Objective: To demonstrate a fermentation process, isolate the ethanol produced by fractional distillation, determine the composition of the ethanol solution recovered, and make stoichiometric and yield calculations.
Weigh out 20.0 g of sucrose and place it into a 250-mL Erlenmeyer flask. Add 100 mL of water and gently shake until all the sucrose has dissolved. To this solution add 0.60 g of dipotassium hydrogen phosphate (K), 1.8 g of sodium phosphate hydrate (Na3PO412HO) and 2.0 g of dried baker’s yeast. Vigorously shake the contents to mix them thoroughly. The Erlenmeyer flask is fitted with a one-hole rubber stopper containing a short piece of glass tubing. Latex tubing (8 -12 in.) is attached to the glass tubing. An overhand knot is loosely tied in the tubing. The low part of the loop is filled with just enough water so that the passage is blocked, but gas from the fermenting chamber will be able to push the water out of the way and escape (brewers call this an airlock). This setup excludes air (and oxygen) from the system (which allows anaerobic oxidation) and prevents further oxidation (by aerobic oxidation) of the ethanol to acetic acid. Label the fermentation setup with your name and place the flask in the incubator chamber
Isolation by Fractional Distillation
Do not shake the flask; avoid disturbing the sediment on the bottom! Get your flask from the incubator bath or chamber. Carefully remove the rubber stopper from the 250-mL Erlenmeyer flask. Prepare a vacuum filtration assembly using two 250-mL side-arm filter flasks, a 5.5-cm Buchner funnel (with a Filtervac or neoprene adapter), and two lengths (each 12 in.) of vacuum tubing. [N.B. We use the second flask so between the aspirator and our filter flask so that the filtrate will not become contaminated if tap water is pulled back through the hose.] Place a piece of filter paper into the Buchner funnel so that it covers all the holes and lies flat. Into a 250-mL beaker, place 100 mL of water and one tablespoon of Celite. Stir vigorously and pour the mixture into the Buchner funnel while the water is running and a vacuum is applied. A thin layer of the Celite Filter Aid will form on the filter paper. Discard the water collected in the filter flask.
Do not suck too much air through the filter pad; if it dries, it may crack and be unusable. Carefully decant the liquid in the fermentation flask above the sediment through the Celite Filter Aid, using suction. This technique traps the small yeast particles in the Celite Filter Aid but lets through water, ethanol, and any other liquid impurities. This liquid filtrate will be distilled. Obtain a distillation setup and assemble the glassware for distillation. Note the placement of the thermometer bulb in the adapter take-off to the condenser. Securely clamp the apparatus and condenser, and secure joints with plastic clips. Use a small dab of silicone grease on all the standard-taper joints as you connect them. Collect the distillate in a graduated cylinder.
Use a round-bottom distilling flask that will be filled approximately one-half to twothirds full; a 250-mL round-bottom flask should do. Add 2-3 boiling stones to the flask. Use a heating mantle for the heat source and a Variac to control the heat. Your mantle may have a built in voltage controller. Gradually turn up the heat until the liquid in the distillation flask begins to boil. As the vapors rise in the head, you will see liquid condensing; this ring of condensate will rise in the column. Control the setting on the Variac so that the condensate rises slowly through the column and at an even rate. (If the rate is too fast, the column will flood.) The temperature readings at the distillation head will rise; when the temperature reaches (about) 78°C, begin to collect the liquid that distills.
Discard any liquid distilling before this temperature is reached. Collect liquid distilling between 78 and 90°C. Collect 10-15 mL of distillate. Turn off the heat source and remove the heating mantle from the distillation flask. Weigh a 50-mL beaker to the nearest 0.001 g. With a 10-mL volumetric pipet, transfer 10 mL of distillate to the beaker (V). Do not pipet with your mouth; use a pipet bulb. Reweigh the beaker and liquid (5), and by difference, determine the weight of the distillate. Determine the density, and by referring to the graph, determine the percent composition of the ethanol.
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Topic: Fermentation of Carbohydrates: Ethanol from Sucrose
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