Chemiluminescence flow-injection method for the determination of amino acids and proteins based on Schiff base formation in reversed micelles

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A chemiluminescence method involving Schiff base formation was developed for the determination of a series of primary amino acids. The Schiff bases derived from phenylacetaldehyde (PAA) and amino acids in sodium bis(2-ethylhexyl)sulphosuccinate (Aerosol OT. AOT) reversed micellar solution were found to produce strong chemiluminescence when oxidized with iron(II) and hydrogen peroxide (Fenton s reagent). The chemiluminescence signal was measured by means of flow injection system. 100ul of the schiff base sample solution being injected into a methanol carrier and mixed with the Fenton's reagent solution. The determination limit was 1.4X10^<-7>M for alanine and the linear dynamic range for various amino acids was 1 : 10^3-10^5M.Then, as the Schiff bases were fluorescent at 450nm (370nm excitation). rate and equilibrium constants at various water-to-surfactant ratios(Wo) were determined from the pseudo-first-order rate equation. The results showed that the forward reaction (Schiff base for … More mation) was greately increased with decreasing Wo. whereas the backward reaction (hydrolysis of Schiff base) was decreased. The equilibrium constant. consequently. was greately increased with a decrease in Wo. From the measurement of the fluorescent lifetimes of phenylalanine and tryptophan in the AOT reversed micelles and of the relative chemiluminescent intensities of different hydrophobic amino acids. it was concluded that the enhancement of the Schiff base formation in AOT reversed micelles was mainly dependent on the solubilizing location of the amino acids.The chemiluminescent system was also sucsessfully applied as the detection system for a high-performance liquid chromatograph with an ODS micro-column. It might possibly be applicable to the determination of the amino acids from protein hydrolysates.The effect of the presence of AOT reversed micelles was also proved in the chemiluminescence system of bis(2.4.6-trichlorophenyl) oxalate(TCPO)-hydrogen peroxide-fluorescer. The intensity of chemiluminescence in reversed micellar solution was about ten times larger than in a homogeneous medium. Less