Separation of barbiturates and phenylthiohydantoin amino acids using the thermally tuned tandem column concept

There are many more choices of column type than of eluent type for method d evelopment in reversed-phase liquid chromatography. It is common to switch between different column types or between the same type from different supp liers to achieve the desired separations. The key difficulty in modulating band spacing by adjusting the column type is that it is a discontinuous, "h it or miss" proposition. The thermally tuned tandem column ((TC)-C-3) conce pt effectively solves this problem by connecting two columns in series and independently controlling the two column temperatures. The columns are chos en to have distinctively different chromatographic selectivities (band spac ing), so that the unresolved peaks on one column are separated by the other . The optimized separation in the (TC)-C-3 is achieved by simultaneously tu ning the two column temperatures. In this study, we used the (TC)-C-3 combi nation of a carbon and a conventional bonded phase for the separation of ba rbiturates and phenylthiohydantoin amino acids (PTH-amino acids). Good peak shapes and comparable retention times were observed on the two phases at r oom temperature. The selectivities on the two phases are quite different. B aseline separations were easily achieved with the (TC)-C-3 set although nei ther column could individually resolve all the peaks. We further compared t he separation of barbiturates optimized by the (TC)-C-3 approach with that optimized by adjusting the mobile phase. We found that (TC)-C-3 gave a bett er separation. We believe that the (TC)-C-3 combination of a carbon phase a nd a bonded conventional reversed-phase material provides a powerful and ge neral method to optimize the separation of various mixtures.