Study On The Method Of Determination Of Mono-Phenol In Grape Berry And Wine

Phenolic compounds in grape and wine can be divided into two groups: non-flavonoid (hydroxybenzoic and hydroxycinnamic acids and stilbenes) and flavonoid compounds (anthocyanins, flavan-3-ols and flavonols). Phenolis is an important secondary substant in plant, they are closely related to the anti- disease of grape, the transport and storage after pick as well as the color and flavor of the wine (Caffeic acid and Catechin). Simultaneity, the amount and types of phenolics present in grape and wines play an important role in controlling oxidation in the human body (Quercetin and phenolic acid).Presently, HPLC is the main method of measuration of mono-phenols from grape and wine. The resolving power of the method is high, the speed of analysis is quick, repeat is good, and the quantitative analysis is accurate and the accuracy is high, but the HPLC request to reduce the sample impurity to determine material analyzed accurate.But there are two many phenolic compound in grape berry and wine, and the composition of phenolic compound are very complex, we always run into the difficulty of separation when we determined phenolic compound by HPLC in grape berry and wine, so it is very important to preparate sample before determining phenolic compound by HPLC. Presently, the method for determination of Gallic acid and Catechin in grape berry and wines with HPLC was studied .but the method of sample preparation are not unifiedThis study aimed to optimize the extraction and measuration of several mono-phenols from grape and wine.The main results were as follows:1 The method of extraction and measuration of several mono-phenols from grape:(1) The method of extraction of several mono-phenols from grape:The sample (0.5 g grape berry) was rinsed with 25 mL of 70% methanol into the bottle. To this mixture, 10 mL of 6 mol HCL was added by careful mixing (final HCL concentration 1.2 mol) and the solution was sonicated for 10 min. The mixture was shaken in 35 ?C water bath in dark. After 12 h, 12000×g 4℃was acentriced 20min, the filtered was evaporated to got rid of methanol, the residue was extracted with 30mL ethyl acetate one time, then, with 20 mL ethyl acetate two time, the fltrate was evaporated to dryness using rotary evaporator and 40℃water bath. The residue was dis-solved in 5 mL of 50% methanol and filtered through a 0.45μm filtered for organic solvents prior to injection to the HPLC apparatus.(2) The method of measuration of several mono-phenols from grape by HPLC: the chromatogram column: Hibar RT Lichrospher RP- C18 precolumn, 250×4.0 mm, 5μm; flow rate: 1.0 mL/min; column temperature: 30℃. Wavelengh: 280 nm. Eluted gradient: solvent A: water: acetic acid (98:2); solvent B: acetonitrile linear gradient: from 0% to 16% of B in 10 min, from 20% to 40% of B over 10 min to 25 min and from 40% to 0% of B over 25 to 30 min.2 The method of extraction and measuration of several Mono-phenols in wine:(1) The choice of the method of sample preparation:Two different methods for sample preparation as a preliminary stage for the simultaneous determination of 10 mono-phenols are compared with the aim to establish the best conditions for the determination of these compounds in wine samples by high performance liquid chromatography (HPLC). Sample preparation using liquid–liquid extraction and solid-phase extraction were compared. To study best extracte condtion, recoveries, definition, we can get the follow condtion:Better results are obtained when liquid–liquid extraction with ethyl acetate is used, and variables such as extraction time and pH have been studied.Best results are obtained using ethyl acetate (3×50 mL) at pH 2 with 20 min as the extraction time. Through the methods for sample preparation for the simultaneous determination of 10 Mono-phenols by HPLC, the recoveries of 10 mono-phenols between 80.28%~91.69%, the relative standard deviation was from 0.72% to 3.69%.Through the methods for sample preparation for the simultaneous determination of 10 mono-phenols by HPLC, the recoveries of 10 mono-phenols between23.37%~91.26%,the relative standard deviation was from 0.98% to 2.82%.Comparison of different sample preparation treatments for the analysis of wine phenolic compounds by reversed phase high-performance liquid chromatography, The results suggest that: The definition of SPE is high than LLE, but the recoveries of individual mono-phenols is low, the equipment and column of extraction were necessary, so the cost of SPE is high than LLE. When we used liquid–liquid extraction as the methods for sample preparation, best results are obtained using 0.1 mol/L HCL: methanol 1:4 (v/v), (15 mL) at natural pH.Considering recoveries and cost,we choose LLE as sample preparation treatments for the analysis of 10 mono-phenols in wine.(2) The method of measuration of several mono-phenols in wine by HPLC: the chromatogram column: Hibar RT Lichrospher RP-C18 precolumn, 250×4.0 mm, 5μm; flow rate: 1.0 mL/min; column temperature: 30℃. Wavelengh: 280 nm. Eluted gradient: solvent A: water: acetic acid (98:2); solvent B: acetonitrile linear gradient: from 0% to 16% of B in 10 min, from 20% to 40% of B over 10 min to 25 min and from 40% to 0% of B over 25 to 30 min.