8

Experiment 2 (part 1)-Fermentation of Sucrose

What do bread, cheese, alcoholic beverages, and a few other foods have in common? The
answer, of course, is fermentation. Yeast or some mold or culture reacts with sugars and
starches to produce carbon dioxide, ethyl alcohol, or other organic products. The making of
bread, cheese, and alcoholic beverages by fermentation are among the oldest chemical processes
used by people. No one can say for certain when alcoholic beverages were first produced, but it
is known that people fulfilled their desire for intoxication very early in history by making ethyl
alcohol the very first synthetic organic chemical used. Bread has influenced history more than
any othe food and has played an important role in thr rise and fall of nations. Bread riots
unseated emperors in ancient Rome, and the French Revolution was spurred in part when hungry
French people cried out for bread in 1789 and only received the unthinking reply "Let them eat
cake."

Even though fermentation had been known as an art for centuries, not until the 19th century did
chemists begin to understand this process from the point of view of science. In 1810 Gay-Lussac
discovered the general chemical equation for the breakdown of sugar into ethanol and carbon
dioxide. The manner in which the process took place was the subject of much conjecture until
Louis Pasteur began his thorough examination of fermentation. Pasteur demonstrated that
fermentation was the result of the metabolic activity of microorganisms within the yeast, malt, or
mold, and that different kinds of fermentations were caused by different microorganisms. He
was also able to identify other factors that controlled the action of the yeast cells. His results
were published in 1857 and 1866.

For many years, scientists believed that the transformation of sugar into ethanol and carbon
dioxide by yeasts was inseparably connected with the life process of the yeast cell. This view
was abandoned in 1897, when Büchner demonstrated that yeast extract would bring about
alcoholic fermentation in the absence of any yeast cells. The fermenting activity of yeast is due
to a remarkably active catalyst of biochemical origin, the enzyme zymase. It is now recognized
that most of the chemical transformation that go on in living cells of plants and animals are
brought about by enzymes. The enzymes are organic compounds, general proteins, and
establishment of structures and reaction mechanisms of these compounds is an active field of
present-day research. Zymase is now known to be a complex of at least 22 separate enzymes,
each of which catalyzes a specific step in the fermentation reaction sequence.

The sugars and starches utilized in fermentation come from a number of sources. Before
efficient synthetic processes were developed, much industrial or commercial ethyl alcohol comes
from blackstrap molasses, a syrupy mixture of sucrose and impurities remaining after pure table
sugar is crystallized from the extracted juice of sugar cane and sugar beets. After fermentation,
the ethanol is removed by distillation. (Not enough industrial alcohol is produced by this
process, however, and most of it is derived from petroleum.) Industrial alcohol is now
manufactured from reaction of ethylene with concentrated sulfuric acid, giving ethyl hydrogen
sulfate, which is hydrolyzed to ethanol by dilution with water. Ethyl alcohol is used for
nonbeverage purposes. Most commercial alcohol is denatured to avoid payment of taxes, the
biggest cost in the price of liquor. The denaturants (ie. methanol, aviation fuel, and other
substances) render the alcohol unfit for drinking.
 9
Experiment: Fermentation of Sucrose

In part 1 of this experiment, with the use of yeast, you will ferment sucrose or table sugar to
obtain ethanol. Since the reaction takes some time, the reagents must be mixed and then left for
one full week.

Sucrose has the formula C12H22O11 and consists of one molecule of glucose combined with one
molecule of fructose. As the following equations show, the enzyme invertase, found in yeast,
cleaves the sucrose molecule into glucose and fructose, which are then converted by zymase into
ethanol and carbon dioxide.

HO
HO O
H OH OH
HO
HO
HO HO
O H HO
HO O HO
HO
O
invertase H H H
HO
HO H HO + H
H2O HO
OH OH OH
Sucrose Glucose Fructose
C12H22O11 C6H12 O6 C6H2O6

zymase

4 CH3CH2OH + 4 CO2

Besides sucrose and yeast, the fermentation solution will contain a small amount of Pasteur's
nutrient, a mixture of potassium phosphate, calcium phosphate, magnesium sulfate, and
ammonium tartrate. Pasteur found that these salts enhances yeast growth and formation.
Enhancement occurs when the six-carbon sugars couple with phosphoric acid to give a
combination that is more easily degraded into carbon dioxide and ethanol.
 10
PROCEDURE

Place 20 g of sucrose (sugar) into a 250-mL Erlenmeyer flask. Add 150-175 mL of water
warmed to 25-30 oC; 25 mL of Pasteur's salts; and 2 g of dried baker's yeast. Swirl the contents
vigorously to mix them, then fit the flask with a one-hole rubber stopper with a glass tube
leading to a beaker or test tube containing a solution of barium hydroxide (Figure 2.1). Protect
the barium hydroxide from air by adding some mineral oil or xylenes to form a layer above the
barium hydroxide. A precipitate of barium carbonate will form, indicating that CO2 is being
evolved.

