Separation of sulfated bile acids by high-performance liquid chromatography

In order to establish a new method for simultaneous determination of sulfated bile acids without deconjugation, separation of 3-sulfates of unconjugated, glyco- and tauroconjugated bile acids by high-performance liquid chromatography has been undertaken. The preliminary experiment indicated that reversed phase chromatography on an ODS SC-02 column using ammonium carbonate aqueous solution/acetonitrile as a mobile phase would be promising. The ratio of peak width to retention time was varied depending upon the salt concentration. The content of water in mobile phase exerted an influence on the capacity ratio to a certain extent, but not on the resolution. The use of 0.5% ammonium carbonate/acetonitrile (26∶8 and 20∶8, v/v) was found to be suitable for complete resolution of sulfated cholate, chenodeoxycholate, deoxycholate, lithocholate and their glyco- and tauro-conjugates.     

Separation of wax esters from olive oils by high-performance liquid chromatography

To detect adulteration of olive oil with solvent-extracted oils, the determination of the wax ester content has become more important during recent years. Hence, a greater number of wax ester analyses need to be performed by quality control laboratories. The most common method in use requires a liquid chromatographic (LC) separation of the less polar fraction, which contains the wax esters, from the glyceride matter on a hand-filled silica gel column. The aim of this project was to verify the possibility of replacing LC with high-performance liquid chromatography by taking advantage of the greater reliability and repeatability of this technique, as well as of the possibility of making the separation automatic. The paper describes how to perform the analysis and the statistical test that was carried out; furthermore, a comparison has been made with the usual method and results are in good agreement.     

Improved separation of conjugated fatty acid methyl esters by silver ion-high-performance liquid chromatography

Operating from one to six silver ion-high-performance liquid chromatography (Ag⁺-HPLC) columns in series progressively improved the resolution of the methyl esters of conjugated linoleic acid (CLA) isomeric mixtures from natural and commercial products. In natural products, the 8 trans, 10 cis-octadecadienoic (18∶2) acid was resolved from the more abundant 7 trans, 9 cis-18∶2, and the 10 trans, 12 cis-18∶2 was separated from the major 9 cis, 11 trans-18∶2 peak. In addition, both 11 trans, 13 cis-18∶2 and 11 cis, 13 trans-18∶2 isomers were found in natural products and were separated; the presence of the latter, 11 cis, 13 trans-18∶2, was established in commercial CLA preparations. Three Ag⁺-HPLC columns in series appeared to be the best compromise to obtain satisfactory resolution of most CLA isomers found in natural products. A single Ag⁺-HPLC column in series with one of several normal-phase columns did not improve the resolution of CLA isomers as compared to that of the former alone. The 20∶2 conjugated fatty acid isomers 11 cis, 13 trans-20∶2 and 12 trans, 14 cis-20∶2, which were synthesized by alkali isomerization from 11 cis, 14 cis-20∶2, eluted in the same region of the Ag⁺-HPLC chromatogram just before the corresponding geometric CLA isomers. Therefore, CLA isomers will require isolation based on chain length prior to Ag⁺-HPLC separation. The positions of conjugated double bonds in 20∶2 and 18∶2 isomers were established by gas chromatography-electron ionization mass spectrometry as their 4,4-dimethyloxazoline derivatives. The double-bond geometry was determined by gas chromatography-direct deposition-Fourier transform infrared spectroscopy and by the Ag⁺-HPLC relative elution order.     

Determination of physicochemical properties of some fatty acid methyl esters by gas liquid chromatography

Physicochemical data such as vapor pressures (p⁰), heats of vaporization (ΔH_v), activity coefficents at infinite dilution (γ^∞) and excess partial molar entropy (ΔS _e ⁰ ) are considered important for conducting unit processes and designing reactor equipment. Scanty information regarding such data is available in the literature for the higher fatty acid methyl esters. The objective of this research was to determine the physicochemical properties of higher fatty acid methyl esters (C₁₁–C₂₃) by a gas-liquid chromatographic technique with SE-30 and diethylene glycol adipate as stationary phases. Correlations between carbon numbers and various thermodynamic properties have indicated definite trends, which could be useful in predicting the properties of unknown fatty acid methyl esters. The data generated may be useful to chemical engineers in the construction of storage tanks, solvent extractors and distillation columns. IICT communication no. 2993.     

