Analysis of Triglycerides of Soybean Oil by High- Performance Liquid Chromatography in Combination with Gas Liquid Chromatography

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1 Analysis of Triglycerides of Soybean Oil by High- Performance Liquid Chromatography in Combination with Gas Liquid Chromatography Shun WADA, Chiaki KOIZUMI, and Junsaku NONAKA Tokyo University of Fisheries, Department of Food Science and Technology (4-5-7 Konan, Minato-ku, Tokyo) A technique of high-performance liquid chromatography (HPLC) was applied to the analysis of triglycerides of soybean oil. The triglycerides were clearly separated into six peaks by HPLC on a column packed with,ƒê-bondapack C18 using methanol-chloroform mixture as a solvent. From the fatty acid compositions of the triglycerides of these six peaks, it was found that the separation of triglycerides by HPLC under the conditions used was governed by the degree of their unsaturation in addition to carbon number ; that is, the higher the degree of unsaturation of the triglyceride, the earlier the elution. From the results obtained, the possible triglyceride compositions of soybean oil were estimated. As a result, it was suggested that the use of HPLC in combination with GLC and TLC can provide more detailed information concerning triglyceride structure. 1 Introduction TLC, GLC, and liquid liquid chromatography (LLC) are current methods for lipid analysis. Using special techniques, these methods can provide detailed information concerning the structure of triglyceride, a major constituent of lipid. Recently, a technique of high-performance liquid chromatography (HPLC) has been de veloped and applied to lipid analysis. For exc ample, the separation of glycerides into mono-, di-, and triglycerides by HPLC on a Porgel 200 A or 60 A column }"2) and the separation of glycerides, fatty acids, and sterols on a Micro Pack SH-10 column'' have been reported. However, there is little information concerning the analysis of triglycerides of lipid. In this study, HPLC was tentatively applied to the analysis of triglycerides of soybean oil in order to search for a procedure which would yield detailed information concerning the structure of triglyceride. The soybean oil triglycerides were chosen as a sample because of their relatively simple fatty acid composition. This paper deals with a procedure for the analysis of the triglyceride composition of soyv bean oil by HPLC in combination with GLC. 2 Experimental HPLC Hige-performance liquid chromatograph used was a JASCO FLC A 700 equipped with a dif ferential refractometer as a detector and a stainless steel tubing (1' x 1/4" i.d.) packed with ~-Bondapack C18 obtained from Waters Associates Company. The theoretical plates of the column were HPLC was carried out using as a solvent system methanol and chloro= form (9:1) which was filtered by glass fiber filter paper immediately before use. The solvents of HPLC grade were obtained from Kanto Kagaku Co. Aliquots of 10 l of the sample dissolved in the solvent were introduced by a microsyringe through a general injector with solvent flow rate of 1.5 ml/min. The column was kept at 30 C by means of a jacket of circulatory water. GLC A Shimazu model GC-4 BMPF gas chroma tograph equipped with a FID was employed for analyses of triglycerides and their constituent fatty acids. The glass tubing (0.5 m'<3 m i.d.) packed with 106 JXR on Gas Chrom Q (60-

2 $0 mesh) and stainless steel tubing (3 m X 3 mm i.d.) packed with 15% DEGS on Shimalite (60-80 mesh) were used for the analyses of triglycerides and fatty acid methyl esters, respectively. The column temperatures operated were C (programmed at 3 C/min.) for triglycerides and 195 C for fatty acid methyl esters. Sample Triglycerides of soybean oil were chosen because of their relatively simple fatty acid composition and prepared by TLC using petroleum ether : diethyl ether : acetic acid (74 : 15 : 1) as a solvent system4'. Methylation of fatty acid The constituent fatty acids of triglyceride were methylated by means of transesterif ication using EF3-MeOH. 3 Results and Discussion HPLC of soybean oil triglycerides Fig.-1 shows the chromatogram obtained by HPLC of soybean oil triglycerides. As seen in the figure, the triglycerides of soybean oil were Fraction numbers are indicated in the figure. Dotted line in the figure indicates the elution patterns of standard glycerides ; A : Monopalmitin, B :Dipalmitin, C : Tripalmitin. Instrument : 1ASCO FLC A 700 Column: c~-bondapack C1$ (1' x 1/4" i.d.) Condition : Flow rate : 1.5 ml/min Detector : RI : 16 X Eluent : McOH/CHC13 : 9/1 Temperature : 30 C Fig,-1 The separation of triglycerides in soybean oil by HPLC. Table-1 Percentage of triglycerides in each fraction separated by HPLC. Calculated based on the data from HPLC. clearly separated into six fractions, though the soybean oil triglycerides have been found to be separated into three groups of triglycerides, C- 50, C-52, and C-54, by GLC' ~. The percentages of the triglycerides separated were tentatively calculated based on their peak areas on the chromatogram and shown in Table- 1. Each fraction contained triglycerides at percentages ranging from 1.3 to GLC of triglyceride fractions The six triglyceride fractions were collected separately and subjected to GLC. 4n the basis of the peak areas on the chromatograms obv tamed, the percentages of C-50, C-52, and C-54 triglycerides in each fraction were calculated and shown in Table-2. Fractions 1 and 2 revealed only one peak corresponding to that of C-54 triglyceride on the chromatograms and lacked those of C-50 and C-52 triglycerides. Fraction 3 was coma posed of C-52 and C-54 triglycerides, although C-52 triglyceride was a minor component. Fracc tion 4 contained C-52 and C- 54 triglycerides which were almost equal at percentage. Frac= Lions 5 and 6 contained a small amount of the shorter chain length triglyceride, C-50, in addition to C-52 and G54 triglycerides. Teble-2 Triglyceride composition of the fractions separated by HPLC. * Calculated based on the data from GLC.

