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

建立了一种测定一系列伯氨基酸的Schiff碱化学发光法。在双（2-乙基己基）磺基琥珀酸钠（Aerosol OT. AOT）反胶团溶液被Fe（II）和H_2O_2（芬顿试剂）氧化时产生强烈的化学发光。用流动注射系统测定了化学发光信号。将100 μ l的席夫碱样品溶液注入甲醇载体中并与芬顿试剂溶液混合。丙氨酸的检出限为1.4 × 10 <-7>~（-5）M，各种氨基酸的线性范围为1：10 ~（-3）~ 10 ~（-5）M。速率和平衡常数在不同的水与表面活性剂的比例（Wo）确定的伪一级速率方程。结果表明，顺向反应（Schiff碱为 ...更多信息 mation）随Wo的降低而显著增加。而反向反应（Schiff碱的水解）减少。平衡常数。因此。随着Wo值的降低，通过测定苯丙氨酸和色氨酸在AOT反胶束中的荧光寿命以及不同疏水氨基酸的相对荧光强度，结果表明，AOT反胶团中氨基酸的增溶位置对Schiff碱的形成有促进作用，并将其成功地应用于ODS微柱高效液相色谱的检测系统中. AOT反胶团的存在对二（2.4.6-三氯苯基）草酸酯（TCPO）-过氧化氢-荧光剂化学发光体系的影响也得到了证实。在反胶束溶液中的化学发光强度比在均相介质中的化学发光强度大约10倍。少

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