Ion-pair reversed phase liquid chromatography-indirect conductivity detection and response deviations of underivatised amino acids

Abstract

Pairing chromatography is coupled with non-suppressed conductivity detection for the analysis of underivatised amino acids. The use of low concentrations of strong acids (perfluorinated carboxylic acids) as ion pairing reagents permitted indirect conductivity detection of the amino acids due to the high difference in the limiting equivalent ionic conductance between the hydrogen cation of the mobile phase and the ammonium cation of amino acids. A main system peak was induced after equilibrium perturbation. Its retention time and peak area were linearly related to the amount of ion pairing reagent adsorbed on the stationary phase and its concentration in the mobile phase, respectively. Amino acid response deviations relative to the baseline depended on their apparent charge and their elution in comparison to the elution of the main system peak. The choice of the optimum chromatographic conditions was a compromise between amino acid sensitivity, chromatographic separation, and stability of the system (minimum number of system peaks). An industrial fermentation product containing glutamic acid in excess (100–1 000 times more than the other amino acids) is used as a real application. The conductivity response of glutamic acid was almost annihilated by operating with a mobile phase pH close to the isoelectric point of Glu, leading to the successful determination of closely eluted minority amino acids. Linear calibration curves (0.9979 < r 2 < 0.9992) are obtained for two orders of magnitude with good relative standard deviations for peak areas (0.76–1.63%) and retention time (0.06–0.12%). Limits of detection are evaluated from 20 to 50 ng of injected amount (using a 10 μL loop) depending on the amino acid.