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ETHANOL IN BEER

ETHANOL IN BEER. OBJECTIVE. Our objective was to use gas chromatography to determine the amount of ethanol in different samples of beer. FOUR SIMPLE INGREDIENTS. Barley Water Hops Yeast. BARLEY. The seed of a grain that looks like wheat Germination

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ETHANOL IN BEER

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  1. ETHANOL IN BEER

  2. OBJECTIVE • Our objective was to use gas chromatography to determine the amount of ethanol in different samples of beer.

  3. FOUR SIMPLE INGREDIENTS • Barley • Water • Hops • Yeast

  4. BARLEY • The seed of a grain that looks like wheat • Germination • Soaked in water then held at 60 °F for five days • Contains starches that are converted into sugars to feed plant • Germination is stopped when sugar-producing enzymes are present but most starch is unconverted • Enzymes produce sugars that feed yeast • Drying • Intensity of malt flavor and color depends on how high temperature is when dried

  5. HOPS • Flower of hop vine • Contains acids that give bitterness and oils that give flavor • Stops the growth of bacteria that spoil beer

  6. YEAST • Single-celled micro-organism responsible for creating alcohol and CO2 • Two categories • Ale and Lager yeast • Ale yeast is top fermenting • Rises near surface during fermentation • Ferments at 70 °F • Lager yeast is bottom fermenting • Ferment slower • Ferments at 50 °F

  7. BREWING • The mash • Converts starches in barley to fermentable sugars • Boiling wort • Hops and yeast are added • Solids separated • Fermentation

  8. FERMENTATION • Yeast converts glucose in wort to ethanol and CO2 • Glucose enters the yeast and keeps doing so as long as glucose is present • Glucose broken down through glycolysis

  9. GLYCOLYSIS • 10-step process • Product is pyruvate and ATP • ATP supplies energy to yeast so that it can multiply • Pyruvate converted into carbon dioxide and ethanol • C6H12O6 => 2 CH3CH2OH + 3 CO2

  10. GLYCOLYSIS

  11. MEASURING ALCOHOL IN BEER • Can be reported by mass or by volume • Beer sold in stores is labeled by volume • Most beer is about 5% alcohol by volume

  12. PERCENT ALCOHOL BY MASS • Compare the specific gravity of beer before it ferments to after fermentation • Specific gravity is the density of a liquid relative to water • Density of water is 1 kg/L • If specific gravity of a liquid is 1.06, one liter will weigh 1.06 kg

  13. CALCULATION • CO2 that forms bubbles out of fermentation vessel • Beginning spec. gravity = 1.06 • Ending spec. gravity = 1.02 • Subtraction give weight of CO2 that left = .04 kg/L • MWCO2= 44.0098 g MWethanol= 46.0688 g • 46.0688/44.0098 = 1.05 g (for each gram of CO2 that bubbles off, 1.05 grams of ethanol are produced) • .04 x 1.05= .042 kg/L = weight of alcohol in container • .042 / 1.02 = .041 x 100 = 4.1%

  14. PERCENT BY VOLUME • Divide by the density of alcohol (.79 g/ml) • 4.1/.79 = 5.2% alcohol by volume • BREWERY.ORG • Over 200 beers and their percent alcohol listed

  15. Comparison of AOAC Method and Working Procedure AOAC Official Methods of Analysis (1990) Ref.: JAOAC 67, 192(1984).

  16. n-Propanol was used as an internal standard made as a 5% aq. Stock solution Standard Ethanol solutions of 3,4,5,6,7, and 8% aq. EtOH were required. No n-Propanol internal standard was used Standard Ethanol solutions were made at 3,4,5,6,7,8 and 10% aq. EtOH Reagents AOAC Procedure Working Procedure

  17. Standard concentrations were to be confirmed by pycnometer, hydrometer or refractometer. OR standards can be created by dilution of concentrated Ethanol. Keep solutions refrigerated Standards were created from dilution of 95% Ethanol in 100 mL volumetric flasks with distilled water. Solutions were kept refrigerated Reagents Cont. AOAC Procedure Working Procedure

  18. Flame Ionization Detector Stainless steel or glass column containing Chromosorb mesh Carrier gas of He or N with a flow rate of 20 mL/min. Flame Ionization Detector A Carbowax Column was used The carrier gas used was He The flow rate was 10 ml/30 sec. according to the soap-bubble meter Gas Chromatography AOAC Procedure Working Procedure

  19. Injector Temp. needed to be at 175oC. Detector Temp. needed to be at 250oC. Column Temp needed to be at 185oC. Set Attenuation as required Injector Temp. was set at 45% (195oC) Detector Temp. was set at 50% (150oC) Column Temp. was set at 40% (190oC) Attenuation equaled 8 Current was set to Maximum Gas Chromatography Cont. AOAC Procedure Working Procedure

  20. Printer Speed =0.2in./min. Each Ethanol standard solution was to be added (5.0 ml) to the n-Propanol internal standard (5.0 ml) solution into separate flasks. Mix flasks well. Inject 0.2 uL of each solution twice. Printer Settings: Speed = 0.2 mm/sec Voltz = 5 There was No mixing with an internal standard 2 uL were injected for each standard solution 5 times Calibration AOAC Procedure Working Procedure