Gas liquid chromatography of the hydroxy-, acetoxy-and oxo-stearic acid methyl esters

Carbon numbers have been determined for all the 17 isomeric methyl hydroxy- and acetoxystearates and for 15 of the 16 isomeric methyl oxostearates using silicone SE-30, silicone QF-1, and ethylene glycol succinate (EGS) as liquid phases. The carbon numbers of the isomers increase with increasing distance of the point of substitution from the carboxyl end of the fatty acid chain (the increases for the 4- and 5-hydroxy esters are unexpectedly large). The change in carbon number from one isomer to the next is greatest when the substituents are attached near either end of the chain. The best separation of isomers with the substituent near the carboxyl end is obtained for oxo esters using the QF-1 column, and that of isomers substituted near the methyl end is obtained for acetoxy esters using the EGS column. Isomers with substituents at carbons 9<13 are not distinguishable on any of the columns used, but the other isomers are partly or completely separated. The quantitative aspects of the separations have not been investigated. Presented at the AOCS Meeting in Minneapolis, 1963. Issued as NRC No. 8095.     

Separation of monoacylglycerols by high-performance liquid chromatography on nitrile-bonded phase

Monoacylglycerol molecular species, as their di-3,5-dinitrophenylurethane derivatives, were well separated by normalphase high-performance liquid chromatography on nitrilebonded phase. The peaks emerged in the order 20∶0, 18∶0, 16∶0, 18∶1, 16∶1, 18∶2, and 18∶3. The peaks of 1- and 2-monoacylglycerols with the same acyl group showed complete overlapping. This method could be applied to get acyl compositions in the three positions of triacyl-sn-glycerols in their stereospecific analysis. Presented partially at the 83rd AOCS Annual Meeting held in Toronto, Canada, May 10–14, 1992.     

Argentation high performance liquid chromatography of methyl esters

The technique of argentation chromatography with silver ion on a macroreticular exchange resin has been applied to high performance liquid chromatography (HPLC) to separate fatty methyl esters and their isomers. Elution of methyl linolenate from the column and more rapid separation of dienes are made possible by programming column temperature from 25 to 70 C. Samples from ca. 0.025 μ to 8 μl can be analyzed on a 2-mm id x 61-cm column. Two 7-mm id x 61-cm columns in series have been used to separate 100-μl samples into fractions for further analysis by capillary gas chromatography. Various forms of argentation chromatography have been widely used for analysis and for separation of compounds from oils and fats including hydrogenated soybean oil. This paper describes the application of argentation procedures to modern HPLC to obtain faster, more efficient separations. It also describes the application of temperature programming to give more rapid separations and to extend our previous method to include methyl linolenate and its isomers. Presented at 1980 ISF-AOCS Congeress, New York.     

Silver nitrate-high performance liquid chromatography of fatty methyl esters

Long chain fatty methyl esters have been separated by high performance liquid chromatography on the basis of number, position, and geometric configuration of double bonds with a silver nitrate-silicic acid column and benzene solvent. Saturated esters are eluted first, followed by methyl elaidate and then methyl oleate. Geometric isomers of methyl 9,12-octadecadienoate and of methyl 9,15-octadecadienoate are also well separated. Methyl linolenate is retained strongly on the column and its elution has not been observed, but thetrans, trans, trans andtrans, cis, trans isomers are separated.     

Separation of fatty acid methyl esters from tall oil by selective adsorption

Fatty acid methyl esters (FAME) and resin acids (RA) were separated from tall oil by selective adsorption. Commercial nonmodified molecular sieve 13X was used as adsorbent. The adsorption isotherms of fatty acids (FA), FAME, and RA on molecular sieve 13X at 25°C were determined using various solvents. The solvents were methanol, ethanol, isopropanol, acetone, benzene, hexane, isooctane, petroleum ether (40–60°C), and petroleum naphtha (80–180°C). With each solvent, FA and RA were adsorbed to a greater extent than FAME. Adsorption isotherms for RA and FAME in binary adsorption systems were also determined using petroleum ether, petroleum naphtha, benzene, and isopropanol. For each component in the binary adsorption, the equilibrium amounts are lower than the values for pure component adsorption. The adsorption of FAME decreased in the presence of RA markedly in petroleum ether and petroleum naphtha. This fact may be the indication of the phenomenon of selective adsorption. Separation was accomplished by adding a solution of esterified tall oil in solvents used in the binary adsorption systems, through a column packed with molecular sieve 13X. With petroleum naphtha, FAME and RA were recovered in yields of 93 and 94%, respectively, from esterified tall oil. Petroleum naphtha gave the best results. The effects of particle size of adsorbent and flow rate of solvent on the efficiency of the separation were also investigated in fixed-bed column studies. The particle size of adsorbent did not apparently alter the results. Changes in the particle size should not significantly change the number of available adsorption sites in a microporous molecular sieve.     

