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 development in reversed-phase liquid chromatography. It is common to switch between different column types or between the same type from different suppliers to achieve the desired separations. The key difficulty in modulating band spacing by adjusting the column type is that it is a discontinuous, "hit or miss" proposition. The thermally tuned tandem column (T³C) concept effectively solves this problem by connecting two columns in series and independently controlling the two column temperatures. The columns are chosen to have distinctively different chromatographic selectivities (band spacing), so that the unresolved peaks on one column are separated by the other. The optimized separation in the T³C is achieved by simultaneously tuning the two column temperatures. In this study, we used the T³C combination of a carbon and a conventional bonded phase for the separation of barbiturates and phenylthiohydantoin amino acids (PTH-amino acids). Good peak shapes and comparable retention times were observed on the two phases at room temperature. The selectivities on the two phases are quite different. Baseline separations were easily achieved with the T³C set although neither column could individually resolve all the peaks. We further compared the separation of barbiturates optimized by the T³C approach with that optimized by adjusting the mobile phase. We found that T³C gave a better separation. We believe that the T³C combination of a carbon phase and a bonded conventional reversed-phase material provides a powerful and general method to optimize the separation of various mixtures.