| High Performance Liquid Chromatography (HPLC) is an important analysis and separation modern technology, which is developed on the basis of column Chromatography and gas Chromatograph. Due to high-pressure pump, high sensitivity detector, highly active filler of column and micro-injecting syringe, it has achieved good separation efficiency, high sensitivity, high-speed analysis, wide range of applications and simple operation. The mobile phase which is formed by different polarity single or mixed solvent, or buffer, flows to column filled by stationary phase by high-pressure pump. Different composition of sample is separated in column, and then is detected by flowing into detector, that is the process. This way has been widely used in the fields of chemistry, pharmacy, environment, food industry, petrochemical processing and legal inspection.Sample preparation affects the efficiency of extraction of the composition of sample and change of property of them as a essential process before injecting. In recent years, it has got wide attention. Many new extraction ways have appeared. This thesis experiments used non-ionic surfactant as the extraction solvent to extract the active components from sample, the result has indicated that this way is easy, fast, effective and environment-friendly.This thesis mainly includes two parts, review and research report. In the review section, it has introduced the characteristic and application, the advancement of sample preparation way, the importance of studying research. In the next research report section, it has reported four experiments. They are:determination of chlorogenic acid, quercetin and kaempferol in Folium Eriobotryae, determination of cholesterol in oil and fat using cloud point extraction by HPLC-UV, determination of Chlorogenic Acid, Rutin and Quercetin in Zanthoxylum schinifolium Zucc., and Zanthoxylum bungeanum by high performance liquid chromatography and determination of resveratrol in polygonum cuspidatum using the non-ionic surfactant as the extraction solvent followed by high performance liquid chromatography.1、Simultaneous determination of chlorogenic acid, quercetin and kaempferol in Folium EriobotryaeThe non-ionic surfactant Triton X-100was chosen as the extraction solvent; three compounds including chlorogenic acid, quercetin and kaempferol in the leaves of Folium Eriobotryae were determined by high performance liquid chromatography (RP-HPLC) with ultrasonic extraction. The samples were analyzed on a Phenomenex C18column with methanol-0.25%acetic acid glacial as a mobile phase using a gradient elution. The flow rate was1.0mL·min-1, and UV detection wavelength was set at350nm, and the contents of chlorogenic acid, quercetin and kaempferol were determined by the external standard method. The calibration curves for chlorogenic acid, quercetin and kaempferol were linear between2.20～220μ/mL (r=0.9997),0.65～130μg/mL (r=0.9999),0.68～136μg/mL (r=0.9999), respectively. The limit of detection (LOD) was0.0.94μg/mL,0.055μg/mL,0.045μg/mL (S/N=3) respectively; The spiked recoveries ranged from90.71%to94.88%. The method is simple, rapid, easily operational, sensitive, environment-friendly, and thus provides a new and scientific means for assaying the contents in the leaves of Folium Eriobotryae.2、determination of cholesterol in oil and fat using cloud point extraction by HPLC-UVA fast, simple, and sensitive sample preparation procedure based on cloud point extraction (CPE) is proposed for the determination of cholesterol in oil and fat using reverse phase high performance liquid chromatography (RP-HPLC) and UV detection. The non-ionic surfactant Triton X-114was chosen as the extract solvent. The samples were analyzed on a Phenomenex C18column with ethanol-methanol as the mobile phase using isocratic elution. The flow rate was0.8mL·min-1, and UV detection wavelength was set at206nm. Optimized conditions for the pretreatment of oil and fat were the concentration of surfactant Triton X-114, the concentration of sodium chloride, equilibration temperature, and equilibration time. There was a good linear correlation between the concentration and the peak area of cholesterol in the range of0.10-150 μg/mL(r=0.9998), the limit of detection (LOD) was0.03μg/mL(S/N=3). The average recoveries were86.00%～93.50%. This new method is fast, easily operational, sensitive, environment-friendly, and can meet the requirements of determination of cholesterol of oil and fat.3、determination of Chlorogenic Acid, Rutin and Quercetin in Zanthoxylum schinifolium Zucc. and Zanthoxylum bungeanum by high performance liquid chromatographyA method for the determination of three compounds including chlorogenic acid, rutin and quercetin in Zanthoxylum schinifolium Zucc. and Zanthoxylum bungeanum by ultrasonic extraction coupled with high performance liquid chromatography (HPLC) was developed. The non-ionic surfactant Triton X-100was chosen as the extract solvent, the samples were extracted at45℃with30min, and centrifuged, then the solution of the upper layer was injected and successfully separated on a Phenomenex C18column with methanol-0.25%acetic acid glacial as a mobile phase using a gradient elution with a flow rate of1.0mL·min-1, UV detection wavelength of350nm, and injection volume of10μL. Under the optimal conditions, the calibration curves for chlorogenic acid, rutin and quercetin were linear between0.022～110μg/mL (r=0.9997),0.016～80μg/mL (r=0.9996),0.026～130μg/mL (r=0.9999), respectively. The spiked recoveries ranged from89.35%to92.57%with relative standard deviation (RSDs) of0.79%-1.70%. The content of chlorogenic acid in Zanthoxylum schinifolium Zucc. was twice as much as that in Zanthoxylum bungeanum, the content of rutin in Zanthoxylum bungeanum was twice as much as that in Zanthoxylum schinifolium Zucc. and the content of quercetin in Zanthoxylum bungeanum was much higher than that in Zanthoxylum schinifolium Zucc. This conclusion provides a valid reference standard for comparing the content difference of main substance in Zanthoxylum schinifolium Zucc. and Zanthoxylum bungeanum.4、determination of resveratrol in polygonum cuspidatum using the non-ionic surfactant as the extraction solvent followed by high performance liquid chromatography.A simple and green method was developed for the determination of resveratrol in polygonum cuspidatum, nonionic surfactant Triton X-100was used to extract resveratrol from polygonum cuspidatum prior to their determination by high performance liquid chromatography (HPLC). The experimental conditions were optimized by single-factor experiment and L9(43) orthogonal array design (OAD) using the extraction yield of resveratrol as the index determined by HPLC. The samples were analyzed on a Phenomenex C18column with methanol-0.25%acetic acid glacial as a mobile phase with isocratic elution. The flow rate was0.8mL-min-1, and UV detection wavelength was set at306nm, and the concentration of resveratrol was determined by the external standard method. Under optimized conditions:8%Triton X-100(w/v), liquid/solid ratio of l:15(g/mL), ultrasonic-assisted extraction for15min at50℃, the extraction yield of resveratrol reached the highest value. The results showed that there was a good linear correlation between the concentration and the peak area of resveratrol in the range of0.12-200μg/mL(r=0.9998), the limit of detection (LOD) was7.60ng/mL (S/N=3). The spiked recoveries were93.50%-104.10%. |
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