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Anal. Chem. 1989, 6 1 , 2076-2078
Supercritical Fluid Chromatographic Determination of Fatty Acids and Their Esters on an ODs-Silica Gel Column Akira Nomura,* Joseph Yamada, Kin-ichi Tsunoda, Keiji Sakaki, and Toshihiro Yokochi National Chemical Laboratory for Industry, Tsukuba, Ibaraki 305, Japan
Free fatty aclds and thelr esters were determlned by supercritlcal fluid chromatography (SFC) on a Inert packed column uslng supercritlcal carbon dioxide as a moMle phase wlthout addition of any modlfler. The column used was ODSsUka gel having a pore diameter of 300 A and end-capped as enough as possible to decrease resldual sllanol groups on the slllca surface. The retention behavior of free fatty acids and their esters was Investigated In terms of the density of a mobile phase, and they were separated according to a reversedphase mode like In llquld chromatography. Llplds extracted from fungus and their esterifled products were separated by the SFC using a pressure programming mode and a constant pressure mode, respectively, and the chromatograms obtained with both F I D and UV detector were compared.
Supercritical fluid chromatography (SFC) continues to attract considerable attention after several years of rapid growth. A wide variety of its applications have been investigated in analytical, as well as in industrial sectors (1). The analysis of fatty acids and their esters has been of great interest in the field of food, chemical, and pharmaceutical industries. Fatty acids and their esters have been usually determined by either gas chromatography (GC) or high-performance liquid chromatography (HPLC). However, nonvolatility of longer-chain acids and thermally labile property of unsaturated acids make analysis by GC much more difficult. Poorer detection limits have been the drawback in the HPLC analysis because UV absorption and fluorescence detection are impossible for saturated compounds (2). Free fatty acids were analyzed by capillary SFC using supercritical C 0 2 as a mobile phase, and the effects of temperature and stationary-phase polarity on efficiency and chromatographic peak shape were investigated ( 3 , 4 ) . Direct coupling of supercritical fluid extraction (SFE) and capillary SFC was applied to the separation of free acids (5). The separation of mixed mono-, di-, and triglycerides by capillary SFC was performed by using a carbon dioxide mobile phase without thermal degradation (6). The capillary SFC is a preferred method for high-resolution separation of thermally labile compounds, and a flame ionization detector (FID) gives the same high sensitivity in capillary SFC as is found in capillary GC. The capillary SFC, however, has some drawbacks in, such as, very small amounts of sample introduction and lack of capability for scale up to preparative separation. The separation and detection of free fatty acids in simple mixtures by SFC/Fourier transform infrared detection were carried out on packed columns using supercritical COz and Freon 23 as a mobile phase (7). Packed column SFC directly coupled with a mass spectrometer was applied to triglycerides (8). Free fatty acids were separated on an ODS-silica column using a modifier control column containing formic acid installed before a injector and were detected by FID (9). Esterified fatty acids from lipids in fungi and neutral lipids containing y-linolenic acid were separated on a ODS-silica column, and their retention behaviors were compared with those in HPLC (10, 11).
We have investigated the chromatographic behavior of ODS-silica gels for SFC having various pore structures by pressure programming SFC (12). We have also reported the preparation of inert silica-based packings and their properties for SFC (13). This paper describes the determination of free fatty acids and their esters on a inert ODS-silica gel column using carbon dioxide as a mobile phase without a modifier.
EXPERIMENTAL SECTION Apparatus. The SFC system used was the same as described elsewhere (22) except that a FID was connected by splitting a mobile phase between the separation column and the W detector. FID of either Shimadzu Model GC-7A or Shimadzu Model GCR1A was used for this purpose. The flow rate of a mobile phase was controlled by changing the size of a restrictor made of fused silica capillary tube. The restrictor made of the capillary tube of 50 pm i.d. x 200 mm was usually llsedunless otherwise specified. The flow rate of COPintroduced to the FID was controlled by the restrictor made of the capillary tube of 25 pm i.d. X 200 mm having a small orifice at the end. Column temperature was held at 45 O C throughout the experiment. Materials. Kaseisorb ODS-300-5 for SFC (4.6 mm i.d. X 250 mm; particle size, 5 km; specific surface area, 100 m2/g; pore diameter, 300 A; carbon content, 6.0%) from Tokyo Kasei Co., Ltd., Japan, was used as a test column for SFC, which was based on Kaseisorb LC ODS-300-5 for HPLC, and was end-capped as completely as possible. Kaseisorb LC ODS-100-5 (4.6 mm i.d. X 250 mm; particle size, 5 pm; pore diameter, 100 A) and Kaseisorb LC ODs-300-5 (4.6 mm