  21. Calculate peak height and use an integrator if preferred. Calculate ratio of peak height for ethanol to n-propanol. Create Calibration Curve for this Ratio against Concentration For each day repeat analysis of 5% ethanol standard solution. Peak area was measured by mass. The peaks for each standard were cut out and weighed to obtain an average mass for each solution. A Calibration Curve was created for Mass against Concentration Calibration Cont. AOAC Procedure Working Procedure

  22. Decarbonate beer samples by pouring it through S&S 560 or comparable filter paper Add 5.0 ml of internal standard to 5.0 ml of Beer sample and Mix Inject 0.2 uL in GC Measure Ratio of ethanol to n-propanol peaks Decarbonation was achieved with the sonic vibrator 5 Beer Samples were evaluated Each Beer Sample was injected 3 times and in a volume of 2 uL Peaks were weighed and averaged for each sample Determination AOAC Procedure Working Procedure

  23. Ethanol % is the Peak Height of Ethanol divided by the Peak Height of n-Propanol and this quantity Divided by the Slope of the Line from the Calibration Curve %EtOH = (Ht.EtOH/Ht.n-PropOH) Slope Ethanol % was Calculated from the Slope of the Line Equation derived from the Calibration Curve. Solve for X: y = mx + b Calculations AOAC Procedure Working Procedure

  24. Results and Troubleshooting 1 • During the experimentation, we were successful after many troubleshooting trials. • Our first obstacle was with the GC. We had to bring up a GC from the organic lab because we weren’t getting any results. • With the GC, we were using inlet A until we decided to use inlet B so we were only using the column, which seemed to help. • Printer problems: The printer gave us many problems. We had trouble synchronizing the printer speed with the GC to get peaks we could work with.

  25. Results and Troubleshooting 2 • Printer problems: We had to also set the voltage to match the speed. The printer would also magically print on angles and we overcame that problem by simply messing around with it. • Adjusting the flow rate was very difficult. The procedure said to set it at 20 ml/min, which we translated into 10ml/30sec meaning it would take 1 bubble 30 seconds to get from 0 to 10 ml. • To set the attenuator and voltage, we tried many combinations. The perfect combination was setting the attenuator at 8 and the voltage at 50.

  26. Results and Troubleshooting 3 • Our standards were from 3% ethanol to 8% ethanol. Our beer samples were IC Light, Pocono Pale Ale, Dunkels, and Natural light. • We figured we would get more peak height with more % ethanol. But no pattern seemed to formulate. • So we cut out our peaks and massed them. We got a general incline in weight with increased percent ethanol. We then could compare our beer samples to our calibration curve.

  27. Calibration Curve • Equation: y= .0009x + .006

  28. Results

  29. Conlcusions • Percent alcohol is usually around 5% • Dead Guy Ale, Dunkels and I.C. Light seem very high • We found the recipe for Pocono Pale Ale and determined the % alcohol by volume • Starting spec. gravity: 1.048 • Ending Specific gravity: 1.012 • 1.048-1.012= .036 x 1.05= .0378/1.012 x 100= 3.7 • 3.7/.79= 4.7% by volume

  30. Conclusions • We looked up the % alcohol for Dead Guy Ale and Natural Light: 6.5% and 4.4% respectively • Could not find data on IC Light, Pocono Pale Ale or Dunkels • The only beer that was close to found data was Natural Light • This method was not very effective in finding the percent alcohol in different brands of beer

  31. Error • Air bubbles in syringe • Human error in injection • Syringe was not properly cleaned • The GOW-MAC

  32. More than 90% of beer consists of water, one of the most essential ingredients. Most modern breweries use water treatment plants that filter impurities and add chemicals as needed. Methods such as particulate filtration, ultraviolet radiation, ozone treatment, precipitation, ion exchange and membrane filtration are used to prepare water before it is used in brewing. Following is a list of ions present in water: Calcium chloride Calcium ions Carbonate ions Copper Iron Iron salts Magnesium chloride Magnesium ions Manganese Nitrate Potassium Sodium Sodium chloride Sulfates Zinc Metals in Beer

  33. Potassium • Potassium is the third most abundant mineral in the body, after calcium and phosphorus. It is critical to maintain proper levels in the body. Potassium works closely with sodium and chloride to maintain fluid distribution and pH balance and to augment nerve-impulse transmission, muscle contraction, and regulation of heartbeat and blood pressure. Potassium is also required for protein synthesis, carbohydrate metabolism, and insulin secretion by the pancreas.

  34. The Effect of Potassium in Beer • Potassium is required for yeast growth, inhibits certain mash enzymes, and is required at trace levels for satisfactory fermentations. • Potassium rarely has any effect on beer flavor.

  35. Calibration Curve: Sodium

  36. Calibration Curve: Potassium

  37. Sodium • No chemical effect • Contributes to the flavor by enhancing its sweetness • Excess levels imparts a sour and salty taste • In presence of sulfates, it creates an unpleasant harshness

  38. Results

  39. Conclusion • Looked for correlation between beer and concentration • Easier to draw conclusion from Na than K • K concentrations – no similarities between the different types • Na concentrations • Ales were similar to each other • Lights were similar to each other

  40. Error • Samples from different areas • Measuring techniques • AA flame not consistent

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