8
Experiment 2 (part 1)-Ferm 1 )-Fermentation entation 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. distillati on. (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 C 12H22O11 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 HO
O HO O H
H
OH
O HO
HO
H
OH H
invertase OH
H2O
HO HO HO
O HO
HO H
H
+ OH
O HO H
HO
Sucrose
Glucose
Fructose
C12H22O 11
C6H12O 6
C6H2O 6
H OH
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 o warmed to 25-30 C; 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 CO 2 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 o about 30-35 C 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% o o of water boils below (78.2 C) the boiling point of pure ethanol (78.5 C). 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 o o mixture of 32.6% ethanol and 67.4% benzene (80.1 C) boils at 68.2 C. A ternary azeotrope o (64.9 C) 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 o o formic acid (100.8 C) and water (100 C), form maximum-boiling-point azeotropes (Figure 2.3). o For these two compounds, the azeotrope boils at 107.2 C. 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-boilingpoint 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 o
o
o
Component A
Bp ( C)
Component B
Bp ( C)
weight % A
weight % B Bp ( C)
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 o
o
o
Component A
Bp ( C)
Component B
Bp ( C)
weight % A
weight % B Bp ( C)
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 L 2. 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 L 3; 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, V 3, 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. o
Fractionating columns that can be used to separate two liquids boiling at least 25 C 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 o separate liquids with boiling points differing by about 70 C; an eight theoretical plate column o can be used to separate liquids boiling 25 C 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 o condensate will appear when the temperature is very close to 36 C, the boiling point of pure pentane. The observed boiling point will remain essentially constant while all the pentane o distills. Then the boiling point will rise rapidly to 69 C, 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 a nd 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 o head will increase to about 78 C and then rise gradually until the ethanol fraction is distilled. o Collect the fraction boiling between 78 and 88 C 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 o
Table 2.3 Density, Percentage by Weight, Percentage by Volume of Ethanol in H 2O at 20 C Density 0.9982 0.9963 0.9945 0.9927 0.9910 0.9893 0.9878 0.9862 0.9847 0.9833 0.9819 0.9752 0.9687 0.9617 0.9539 0.9450 0.9352 0.9248 0.9139
% by Weight 0 1 2 3 4 5 6 7 8 9 10 15 20 25 30 35 40 45 50
% by Volume ----------6.2 --------12.4 18.5 24.5 30.4 36.2 41.8 47.3 52.7 57.8
Density 0.9027 0.8911 0.8795 0.8676 0.8557 0.8436 0.8310 0.8180 0.8153 0.8125 0.8098 0.8070 0.8042 08013 0.7984 0.7954 0.7923 0.7893
% by Weight 55 60 65 70 75 80 85 90 91 92 93 94 95 96 97 98 99 100
% by Volume 62.8 67.7 72.4 76.9 81.3 85.5 89.5 93.3 94.0 94.7 95.4 96.1 96.8 97.5 98.1 98.8 99.4 100.0
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 _______ g
(b)
_______ density _______ g
_______ % by weight
_______ % by volume
_______ mL _______ % by weight
_______ % by volume
_______ 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?
o
5. Estimate the boiling point of a mixture of 95% water and 5% ethanol (L 1). _____ C (Figure 2.2) Estimate the composition of the vapor (V 1) above L1. _____ % ethanol _____ % water This liquid is then condensed (L 2) and revaporized in a second cycle (V 2). Estimate the composition of the liquid collected after the second cycle (L 3). _____ % 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? _____
