| A novel precolumn N-methylation derivative method for the quantification of α-amino acids was established using formaldehyde and sodium borohydride as main derivatization reagents.In a mild aqueous medium conditions,both primary and secondary α-amino acids could be converted to their N,N-dimethylation derivatives through a series of chemical reactions including nucleophilic addition,dehydration and chemical reduction processes.And,these α-amino acid derivatives were the effective coreactant of tris(2,2’-bipyridine)ruthenium(II)(Ru(bpy)32+)which could produce the enhanced electrochemiluminescence signal.On the basis of experimental results relating to the derivative reaction conditions and reaction pathway,a new method was developed for quantitative determination of some free α-amino acids by precolumn derivation capillary electrophoresis coupled with electrochemiluminescence detection technique(CE-ECL).With the homemade cross-linking chitosan modified capillary as separation capillary,after the experimental parameters about the electrochemiluminescence(ECL)measure and electrophoresis separation conditions were optimized,several proteinogenic α-amino acids had been rapidly determined with the high efficiency and selectivity.In addition,the content of theanine in different commercial tea samples was analyzed with the same method.This paper consists of four chapters.Chapter 1: A literature review on this research.Firstly,a brief review of definition,properties,classification and practical applications of amino acids were afforded,and the routine analytcal methods and the recent trends about amino acid analysis also were summarized.Moreover,we mainly introduced the chemical derivatization techniques and CE-ECL technique for the analytical application of amino acids.Specially,some actual documentation about the determination of α-amino acid in different biologic samples,which adopted the CE-ECL method relied on the Ru(bpy)32+-based ECL detection mode,were reviewed in detail.Chapter 2: Study on a precolumn N-methylation derivative method for the quantification of α-amino acids.By using alanine(Ala),valine(Val),phenylalanine(Phe)and histidine(His)as model compounds,CE-ECL as a technical means,we studied the N-methylation derivative reaction of α-amino acids in detail.According the survey of ECL intensity of thses α-amino acid derivatives in the different experimental conditions,the influencing factors and reaction pathway relating to the derivatization reaction were validated,and the reaction parameters such as the added amount of derivatization reagents,reaction temperature,reaction time,solution acidy and the dosage of catalyst were optimized.The results showed that in a weakly acidic aqueous medium,α-amino acids containing primary or secondary amino groups could undergo an initial nucleophilic attack on formaldehyde,a successive dehydration step to form imine intermediates and an ultimate reductive reaction with sodium borohydride to the stable N,N-dimethylation products.Thus,the mechanism about the N-methylation derivatization reaction of α-amino acids was inferred from the experimental evidences.Under the optimized experimental conditions,the ECL peak intensity of four α-amino acid derivatives could clearly exhibite a significant increase of approximately 35-115 times.The linear ranges was attained to 5-250 μM for Ala and Val,10-100 μM for Phe and 10-500 μM for His,with correlation coefficients(R2)between 0.9965 and 0.9994.The evaluated detection limits(S/N=3)were 2.2 μM for Ala,4.3 μM for Val,7.2 μM for Phe and 9.8 μM for His.Apparently,this derivatization strategy might be expected to be implemented into CE-ECL analysis for α-amino acids in complex samples with high selectivity and sensitivity.Chapter 3: Experimental study on the separation and determination of five essential α-amino acids using pre-column derivatization CE-ECL method.With the homemade cross-linking chitosan coating capillary as separation capillary,the separation and analysis conditions of five kinds of essential α-amino acids(Arg,Lys,Leu,Met,His)were studied by CE-ECL method.Many influencing factors impacting on the ECL intensity of five amino acid derivatives,including detection potential,separation voltage,injection voltage and injection time of sample,the concentration and p H of running buffer,amount of separation additives were investigated.In particular,the action of two separation additives,sodium tripolyphosphate(STPP)and sodium dodecyl benzene sulfonate(SDBS),was discussed again.Under the optimal experimental conditions,the derivatives of five essential α-amino acids were attained to baseline separation within 5 min,and their ECL peak intensities had a good linear relationship with the concentration of five α-amino acids between 2-500 μM(R2=0.9932-0.9994).The evaluated detection limits(S/N=3)were 5.5 μM for Arg,4.6 μM for Lys,4.8 μM for Leu,7.2 μM for Met and 9.8 μM for His.These results would offer a substantial basis for the quantitative CE-ECL determination of five α-amino acids in practical samples.Chapter 4: Determination of theanine content in tea samples by pre-column derivatization CE-ECL method.With the homemade cross-linking chitosan coating capillary as separation capillary,the content of theanine in six kinds of marketed tea samples was taken separation and determination by CE-ECL method.The effect of some analytical conditions on the ECL peak intensity of theanine derivative,including detection potential,separation voltage,the PBS concentration and p H in the detector cell and the amount of separation additives were evaluated.Under the optimal experimental conditions,the rapid separation of theanine derivative could be attained within 5 min.The linear ranges was 5-200 μM for theanine,and its detection limit(S/N = 3)was 2.8 μM.The repeatability of this method was investigated by six consecutive injections of a standard derivative solution containing 75 μM theanine,and the RSD values of both migration time and ECL peak intensity were 1.14 % and 2.49 %,respectively.By using standard addition method,the content of theanine in the six original tea samples was successfully obtained,while the specific data of theanine content in the tea samples were 1.53 %、0.91 %、0.61 %、0.37 % 、0.28 % and 0.14 %(by dry weight measure)for White tea,Black tea,Longjin tea,Green tea,Pu’er tea and Tie guanyin tea,respectively.Meanwhile,the average recoveries of theanine in the actual White tea samples were attainable 105.3 %.This method was much simple and rapid,and could be suitable to the different tea products analysis. |
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