NOTE: Calcium hydroxide could be used in place of barium hydroxide. Alternatively, a balloon
may be substituted for the barium hydroxide trap.

Oxygen from the atmosphere is excluded from the chemical reaction by these techniques. If
oxygen was allowed to continue in contact with the fermenting solution, the ethanol could be
further oxidized to acetic acid or even all the way to carbon dioxide and water. As long as
carbon dioxide continues to be liberated, ethanol is being formed. Allow the mixture to stand at
about 30-35 oC until fermentation is complete, as indicated by the cessation of gas evolution.
This usually requires about 1 week.

Figure 2.1 Apparatus for fermentation of sucrose.

 11
Experiment 2 (part 2)-Fractional Distillation of Ethanol

Azeotropes

Not all liquids form ideal solutions and conform to Raoult's law. Ethanol and water are such
liquids. Because of molecular interaction, a mixture of 95.6% (by weight) of ethanol and 4.4%
of water boils below (78.2 oC) the boiling point of pure ethanol (78.5 oC). Thus no matter how
efficient the distilling apparatus, 100% ethanol cannot be obtained by distillation of a mixture of,
say, 75% and 25% ethanol. A mixture of liquids of a certain definite composition that distills at
a constant temperature without change in composition is called an azeotrope; 95% ethanol is
such an azeotrope.

Figure 2.2 Phase diagram for ethanol and water,

showing the formation of an azeotropic mixture where the liquid and vapor curves meet.

The boiling-point-composition curve for the ethanol-water mixture is seen in Figure 2.2. In
order to prepare 100% ethanol the water can be removed chemically (reaction with calcium
oxide) or by removal of the water as an azeotrope (with still another liquid). An azeotropic
mixture of 32.6% ethanol and 67.4% benzene (80.1 oC) boils at 68.2 oC. A ternary azeotrope
(64.9 oC) contains 74.1% benzene, 18.5% ethanol, and 7.4% water. Absolute alcohol (100%
ethanol) is made by addition of benzene to 95% alcohol and removal of the water in the volatile
benzene-water-alcohol azeotrope.
 12

Figure 2.3 Phase diagram for water and formic acid,

showing a maximum-boiling-point azeotrope.

The ethanol and water form a minimum-boiling-point azeotrope. Other substances, such as
formic acid (100.8 oC) and water (100 oC), form maximum-boiling-point azeotropes (Figure 2.3).
For these two compounds, the azeotrope boils at 107.2 oC. Some examples of minimum- and
maximum-boiling azeotropes are given in Tables 2.1 and 2.2. Minimum-boiling-point
azeotropes always result when the combined vapor pressure exceeds that of either pure
component. Maximum-boiling-point azeotropes always result when the combined vapor
pressure is less than that of either pure component.

One key difference in the two types of azeotropes is where the azeotropic mixture will be formed
during a fractional distillation. From Figure 2.2, it should be evident that a minimum-boiling-
point azeotrope will always be formed in the fractionating column and is what will first be
collected in the distillate. However, analysis of a phase diagram for a maximum-boiling-point
azeotrope shows the exact opposite. The azeotrope is what remains in the distilling flask after all
the lower-boiling-point material has been removed.

A pure liquid has a constant boiling point. A change in boiling point during distillation is an
indication of impurity. The converse proposition, however, is not always true, and constancy of
a boiling point does not necessarily mean that the liquid consists of only one compound. For
instance, two miscible liquids of similar chemical structure that boil at the same temperature
individually will have nearly the same boiling point as a mixture. And, as noted previously,
azeotropes have constant boiling points that can be either above or below the boiling points of
the individual components.
 13
Table 2.1. Minimum-Boiling-Point Azeotropes

Component A Bp (oC) Component B Bp (oC) weight % A weight % B Bp (oC)

Water 100.0 Acetonitrile 81.5 14.2 85.8 76.0
Water 100.0 Ethanol 78.5 4.4 95.6 78.2
Water 100.0 t-Butanol 82.5 11.8 88.2 79.9
Water 100.0 2-Propanol 82.3 12.6 87.4 80.3
Water 100.0 1-Propanol 97.3 28.3 71.7 87
Water 100.0 Dioxane 101.3 18 82 87.8
Water 100.0 Butyl acetate 126.5 27.1 72.9 90.7
Water 100.0 Pyridine 115.5 57 43 94
Water 100.0 Propanoic acid 141.4 82.2 17.8 99.1
Water 100.0 Phenol 181.8 90.8 9.2 99.5
Methanol 64.7 Acetone 56.2 12 88 55.5
Methanol 64.7 Benzene 80.1 39.1 60.9 57.5
Methanol 64.7 Toluene 110.6 72.4 27.6 63.7
Ethanol 78.5 Benzene 80.1 32.6 67.4 68.2
Ethanol 78.5 Ethyl acetate 77.1 31 69 71.8
Ethanol 78.5 Toluene 110.6 68 32 76.7
Acetic acid 118.5 Toluene 110.6 28 72 105.4

