The development of electroanalytical techniques

Abstract

This thesis describes the development of an electroanalytical technique for the estimation of compounds of biochemical interest. The technique involves titration with hypobromite using double electrode systems - the rotating ring disc electrode (RRDE) and the flow-through tubular double electrode (TDE). Hypobromite is continuously electregenerated at the upstream disc (generator) electrode and is transported by convection and diffusion to the downstream ring (detector) electrode where it is detected amperometrically. The presence of a reactive substrate in solution decreases the amount of titrant which reaches the ring, and from measurements of the generating and detecting currents the bulk concentration of substrate may be estimated. At pH 9.2 the amine group of amino acids reacts with two hypobromite molecules and some side chains (e.g. cystinyl, tyrosinyl, tryptophanyl) will also react. The reactivity of these side chains permits the titration of proteins. The titration response may be described theoretically which enables the calculation of the number of hypobromite molecules which react with one protein molecule ( ~500 for haemoglobin). With this chemical amplification the detection limit of the technique is ~10-8 g ml-1 for proteins and ~10-8 M for amino acids. An electronic circuit has been developed which enables the detector electrode to control the rate at which titrant is generated. This autotitrator produces a generator current which is proportional to substrate concentration, and with the TDE should enable the continuous estimation of proteins as they are eluted from a chromatographic column. Proteins have been titrated at pH 9.2 and pH 5; the ratio of a protein's titration responses reflects its amino acid composition and, when combined with the extinction coefficient, enables the identification of proteins. Patent applications for this method of identification and for the autotitration technique have been submitted.</p