3 research outputs found
The development of electroanalytical techniques
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