From these results, it is clear that the factor governing the separation of soybean oil tri glycerides by HPLC is different from that by GLC in which the triglycerides are separated on the basis of

3 From these results, it is clear that the factor governing the separation of soybean oil tri glycerides by HPLC is different from that by GLC in which the triglycerides are separated on the basis of their carbon numbers. Fatty acid composition of triglyceride fractions Each triglyceride fraction collected by means of HPLC was subjected to GLC after transz esterification with BF3-MeOH, in order to examine its fatty acid composition. The results obtained are shown in Table-3. Fractions 1 and 2 which contained only C-54 triglyceride were mainly composed of unsaturated fatty acids, C18:2 and C18 ; 3 Fraction 3 contained C18:2 as a major constituent fatty acid and lacked C18: Fractions 4 and 5 were mainly composed of C1872f C18.1, and C16: p. Fraction 6 contained the saturated fatty acids, C1610 and C18:o, at relatively high percentages in addition to C1811 and C18:2. Both fractions 5 and 6 similarly contained trace amounts of C18:3. Table-3 also shows the iodine number of each triglyceride fraction tentatively calculated on the basis of its fatty acid composition and the following iodine numbers of fatty acids ; C18:1 : 89.9, C18: 2 : 181.0, C1813: The iodine number thus calculated is the highest in fraction 1 and decreased with increasing fracc tion number, as can be deduced from the fatty acids composition of each triglyceride fraction shown in the table. It is clear that in HPLC under the chromatographic conditions employed Table-3 Fatty acid composition of triglyceride fractions separated by HPLC. * Calculated based on the data from GLC. ** Calculated on the basis of each fraction's fatty acid composition and the following iodine numbers of fatty acids; C18:1: 89.9, C18:2: 181.0, C18:3: here the separation of triglycerides is mainly governed by the degree of their unsaturation; that is, the higher the degree of unsaturation of triglyceride, the earlier the elution. The iodine number of total fatty acid com posing the triglyceride fraction of soybean oil can be calculated on the basis of the calculated iodine number shown in Table-3. The value calculated in this manner is 137.8, which coin cides well with that of the triglyceride fracc tion of soybean oil (137.2). This suggests that no triglyceride of soybean oil is lost during the course of HPLC analysis. Estimation of triglyceride composition Based on the data shown in Table-2 and -3, the possible fatty acid composition of triglycev rides in each fraction can be estimated, assum= ing that the fraction exclusively contains tri glycerides having the same partition numbers'"''. Thus, the triglyceride composition of soybean oil was calculated. The results obtained are shown in Table-4. Fraction 1 contained only C-54 triglyceride which was mainly composed of C1813 and C18:2. From these data, the fatty acid compositions of triglyceride in fraction 1 are calculated to be (1 x C18: 2, 2 x C1B: 3) and (2 x C18: 2, 1 x C18, 3) at percentages of 70 and 24, respectively. The partition number of the triglycerides having the group (1 x C18: 2, 2 x C18:3) is calculated to be 38 and that having the group (2 X C18:2, 1 X C18: 3) to be 40 because the former triglycerides have eight double bonds and the latter seven. Therefore, the triglycerides having the group (2 x C182, l x C18:8) should be included in fraction 2 of which the partition number is calculated to be 40 as mentioned later. This implies that fraction 1 was indelibly contaminated with a part of fraction 2 in the course of collection. Fraction 2 also contained only C-54 triglyc ceride which was composed of C18:2 and C1815 in the percentage ratio of 2:1. The only possible fatty acid composition for triglycerides of this fraction is (2 x C1812, 1 x C18, 3) having partition number of 40. Fraction 3 contained a small amount of C-52 triglyceride in addition to major triglyceride, C-54. The fatty acid composition of this trim glyceride fraction included a small amount of C, 8 : o derived from C-52 triglyceride aside f rozn 13

