E x p e r i m e n t 6 Fermentation of Sucrose bjectives To produce ethanol from sucrose via a bioloical anaerobic process. To purify the ethanol usin both fractional and simple distillations. To evaluate the two distillation types by assayin the concentration of ethanol after distillation. In the Lab Lab Quiz over backround information about ethanol Students work in pairs After Lab Complete the Chem21 assinments Prepare for the Fisher Proof quiz (next lab period) Waste All solutions produced in this lab can be placed down the sink with water when you clean your lassware with soap and water. Safety Students must wear oles for this experiment.
Reaction H H CH 2 H CH 2 Sucrose CH 2 H 2 invertase H H Glucose H CH 2 + H CH 2 CH 2 zymase 4CH 3 CH 2 + 4C 2 Fructose The fermentation process is the oldest chemical process known. Its use in makin breads, wines and beers has been practiced in an artful way for many millenniums. However, in the nineteenth century chemists bean to make reat ains in understandin the science behind the art. A French chemist, Louis Pasteur, was studyin the chemistry of breads and wines and discovered that yeast was required in the fermentation process. In today s lab, we will use Pasteur salts a mixture of cations (Ca 2+ and M 2+ ) and anions (P4 3- ) that provide an optimum nutrient medium for the reproduction and livin environment of yeast cells. Büchner proved some years later that it was the enzymes present in yeast cells that were responsible for the fermentation process you don t have to have yeast cells. Thus bean the idea that chemical transformations in livin cells are mediated by enzymes macromolecules formed by linkin amino acids toether throuh peptide bonds. Chemical Name Table of Physical Constants Chemical Formula Molecular Weiht Meltin Point Sucrose C12H2211 342.30 185-187 Boilin Point Density 20 n D Ethanol C2H6 46.07 78 0.785 1.3665 Potassium carbonate K2C3 138.21 Water H2 18.02 0 100 1.00 1.3333 Table 1
Start at Step 7 if the fermentation has been completed for you. Procedure: 1. Place 40.0 of sucrose in a 500 Erlenmeyer flask. 2. Add 350 of water. 3. Add 35 of the solution of Pasteur's salts and half a packae of yeast (3.5 ). 4. Shake viorously and in the top of the flask place a one-hole rubber stopper with bent lass tubin in it. The end of the tubin should be well above the surface of the fermentation solution. 5. Place about 150 of water in a 250 beaker. Attach a short piece of rubber tubin to the bent lass and place the rubber tubin in the beaker under the water level. The rubber tubin must remain under the water throuhout the experiment to ensure anaerobic conditions. 6. Allow the mixture to stand until fermentation has ceased (no more C2 as bubbles from the end of the rubber tubin). This usually takes about 5 days. Go to Step 9 if the fermented liquid has been filtered. 7. Set up a Büchner funnel. Make sure the filterin flask is clean, since the liquid is what you want to keep. Seat the filter paper with a small amount of water. Add a ¼ inch pad of Celite. Filter the liquid makin sure no foam exits the flask. Place the Büchner funnel with its rubber stopper on top of the filter flask. Secure the top of the filter flask with a clamp. Attach the hose from the vacuum trap to the side of the filter flask. Place an appropriately sized filter paper in the Büchner funnel and turn the vacuum on. Seat the filter paper with Water. Filter the fermented liquid makin sure no filtrate (or foam) exits the flask. As soon as the filtrate has collected in the filter flask, TURN THE VACUUM FF. 8. If the bubbles reach the arm of the filter flask, quickly disconnect the hose, pour the liquid into a different 500 Erlenmeyer flask and reattach the hose. If you are startin here, obtain 350 of fermented liquid the amount of suar in the liquid is 0.1333 /. 9. btain two 500- RBFs. Add 20 NaCl and two boilin stones to each RBF. Lihtly rease the upper 1/3 of the RBF s outer joint. 10. Use a raduated cylinder to divide the filtrate evenly between the two 500- RBFs. If any salt crystals are on the round lass part of the 500 RBF, make sure the filtrate washes them into the bottom of the RBF. 11. Set up a simple distillation apparatus as shown collect the distillate (Step 13) in a weihed vial cooled in an ice water bath. 12. Turn the heatin mantle on 10 and take a picture. 3
