Effects of Trifluoroacetic Acid Concentrations in Mobile Phases on HPLC Retention of Zwitterionic and Weakly Basic Triazole Derivatives

Abstract

Minor changes in the concentration of trifluoroacetic acid (TFA), a popular acidic HPLC mobile phase modifier, resulted in dramatic affects on hydrophobic retention. Two 1,2,4‐triazole carboxylic acids and the corresponding decarboxylated 1,2,4‐triazoles were chromatographed using a reversed‐phase column with 0.01% to 0.4% TFA in the mobile phases. Retention time shifts and reversed elution orders were observed. At low TFA concentrations, the decarboxylated 1,2,4‐triazoles eluted earlier, whereas at high TFA concentrations the two 1,2,4‐triazole carboxylic acids eluted earlier. An increased capacity factor, when TFA concentrations exceeded 0.05%, exhibited an increased hydrophobicity as a result of TFA‐associated chaotropic effects. Formic acid, acetic acid, or phosphoric acid were not as effective as TFA and attenuated the retention shifts significantly such that the reversed order of elution was no longer observed. The use of a combination of TFA and triethylamine (TEA) generated similar chromatographic profiles to those obtained when TFA was used alone. In conclusion, ideal HPLC separations of zwitterionic and weakly basic compounds could sometimes be obtained simply by optimizing TFA concentration in mobile phases.