i.d. X 250 mm; 5 pm; 300 A), both from Tokyo Kasei were used to compare the column properties for SFC. Free saturated fatty acids (C,-C,; 60-26:0), free unsaturated fatty acids (18:1, 182, 18:3, 204), methyl palmitate (16:0-Me),methyl stearate (18:0-Me), methyl oleate (18:1-Me), methyl linolate (18:2-Me),and methyl linolenate (183-Me)were all obtained from Tokyo Kasei. Methyl y-linolenak was obtained from Wako Pure Chemicals. All these standard samples were dissolved in chloroform from Kanto Chemical. The retention time of chloroform was used as toto calculate the capacity factor k’. Lipids produced in Mortierella ramanniana var. angulispora cultured in our laboratory were extracted from the fungus with hexane and were conventionally esterified to fatty acid methyl esters. The lipids and the methyl esters were dissolved in chloroform to be applied to SFC determination. Other reagents were of reagent grade and used without purification. RESULTS AND DISCUSSION In packed column SFC, silica-based packings such as ODS-silica gel are packed into a conventional HPLC column or microcolumn. When carbon dioxide is used as a mobile phase without a modifier, the residual silanol groups of silica gel base after surface modification interact with polar compounds and cause serious problems, such as irreversible or nonspecific adsorption leading to peak tailing or considerable delay of sample elution. Thus, we have proposed the utilization of silica gel having larger pore diameter (pore size, lo00 A) and sufficient end-capping of residual silanol groups after primary surface modification in order to prepare inert packings for SFC (13). Although the silica base with larger pore diameter has less silanol groups and less micropores in the pore structure, which makes the end-capping much easier, it is still necessary to develop the inert packings for SFC having smaller
0003-2700/89/0361-2076$01.50/00 1989 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 18, SEPTEMBER 15, 1989 1.5 r
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Figure 1. Retention behavior of free fatty acids including essential fatty acids: (0)18:O; (A)18:l: (0) 18:2; (0)18:3; (A)20:4.
pore size and possessing larger capacity for sample loading when applying to analytical and, particularly, preparative scale SFC separation. Kaseisorb LC ODs-300-5 has a relatively larger pore diameter of 300 A and usually is used for the separation of proteins in HPLC. This column was further end-capped for the SFC application (Kaseisorb ODs-300-5 for SFC) and was applied to the determination of free fatty acids and their esters using carbon dioxide as a mobile phase without the addition of a modifier. The retention behavior of essential fatty acids (linoleic, y-linolenic, and arachidonic acid) as well as stearic and oleic acid according to the density of the mobile phase was investigated on the ODS column for SFC (Kaseisorb ODs-300-5 for SFC) as shown in Figure 1. The separation mode of these fatty acids seems to be reversed phase in terms in LC, that is unsaturated fatty acids having more double bonds eluted faster than those having less double bonds, but that of arachidonic acid was different from others presumably because of its specific molecular structure. A linear relationship between the density of COz and the log k’value of samples was obtained above the density of 0.5 g/cm3 (110 atm). The mixture of standard free saturated fatty acids from 8:O to 24:O was applied to the three types of ODs-silica columns, that is Kaseisorb LC ODs-100-5 (A), LC ODs-300-5 (B), and ODs-300-5 for SFC (C), by linear pressure programming mode from 100 to 300 atm, from 100 to 240 atm, and from 100 to 160 atm, respectively, for 20 min, and the chromatograms are shown in Figure 2. Chromatogram A provided almost no peak separation because of large amounts of silanol groups remaining in the smaller pore structure (100 A), and chromatogram B obtained by the wider pore (300 A) ODS column gave better separation profiles than A, but the peaks showed unsatisfactory tailing patterns. In the case of chromatogram C obtained by the test column for SFC, the peak shape of the individual fatty acid was much more improved than that of B, but was not sharp enough to provide base-line separation presumably because free carboxyl groups in the molecules interact with the silanol groups still remaining on the surface of the silica base. However, it may probably be the first time that these free fatty acids could be separated to a considerable extent on a packed column using carbon dioxide as a mobile phase without a modifier. The retention behaviors of free fatty acid and fatty acid methyl esters on the column for SFC were compared to demonstrate the interaction between free carboxyl group and residual silanol group as shown in Figure 3. In reversed-phase LC, stearic acid (180) should be retained on a column weakly
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Figure 2. Chromatogram of standard free fatty acid mixture: A, Kaseisorb LC ODS-100-5; B, Kasisorb LC ODs-300-5; C, Kaseisorb ODS-300-5 for SFC. Number upon the peak shows the carbon number.
