Abstract: A solution of 0.25 M KBr in 0.25 M sodium phosphate of pH 5 was used to prepare 10 mM standard solutions of amino-acids and proteins. A thin-layer dual electrode cell (DEC) was employed for both the FIA-electrochemical detector system (FIA-ECD) and the HPLC-ECD system. The DEC cell (7 mm x 4 mm) contained two Pt planar working electrodes (3 mm x 4 mm) in series along the flow path, separated by 1 mm. The PTFE block with the two working Pt electrodes was separated from a stainless-steel counter electrode block which held a Ag/AgCl reference electrode with a 0.045 mm PTFE spacer. The potential of the dual working electrodes was controlled with a bipotentiostat with a common reference electrode. Br2 generated at the upstream electrode was detected at the downstream electrode. Amino-acids and proteins lowered the downstream current but had no apparent effect on the upstream current. A SAX-300 Synchropak column (25 cm x 4.6 mm i.d.) linked to a DEC was employed for HPLC separations. A 100 l portion was used for both procedures with a mobile electrolyte flow rate of 0.5 ml/min. Calibration graphs were linear from 1-1000 M solutions of cysteine, tyrosine, lysine, tryptophan, glycine, methionine and arginine and for 0.1-1000 M solutions of a number of total proteins (listed) with an upstream electrode potential of 1 V.

Abstract: In order to facilitate the online electrochemical detection of aromatic amino acids, peptides and proteins in HPLC, a post-column photolytic derivatization technique was developed (cf. Anal. Chem., 1990, 62, 2599) to convert non-electroactive compounds into electroactive derivatives. Further experiments have been carried out on model compounds (e.g. phenylalanine, tyrosine, cystine, tyrosine-glycine, phenylalanine-glycine, insulin and ribonuclease A) in order to understand the detection mechanisms. Photolysis was carried out offline and the decomposition products were identified mainly by HPLC. Photolytic processes that occurred in proteins included disulfide bond cleavage, photolysis of side-chain amino acids, and conformational changes. In most instances the electrochemical activity of proteins can be improved by photolysis thereby providing a means of improving the electrochemical detection of such compounds.