Heatin Mantle Labjack Thermometer Tip of thermometer below sidearm Rubberband H2 column with a 3 rd clamp. 20. Reattach the condenser, distillate takeoff, and collection vial. Take a picture. 21. Wrap the RBF with Al foil and place an insulatin foam tube around the fractionatin column. Distill the contents of the second 500 RBF into a weihed vial cooled in an ice water bath. Follow the procedure in Steps 12 17. 22. Record the weiht of each vial and its contents [Data Sheet]. 23. Place a label on each vial and record the mass of ethanol / water on the label. 13. Wrap the top half of the RBF with Al foil to speed the distillation. Record the temperature when the first drop enters the vial. Collect all ethanol that distills below 95 C. 14. nce the temperature reaches 95 C lower the heatin mantle for 3 minutes. 15. Raise the heatin mantle and continue to collect the ethanol that distills below 95 C a second time. 16. Repeat Steps 14 and 15 one more time. 17. Cap the vial containin your ethanol and set it aside. Don t clean any of the lassware. 18. Remove the distillin flask (the 500 RBF) and replace it with the other 500 RBF. 19. Unclamp the condenser and place it on your lab bench. Then, insert a fractionatin column between the RBF and distillation head. Secure the fractionatin Glass bead Density 24. The mass % ethanol will be determined from its density usin the polynomial equation shown below. If you have more than 10 of liquid, follow Steps 25 33. If not, follow the directions below. btain a dry 10.0 raduated pipette, a suction bulb and a dry 150 beaker. Tare the beaker on the balance. Use the suction bulb to pull the distilled ethanol into the pipette leave about 0.5 1 of liquid in the vial (do not allow air bubbles to enter the pipette). Record the volume of ethanol in the pipette to the nearest 0.01 [Data Sheet]. Transfer the ethanol in the pipette to the tared beaker and record the mass of the ethanol [Data Sheet]. Return the ethanol to the labeled vial. Repeat for fractional distillation. Rinse the 10.0- raduated pipette 4
with acetone and return it to the instructor s table. 25. btain a dry 10.0- volumetric flask from the instructor s table with its round lass stopper. 26. Weih the volumetric flask with its stopper (to the nearest 0.001 ) record this mass. 27. Use a pipette to fill the flask with ethanol (from simple distillation) so that the meniscus of the liquid is even with the line scored in the neck of the volumetric flask (place the pipette tip below the line scored in the neck of the flask while addin the unknown liquid to prevent drops of liquid adherin to the flask above this line). 28. Weih (on the same balance as before) the volumetric flask with its stopper and oranic liquid and record the mass. Subtract the mass of the empty volumetric flask / stopper from Step 26 and enter the mass of the ethanol on your data sheet [Data Sheet]. 29. Enter the volume of Ethanol as 10 [Data Sheet]. 30. Pour the ethanol back into the vial labeled simple distillation. 31. Shake-dry the 10.0- volumetric flask and repeat with the ethanol from the fractional distillation. 32. Pour the ethanol back into the vial labeled fractional distillation. 33. Rinse the 10.0- volumetric flask with acetone and return it to the instructor s table inverted and unstoppered. 34. Determine the density (in /) of the ethanol from the simple and fractional Density to Mass Percent Equation Mass % (Ethanol) = 3349 11008 D + 12764 D 2 5108 D 3 distillation [nline Report Sheet]. 35. Determine the mass % (see equation above) of the ethanol from the simple and fractional distillation [nline Report Sheet]. 36. Determine the overall percent yield of your fermentation [nline Report Sheet]. 37. Turn in to your Instructor the two labeled vials of ethanol. Purity In addition to the density of the ethanol samples, the purity will also be determined (by your Instructor after lab) by placin it on a watch lass and burnin it. If the water content is too lare, it will not completely burn up. The mass of the remainin liquid will also be used to determine the alcohol s purity. Name: Partner s Name: Mass: Name: Lab 6 Ethanol (simple distillation) Lab 6 Ethanol (fractional distillation) Partner s Name: Mass: 5
Volume of Fermented Liquid (~350 ) Fermentation of Sucrose Student Data Sheet Simple Distillation Boilin Point Rane - 95 C Fractional Distillation Boilin Point Rane - 95 C Fermentation of Sucrose Instructor Data Sheet Name: Partner: Volume of Fermented Liquid (~350 ) Simple Distillation Boilin Point Rane - 95 C Fractional Distillation Boilin Point Rane - 95 C 6