on their degree of unsatu- ration was carried out by developing with benzene:hexane (1:1 v/v) on a AgNO3-silicic

354

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LIPIDS, VOL. 11, NO. 4

356 SHORT COMMUNICATION

acid (Wakogel B-10) plate. Spots were de tec ted unde r ul t raviolet light af ter spraying wi th

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RESULTS A N D DISCUSSION

The gas chromatograms of the to ta l wax esters of sperm whale head oil and the subfrac- t ions ob ta ined by AgNO3-TLC on A P O L A R 10C are shown in Figure 1. The average of the s e p a r a t i o n f ac to r s , monoene / sa tu ra t e and d iene /monoene , are 1.15 and 1.16, respect ively. In spite of the appropr ia te separat ion factors, partial overlapping of a few peaks occurs as shown in Figure I A. This can be a t t r ibu ted to the broadening of the unsa tura ted ester peaks due to small differences in the re ten t ion t imes of wax esters having the double bonds in the various posi t ions of the acid and a lcohol moieties. The double bonds are dis t r ibuted in posit ions 5, 7, 9, 11, and 13 in the acids and alcohols of the sperm whale oil (10). The composi t ions ob ta ined by GLC of the original wax esters on A P O L A R 10C and by GLC of the subf rac t ions separated by AgNO3-TLC are compared in Table I. The good agreement indi- cates the sat isfactory accuracy for direct GLC on A P O L A R 10C. It was repor ted previously that the observed compos i t ions of wax esters in spe rm whale head oil were in reasonable agreement with those calculated under the

a s s u m p t i o n o f r andom ester i f icat ion (8). R a n d o m ester i f icat ion is borne ou t by the results of the present study.

T O R U T A K A G I Y U T A K A ITABASHI T O R U OTA KENJI H A Y A S H I Depar tment of Chemis t ry Facu l ty of Fisheries Hokkaido University, Japan

REFERENCES

1. Myher, J.J., and A. Kuksis, J. Chromatogr. Sci. 13:138 (1975).

2. Tulloch, A.P., JAOCS 50:367 (1973). 3. Ibid. 49:609 (1972). 4. Richard, F.L., J.C. Nevenzel, and A.G. Lewis,

Lipids 9:891 (1975). 5. Iyengar, R., and H. Schlenk, Biochemistry 6:396

(1967). 6. Streibl, M., J. Jirousov~, and K. Str~lnsk3~, Fette

Seifen Anstrichrn. 73:301 (1971). 7. Ackman, R.G., J.C. Sipos, C.E. Eaton, B.L.

Hilaman, and C. Litchfield, Lipids 8:661 (1973). 8. Challinor, C.J., R.J. Hamilton, and K. Simpson,

Chem. Phys. Lipids 3:145 (1969). 9. Nevenzel, J.C., W. Rodegker, J.S. Robinson, and

M. Kayama, Comp. Biochen~ Physiol. 31:25 (1969).

10. Spencer, C.F., and W.H. TaUent, JAOCS 50:202 (1973).

[ Received Sep tember 12, 197 5 ]

LIP1DS, VOL. 11, NO. 4