Analysis of amino acids using serially coupled columns

Single conventional columns in reversed-phase liquid chromatography are insufficient for analysing the isoindoles of primary amino acids due to their limited functionality. An interesting possibility for increasing the separation power is the combination of several columns of different nature, where the length is modified by coupling small segments. This approach may require a considerable investment to have multiple lengths for each stationary phase. However, the combination of only two columns of fixed length can be enough to resolve satisfactorily relatively complex mixtures, provided that an optimised gradient program is applied. In this work, a mixture of 19 primary amino acid isoindoles found in proteins was analysed. Four stationary phases were assayed: C18, pentafluorophenyl-C18, C4 and cyano. The mixture of isoindoles was successfully resolved in practical times using a pentafluorophenyl-C18 column coupled to a C4 column, in spite of the extremely poor performance obtained when each column is used isolatedly, independently of the length. The extreme diversity in the polarities of the isoindoles and the need of extrapolating the retention behaviour in certain regions of the solvent content domain makes the modelling of the retention behaviour of the isoindoles particularly difficult. Nevertheless, the predicted optimal separations were very satisfactory.