Study On The Determination Method Of Sorghum Tannin

Due to their complex structure,sorghum tannins are difficult to be analyzed quantitatively.The current determination method of sorghum tannins is Ferric ammonium citrate assay described in ISO and National standard.However,the standard tannic acid(TA)used in the ISO 9648 method have significant difference with tannins in sorghum from both structure and kinetics and therefore would cause inaccurate quantitative result.In this study,we purified and identify high-tannin substances(ST)by Sephadex LH-20 chromatography in combination with normal phase high pressure liquid chromatography(NP-HPLC).The ST with higher purity were obtained according to the different polymerization degrees,which can provide reference for the subsequent exploration of spectrophotometric method to quantify the commercially available standard TA,epicatechin(EA)and procyanidin(PA)of sorghum tannin.Then,the experimental steps and standards of quantitative determination of tannin content by Ferric ammonium citrate assay and Vanillin method were improved,and then explore the Prussian blue assay,Acid n-butanol assay and Protein precipitation assay to quantify the sorghum tannin experimental procedures and standards.Finally,the comparative analysis of the advantages and disadvantages of the five spectrophotometers was carried out.The purpose of the experiment in this study is to select a good specificity,high sensitivity,simple and fast according to the revised work requirements of ISO 9648-1988 "SorghumDetermination of tannin content" international standard,which can be universally applied to the spectrophotometric method for quantifying tannin content in sorghum among international laboratories.(1)Extraction,isolation and purity identification of sorghum tannins.The results showed that: 95% ethanol was used to extract sorghum tannins,and Sephadex LH-20 was used for separation and purification,the main substances eluted by mobile phase ethanol were nontannin phenols and some tannins and the main substance in the eluted fraction of acetone: water(1:1)solution was ST.Through chromatographic analysis,ST with a purity of 98.62% was obtained,which provided a reference for the selection of standard products in subsequent methods.(2)Improvement of the spectrophotometric method for quantifying sorghum tannins by Ferric ammonium citrate assay.The results show that the standard product PA was better than TA and EA as the standard product,and its structure and color yield were closer to ST.Compared with TA,it could improve the accuracy of the method.The 70% acetone aqueous solution as an alternative extraction solvent had the extraction effect second only to the 75% dimethylformamide(DMF)aqueous solution,with low toxicity and improved method safety.The extraction conditions were optimized to the material-liquid ratio: 0.5:20(V/V),the extraction time was 30 min,and the extraction temperature was 20°C.Compared with the original method,it could not only save materials,shorten the extraction time,but also be more suitable for large-scale sample measurement.It was verified that the recovery rate and repetition rate of this method can reach the range specified by AOCO.(3)Improvement of spectrophotometric method for quantifying sorghum tannins by Vanillin assay.The results showed that the reaction kinetics of the standard PA in the method was closer to ST than that of EA,and the extraction conditions were optimized to a materialliquid ratio of 0.5:20(V/V),the extraction time was 30 min,the extraction temperature was 20°C,and the reaction conditions were optimized to 2.5 m L mass fraction of 1% vanillin methanol solution and 2.5 m L volume fraction of 15% methanol sulfuric acid solution improved the sensitivity of the method.It was verified that the recovery rate and repetition rate of this method can reach the range specified by AOCO.(4)Explore the Prussian blue assay,Acid n-butanol assay and Protein precipitation assay to quantify sorghum tannins.The standard PA was also applicable to Prussian blue assay,Acid n-butanol assay and Protein precipitation assay.The comparison analysis results of the five methods showed that the Ferric ammonium citrate assay was similar to the Prussian blue assay and was a method for quantifying the total phenols in sorghum.It cannot distinguish between tannin and non-tannin phenols,and the specificity was poor.The Vanillin assay was specific for flavanols,not total phenols,with high sensitivity and simple operation.The Acid n-butanol assay had strong specificity for tannin,but it may have shortcomings such as incomplete acidolysis,poor stability,and side reactions.The Protein precipitation assay was more specific,but the precipitation ability was poor,there was a possibility of underestimation,and the stability and repeatability of the method were poor.Compared with the ISO 9648-1988 "Sorghum-Determination of tannin content" method,the Vanillin assay had better specificity and the use of PA as a standard improves the accuracy of the method.In summary,the Vanillin assay was selected as the ISO 9648 standard: Sorghum-Determination of tannin content.