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Separation of Water-soluble Glycoprotein from Sweet Potato and Determination by High-performance Gel Chromatography

Author: LiMei
Tutor: GanChunZuo
School: Fujian Agriculture and Forestry University
Course: Biochemistry and Molecular Biology
Keywords: Sweet potato Water - soluble glycoprotein High performance gel chromatography Diode array detector Evaporative light scattering detector
CLC: S531
Type: Master's thesis
Year: 2009
Downloads: 164
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Abstract


The glycoprotein is a complex structure of the class of polymer compounds, having a variety of biological activities, and plays an important role in vivo. In this thesis, the sweet potato as raw material, using chromatography, gel electrophoresis and infrared scanning technology to study the separation and purification of the water-soluble glycoprotein of the sweet potato, the physical and chemical properties and structure; bovine serum albumin (BSA), dextran labeled efficient gel chromatography (HPGC) samples (DT70) and extract the sweet potato water soluble glycoprotein samples diode array detector (DAD) and evaporative light scattering detector (ELSD) series to determine the water-soluble glycoprotein . Conclusions: (1) through the single factor experiment and orthogonal experiment to determine the optimum extraction conditions of the sweet potato soluble glycoprotein: solid-liquid ratio 1:25, extraction temperature 40 ℃, extraction time of 1 h at pH 9, protein The extraction rate is 2.49%. The water-soluble glycoprotein white flocculent, easily soluble in water, insoluble in organic solvents such as methanol, ethanol, acetone; molecular weight of 66,000 Da; 88.23% protein content, sugar content of 11.75%; it contains non-starchy polysaccharides , acetylamino, pyranose ring, and the α-glycosidic bond. β-elimination reaction, the UV scans show significant absorption peak at 240nm at its O-sugar protein. 0.1um pore size of the membrane may be sweetpotato water extract most of the glycoprotein interception. Sweet potato soluble glycoprotein biological activity in vitro experiments showed that: It has good resistance to lipid peroxidation. (2) HPGC-DAD-ELSD chromatographic conditions for the detection of water-soluble glycoprotein series: pure water as mobile phase flow rate was 0.5ml/min, column temperature temperature of 30 ° C, the drift tube temperature of 80 ° C, the carrier gas pressure For 0.35Psi. (3) using HPGC-DAD-ELSD series of BSA and DT70 qualitative and quantitative analysis showed that: For the purposes of the non-bound proteins and polysaccharides, after separation by HPGC ELSD chromatograms show two peaks, but according to the DAD chromatography whether the peak retention time, the qualitative determination of BSA and DT70. Quantitative analysis showed that the BSA at 280nm, the peak height and concentration to take the highest correlation coefficient on the number of linear equations: R2 = 0.9999, the formula: logA = 0.9488LogC 3.4377; ELSD chromatogram peak area and concentration to take on good correlation to the number of linear equations: R2 = 0.9981, the formula: logA = 1.3649LogC 6.6932. DT70 ELSD chromatogram peak height and concentration to take a good correlation to the number of linear equations: R2 = 0.999, the formula: logA = 0.9139LogC 6.2925. BSA and DT70 spiked recoveries in the range of 95% to 103%, the minimum detection limit were 50ng, 24ng, better precision and variability were smaller. Detector series sequence of cause bovine serum albumin in the ELSD chromatogram retention time than DAD chromatogram retention time lag of 0.13 s. (4) HPGC-DAD-ELSD tandem DAD and ELSD chromatograms after separation by HPGC received only one chromatographic peak, indicating that the substance is the covalent binding of the protein and sugar purified sweetpotato water-soluble glycoprotein qualitative and quantitative analysis showed that: states. Sweetpotato a water-soluble glycoprotein in the concentration range given in the DAD chromatogram peak height difference of 215-225nm and the concentration to take the number of the resulting linear equations: logA = 1.4051LogC 1.124, the ELSD peak area on the chromatogram and the concentration of The linear equation: y = 2E 09x-5E 06, the correlation coefficient R2 = 0.9998 and R2 = 0.9982, respectively, were greater than 0.99, a good linear relationship. Sweet potato soluble glycoprotein spiked recoveries in the range of 92% to 103%. The minimum detection limit of 18ng. The precision of the method is better, smaller mutation rate. The retention time of the the sweet potatoes water-soluble glycoprotein ELSD chromatogram relative to the the DAD chromatogram lag 0.13 s. In addition, HPGC-DAD-ELSD series can intuitively reflect the effect of deproteinized, and the purity of the water-soluble polysaccharide prepared to do further verification.

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