Submitted to: Journal of Chromatography A
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/13/2007
Publication Date: 3/21/2007
Citation: Adlof, R.O. 2007. Analysis of triacylglycerol and fatty acid isomers by low-temperature silver-ion high performance liquid chromatography with acetonitrile in hexane as solvent: limitations of the methodology. Journal of Chromatography A. 1148:256-259. Interpretive Summary: Silver ion high performance liquid chromatography (Ag-HPLC) has shown itself to be a useful technique for analyzing a variety of fats and oils. The authors demonstrated that, using but a single solvent and a chiller/heater to control column temperatures, fatty acids and triglycerides actually eluted more rapidly at lower column temperatures. The temperature range of this effect was studied and, using chromatography equipment available in most analytical laboratories, was found to be +70 deg C to -25 deg C. This discovery could potentially be utilized to develop a single-solvent, temperature-programmed (as in gas chromatography) Ag-HPLC system for the rapid analysis of a wide variety of samples without requiring a change of solvent. The improved reproducibility (a problem with many HPLC systems) and sample separation(s) noted in this method will be extended to HPLC using other solvents and to develop standard methods of fat analysis. The latter development will let HPLC become more of a standard method and to be used in more analytical laboratories.
Technical Abstract: Silver ion HPLC (Ag HPLC), utilizing columns containing silver ions bonded to a silica substrate and acetonitrile in hexane as solvent, has proven to be a powerful technology for the analysis of geometric (cis or trans) or positional fatty acids, fatty acid ester (primarily methyl ester; FAME), or triacylglycerol (TAG) isomers. Previous studies had demonstrated that, unlike gas chromatography, samples eluted more rapidly at lower temperatures (at 20 deg C vs. 40 deg C, for example). A low-temperature bath [dual-column Ag-HPLC; isocratic solvent systems of 0.6% and 0.7% acetonitrile (ACN) in hexane] was utilized to study the limitations of this system at low (below 0 deg C) temperatures for analysis of FAME (zero to six double bonds) and TAG [SSS, OOO and LLL, where S = stearic acid (18:0), O = oleic acid (9c-18:1), and L = linoleic acid (9c,12c-18:2)] isomers. While FAME elution times continued to decrease from 0 deg C to -10 deg C, they began to increase at -20 deg C. A similar situation was noted for the TAG isomers, except that retention times began to increase below 0 deg C. While temperatures between 0 deg C to -20 deg C could be utilized to analysis or improve resolution of FAME and TAG samples, increasing pump pressures limited the Ag-HPLC/ACN in hexane system system lower temperature limit to ca. -25 deg C. Equilibration times at each temperature could be reduced to ca. 15 minutes without loss of resolution and with retention times of +/- 2%.