Analysis of amino acids using serially coupled columns

Abstract

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.

Recent applications of retention modelling in liquid chromatography

“…Such high S LSS values are one of the reasons why gradient elution is indispensable in RPLC of proteins. Selectivity could be improved by serially coupling columns with increasing retentive capacity and applying multistep gradients [49,50].…”

In Silico Method Development of Achiral and Chiral Tandem Column Reversed-phase Liquid Chromatography for Multicomponent Pharmaceutical Mixtures

“…Previous works have demonstrated the improved efficiency and selectivity when columns are connected in series. 23,26,28,29 The increased length translates into higher peak capacity, especially when more columns packed with the same stationary phase are serially installed. 30−32 different stationary-phase columns.…”

“…It has been demonstrated that serially connected columns can outperform the same columns used separately, suggesting that this tandem column approach has a unique advantage to improve selectivity. 21,23,26,28,31,33,34 Welch and co-workers reported the use of tandem supercritical fluid chromatography (SFC) screening, which facilitates identification of column and modifier combinations for separation of small-molecule mixtures. 34 A challenege associated with tandem column separation is to select ideal method parameters such as pH, temperature, eluent composition, and gradient programs as they all need to be compatible for all the column combinations.…”

Updating chromatographic predictions by accounting ageing for single and tandem columns

“…Our laboratory has been involved since 2012 in the improvement of separations in RPLC using serially coupled (tandem) columns [8,[11][12][13][14][15]. The successful development of such column systems required solving technical problems related to the robustness of connections, reduction of extra dead volumes, or deterioration of column packings.…”

“…Tandem columns are powerful, but they may require considerable investment, since multiple columns of different lengths are needed for each stationary phase. Recently, we have demonstrated that the combination of only two columns of fixed length may be enough to resolve relatively complex samples satisfactorily, provided that an optimized gradient program is applied . It should be also considered that the search of the best separation conditions requires modeling the chromatographic behavior of two or more stationary phases, although there is no need to develop models for each column length, since these can be derived from the models obtained for a column with a specific length taken as reference .…”