Separation of Lipides by Gas-Liquid Chromatography S. R. LIPSKY, R. A.
LANDOWNE, and J. E. LOVELOCK
Department o f Internal Medicine, Yale University School o f Medicine, New Haven, Conn.
b A gas-liquid chromatographic method which utilizes an ionization detection system and high efficiency capillary columns has been developed for the analysis of complex mixtures containing long-chain saturated and unsaturated acid esters. The theoretical plate efficiency of certain capillary columns approached 1000 plates per foot. The separation of certain cistrans isomers-i.e., methyl elaidate from methyl oleate-was accomplished. The minimum quantity of organic vapor detectable was approximately 1 O - l j mole.
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technique of gas chromatography has made possible, for the first time, the convenient separation, isolation, and analysis of certain complex mixtures of fatty acids of biological origin ( 3 ) . Conventional packed columns coated with relatively nonpolar heat-stable hydrocarbon or silicone compounds were employed in early studies concerned with the composition of substances containing long-chain saturated and unsaturated fatty acid methyl esters. Under these circumstances, the more polar componentsLe., fatty acids that contained the greatest degree of unsaturationemerged from the column before the corresponding saturated acid ester of the same carbon chain length. Certain of the C-18 series of esters were satisfactorily separated only when many of the operational parameters involved in the technique of high temperature gas chromatography were stringently controlled. The recent introduction of a new class of stationary liquids has greatly simplified the resolution of closely reljted long-chain acid esters (6, 6 , 13) and has extended the range of analysis to include compounds containing 26 or more carbons (4, 7 ) . Following the screening of a variety of polar polymers synthesized under varying laboratory conditions, it became possible to draw some degree of correlation between the nature of the chemical composition of the liquid phase and the facility and speed with which the individual components of higher molecular weight saturated and polyunsaturated acids could be resolved. When the sebacate and azelate polyesters of propylene, butylene, and diethylene HE
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ANALYTICAL CHEMISTRY
glycol were used as stationary liquids, the long-chain acid esters rapidly emerged from the column. There was complete separation of linoleate and linolenate bands. Peculiarly, the resolution of stearate from oleate did not readily occur. Khen additional ester and ether linkages were introduced into the molecule by reaction of the shorter chain dicarboxylic acids-i.e., adipate, glutarate, and succinate-with diethylene glycol to form more polar polymers of molecular weights extending from 3200 to 30,000, unsaturated acid esters were easily separated from the saturated members of the same carbon length under a wide variety of operating conditions ( 7 ) . Examples of the unique versatility of certain of these polymers to function as effective stationary liquids for the analysis of fatty acids can be noted in Figures 1 to 4. I n all instances, Celite 545, 60 to 80 mesh, was coated by mixing with a 10% chloroform solution of a succinate polyester of diethylene glycol (laboratory batch LAC-4R777; molecular weight 4480; acid number, 0.21; and viscosities of 1292 and 61,500 centistokes a t 100" and 38" C., respectively,) At the unusually low column temperature of 125' C. (Figure 1) the unsaturated components were retarded and emerged from the coIumn after the corresponding saturated acid ester, Under thrse conditions, the time required for analysis was relatively long and stearate was not separated from oleate. TI-hen the operating temperatures and the column length were increased (Figures 2 to 4), the retention times of the various components were appreciably shortened and stearate was now readily separated from oleate. Batches of the same polymer may vary greatly in their performance as stationary liquids for the analysis of fatty acid esters. The purity of starting materials, the mode of preparation, and experimental conditions during synthesis are factors which can lead to variations in the physical and chemical properties of these substances. Excellent results were obtained when columns containing this type of liquid phase were "preconditioned" for 48 t o 72 hours prior to use and the operating temperatures of the column were maintained below 190" C. Under these conditions, the interaction of the sample vapor with the stationary phase (transesterification)
mas minimal and the columns could be used for 3 to 4 weeks before losses in resolving power were noted. Despite advances in the analysis of fatty acids by gas chromatography, the resolution obtained by the use of the conventional packed column is still limited and the separation of certain interesting fatty acids-Le., the positional and stereo isomers-is difficult or impossible. A new concept in gas chromatography was recently introduced by Golay ( g ) , who suggested the use of columns made by coating the inner surface of long narrow-bore capillary tubing with a thin layer of stationary phase. Such columns are reported to have efficiencies far exceeding those of packed columns. Their use, however, requires a highly sensitive detector, because the maximum load of any single substance which can be applied without loss of resolving power is in the order of 0.1 y. One of the few devices known a t this time to possess sufficient sensitivity for use with these columns is that developed by Lovelock (11). The present study was conducted in an effort to obtain high efficiency capillary columns for the analysis of complex mixtures of fatty acids by using a modified form of this detector. The operational characteristics of this type of sensing system will be described elsewhere (12 ) . THE DETECTOR
The operation of the argon detector depends upon the ionization of organic molecules by collision with metastable argon atoms. The original device (10) as developed for use with packed columns consisted of a small metal ionization chamber to which a high potential was applied. It contained an CY or p source of ionizing radiation and had a volume b e h e e n 3 and 10 ml. When argon was used as the carrier gas and entered the chamber from the column, the primary electrons set free in the argon were accelerated by the applied potential to velocities sufficient to excite large numbers of the argon atoms to their metastable state. rirgono a- or &rays argon' e - (primary) Argon" e high voltage --c argon* (11.6 e.v.) (metastable state)
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4 Figure 1. Separation of fatty acid esters on 6-foot column of 10% diethylene glycol succinate polymer on 60-80 mesh Celite 545 Temp. 125' C.
Flow 50 ml./min. argon
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Figure 2. Separation of fatty acid esters on 9-foot column of 10% diethylene glycol succinate polymer on 60-80 mesh Celite 545 I
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Temp 1.58'
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