Experiment 37

Fermentation of a carbohydrate;
ethanol from sucrose

Background

Ethanol is the least toxic of the alcohols, and as such, has been used as a beverage. It is an
alcohol that has been known since ancient times. How ancient people first discovered the
process of making ethanol (ethyl alcohol or grain alcohol) by fermentation is lost in time.
However, it is a process known for more than 5000 years; evidence exists from Egyptian
tombs that beer preparation can be dated to the third millennium B.C. No doubt the basic
chemistry of alcohol fermentation came about completely by accident.
An impure form of ethanol results from the fermented juice of fruits, such as grapes
and apples, and grains, such as hops and barley. These beverages are known as wine,
cider, beer, and ale. Alchemists further learned that from these juices relatively
concentrated ethanol solutions could be had by using the technique of distillation.
In fermentation, any carbohydrate, either a complex polysaccharide or simple sugar,
can be used as a starting material. In the present experiment, sucrose, a disaccharide, is
converted to ethanol by the action of yeast. Sucrose has the formula C 12H22O11 and is
composed of a glucose molecule and a fructose molecule. Enzymes in yeast convert these
sugars to ethanol and carbon dioxide. First, invertase catalyzes the hydrolysis of sucrose to
glucose and fructose; second, zymase converts the hydrolyzed sugars to ethanol and CO2.
Addition of phosphate salts to the nutrient medium increases the rate of fermentation.

CH2OH
HOCH2
HO O O
+ H2O
HO HO
O invertase
OH CH2OH
O
H
Sucrose
CH2OH CH2OH
O O
HO HO CH OH + HO zymase 4 CH CH OH + 4 CO
2 HO 3 2 2

OHOH
OH
Fructose Glucose

The fermentation process produces solutions of only 10–15% ethanol. This is because
the end-product ethanol inhibits the action of the enzymes in the yeast. In order to get
more concentrated solutions, distillation is necessary. It is a method that is based on
differences in boiling points of the components of a mixture. If one component is

Experiment 37 383
nonvolatile, or is a liquid which boils much higher or lower than the component desired, a
simple distillation is used. (See Experiment 5 for an application of this technique.) Further
purification can be done by fractional distillation. Here the liquids to be distilled are
heated in a boiling flask and the vapors are passed through a fractioning column. This
column allows for many condensations and vaporizations to take place before pure vapors
finally enter a cooling condenser. The purified material is collected in a clean container.
The ethanol prepared in this way is a constant boiling mixture of 95% ethanol and 5%
water. This constant boiling mixture is called an azeotrope. Distillation is unable to
concentrate and purify further. Pure, or 100%, ethanol is called absolute ethanol and is
obtained by adding benzene to the water-ethanol mixture. Benzene-water-ethanol forms a
low-boiling azeotrope that distills before pure ethanol. Thus, after this three-component
azeotrope distills, a 100% solution of ethanol is left. Treatment with calcium oxide
removes the last traces of water, and a final distillation gives pure ethanol.
Proof is the usual designation of the alcohol content of wines and liquors. Proof is
twice the percentage of alcohol. Thus, absolute ethanol is 200 proof, and eighty proof
vodka has a 40% alcohol content.

Objectives

To demonstrate a fermentation process.

To isolate the ethanol produced.
To determine the percent composition of the ethanol solution recovered.

Procedure

This is a two-part procedure that is to be carried out in two successive weeks. Part A,
Fermentation, is to be done the first week. Part B, Distillation, is to be done the
second week.

Part A. Fermentation

1. Weigh out 20.0 g of sucrose and place it into a 250-mL Erlenmeyer flask.
2. Add 100 mL of water and gently shake until all the sucrose has dissolved.
3. To this solution add 0.50 g disodium hydrogen phosphate (Na 2HPO4), 1.0 g potassium
phosphate (K3PO4), and 2.0 g dried baker’s yeast. Vigorously shake the contents to mix
them thoroughly.
4. Figure 37.1 shows the setup for the fermentation experiment. The Erlenmeyer flask is
fitted with a one-hole rubber stopper containing a short piece of glass tubing. Latex
tubing (approx. 12 in.) is attached to the glass tubing; at the other end is a short length
of glass tubing or a Pasteur pipet. The opened end is placed into a 50-mL Erlenmeyer
flask [or a test-tube (15 x 150 mm)] containing 30 mL of calcium hydroxide [Ca(OH) 2]
solution; be sure the glass opening is at least 1 cm below the surface of the liquid.
Carefully cover the top of the calcium hydroxide solution with 1 cm of mineral
(paraffin) oil. Hold the glass tubing vertically in place with a clamp attached to a ring
stand; plug
 Ring stand

Rubber tubing

One-hole Pasteur pipet

rubber stopper (or glass tubing)

Clamp

250-mL
Erlenmeyer Cotton
50-mL Erlenmeyer

Fermentation
mixture Mineral oil
Solution of
Ca(OH)2

Figure 37.1 • Fermentation setup.

the opening of the Erlenmeyer loosely with a small piece of glass wool or cotton. 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).
5. Label the fermentation setup with your name. Your laboratory instructor will instruct
you where to place your experiment for the week. The fermentation process requires
from 5–7 days at 30–35ºC to be complete.