Ion-pair high-performance liquid chromatography of bile salt conjugates: Application to pig bile

The high-performance liquid chromatographic separation and quantitation of conjugated bile salts from pig bile is reported. Synthetic standards and bile samples were chromatographed on a C₁₈ reversed phase column using acetonitrile/water/tetrabutyl ammonium phosphate as an isocratic mobile phase at a flow rate of 1 mL/min. Detection of the ion-pairs was at 214 nm. The method permits efficient separation of all conjugated pig biliary bile salts without prior modification or treatment of the samples. Analysis of 12 pig biles showed that 85% of the bile salts are conjugated to glycine. The three main conjugated bile salts were glyco-3α,6α,7α-trihydroxy-5β-cholanoic acid (GHC), glyco-3α,7α-dihydroxy-5β-cholanoic acid (GCDC), and glyco-3α,6α-dihydroxy-5β-cholanoic acid (GHDC). Glyco-3α-hydroxy-6-oxo-5β-cholanoic acid (G3α6oxo), tauro-3α,7α-dihydroxy-5β-cholanoic acid (TCDC), tauro-3α,6α,7α-trihydroxy-5β-cholanoic acid (THC), and tauro-3α,6α-dihydroxy-5β-cholanoic acid (THDC) were found to contribute each for 4 to 5% ot the total. An excellent correlation was found between the sum of conjugated bile salts quantitated by high-performance liquid chromatography (HPLC) and values obtained by conventional enzymatic assay. Simplicity, efficiency and relative rapidity of the method render it suitable for routine analyses.     

Separation of stigmasta-3,5-diene, squalene lsomers, and wax esters from olive oils by single high-performance liquid chromatography run

To ascertain the authenticity of olive oils, the European Community Regulation requires the stigmasta-3,5-diene and wax ester contents to be determined. The official methods are time-consuming and not suitable for many daily analyses, as quality-control laboratories need. A method is presented here that allows single high-performance liquid chromatography separation of stigmasta-3,5-diene and wax esters, as well as of the squalene isomers, which give further information on the oil’s authenticity. For stigmasta-3,5-diene, the comparison with results obtained with the official method is good. Also for wax esters, the agreement was good, even if they were compared with results obtained from a quicker method as reliable as the official one. The possibility of separating the squalene isomers also at the same time makes the proposed method more advantageous. On the whole, the method, which is suggested for routine and quick screening but not for the exact evaluation of the analyte contents, seems to be a convenient choice for ascertaining on a daily basis the samples’ legal compliance (i.e., whether the analyte content is or is not below the legal value).     

Separation and quantitation of hydroxy and epoxy fatty acid by high-performance liquid chromatography with an evaporative light-scattering detector

A new high-performance liquid chromatography technique with an evaporative light-scattering detector (ELSD) has been developed for the separation and quantitative analysis of hydroxy and epoxy fatty acids. This method employs a gradual binary gradient (hexane/isopropanol) and ELSD detection. The minimum limit of detection is about 1 μg and ratio of mass to signal is essentially linear in the range of 10 to 200 μg. This high-performance liquid chromatography (HPLC) technique is able to separate various positional isomers of mono-hydroxy and dihydroxy fatty acids and can also discriminate between monohydroxy, epoxy, epoxyhydroxy, dihydroxy and trihydroxy fatty acids.     

High performance liquid chromatography of fatty methyl esters: Analytical separations

Fatty methyl esters are separated on the basis of unsaturation and chain length on an analytical scale by high performance liquid chromatography with a C18/Corasil column and aqueous acetonitrile solvent. Analysis by this method includes polymerized and oxidized esters which may not be detected by gas chromatography.     

