ATTOMOLE DETERMINATION OF PHENYL THZOHYDANTOrN DERNATNES OF AMINO ACIDS BY CAPILLARY ZONE ELECTROPHORESLS SEPARATION AND ULTRAVIOLET- LASER EXCITED THERMO.OPTICAL ABSORBANCE DETECTION

Capillary zone electrophoresis provides a rapid method for the analysis of minute amounts of amino acids. A laser-based absorbance technique is used to determine phenylthiohydantoin amino acids with detection limits of 10.15 mot; separation of 20 amino acids, based on micelle modified zone electrophoresis, requires 13 minutes. The amino acid sequence of a protein is of fundamental value in the study of the structure and function of the molecule. The Edman degradation procedure is commonly employed to determine the amino acid sequence of proteins [1, 2]. In this procedure, phenyl isothiocyanate reacts with the amine group on the terminal amino acid of the protein under basic conditions to produce a thiocarbamyl protein derivative. Under acidic conditions, the phenyl thiocarbamyl (PTC ) cyclizes with the carboxylic acid group to cleave the terminal amino acid from the protein and to produce a thiohydantoin amino acid derivative. The phenyl thiohydantoin (PTH) derivative is collected and the degradation is repeated to cleave the next amino acid from the protein. Liquid chromatography, with ultraviolet absorbance detection, is usually used to identify the cleaved amino acid. Absorbance detection limits of 200 femtomole are produced for PTH-amino acids in liquid chromatography [2]. A number of alternative Edman degradation reagents, such as dimethylaminoazobenzene isothiochyanate and fluorescein isothiocyanate have been developed. Although the reagents can be detected with exquisit sensitivity [3], there has been very little application of the modified reagents. It appears that limited reaction efficiency during the formation of the thiocarbamyl has discouraged application of the modified Edman reagents. Capillary electrophoresis is a useful technique for the rapid and efficient separation of minute amounts of analyte [3]. Zone electrophoresis has been used to determine phenylthiocarbamyl (PTC) amino acids; ultraviolet absorbance produces detection limits of -0.2 picomoles of PTC amino acids [4]. However, zone electrophoresis does not separate the neutral PTH-amino acids. Otsuka, Terabe, and Ando reported the use of micellar capillary electrophoresis for the separation of PTH-amino acids [5]. Surfactant, such as sodium dodecyl sulfate, is added to the separation buffer; distribution of the PTH-amino acids between the micelles and aqueous separation buffer leads to separation. Unfortunately, no data was provided on PTH-amino acid detection limit, but it is expected to be similar to that for the PTC-amino acids. Because of the short optical path length across the capillary, conventional absorbance detection leads to limited sensitivity.