Part B. Isolation by Fractional Distillation

1. Carefully remove the rubber stopper from the 250-mL Erlenmeyer flask. Do not
shake the flask in order to avoid disturbing the sediment on the bottom.
2. Prepare a vacuum filtration assembly using two 250-mL side-arm filter flasks, a 5.5-cm
Büchner funnel (with a Filtervac or neoprene adapter), and two lengths (each 12 in.) of
vacuum tubing as assembled in Fig. 37.2. Place a piece of filter paper into the Büchner
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 (Filter Aid, which is diatomaceous earth). Stir
vigorously and pour the mixture into the Büchner 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.
3. 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.
 Layer of Celite
Filter paper

One-hole
rubber stopper (#6)

Splashgon Heavy-walled
tubing

Büchner funnel
Filter adapter
250-mL
Filter flask

250-mL
Filter flask

Figure 37.2 • Vacuum filtration with a Büchner funnel.

4. Obtain a distillation setup and assemble the glassware as shown in Fig. 37.3. Use a
small dab of silicone grease on all the standard-taper joints as you connect them.
a. Use a round-bottom distilling flask that will be filled approximately one-half
to two-thirds full; a 250-mL round bottom flask should do. Add 2–3 boiling
stones to the flask.
b. Use a heating mantle for the heat source and a Variac to control the heat.
c. Pack the fractionating column loosely, but uniformly, with soap-free stainless
steel cleaning pad material.
5. Gradually turn up the heat until the liquid in the distillation flask begins to boil. As the
vapors rise in the column, 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 78ºC, begin to collect the liquid that distills. Discard any liquid distilling before
this temperature is reached.
6. Collect liquid distilling between 78 and 90ºC. Collect 10 to 15 mL of distillate.
7. Turn off the heat source and remove the heating mantle from the distillation flask.
8. Weigh a 50-mL beaker to the nearest 0.001 g (3). With a 10-mL volumetric pipet,
transfer 10 mL of distillate to the beaker (V distillate). Reweigh the beaker and liquid (5),
and by difference, determine the weight of the distillate (6). Determine the density (7),
and by referring to Fig. 37.4, determine the percent composition of the ethanol.
 Thermometer

Thermometer adapter

Distilling head

Condenser

Vacuum adapter

Fractional
distillation
column

Stainless Open to air

steel Water out
packing

No water Water in
Graduated
cylinder
Distilling flask

Heating mantle

To Variac

Figure 37.3 • Fractional distillation apparatus.

Figure 37.4
100
Percent composition of aqueous
ethanol as a function of density.

95
Percentage Ethanol by Weight

90

85

80

75

70
0.75 0.80 0.85 0.90
Density at 20C (g/mL)

9. Clean up: solid waste can be disposed in solid waste containers; liquid waste can be
flushed down the sink with plenty of water.
 Chemicals and Equipment

Calcium hydroxide solution, Ca(OH)2

Disodium hydrogen phosphate, Na2HPO4
Dried baker’s yeast
Mineral oil
Potassium phosphate, K2PO4
Sucrose
Boiling stones
Celite (Filter Aid)
Clamps
Heating mantle (or Thermowell)
Kit for fractional distillation
Ring stands
Silicone grease
Steel wool, coarse
Variac
NAME SECTION DATE

PARTNER GRADE

Experiment 37 PRE-

LAB QUESTIONS
1. Define the term azeotrope.

2. Wine usually contains about 12% ethanol. What is its proof ? How is proof defined?

3. Give the complete, balanced equation for the conversion of glucose (C6H12O6) to ethanol.

4. The fermentation process requires the presence of catalysts. What is the name of these
fermentation catalysts?
NAME SECTION DATE

PARTNER GRADE

Experiment 37

REPORT SHEET
1. Distilling temperature range, ºC

2. Volume of distillate collected

3. Weight of 50-mL beaker: W1

4. Volume of distillate: Vdistillate 10.00 mL

5. Weight of beaker and distillate: W2

6. Weight of distillate: (5) — (3)

7. Density of distillate
d = (W2 — W1)/Vdistillate = [(5) —
(3)]/(4)

8. Percentage ethanol

Experiment 37 391
POST-LAB QUESTIONS
1. If the air trap was not used in the fermentation setup, would ethanol be the end
product? If not, then what would be the end product?

2. During fermentation, bubbles can be seen emerging from the glass tubing. Identify
the gas causing the bubbles.

3. A solid forms in the calcium hydroxide trap during the fermentation. What is the
solid? Write a balanced equation for the formation of this solid.

4. From the percentage ethanol in your experiment, determine the proof.

5. Write the balanced equation for the conversion of sucrose to ethanol. Using the
quantity of sucrose from this experiment, calculate the number of grams of ethanol and
carbon dioxide you expect. How many liters of carbon dioxide would this be at 20ºC
and a pressure of 1 atmosphere?