Optimization of in-needle derivatization in HPLC

HPLC is one of the most widely used separation technique today. There are several reasons for this; its reproducibility, practicality, wide range of usability, sensitivity and quickness. Proteins play a role in all the vital reactions of the cell. They consist of amino acid chains. The most used method for the acid determination is HPLC analyze after derivatization. The derivatization increases the detectability on HPLC, changes the molecular structure and polarity of the analyte for better chromatogram, and increases the stability of the assay. However, there are many steps in the procedures applied to derivatization. Because of that, numbers of errors are made by analysts who apply those steps. Most frequent errors that are made are measurement errors and standardization of waiting times. In addition to that if small amount of samples are used or if sample's derivatization gives a rapid degraded result the determination becomes really difficult. These errors can get eliminated by derivation with the commands to the needles of the autosampler. By doing this the time spent for each sample can be reduced and repeatability increases. In this study, we first separated our amino acids by derivatization in the needle of the autosampler of the HPLC in our laboratory. We used o-phthalaldehyde (OPA) and mercapto ethanol as the derivatization agents. The model amino acids, used for this purpose, were L-glycine, L-arginine, L-alanine and DL-lysine. The effect of various experimental parameters on the chromatograms were investigated with the method we created.