Simultaneous Determination Of 20 Amino Acids By Core-shell Column HPLC With Evaporative Light Scattering Detector

Amino acid is one of the essential substances in life,and it is also a common food additives,feed additives,and pharmaceutical ingredient.For amino acid analysis,there are many problems such as derivatization,derivative instability,high cost,expensive instrumentation,high operating costs,long operating time and so on.Evaporative light-scattering detector(ELSD)is a kind of universal detector,by which all samples with lower volatility compared to mobile phase can be detected.It is not restricted by the structure of the material itself,which is applied widely in the fields of food safety,analysis of pharmaceutical and traditional Chinese medicine.The Halo core shell column can be used to obtain the separation speed and effect of UPLC on the common liquid chromatograph,which greatly improves the separation degree of the complex products.This study,based on the above background,we have carried out an analytical method for the determination of 20 underivatized amino acids using HPLC with HALO column coupled with an ELSD.Five chapters are concluded in this paper :Chapter 1 first briefly introduced the background and significance of the thesis,including kinds and the application of amino acids,the HALO column and evaporative light scattering detector and advances in studies on separating amino acids.In Chapter 2,the separation of amino acids was carried out by HILIC column,HALO C18 column,reverse C18 column,and HALO Amide column.The run time was at least 40 minutes on separation of amino acids by conventional C18 column.The separation effect of Amide HALO column was not obvious because of the poor column capacity and separation ability.In comparison,15 HALO C18 cm column can meet the separation requirements of amino acids.In Chapter 3,Through detecting 20 kinds of amino acids Overlay of chromatogramsed standard solutions,we optimize chromatographic conditions,such as the organic mobile phase,ionic additives(TFA?),gradient program,flow rate.Referring the peak signal to noise ratio of taurine(Tau),hydroxyproline(Hyp)and Su-ammonia acid(Thr),ELSD parameters are also optimized.We finally use a core-shell C18 stationary phase(4.6 mm×150 mm,2.7 ?m),A solvent gradient elution was performed with nonafluoropentanoic acid-heptafluorobutyric acid containing trifluoroacetic acid as mobile phase A and methanol as mobile phase B.The temperature of the drift tube in ELSD was set at 40? and the flow rate of carrier gas was 3.0 L/min.The separation and detection can be completed in 30 min.In Chapter 4,analytical methods of amino acids were verified.The logarithm of peak area and logarithm of quality of amino acids had a good linearity between 0.01~1.0 mg/ml.The lowest detection limits of amino acids are between 20 ng and50 ng when the peak signal to noise ratio is 3.The average recoveries of the 20 amino acids were between 89.0%and 111.2%.The precision and intermediate precison are compied.This method of amino acids without derivatization is simple and reliable.Chapter 5 summarizes three part experiments of this study.Further experiments were proposed and future prospects were speculated.