Study On Separation And Purification Of Gallic Acid By Biological Method

In this study,the fermentation broth was prepared by immobilized Aspergillus niger.There were tannic acid,gallic acid,the residue of the culture medium and the cell protein.In this study,gallic acid was separated and purified by resin method.According to the composition and characteristics of fermentation broth,a detailed separation and purification process of gallic acid was developed.A high performance liquid chromatography(HPLC)method was established for the simultaneous determination of gallic acid and tannic acid in fermentation broth;The reflux of fermentation broth was studied;The separation and purification of gallic acid by resin method was optimized and the preliminary study on membrane separation.get the following conclusion:(1)The method for determination of tannic acid and gallic acid coexistence in fermentation broth.Determination of gallic acid and tannic acid by UV spectrophotometry.Gallic acid and tannic acid had a large absorption peak at 260 nm to 280 nm,when both are present in the solution the two substances interfere with each other by UV spectrophotometer.Therefore,the use of UV spectrophotometric determination of tannic acid will cause great error.In this study,gallic acid and tannic acid were determined by HPLC.Two chromatographic conditions were determined.(1)Chromatographic column:VP-ODS(150 mm×4.6 mm),Flow matching ratio:A:B:D=Ultra pure water(55%): methanol(30%): 0.5% glacial acetic acid(15%),Current speed:1 ml·min-1,Sample size:20 ?l,Column temperature:30 ?,Detection wavelength:264 nm.(2)Chromatographic column:VP-ODS(150 mm×4.6 mm),Flow matching ratio:A:B:D=Ultra pure water(20%): methanol(65%): 0.5% glacial acetic acid(15%),Current speed:0.8ml·min-1,Sample size:20 ?l,Column temperature:30 ?,Detection wavelength:275nm.Two kinds of chromatographic conditions have good stability,high accuracy and sensitivity.(2)To explore the reflux of fermentation broth.In this experiment,the fermentation broth was reflux in two ways: tannic acid content and gallic acid content.By making gallic acid content constant and reduce the dosage of tannic acid can effectively reduce costs.The reflux of the fermentation broth was 100%.Repeat batch fermentation in 6 batches.Total tannin content:708 g·L-1,7 batches of tannic acid conversion rate reached 91.97%,The total yield of gallic acid reached89.68%,Residual tannic acid concentration decreased from 65.46 g·L-1 to 18.31 g·L-1.(3)Selection of resin,the static adsorption of several resins was studied in this paper.The separation and purification of gallic acid was determined by the fractional step method.The fermentation broth was first treated with resin XDA-7 and then treated with A-722 MP.Where the XDA-7 resin is used to adsorb tannic acid and the A-722 MP resin is used to adsorb gallic acid.The advantage of this method is that the tannic acid and gallic acid can be recovered step by step.(4)The separation and purification process of XDA-7 resin and A-722 MP resin were optimized.Process conditions for separation and purification of gallic acid by XDA-7 resin,Through single factor and response surface optimization.The single factor optimization results are consistent with the response surface optimization results.The optimum adsorption conditions:gallic acid concentration 2 g·L-1,solution pH 5,Liquid-solid ratio 10:1,Sample velocity 2.0 BV·h-1.In this condition the adsorption rate of gallic acid 16.51% and the adsorption rate of tannic acid97.18%.The fermentation broth was treated with XDA-7 resin and then treated with A-722 MP resin.Process conditions for separation and purification of gallic acid by XDA-7 resin,Through single factor and response surface optimization.The single factor optimization results are consistent with the response surface optimization results.The optimum adsorption conditions:gallic acid concentration 2 g·L-1,solution pH 6,Liquid-solid ratio 5:1,Sample velocity 2.0 BV·h-1.In this condition theadsorption rate of gallic acid 99.03%.Optimum elution condition:Sample velocity 2.0BV·h-1,NaCl concentration 10%,Liquid-solid ratio 25:1.In this condition the elution rate of gallic acid 89.52%.Final yield of gallic acid 88.65%.The purity increased from85.95% to 97.26%.Resin XDA-7 and A-722 MP can be used repeatedly.(5)Preliminary study on membrane separation.The membrane which molecular weight of 300 and 1000 had a significant effect on the separation and purification of gallic acid.