Table 2.2. Maximum-Boiling-Point Azeotropes

Component A Bp (oC) Component B Bp (oC) weight % A weight % B Bp (oC)

Water 100.0 Formic acid 100.8 77.4 22.6 107.2
Water 100.0 Hydrofluoric acid 19.5 64.4 35.6 111.4
Water 100.0 Ethylenediamine 116 77 23 118
Water 100.0 Nitric acid 86 32 68 120.5
Water 100.0 Perchloric acid 110 28.4 71.6 203
Water 100.0 Sulfuric acid dec. 1.7 98.3 338
Acetic acid 118.5 Dioxane 101.3 77.0 23.0 119.5
Acetic acid 118.5 Pyridine 115.5 47 53 140
Benzaldehyde 178.1 Phenol 181.8 49 51 185.6
 14
Fractional Distillation

In a simple distillation only one vaporization and condensation occurs, corresponding to points
L1 and V1 (Figure 1.2). This process could not effectively separate a mixture of pentane and
hexane, nor would it for a mixture of ethanol and water. The process of repeated vaporizations
and condensations, fractional distillation, would have been needed in order to separate pentane
and hexane and will be employed in the purification of ethanol.

In a fractional distillation, the use of a fractionating column allows repeated vaporizations and
condensations to occur. A fractionating column in the distillation apparatus provides the large
surface area over which a number of separate liquid-vapor equilibria can occur. As vapor travels
up a column, it cools, condenses into a liquid, revaporizes as more heat reaches it, and repeats
the process many times. Each successive equilibrium enriches the condensate returning to the
boiling flask in the component with the higher boiling point. If the fractionating column is
efficient, the vapor that reaches the distilling head at the top of the column will be composed
entirely of the component with the lower boiling point.

The efficiency of a fractionating column is given in terms of theoretical plates. It is simplest to

define this term by referring back to Figure 1.2. Let us assume that the original solution being
distilled has a 1:1 molar ratio of pentane to hexane. A column would have one theoretical plate
if the liquid that distills from the top of the fractionation column has the composition L2. In
other words, a column has one theoretical plate if one complete vaporization of the original
solution followed by recondensation of the vapor occurs in the column. The column would have
two theoretical plates if the liquid that distills has the composition L3; notice that L3 is already
98% pentane and only 2% hexane. Starting with a 1:1 solution, a column with three theoretical
plates would seem sufficient to separate pure pentane, V3, from hexane. However, as the
distillation progresses, the residue becomes richer in hexane, so more theoretical plates are
required for complete separation of the two compounds.

Fractionating columns that can be used to separate two liquids boiling at least 25 oC apart are
shown in Figure 2.4. The larger the surface area on which liquid-vapor equilibria can occur, the
more efficient the column will be. The fractionating columns shown in Figure 2.4 has from two
to eight theoretical plates. A fractionating column with two theoretical plates can be used to
separate liquids with boiling points differing by about 70 oC; an eight theoretical plate column
can be used to separate liquids boiling 25 oC apart.

More efficient columns can be made by packing a simple fractionating column with a wire spiral,
glass helixes, metal sponge, or thin metal strips. These packings provide additional surface area
on which liquid-vapor equilibria can occur. Some care must be used with metal packings,
because they can become involved in chemical reactions with the hot liquids in the column.

Figure 2.5 shows a distillation curve for the simple vs. fractional distillation of a 1:1 solution of
pentane and hexane. If the fractionating column has enough theoretical plates, the initial
condensate will appear when the temperature is very close to 36 oC, the boiling point of pure
pentane. The observed boiling point will remain essentially constant while all the pentane
distills. Then the boiling point will rise rapidly to 69 oC, the boiling point of hexane. The abrupt
temperature increase from the boiling point of pentane to that of hexane demonstrates the greater
efficiency of fractional distillation.
 15

Figure 2.4 Examples of fractionating columns.

Figure 2.5 Distillation curve for the fractional distillation of a 1:1 molar solution of pentane and hexane.
The dotted line represents the distillation curve for a simple distillation of the same solution.

