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

基于反胶束席夫碱形成的化学发光流动注射法测定氨基酸和蛋白质

基本信息

批准号：
02650546
负责人：
HOBO Toshiyuki
金额：
$ 1.28万
依托单位：
TOKYO METROPOLITAN UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

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

开发了一种涉及席夫碱形成的化学发光方法，用于测定一系列伯氨基酸。研究发现，源自苯乙醛 (PAA) 和双(2-乙基己基)磺基琥珀酸钠 (Aerosol OT. AOT) 反胶束溶液中氨基酸的席夫碱在被铁 (II) 和过氧化氢（芬顿试剂）氧化时会产生强烈的化学发光。通过流动注射系统测量化学发光信号。将100ul希夫碱样品溶液注入甲醇载体中并与芬顿试剂溶液混合。丙氨酸的测定限为1.4X10^-7M，各种氨基酸的线性动态范围为1:10^3-10^5M。然后，由于席夫碱在450nm处发出荧光(370nm激发)。不同水与表面活性剂比例（Wo）下的速率和平衡常数由伪一级速率方程确定。结果表明，正向反应（希夫碱）随着 Wo 的减少而大大增加。而逆向反应（希夫碱的水解）则减少。平衡常数。最后。随着Wo的减少而大大增加。通过测量 AOT 反相胶束中苯丙氨酸和色氨酸的荧光寿命以及不同疏水性氨基酸的相对化学发光强度。结论是AOT反胶束中席夫碱形成的增强主要依赖于氨基酸的增溶位置。该化学发光系统也成功应用于ODS微柱高效液相色谱仪的检测系统。它可能适用于蛋白质水解物中氨基酸的测定。AOT反胶束存在的影响也在双(2.4.6-三氯苯基)草酸酯(TCPO)-过氧化氢-荧光剂的化学发光体系中得到了证明。反胶束溶液中的化学发光强度比均匀介质中的化学发光强度大约高十倍。较少的