Resolution ofvic-dihydroxy acid diastereomers to four enantiomers by high-performance liquid chromatography

A chromatographic method is described for resolution of 9,10-vic-dihydroxyoctadecanoic acid. Thethreo anderythro isomers are both resolved into two enantiomers as di-3,5-dinitrophenylurethane derivatives by chiral-phase highperformance liquid chromatography on (S)-proline, (R)-1-(α-naphthyl) ethylamine stationary phase. Simultaneous separation of thethreo anderythro dihydroxy acid mixtures to the four enantiomers is possible by this method.     

Separation of tripalmitin from its hydrolysis products by simple isocratic reversed-phase high-performance liquid chromatography

An isocratic reversed-phase high-performance liquid-chromatographic method was developed for monitoring the lipase-catalyzed hydrolysis of solid triglycerides in supercritical carbon dioxide. The reaction products obtained consist of free fatty acids, monoglycerides, diglycerides, and unreacted triglycerides. For the method, developed with tripalmitin, a mobile phase of acetonitrile/chloroform/acetic acid (50:50:1, vol/vol/vol), a C₁₈ column, and refractive index detection were used. Analysis time is 7 min. Response factors were determined for each neutral lipid class, which permitted quantitation of the hydrolysis product mixture.     

Separation of molecular species ofcis- andtrans-triacylglycerols intrans-hardened confectionery fats by silver-ion high-performance liquid chromatography

Silver-phase high-performance liquid chromatography (HPLC) on silver nitrate-loaded silica achieves incomplete separation of major triacylglycerol (TAG) classes present intrans-hardened fats. The “ChromSpher Lipids” silverloaded cation exchange HPLC column has been found to yield good separations oftrans-hardened TAG, with molecular species well resolved. Separations comparable to those previously possible for nonhardened fats are now possible fortrans-hardened fats. The separation is on the basis of number and type (i.e.cis/trans) of double bonds only; the position of the double bond along the acyl group appears not to influence the separation significantly. The analysis of a palm fraction, hardened to a slip melting point of 37°C and chemically randomized, is presented as an example. This technique offers a new approach to understanding and controlling the hydrogenation and processing oftrans-hardened fats.     

Influences of operating conditions on determination of fatty acid methyl esters by gas chromatography

The compositions of standard mixtures of methyl laurate, myristate, plamitate and stearate were determined by gas liquid chromatography, varying the operating conditions such as sample size, column temperature and flow rate of the carrier gas. The influences of these operating conditions on analytical values were examined. The sample size had a significant influence on the analytical values. The degree of influence was dependent on the column temperature and the composition of the sample. It was also observed that the flow rate of the carrier gas affected the analytical values. Presented at the JOCS-AOCS Joint Meeting, Los Angeles, April 1972.     

Monitoring monohydroperoxides in docosahexaenoic acid using high-performance liquid chromatography

The oxidation of free DHA has been investigated with respect to monohydroperoxides and polyhydroperoxides, which were analyzed with a novel HPLC method. The temperature and physical system, i.e., bulk and liposome, were varied. We have also studied the effects of antioxidants such as α-tocopherol, ascorbic acid, and juice from sea buckthorn on DHA. The HPLC method, which was performed isocratically, eluted eight peaks, each containing one or two isomers of monohydroperoxy-DHA. This method showed that the double bond farthest from the carboxyl group was easily oxidized, as shown by the rapid increase in the amount of C20-monohydroperoxy-DHA, which always provided the largest contribution to the total amount of monohydroperoxides. The monohydroperoxy-DHA containing the hydroperoxy group located on the double bond nearest the carboxyl group also was shown to incease considerably during an increase in the total amount of monohydroperoxides. This demonstrates that the double bonds located nearest and farthest from the carboxyl group were the most prome to hydroperoxide formation. DHA was more stable when stored in liposomes than as bulk. Addition of α-tocopherol to the DHA-containing liposomes reduced the oxidation of these double bonds. The antioxidant effect of α-tocopherol was prolonged when combined with ascorbic acid, since α-tocopherol was regenerated.     

Separation and identification of eugenol in ethanol extract of cloves by reversed-phase high-performance liquid chromatography

A method has been developed for the separation and identification of eugenol in the alcohol extract of cloves directly without having to previously separate other components. Extraction of eugenol with alcohol is followed by analysis with high-performance liquid chromatography. Separation by reversed-phase chromatography on low-polarity μBondapak C₁₈ was achieved with isocratic elution. Tentative identification is based on chromatographic appearance of a peak with retention time 5.54 min for pure standard reference eugenol. The identification of eugenol was confirmed by gas chromatography/mass spectrometry analysis.