Plastic Film Ring-Disk Carbon Electrodes for the Indirect Amperometric Detection of Underivatized Amino Acids in Liquid Chromatography
The analysis of amino acids is of great importance in the biological and food fields. Over the past decade, numerous procedures involving high performance liquid chromatography have been successfully developed for the determination of amino acids in biological and food samples. However, because the most used detection mode, UV-visible, often lacks sensitivity due to the absence of strong absorbing chromophore or fluorophore groups in most of the amino acids, pre-or postcolumn derivatizations
... derivatizations are necessary to make the analytes UV or fluorescence active. The reaction between primary alkyl amines and o-phthalaldehyde (OPA) in the presence of an alkyl thiol is quite frequently used for the detection of amino acids in liquid chromatography (LC) with high sensitivity. 1-3 However, the derivatizations generally introduce sources of contamination in the analytical procedure. Electrochemical detection is an attractive approach for the determination of many compounds in LC owing to its high sensitivity and relative low cost. However, because there are only six amino acids, such as tyrosine, tryptophan, histidine, cysteine, cystine and methionine, which show electroactivity at the carbon electrode, procedures have been investigated for the purpose of preparing suitable electroactive derivatives for different classes of amino acids. For example, the OPA derivatives are electrochemically active and can be amperomertically detected. 4,5 Recently, several transition metal electrodes have also been developed for a direct amperometric detection mode based on the electrocatalytic oxidation of amino acids in an alkaline medium at these electrodes. Johnson et al. reported on the detection of trace underivatized amino acids with gold and platinum electrodes. 6,7 Baldwin et al. demonstrated the utility of the copper-based electrode for the constant amperometric detection of amino acids in LC. 8 The most serious defect of these metal electrodes is that they are subject to rapid surface fouling and, as a result, required a continuously pulsed potential waveform to maintain the optimum operation conditions. On the other hand, copper ions, resulting in the dissolution of the electrode substrate have shown a chelating ability with amino acids, and thereby lead to an increase in the anodic current. The use of electrogenerated reagents for the indirect detection of some nonelectroactive species is an interesting method that has gained practical use. King and Kissinger first reported on a postcolumn reaction for the detection of fatty acids with electrogenerated bromine. 9 Bromine is used as the reagent because it may be electrogenerated directly in the mobile phase, and reacts rapidly with a large number of compounds 957 ANALYTICAL SCIENCES OCTOBER 1999, VOL. 15 1999 An indirect amperometric detection of underivatized amino acids has been developed based on the use of a plastic film ring-disk carbon electrode in liquid chromatography. Bromide present in the postcolumn addition reagent is oxidized to bromine at the upstream (disk) electrode, and is subsequently detected at the downstream (ring) electrode. Most of the underivatized amino acids that are electroinactive under conventional amperometric conditions can react rapidly with the electrogenerated bromine. The concentration of amino acids can therefore be indirectly determined by continuously monitoring the reduction current of bromine. The signal monitored at the downstream electrode was largely dependent on the pH of the mobile phase, the generation and collection potentials, and the bromide concentration in the mobile phase. Under the optimized conditions, the detection limits of the 0.5 µM level were obtained for a number of amino acids with a linear correlation coefficient of 0.990 -0.998 over the concentration range 1 -100 µM. The primary advantage of this approach lies in the capability that it affords a simple and sensitive determination of underivatized amino acids. In addition, it was almost unaffected by fouling effects normally arising from the anodic oxidation of the sulfur-containing amino acid.