| Life science is one of the most important research domains in this century, and amino acid analysis is one of the important analytical techniques in the investigation of life science. Therefore, it is necessary to develop accurate, sensitive, simple,and rapid methods for the analysis of amino acids.Chemiluminescence (CL) method is an attractive and powerful detection technique due to its analytical advantages including low detection limit, very high sensitivity, wide linear dynamic range, and fast response, and it has been used in life science, clinical medicine, and environmental science.Flow injection analysis (FIA) is well-established technique for rapid automated solution treatment and detection. Sequential injection analysis (SIA), as a branch of sampling means of FIA or second generation FIA, the principles upon which SI is based are similar to those of FIA, namely controlled partial dispersion and reproducible sample handing. In contrast to FIA, the flow programming in SIA allows the employment of complex chemistry without reconfiguration of the manifold, and the instrumentation is simpler and intrinsically robust, easy to automate and detect on-line with very low reagent consumption.When chemiluminescence detection was combined with SIA analysis, its features were reserved, and the shortcomings, such as inconvenience of reaction process control and poor repeatability caused by fast reaction, were overcame. Combination CL with SIA remarkably improved the repeatability, accuracy and analytical frequency of the determination results.In this paper, some new CL reaction systems were proposed and studied. Three new SIA-CL methods were developed for selective determination of amino acids. In addition, the possible reaction mechanisms of the CL reaction systems were discussed. The dissertation is divided into four chapters:Chapter 1 briefly reviewed main methods for the determination of amino acids in recent years, and these methods include chromatography, chemometrics procedure, and photometric measurement method without chromatograph.In Chapter 2 of the dissertation, a simulation experiment was proceeded to explain the reason in which various zone stacking sequences influence the CL intensity in sequential injection sampling. As a result, physical dispersion caused by sampling sequences for rapid CL reaction is the major factor that influenced luminescent intensity. In addition, a SIA-CL method for the determination of histidine was developed, based on the reaction that histidine increased the catalytic activity of manganese (Ⅱ) salts in luminol-hydrogen peroxide reaction system in the presence of dioximes in sodium borate medium. At optimized conditions, histidine can be determined in the linear range from 5.0×10-7 to 1.0×10-3 mol/L with a detection limit (3σ) of 2×10-7 mol/L for 60μL sample. The relative standard deviation (R.S.D.) for eleven repeated measurements of 4×10-5 mol/L histidine was 0.97%, and the sampling frequency was 80 h-1, and the recoveries were 90.0～103.3%. The proposed method has been successfully applied to the determination of histidine in some beer.In Chapter 3 of the dissertation, it was observed that the chemiluminescence intensity from the reaction of acidic Ce(Ⅳ) and Tween20 was greatly enhanced by tryptophan. Based on Ce(Ⅳ)-Tween20-Trp reaction, a sequential injection procedure with chemiluminescence detection was proposed for the determination of tryptophan. The influences of several physical and chemical parameters were assessed.Under the selected working conditions, a linear dynamic range of 2.0×10-7～8.0×10-6 mol/L, a 3σdetection limit of 1.5×10-7 mol/L and a coefficient of variation of 0.7% at 3.0×10-6 mol/L level were obtained. The procedure was applied to the determination of tryptophan in beer and amino acid injections, and the recoveries were 91.4～106.9%. At the same time, the possible reaction mechanism was studied, the emitter in the CL reaction is probably a transient intermediate product of Tween20 oxidized by Ce(IV), and tryptophan is an enhancer for the reaction of Ce(IV) and Tween20.In Chapter 4 of the dissertation, two new chemiluminescence reaction systems, namely Ce(Ⅳ)-quinine-Cys-(Trp+Tyr) and Ce(Ⅳ)-Tween40-Trp were studied. The possible reaction mechanisms were also investigated. Furthermore, a SIA manifold with double-pump and double-multiposition valve was developed for simultaneous determination of tryptophan and tyrosine. The total amounts of tyrosine and tryptophan could be obtained by their quantificational inhibition to the CL reaction of Ce(Ⅳ)-quinine-Cys, and the accurate content of tryptophan was obtained from its quantificational enhancement to the CL reaction of Ce(Ⅳ)-Tween40, and the content of tyrosine was obtained by subtracted content of tryptophan from the total content of tyrosine and tryptophan.To the CL reaction of Ce(Ⅳ)-quinine-Cys-(Trp+Tyr) and Ce(Ⅳ)-Tween40-Trp, the dynamic range of 3.0×10-7～1.2×10-5 mol/L and 2.0×10-7～1.4×10-5 mol/L, the detection limits (3σ) of 2.9×10-7 mol/L and 9.0×10-8 mol/L, and the relative standard deviation (R.S.D.) of 1.1% and 1.3% were obtained, respectively. The method was applied to determination of tyrosine and tryptophan in amino acid injection, beer, serum, soybean milk and milk.A microwave-assistance treatment procedure was used to hydrolyze soybean milk and milk samples with 5.0 mol/L sodium hydroxide for 50 s, and the procedure dramatically improved the treatment efficiency.In conclusion, this dissertation demonstrated selective determinations of amino acids by sequential injection with chemiluminescence detection. These proposed methods are sensitive, simple, and less reagents consumption and waste output, and the methods were applied to determination of three amino acid contents in food beverage, blood serum, and amino acid injection. |
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