The Application Of Capillary Electrophoresis In The Research Of Er-tang Finger-Print And The Single-Cell Analysis Of Vegetal Cell

Objective: To study the the application of capillary electrophoresis in the research of TCM (Traditional Chinese Medicine) modernization. 1. To study the application of capillary electrophoresis in the research of finger-print of Chinese traditional medicine, and establish the analytical method for finger-print of er-chen tang. 2. To study the application of capillary electrophoresis and Laser-induced fluorescence in single-protoplast analysis in order to analyze amino acids in wheat embryonal cell.Methods:1.The decoction of er-chen tang from Tai Ping Hui Min He Ji Ju Fang was analyzed by capillary electrophoresis. Separating buffer: 35mmol/L borax (with acetonitrile 10% pH =9.0) was chosen as buffer solution (pH 9.0). 3D spectrogram was obtained by scanning spectrogram using diode array detector in the wavelength of 190nm～300nm. And 200nm was determined as the best wavelength. Then sample of er-chen tang was detected by ultraviolet detector. CE condition: Voltage:25kV, from anobe to cathode; capillary cartridge temperature: 25℃; enter sample by pressure: 0.5psi×5s. 2. Amino acids analysis in single wheat embryonic protoplast was performed using capillary electrophoresis equipped with laser-induced fluorescence (CE-LIF). Reagent fluorescein isothiocyanate (FITC) was introduced into living protoplasts by electroporation for intracellular derivatization. CE-LIF detection was made after the intracellular derivatization finished. Separate buffer: 25×10-2mol/L borax/1.25×10-2 mol/L NaOH-1.20×10-4 mol/L spermine (pH 9.45).CE condition: Voltage: 25kV; from anobe to cathode;capillary cartridge temperature: 25℃.Results:1. The best buffer and electrophoresis condition of er-chen tang were determined. Er-chen tang fingerprint was initially established. The separation of more then 20 peaks in the formula was realized in one electrophoresis condition, which also showed considerable repeatability and stability. 2. Reagent of derivatization was introduced into living protoplasts by electroporation for intracellular derivatization so that the separation of amino acids could occur in cell, which could minish the dilution of sample. Analysis of ingredients in vegetal cell was realized by combining with high saitivity of LIF, and 10 amino acids in single wheat embryonal cell were considerably separated combing with the high sensitivity of LIF detection.Conclusion:Capillary electrophoresis had excellent characteristics such as multiple separating mode, high separating efficiency, rapidness, wide using range, less requirement of sample and reagent, direct analyzing water liquor. CE showed its advantage in the analysis of finger-print and amino acids in single cell. We could foresee that the technology of capillary electrophoresis would have broad future.