Application Of Sample Pre-treatment Technique Before Chromatographic Analysis

High performance liquid chromatographic method(HPLC)is becoming widely used in analytical chemistry field,such as medicine analysis,environment protection,life science and petrochemical industry.However,many samples for HPLC were complex and lower concentration.So,removing interferents from samples before HPLC analysis and improve the sensitivity of HPLC methods are important.Up to now,there are many samples pretreatment techniques had been developed for HPLC analysis.Two kinds can be summarized for these pretreatment techniques.The first kind of technique is derivatization reaction.For some compounds,they can't be detected by HPLC-UV detector or HPLC-Fluorescence detector.These compounds can be detected after derivatization through chemical reaction.The second is pre-concentration technique.This kind of technique includes liquid-liquid extraction(LLE),microwave assistant extraction, solid-phase extraction(SPE),solid-phase microextraction(SPME)and liquid-phase microextraction(LPME),et al.In this dissertation,we have carried out some original studies as follows:(1)A rapid and sensitive pre-column derivatization-HPLC method for analysis of insulin was accomplished using NBD-Cl as fluorescent derivatization reagent.(2)A rapid and sensitive pre-column derivatization-HPLC method for analysis of free amino acids in islets of Langerhans was accomplished using NBD-Cl as fluorescent derivatization reagent.(3)A simple,inexpensive and efficient liquid-phase microextraction(LPME) technology combined with high-performance liquid chromatography(HPLC) was developed for the simultaneous analysis of tetrandrine and fangchinoline in plasma samples.(4)Using mixed traditional sorbents to collect air samples in less volume than traditional method and to desorb the substances via heat desorption and concentration injector or SPE,a novel method for qualitative analysis pollutant in air was developed.Five parts are included in the dissertationChapterⅠ:Sample preparation was summarized from two sides:(1)Derivatization technique:UV and Fluorescence derivatization were introduced including the choice of derivatizative regents and derivatizative conditions.(2)Isolation,purification and enrichment technique:liquid phase microextraction, solid phase microextraction and solid phase extraction were introduced here with liquid phase microextraction as the emphasis including its theory,operation and application.ChapterⅡ:Determination of insulin with HPLC and precolumn derivatization using NBD-Cl as derivatizative reagent.All kinds of conditions which affected the derivative reaction including temperature,time,pH and buffer et al were studied.The optimal conditions are:50℃,2h,0.1M phosphate buffer with pH 9.0 and the derivative reaction was carried out in the dark.The HPLC conditions are:C18,mobile phase:ACN- 0.1% TFA(30:70,v/v).ChapterⅢ:Free amino acids in islets of rat were measured by HPLC with NBD-Cl as derivatizative reagent for precolumn derivatization.15 amino acids were separated and analyzed with gradient elution.The matrix and all kinds of conditions affecting separation and derivatization were studied,the results are satisfied.ChapterⅣ:Determination of tetrandrine and fangchinoline in plasma samples using hollow fiber liquid-phase microextraction combined with high-performance liquid chromatography was studied.Several parameters influencing the efficiency of LPME were investigated and optimized including organic solvent,stirring rate,extraction time, salt concentration,organic modifier and pH.The method set up was compared with other techniques and the results showed that the method is prior to other techniques on the sample cleaning and enrichment factor.ChapterⅤ:The pollutants in air of Lanzhou city were qualitatively and quantitatively analysed by GC-MS with solid phase extraction using a mixed absorbent and heat-desorption.The sample amount,organic solvent amount used and the analysis time of the experiment were less than with other methods.