Submitted to: New Techniques and Applications for Lipid Analysis
Publication Type: Book / Chapter
Publication Acceptance Date: 8/10/2005
Publication Date: 1/15/2006
Citation: Adlof, R.O. 2006. Silver-ion hplc utilizing acetonitrile in hexane as solvent: Temperature effects on lipid elution orders/resolution. In: Mossoba, M., Kramer, J., Brenna, W., McDonald, R., editors. Lipid Analysis and Lipidomies, New Techniques and Applications. Champaign, IL: AOCS Press. p. 175-190. Interpretive Summary:
Technical Abstract: Silver-ion HPLC (Ag-HPLC), utilizing columns packed with silver ions bonded to a silica or similar substrate and isocratic solvent systems of acetonitrile in hexane, has proven to be a tremendously powerful technique for the analysis geometric and/or positional fatty acid (FA) esters and triacylglycerol (TAG) isomers. While temperature control and programing have become important and integral parts of gas chromatography the technology has, until recently, found only limited application(s) in Ag- and other forms of HPLC. Recent studies on the effects of temperature on FAME and TAG retention and resolution, utilizing commercially-available Ag-HPLC columns and isocratic solvent systems of ACN in hexane, unexpectedly demonstrated that unsaturated FAME and TAG samples eluted more rapidly at lower column temperatures [a result just the opposite to the temperature effects (where samples elute more rapidly at higher temperatures) noted in gas and liquid (reversed phase or silica gel substrates) chromatography]. A number of temperature-dependent factors which may influence retention are discussed, including: a) the equilibrium between Ag ion(s) and sample FA double-bonds/unpaired carbonyl oxygen electrons, b) the solubility of ACN in hexane, c) changes in sample flexibility/structure(s) at higher temperatures (steric argument/sample), d) changes in flexibility of phenyl-/ butylsulfonic acid or residual silanol groups on silica backbone of the Ag-HPLC column (steric argument/substrate), e) solubility of TAG or FAME sample(s) in the ACN/hexane solvent, f) the solvent viscosity, and/or g) the dielectric constant of the solvent(s). Other topics reviewed include the use of temperature to improve the resolution of lipid isomers, the relationship(s) between sample size injected/unsaturation and sample retention, temperature-induced changes in elution orders, day-to-day method reproducibility, and the effects of co-solvents such as diethyl ether.