4 Table-4 Estimated triglyceride composition of soybean oil. * Calculated from the following equation ; Partition number = ** See text *** Calculated using the data in Table-1. Carbon number -2 Number of double bond The values are approximate, since the refractive index of each fraction was not determined. C18:,, C18:2, and C18:3. From these results, the triglycerides in fraction 3 are considered to have five or six double bonds. On the basis of this, the partition number of triglycerides is calculated to be 42, Thus, the fatty acid come position of the triglycerides having the partition number of 42 is estimated to be (1 X C,,, o, 1 x C,8:2, 1 XC18:3; 15%), (1 xc18:,, 1 XC,,:2, 1 x C,,::; 14%), and (3 x C,8:2 ; 69%) Fraction 4 consisted of C-52 and C-54 tri glycerides, which are deduced to have four or five double bonds from their fatty acid compo sitions and the calculated iodine number. On the basis of the partition number obtained, 44, the triglycerides in fraction 4 are calculated to have the following fatty acid compositions, (1 x Cle: o, 2 x C18: 2 ; 42%), (2 x C18:, 1 x C18: 3 ; 6%), and (1 x C36:1, 2 x C18: 2 ; 45%). In a similar manner, the partition numbers are calculated to be 46 for fraction 5 and 48 for fraction 6. In consideration of the partition number and the fatty acid composition of the triglyceride fractions, the fatty acid compositions are considered to be (2 x C,6: o, 1 x C18:2 ; 6%), (1 xc16:,, 1 xc18:1, 1 xc,,:2, 48 o), (2xC18:,, 1XC18:2; 30%), and (lxc18s0, 2xC,s:2; 15%) for fraction 5 and (2 x C,,: o, 1 x C,,,,; 12%), (l X C,,: o, 2x0,8:,; 12%), (1 x C,e: o. 1 X C18: o, 1 x C18: 2 ; 12%), (3 x C,,:1 ; 30%), and(1 x C,,: o, 1 x C18:1, 1 ><:,; C1833%) for fraction 6. The triglyceride composition of soybean oil obtained here almost coincides with that reported by Coleman'". The results obtained in this study indicate

5 that the use of HPLC in combination with GLC and TLC provides more detailed information concerning triglyceride structure. (Received Oct. 8, 1976) References 1) J.G. Lawrence, J. Chromatogr., 84, 299 (1973). 2) M.J. Cooper and M.W. Anders, J. Chrom. Sci., (1975). 3) K. Kiuchi, T. Ohta, and H. Ebine, J. Chrom. Sci, 13, 461 (1975). 4) N. Ikekawa, M. Matsui, T. Yoshida, and T. Watanabe, Nippon Suisan Gakkaishi, 38, 1267 (1972). 5) M. Matsui, T. Watanabe, and N. Ikekawa, Nippon Suisan Gakkaishi, 39, 367 (1973). 6) H.P. Kaufman and Z. Makus, Fette, Seifen, Anstrichm., 62, 125 (1960). 7) J. Hirsch, J. Lipid Research, 4, 1 (1963). 8) A.G. Vereshchagin, J. Chromatogr., 14, 184 (1964). 9) C. Litchfield, Lipids, 3, 170 (1968). 10) M.H. Coleman, J. Am. Oil Chem. Soc., 38, 685 (1961).

FATTY ACID COMPONENTS OF BLACK RIGHT WHALE OIL BY GAS CHROMATOGRAPHY HIDEO TSUYUKI* AND SHINGO ITOH* INTRODUCTION

FATTY ACID COMPONENTS OF BLACK RIGHT WHALE OIL BY GAS CHROMATOGRAPHY HIDEO TSUYUKI* AND SHINGO ITOH* INTRODUCTION There have been a number of studies on whale oil. However, there are a few studies on black