Experiment: Fractional Distillation of Ethanol

Consider how much ethanol can be generated from a week-long fermentation of sucrose. The
fermentation of sucrose does not provide a 100% yield of ethanol. Increasing amounts of ethanol
poison and kill the yeast. It is well-known that the alcohol content of most beers is not 100% but
more reasonably at 7%.

The density of the fermentation solution will be used to determine the percentage of alcohol
synthesized. The density of the distillate will be used to determine the efficiency of the
fractional distillation.
 16

PROCEDURE

After the fermentation, without disturbing the solution, transfer by decanting (pouring off)
slowly 50 mL to a weighed 100-mL graduated cylinder. Calculate the actual weight and record
the volume of the 50-mL fermentation solution.

Pour the 50-mL fermentation solution into a 100-mL distilling flask, equipped with a magnetic
stirring bar. Set up the apparatus according to Figure 2.6. Pack your distilling column with the
glass beads provided. Don't forget to grease the joints connecting the distilling column to the
reaction flask and the still head. Make sure the thermometer is in the right position to accurately
take the temperature of the vapor before condensation and collection (Figure 1.5).

Stir the mixture and heat gradually to distill the liquid slowly through the fractionating column to
get the best possible separation. Once the distillation begins, the temperature in the distillation
head will increase to about 78 oC and then rise gradually until the ethanol fraction is distilled.
Collect the fraction boiling between 78 and 88 oC in a weighed 10-mL graduated cylinder. You
should collect about 4-6 mL of distillate. The distillation should then be interrupted by removing
the apparatus from the heat source.

Re-weigh the 10-mL graduated cylinder with the distillate to determine the weight of the
distillate. With the volume and the weight of the distillate, the density could be calculated and
used to determined the percentage of ethanol in the distillate.

Figure 2.6 Fractional distillation apparatus.

 17
Data Sheet NAME: ______________________________ DATE: __________

Experiment 2-Fermentation of Sucrose & Fractional Distillation of Ethanol

Table 2.3 Density, Percentage by Weight, Percentage by Volume of Ethanol in H2O at 20 oC

Density % by Weight % by Volume Density % by Weight % by Volume
0.9982 0 --- 0.9027 55 62.8
0.9963 1 --- 0.8911 60 67.7
0.9945 2 --- 0.8795 65 72.4
0.9927 3 --- 0.8676 70 76.9
0.9910 4 --- 0.8557 75 81.3
0.9893 5 6.2 0.8436 80 85.5
0.9878 6 --- 0.8310 85 89.5
0.9862 7 --- 0.8180 90 93.3
0.9847 8 --- 0.8153 91 94.0
0.9833 9 --- 0.8125 92 94.7
0.9819 10 12.4 0.8098 93 95.4
0.9752 15 18.5 0.8070 94 96.1
0.9687 20 24.5 0.8042 95 96.8
0.9617 25 30.4 08013 96 97.5
0.9539 30 36.2 0.7984 97 98.1
0.9450 35 41.8 0.7954 98 98.8
0.9352 40 47.3 0.7923 99 99.4
0.9248 45 52.7 0.7893 100 100.0
0.9139 50 57.8

Record the densities of your (a) fermentation solution and (b) distillate. Use Table 2.3 to
determine the percentages by weight and volume of ethanol. Then calculate the weight and
volume of ethanol for each solution.

(a) _______ density _______ % by weight _______ % by volume

_______ g _______ mL
(b) _______ density _______ % by weight _______ % by volume
_______ g _______ mL
How much water was azeotroped with ethanol in your distillate? _______ mL

Provide a brief discussion on how to improve the efficiency of your fractional distillation of ethanol.
 18
Questions

1. After the fermentation, what is the precipitate in the Ba(OH)2 solution?

Write the balanced equation for this reaction.

2. Write the balanced equation for the reaction of ethanol with oxygen to give
carbon dioxide and water.

3. Explain why a packed fractionating column is more efficient than an unpacked one.

4. In fractional distillation, liquid can be seen running from the bottom of the distillation column
back into the distilling flask. What effect does this returning condensate have on the
fractional distillation?

5. Estimate the boiling point of a mixture of 95% water and 5% ethanol (L1). _____ oC (Figure 2.2)
Estimate the composition of the vapor (V1) above L1. _____ % ethanol _____ % water
This liquid is then condensed (L2) and revaporized in a second cycle (V2). Estimate the composition
of the liquid collected after the second cycle (L3). _____ % ethanol _____ % water

6. Which component will be collected after fractional distillation of a mixture of: (Figure 2.3)
20% water and 80% formic acid? ____________________
How many theoretical plates were needed? _____
80% water and 20% formic acid? ____________________
How many theoretical plates were needed? _____