The Use of Large Volume Injections for the Isocratic Separation of Phenylthiohydantoin Amino Acids by Microbore Liquid Chromatography

The use of microbore columns in high performance liquid chromatography (HPLC) has remained limited primarily due to difficulties in adapting conventional HPLC analysis to the smaller volumes and lower flow rates of microbore chromatography while maintaining high quality chromatographic results. Although conventional HPLC techniques are the method of choice for PTH-amino acid analysis, these techniques are rapidly approaching their useful limits in the field of microsequencing. A suitable method for the isocratic room temperature separation of PTH-amino acids employing microbore liquid chromatography is discussed herein. As described, the system employed for this type of analysis makes it readily adaptable to the field of protein sequencing. A comparison of a large volume mobile phase injection and a large volume "non-eluting" solvent injection with microbore columns is also presented.