Determination of Ethanol in Wine

This lab introduced students to enzymatic and distillation techniques for determining ethanol content in wine. Virginia Smith 983976633 Lab Partner: Jeff Martz Due Date: 3-29-11

Introduction The objective of this experiment was to introduce enzymatic and distillation techniques in order to determine the content of ethanol in wine. In the food industry, enzymes are used to determine specific components of a food. Enzymatic test procedures are very rapid and easy to perform. This experimental technique also used ultraviolet light and absorption measurements taken with a spectrophotometer. Distilling wine is also used in order to determine ethanol content. This method takes into account that ethanol has a lower boiling point than water, and the ethanol vapor can be collected and measured. Materials and Methods This experiment was conducted as described in the laboratory manual (Roberts 2011) with the following minor modifications. Firstly, the unknown wine at the lab bench was Sample 6. Secondly, the ethanol content of the wine is at 37C, not 15.55C. Results Table 1. Summary of determining ethanol in wine using enzymatic techniques. Absorbance is at 340 nm. A1 New A1 A2 New A2 A C (g EtOH/ L %EtOH solution) Blank 0.2640 0 0.3583 0 0 0 0 Control 0.2571 -0.0069 0.8382 0.4799 0.2228 0.02525 0.0032 Sample 0.2381 -0.0259 0.9784 0.6201 0.3820 43.2933 5.4854 1 Sample 0.2284 -0.0356 0.9266 0.5683 0.3399 38.522 4.8809 2 ave of 0.36095 40.9077 5.1832 samples std of 0.02976 3.3738 0.4275 samples %CV 8.2475 8.2475 8.2475

New %EtOH 0 2.37617 2.37617 2.37617 2.37612 0 0

Table 2. Summary of determining ethanol in wine using a distillation method. % ethanol was found using Table AOAC 913.02. Average Average std %CV SG @ weight with weight 37C flask (g) without flask (g) empty 64.5620 ----flask water 163.6478 99.0858 0.05005 0.050514 -wine 162.3273 97.7653 0.05283 0.054037 0.9867 Calculations Enzymatic Determination Treatment Blank Control Sample 1 Sample 2 A1 B1 C1 1S1 1S2 New A1 B1-B1= 0 C1-B1=C1 S1-B1=S1 S2-B1=S2 A2 B2 C2 2S1 2S2 New A2 B2-B2= 0 C2-B2=C2 2S1-B2=S1 2S2-B2=S2

%EtOH

--5.184

Delta A (A2-A1) 0-0= 0 C2-C1 S1-S1 S2-S2

c = (0.714 / ) * A * F = (0.714 / ( 6.30 mmol-1 x cm-1 ) ) * 0.36095 * 1000 = 40.9077 g ethanol / L sample where, F = dilution factor (g ethanol / L sample) = extinction coefficient of NADH at 340 nm = 6.30 (l x mmol-1 x cm-1) % Ethanol = (C g EtOH / L soln) / (10 * 0.78924) = 40.9077 g/L sample / (10 * 0.78924 g/mL) = 5.1832 % Correction Factor New %EtOH = % EtOH * (0.06/c Control) = (0.06 / 0.02525) = 2.376%

Determination of Dilution of Sample #6 Dilution = (New %EtOH / 11.2%) * 100 = (2.376%/11.2%) * 100 = 21.2% Distillation Determination Specific Gravity @ 37C = Weight of Sample / Weight of Water = 97.7653 g wine / 99.0858 g water = 0.9867 Discussion The theoretical value of ethanol for wine samples was 11.2%. At each lab bench, the wine samples were diluted to different concentrations. For wine sample #6, the percent ethanol for the enzymatic determination of ethanol was found to be 5.1832%, but the NEW %EtOH was calculated to be 2.376%. This can be seen in Table 1. The percent ethanol for distillation determination of ethanol was found to be 5.184%. This can be seen in Table 2. When comparing the two methods of ethanol content determination, it is clear that the results before calculating the new value of %ethanol show very similar values. However, it is concluded that the Distillation Method is more accurate than the enzymatic method because of the lower %CV in the Distillation Method. The higher %CV in the enzymatic method may be due to pipetting error or improperly vortexing. If the sample is improperly vortexed, then it could cause a higher absorbance reading. The improper weighing of the pycnometers could cause error in the distillation method. Also, if the water in the flask was not tempered to 37C, then this could cause a higher weight and ultimately change the specific gravity. Specific Gravity was found to be 0.9867, as seen in the Calculations section. Wine sample #6 was diluted to a 21.2% dilution, as seen in the Calculations section of the results.

Questions 1. % Error = [(theoretical value experimental value) / theoretical value] * 100 = [(11.2% 5.1832%)/(11.2%)] * 100 = 53.72% The high error of 53.72% may be due to pipetting error during enzymatic determination of ethanol in the wine sample. Also, improper vortexing of the samples may also lead to error. 2. For the enzymatic analysis, some samples must be discolored or clarified prior to analysis because enzymatic analysis uses spectrophotometry to find the absorbances. If the samples were not clear, then this would affect the absorbance values of the samples. Absorbance readings may be higher then they should be. Calculations for the percent alcohol would therefore be incorrect due to the turbidity of the sample.

References Roberts, R. and Elias, R. Lab 9-Determination of Ethanol in Wine. Spring 2011. Official Methods of Analysis. 18th ed. 2009. 913.02. Appendix Table 3. Absorbance data using the enzymatic technique. Reaction 1 Blank 0.2640 Control 0.2571 Trial 1 0.2381 Trial 2 0.2284 Table 4. Weight of Pycnometer during Distillation weight of weight of weight of pycnometer pycnometer water (g) & water (g) (g) 1 64.1878 163.2183 99.0305 2 61.6753 160.7742 99.0989 3 67.8229 166.9509 99.1280 Ave 64.5620 163.6478 99.0858 Reaction 2 0.3583 0.8382 0.9784 0.9266

weight of pycnometer, water and wine (g) 161.9025 159.4363 165.6430 162.3273

weight of wine (g) 97.7147 97.7610 97.8201 97.7653