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Figure 3. Retention behavior of free fatty acid and fatty acid methyl esters.
and be eluted faster than its methyl ester (180-Me) according to less carbon atoms, but the result shows that the stearic acid retained on the column longer than its methyl ester by the interaction between the polar carboxyl group and the residual silanol group. The retention behavior of the methyl esters was reversed phase like in free fatty acids as shown in Figure 1, that is, methyl linolate and methyl palmitate eluted faster than methyl stearate according to the number of double bond and the carbon number, respectively. The column efficiency, expressed as HETP, was measured by using stearic acid and methyl stearate as samples, and the results are shown in Figure 4. The efficiency measured by methyl stearate was high and comparable to that in HPLC. On the contrary, the efficiency measured by stearic acid was very low, especially in the low density region, because the free carboxyl group interacts with the residual silanol group, which makes the peak wider. The efficiency increases to some extent as the density increases, presumably because the solubility of C02 increases and the interaction between the carboxyl group and the silanol group decreases. To show the applicability of this system to real analytical problems, the present packed type SFC was applied to the separation of lipids extracted from Mortierella ramanniana with hexane and their esterified products. Figure 5 shows the
2078
ANALYTICAL CHEMISTRY, VOL. 61, NO. 18, SEPTEMBER 15, 1989
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Figure 6. Chromatogramsof lipids produced by fungus using F I D and UV detection: pressure programming, from 150 to 250 atm for 30 min (hold at 150 atm for 5 min); UV detector, 190 nm.
Figure 4. HETP vs density of C02 mobile phase: (0)stearic acid: (0) methyl stearate. FID
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Figure 5. Chromatograms of fatty acid methyl esters from lipids produced by fungus using FID and UV detection: (1) 16:0-Me, (2) y-18:3-Me, (3) 18:2-Me, (4) 18:1-Me, (5) 18:O-Me; pressure of COS, 100 atm; UV detector, 190 nm.
chromatograms of the esterified fatty acid methyl esters detected by FID and UV detector. The amount of the individual methyl ester is approximately proportional to its peak area detected by FID. It is assumed from the result that there are larger amounts of methyl palmitate [ 1in Figure 51 and methyl oleate [4] and smaller amounts of methyl y-linolenate [2], methyl linolate [3], and methyl stearate [5]. On the other hand, the peaks of saturated esters such as methyl palmitate and methyl stearate, which have no double bond, disappeared from the chromatogram obtained with UV detector (at 190 nm, in Figure 5). Moreover, although the content of methyl y-linolenate was found to be small according to FID detection, its peak was emphasized in the UV detection because of the
three double bonds in the molecule. Thus, the use of both UV detector and FID at the same time can provide structural information of analytes. The chromatograms of the lipids extracted from Mortierella ramanniana using FID and UV as a detector are shown in Figure 6. Linear pressure programming mode from 150 to 250 atm for 30 min (hold at 150 atm for 5 min) was used for both FID and UV detection. Many peaks were found on both chromatograms and they were grouped into three groups, namely A, B, and C. The profiles of the two chromatograms are similar to each other except that the individual peak response differed according to the sensitivity of FID and UV. Although each peak of these groups was not identified yet, the major peaks in group B and C are considered to consist of diglycerides and triglycerides, respectively. Further study on identifying these peak components should be performed in the future.
LITERATURE CITED (1) Supercritical Fluid Chromatogrephy; Smith, R. M., Ed.; The Royal Society of Chemistry: London,-1988. (2) Stolyhwo, A.; Colin, H.; Guiochon, G. Anal. Chem. 1985, 5 7 , 1342-1354. (3) Markldes, K. E.: Fields, S. M.; Lee, M. L. J . Chromatogr. Sci. 1986, 2 4 . 254-257. (4) . . Doehl, J.; Farbrot, A.; Greibrokk, T.; Iversen, B. J. Chromatoar. 1987, 329, 175-184. (5) Gmuer, W.; Bosset, J. 0.; Pattner, E. J . Chromatogr. 1987, 388, 335-349. (6) White, C. M.; Houck, R. K. HRC CC. J. High Resolut. Chromatogr. Chromatogr. Commun. 1985, 8 . 293-296. (7) Hellgeth, J. W.; Jordan, J. W.; Taylor, L. T.; Khorassani, M. A. J . ChrOmatOgr. SCi. 1986, 2 4 , 183-188. (8) Matsumoto, K.; Tsuge, S.; Hirata, Y. Anal. Sci. 1986, 2 , 3-7. (9) Hirata, Y. SFC Applications ( 1988 Workshop on Supercrltkal FluH Chromatography);Compiled by K. E. Markiis and M. L. Lee; Brigham Young University Press: Salt Lake City, UT, 1988; p 110. (IO) Sakaki, K.; Sako, T.; Yokochi, T.; Sugeta, T.; Nakazawa, N.; Sato, M.; Suzuki, 0.; Hakuta, T. J . Jpn. Oil Chem. SOC. 1987, 36. 943-946. (11) Sakaki, K.; Sako, T.; Yokochi, T.; Suzuki. 0.; Hakuta, T. J. Jpn. Oil Chem. SOC.1988. 3 7 , 54-56. (12) Nomura, A.; Yamada, J.; Tsuncda, K. J. Chromatogr. 1968, 448, 87-93. (13) Nomura, A.; Yamada, J.; Tsuncda. K.; Fukushima, K.; Nobuhara, K. Anal. Sci. 1989, 5 , 335-338.
RECEIVED for review May 19; 1989. Accepted June 15,1989.
