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In the application of modern separation science research, efficient, rapid and high-performance liquid chromatography (High Performance Liquid Chromatography, HPLC) has sensitivity and high purification factor of advantages, has become a powerful tool for the analysis and separation of natural active ingredients. Especially from complex samples, such as an active ingredient of the Chinese herbal medicine, functional proteins, enzymes and the analysis and separation of biological macromolecules increasingly tends to be fast, environmentally friendly, low-cost analysis, separation methods Establishment. Therefore, this study by HPLC determination method of traditional Chinese medicine preparation Rhodiola Dripping Pills Salidroside, while purified spinach superoxide dismutase (SOD) were isolated. This thesis consists of four parts: 1. Literature review: This chapter reviews the different mechanisms by HPLC from the one-dimensional and two-dimensional online / offline LC hyphenated techniques in complex sample analysis and separation and extraction progress and its applications. 2. The rhodiola Dripping Pills Salidroside separation and its content determination: HP-RPLC method of Rhodiola Dripping Pills Salidroside, qualitative and quantitative analysis, including chromatographic elution mode choice, repeat stability, the recoveries, and determination of the investigation, and the quantitative determination of different solvents the rhodiola Dripping Pills Salidroside, the content. The results show that were washed with water and methanol as solvent extraction from Rhodiola Diwan salidroside its extraction rate is 9.25 mg / g and 9.27 mg / g and, therefore, can be washed with water to replace the methanol solvent extraction to take the red King day camp, thereby reducing the environmental pollution caused by the use of organic solvents. Spinach superoxide dismutase crude separation: Optimizing spinach SOD extraction conditions, including the pH of the extract, the heating temperature in the solid-liquid ratio and SOD separation and purification of the crude enzyme solution and the amount of acetone, and measured live The method of screening, from spinach superoxide dismutase crude separation method. The results obtained under optimal conditions, the SOD specific activity of up to 72.38 U / mg, a purification factor of 4.2. Isoenzyme electrophoresis in spinach SOD isoenzyme identification spinach SOD Cu, Zn-SOD; determination of Cu, Zn-SOD in spinach molecular weight by SDS-PAGE, MALDI-TOF-MS subunit molecular weight of 31.8 kDa, 19.8 kDa. 4. HPLC method separation and purification of superoxide dismutase: the use of the method of HPHIC and HPIEC law as well as HPHIC-SEC method SOD crude enzyme solution, pure, and compare several methods. Results after separation with HPHIC, specific activity for 119.82 U / mg, a purification factor of 7.8, the activity recovery was 34.3%; specific activity after the separation of the method of HPIEC 159.92 U / mg, a purification factor of 10.7, the activity rate was 43.6%; while the use of two-dimensional Offline mode HPHIC-SEC separation specific activity was 284.65 U / mg, and a purification factor of 19.0, the activity rate was 60.5%. In offline HPHIC-SEC mode separation Therefore, purified the SOD obtain maximum activity recovery and purification fold.
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