Organic chemistry lab 1: Biosynthesis of ethanol
8/24/04
Introduction
Welcome to your first organic chemistry lab. Our labs this semester will focus on standard organic chemistry techniques: distillation, extraction, chromatography, recrystallization, spectroscopy and synthesis. The first four of these methods are techniques for separating one compound from another. After we cover these separations, we will move on to characterizing our products (i.e. figuring out what we’ve got) through spectroscopy. We will finish up the semester with several reactions. Next semester, you will apply these basic skills to your own project in natural products.
This week and next, we will synthesize and distill ethanol. The fermentation reaction that we will use here to synthesize ethanol has been used for thousands of years in brewing and baking. Most of you have probably had experience with at least one of these.
In this reaction, enzymes in yeast convert sugar to carbon dioxide and ethanol by first catalyzing the hydrolysis of sucrose (common table sugar) into the monosaccharides fructose and glucose. After conversion to their phosphates, the monosaccharides undergo glycolysis leading to ethanol and carbon dioxide (Scheme 1). We will study more about sugars and their reactions next semester.
Scheme 1. Biosynthesis of ethanol from sucrose
Sucrose, a renewable resource, serves as the feedstock for ethanol in this experiment. Renewable starting materials for chemical reactions are gaining in importance but are still unusual. Over 90% of our manufactured chemicals, such as plastics, pharmaceuticals and food additives, come from petroleum. Much of modern organic chemistry is about functionalizing oil by-products: hydrocarbons. However, as concern over the finite supply of petroleum grows, renewable resources such as sugars are becoming increasingly common, along with new techniques for modifying sugars into plastics and other products.
This experiment also illustrates the increasingly common practice of using enzymes to do our synthetic work for us. Through catalysis with enzymes, it becomes possible to quickly produce high yields of a specific product. However, one of the biggest problems with enzymatic catalysis is purification and separation of the product from the (in this case) “mash”. Next week, you will isolate your ethanol by passing the mash over Celite, or diatomaceous earth, a powdered inert material made from the shells of diatoms. The Celite catches the yeast cells. You will then distill your ethanol using a simple distillation apparatus. As you may know, distillation does not yield 100% pure ethanol because the azeotropic mixture of 95% ethanol and 5% water boils at 78.1°C while 100% ethanol boils at 78.4°C. The last bit of water can be removed by passing your product through a molecular sieve.
Procedure
Add one packet of yeast to 50 mL of water in a 500-mL round-bottomed flask. Add 0.35 g Na2HPO4 to the flask and swirl. Add a solution of 51.5 g sucrose in 150 mL water and mix thoroughly. Stopper the flask with a one-hole rubber stopper fitted with a bent glass pipette. Place the tube in a test tube containing a saturated solution of calcium hydroxide, commonly called lime-water. Be sure the tubing is >0.5 cm below the limewater surface. Note the evolution of gas and that the experimental set-up allows gas to escape while preventing air from entering the flask. Leave the apparatus on the benchtop until the next laboratory period, at which time no gas bubbles should be evident.
Prepare a bed of Celite (about 10 g) in a Buchner funnel. Gently rinse with water and discard the rinse from the filter flask. Gently decant the settled fermentation mixture through the Celite bed, using the vacuum at about half-strength. Increase the vacuum to full once all of the solids have been transferred to the Celite bed. The filtrate contains ethanol, water, cellular contaminants and other compounds. Ethanol is isolated through simple distillation of the filtrate and is collected as a 95% ethanol azeotrope boiling at 78°C.
Part of our job as chemists is to think about where the products of our experiments go after we are done with them. Part of each lab will be to learn techniques for treating and disposing of products that you have no need for after the lab. This week is easy because we are preparing and using materials that are non-toxic (except for ethanol) and can be sent to the landfill or sewage treatment plant. All solids are disposable in solid waste (i.e. the trash can) while ethanol can be washed down the sink.
Some weeks will be more complicated and you will learn how to do neutralizations, separations and other techniques to safely send our products on to the next step in their life cycle.
Pre-lab exercises
Before you come to class, write down in your lab notebook the procedure for the work that you plan to do. Make note of any questions about the procedure or materials that you may have.
During lab, discuss with your lab partner the questions that you wrote down in your lab notebook, keeping track of what you learn. Before you begin to work, a few standard methods involving bending glass and using glassware will be presented. Make sure to take notes on them. You'll need to do them again in this year and the techniques are surprisingly easy to forget.
Lab Write-up
Follow the format on lab write-ups. You will hand in the lab write-up in two weeks, at the beginning of our third lab period.
Date: 8/5/04
Contact: rajameton@lcsc.edu
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