| High performance liquid chromatography has the advantage of high pressure, high efficiency, high sensitivity and simple operation. It has wide application in the modern analysis field, such as food sanitation, environmental safety, petrochemical industry, natural products, biological engineering and so on. The principle of high performance liquid chromatography is the adsorption and unadsorption velocity difference of analytes in the chromatographic column. Retention time of analytes in the column is different, the separation of analytes is achieved when the analytes flow successively the column. Sample was appropriate prepared before analysis, and preparation processing quality directly affect the sensitivity and accuracy. These years, many sample pretreatment technology was developed and used for the real sample analysis. Environment friendly, simple, fast sample preparation technology for determination analysis is always the target that the plenty of researchers pursue.The paper mainly includes two parts: review and research report. In the first part, the development and application of high performance liquid chromatography, Chinese herbal medicine and sample preparation was reviewed. In the research report part, the determination method of flavonoids compounds and organic acid in three Chinese traditional medicine and the organophosphorus pesticides residues in water was established.1. Simultaneous separation of chlorogenic acid, quercetin and kaempferol in pyrrosia lingua(thumb.) farwell by high performance liquid chromatographyMethanol was used to extraction solvent, the high performance liquid chromatography(HPLC) method for simultaneous separation and assaying of three compounds(chlorogenic acid, quercetin and kaempferol) in pyrrosia lingua(thumb.) farwell was developed. The separation was obtained on Phenomenex C18 column(250 mm×4.6 mm, 5 μm) with the mixture solution of methanol and acetic acid(p H=3.0). A gradient elution, 1.0 m L/min flow rate, 254 nm UV detection wavelength and 35 ℃ column temperature was set, respectively. The linear curves of three compounds(chlorogenic acid, quercetin and kaempferol) were linear in the range of 0.000 24-3.00 μg(r=0.999 9), 0.000 16-2.00 μg(r=0.999 9), and 0.000 16-2.00 μg(r=0.999 9), respectively. The limits of detection(S/N=3) were 3.29 ng/m L, 0.43 ng/m L, and 0.33 ng/m L, respectively. The spiked recovery for three compounds was 97.73%, 98.07%, and 96.92%, respectively. The developed method was simple, fast, accuracy and sensitive. It provides experimental proof for separation and assaying of chlorogenic acid, quercetin and kaempferol in pyrrosia lingua(thumb.) farwell.2. Ultrasonic assisted extraction of seven flavonoids and organic acids in potentilla discolor bunge by high performance liquid chromatographyThe HPLC method based on ultrasonic assisted extraction was developed for separation and determination of seven flavonoids and organic acids(chlorogenic acid, caffeic acid, hyperoside, quercetin, naringenin, kaempferol and apigenin) in potentilla discolor bunge. The separation of chlorogenic acid, caffeic acid, hyperoside, quercetin, naringenin, kaempferol and apigenin was achieved on Phenomenex C18 column(150mm×4.6mm, 5μm) with gradient elution. Mobile phase was methanol and 0.1% acetic acid, flow rate and UV detection wavelength was set at 1.0 m L/min and 350 nm, respectively. The simultaneous separation of seven flavonoids and organic acids was obtained within 20 min, and satisfactory linear equations(r>0.999 5, n=7) were established. The average recoveries of real sample were between 84.61% and 104.06%(RSD<4.77). The optional extraction conditions of chlorogenic acid, caffeic acid, hyperoside, quercetin, naringenin, kaempferol and apigenin in potentilla discolor bunge were ultrasonication extraction by 50 times 80% ethanol for 20 min. The analytical results of real samples proved that the content of chlorogenic acid, caffeic acid, hyperoside, quercetin, naringenin, kaempferol and apigenin was 12.1μg, 6.0 μg, 12.7 μg, 10.8 μg, 29.4 μg, 6.1 μg and 11.4 μg respectively in per o.1 gram potentilla discolor bunge. It could provide a reference for development and utilization of potentilla discolor bunge.3. Ultrasonic extraction and determination of 8 active ingredients in herba leonuriThe method of ultrasonic extraction and determination of syringic acid, ferulic acid, rutin, daidzein, quercetin, quercetin, quercetin and quercetin in Herba leonuri was developed. The separation of syringic acid, ferulic acid, rutin, daidzein, quercetin, quercetin, quercetin and quercetin in Herba leonuri was performed on Inert Sustain C18(150 mm×4.6 mm, 5 μm) with a gradient elution. Mobile phase consisted of methanol and p H=3.0 acetic acid with the flow rate of 1.0 m L/min. The ultraviolet determination wavelength was maintained at 285 nm. The column temperature was set at 35 ℃. The injection volume was 20 μL. The analytes of syringic acid, ferulic acid, rutin, daidzein, quercetin, quercetin, quercetin and quercetin were successfully separated with good calibration curves and correlation coefficient(r >0.999 1, n=7). The average spiked recovery in the range of 89.93%~102.01%, RSD were between 1.17%~3.11%. The results proved that the method is simple, fast and accuracy, which provides a reference for quality control of Herba leonuri.4. Solid-phase extraction followe d by dispersive liquid-iquid microextraction for the sensitive determination of four organophosphorus pesticides in water matrices by high performance liquid chromatographyA simple approach for determination of four organophosphorus pesticides including isocarbophos, parathion-methyl, triazophos and fenitrothion in water samples was developed. The analytes were rapidly extracted and concentrated from large volumes of aqueous solutions using solid-phase extraction followed by dispersive liquid-liquid microextraction(SPE-DLLME). The obtained extraction solvent then analyzed using high performance liquid chromatography coupled with ultraviolet detector. Variables affecting the extraction performance, including type and volume of elution solvent, volume and flow rate of sample solution, salt concentration, type and volume of extraction solvent and sample solution p H, were discussed. The best results were obtained using methanol as eluent and ethylene chloride as extraction solvent. Under the optimal conditions, 100 m L of water sample was first concentrated using a reversed phase extraction and then targets were eluted with 1 m L of methanol. In the spiked real experiment, an exhaustive extraction for analyte compounds(recoveries >86.9%) and high enrichment factors were obtained. The limits of detection(LODs) were in the range of 0.021-0.15 μg·L-1. The relative standard deviations(RSD) for 0.5 μg·L-1 spiked water sample were between 1.9% and 6.8%(n=5). The proposed approach offers many advantages such as simple operation, rapidity, high enrichment factor and sensitivity. The method was successfully applied to the analysis of four OPPs in real water samples. |
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