Indirect chemiluminescence detection of amino acids separated by capillary electrophoresis

An indirect chemiluminescence (CL) detection method for amino acids following capillary electrophoretic separation is described. Since amino acids can form stable complexes with Cu(II) and complexed Cu(II) is a much poorer catalyst for the CL reaction between luminol and H2O2, indirect detection is based on the measurement of the decreasing catalytic activity of Cu(II) in the presence of amino acids. The degree of CL suppression is proportional to the amino acid concentrations. The optimal conditions for the indirect CL detection were determined with regard to buffer composition and reagent concentration. However, the sensitivity of the indirect CL signal to buffer additives limits the buffer composition and, hence, the resolution of complex mixtures. Due to the sigmoid nature of the calibration curves, a limited working range (less than or equal to 2 orders of magnitude) was generally obtained. Detection limits for the unlabeled amino acids were in the 100-400 fmol range, depending upon the magnitude of the complex formation constants. Precision was between 3 and 6%. Applicability of this method to the analysis of amino acids in relatively simple samples was demonstrated.