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.
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 degrees C versus 40 degrees C, for example). A low-temperature bath [dual-column Ag-HPLC; isocratic solvent systems of 0.3 to 0.7% acetonitrile (ACN) in hexane] was utilized to study the application of this system at low (below 0 degrees 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)] standards. While FAME elution times continued to decrease from 0 degrees C to -10 degrees C, they began to increase at -20 degrees C. A similar situation was noted for the TAG isomers, except that retention times began to increase below 0 degrees C. The lower temperature limit of the Ag-HPLC/ACN in hexane system is thus ca. -25 degrees C. Increasing sample elution times and pump head pressures upon sample injection were noted at temperatures of -25 degrees C to -40 degrees C. Equilibration times at each temperature could be reduced to ca. 15 min without loss of resolution and with retention times of +/-2%. Temperature, rather than solvent composition, can therefore be utilized with the Ag-HPLC/ACN in hexane solvent system to optimize elution times and resolution(s) of FAME and TAG isomers.
Adlof
R
R
Food and Industrial Oil Research, National Center for Agricultural Utilization Research, USDA, Agricultural Research Service, Peoria, IL 61604, USA. adlofro@ncaur.usda.gov
D000097:Acetonitriles / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005227:Fatty Acids / Q000032:analysis* / Q000737:chemistry; D006586:Hexanes / Q000737:chemistry*; D007536:Isomerism; D015203:Reproducibility of Results; D012834:Silver / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D014280:Triglycerides / Q000032:analysis* / Q000737:chemistry
D000097:Acetonitriles; D005227:Fatty Acids; D006586:Hexanes; D012997:Solvents; D014280:Triglycerides; C026385:n-hexane; D012834:Silver; C032159:acetonitrile