Ethanol Determination by Distillation
In this lab, you will use distillation as a means to study the stoichiometry of the chemical reaction involved in alcohol fermentation as performed by yeast.
Many microorganisms, notably yeasts and bacteria, extract energy from their food (glucose) by fermentation. The overall chemical reaction for this is:
C6H12O6 (glucose) → 2CO2 + 2CH3CH2OH (ethyl alcohol)
or, starting from sucrose or maltose,
C12H22O11 + H2O → 4CO2 + 4CH3CH2OH (ethyl alcohol)
It was this process which Louis Pasteur studied, leading to a biochemical understanding of biological processes. Humans have known about and utilized the process of fermentation for many thousands of years. CO2 liberated by yeast cells doing alcohol fermentation causes bread to rise. The Egyptians and many subsequent civilizations have fermented grains such as barley to break the starch down to malt (maltose), then glucose, and finally alcohol. For at least that long, people have known that various fruits, especially grapes, could also be fermented to produce alcoholic beverages. Thus, alcoholic fermentation is the process which is responsible for the production of wine, beer, and other fermented products. It is the toxic nature of ethanol which acts to preserve these brews, and which leads to intoxication upon consumption. In fact, yeasts cannot generally survive in alcohol concentrations in excess of approximately 12 to 14%. In this lab, we will be studying the process of fermentation as performed by the yeast, Saccharomyces cerevisiae (sacchar = sugar; myces = fungus; Ceres = goddess of grain; vis = to see; -ia = state of, condition of, disease).
Stoichiometry of Fermentation
Today’s lab can be used to demonstrate a number of important principles relating to alcoholic fermentation including the stoichiometry (the study of the definite proportions in which chemicals will interact with each other — stoichi = an element, metr = measure) of the conversion of sugar to alcohol. The following conversion factors will be important, here:
|Molecular Weight of Sucrose/Maltose||342.17 g/m|
|Moles of EtOH per Mole of Sucrose/Maltose||4 m|
|Molecular Weight of Ethanol||46.05 g/m|
|Grams of EtOH per Mole of Sucrose/Maltose||4 × 46.05 = 184.20 g|
|Grams of EtOH per Gram of Sucrose/Maltose||184.20 ÷ 342.17 = 0.538 g|
|Density of Pure EtOH at 20° C||0.789 g/mL|
|Density of Pure H2O at 20° C||0.99823 g/mL|
|Weight of One Can of Malt Syrup||1.5 kg (3.3 lb)|
|Percentage of Weight of Maltose in Malt Syrup*||80%|
|Percentage of Weight of H2O in Malt Syrup*||20%|
|Weight of 1 C of Granulated Sucrose†||213.97 g|
|Density of Malt Syrup§||1.426 g/mL|
percentages were found on another Web site |
†This was weighed here in our lab
§This number was found on another Web site as 0.084 gal/lb, and since 0.084 gal
= 318.04 mL and 1 lb = 453.59 g, therefore 453.59 g/318.04 mL = 1.426 g/mL
In this lab, we may be testing samples of “lab brew” and/or root beer made in previous labs. Thus, it would be of interest to reexamine the recipes from those two labs and calculate the hypothetical stoichiometry involved.
When we made our root beer, the recipe called for 1 C of sugar (sucrose) per 2-L bottle. While most of that sugar was included to make the root beer taste sweet, and not all was intended for the yeast to ferment, if hypothetically, 100% of that would be converted to alcohol, that would be
|1 C sucrose||×||213.97 g sucrose||×||0.538 g EtOH||=||115.12 g EtOH|
|2-L bottle||1 C||1 g sucrose||2-L bottle|
|115.12 g EtOH||÷ 2 =||57.56 g EtOH|
|2-L bottle||L of root beer|
|57.56 g EtOH||×||1 mL EtOH||=||72.95 mL EtOH|
|L of root beer||0.789 g EtOH||L of root beer|
For our lab brew, the amount of sugar going in depended on which recipe was followed. The original, Fankhauser recipe calls for 6 C of sucrose + 1⅓ C of canned malt syrup. Alternatively, we sometimes make the lab brew with one, whole, 1.5-kg can of malt, and no added sucrose. As you, hopefully, recall, the recipe makes 5 gal, or about 10 2-L bottles of lab brew. Let’s start with the calculations for the latter situation, first, because they are easier:
|1500 g syrup||×||0.80 g maltose||×||0.538 g EtOH||=||32.28 g EtOH|
|20 L of lab brew||1 g syrup||1 g maltose||L of lab brew|
|32.28 g EtOH||×||1 mL EtOH||=||40.91 mL EtOH|
|L of lab brew||0.789 g EtOH||L of lab brew|
|6 C sucrose||×||213.97 g sucrose||×||0.538 g EtOH||=||34.53 g EtOH|
|20 L of lab brew||1 C sucrose||1 g sucrose||L of lab brew|
|1⅓ C syrup||×||236.64 mL syrup||×||1.426 g maltose||×||0.538 g EtOH||=||12.10 g EtOH|
|20 L||1 C syrup||1 mL syrup||1 g maltose||L of lab brew|
|34.53 + 12.10 g EtOH||×||1 mL EtOH||=||59.10 mL EtOH|
|L of lab brew||0.789 g EtOH||L of lab brew|
Expressing Concentrations of Ethanol Solutions
The concentration of ethanol solutions can be expressed in several ways. Often, molarity is not used, but rather concentration is expressed as either percent-by-weight (%w/w) or percent-by-volume (%v/v). Wine bottle labels show the amount of alcohol in their contents in terms of %v/v. The density of ethanol at 20° C is 0.789 g/mL, so pure ethanol is lighter than water (density of 0.99823 g/mL), but despite knowing that, in the case of alcohol solutions, it’s not easy to convert %w/w to/from %v/v. As you may have learned in chemistry, alcohol-water mixtures take up significantly less volume than the total of the components. For example, if you mix 100 mL of ethanol with 100 mL of water, you’ll end up with only about 192 mL of solution. However, since that 100 mL of ethanol weighs 78.9 g and that 100 mL of water weighs 99.8 g, even though you end up with “only” 192 mL of solution, that much would still weigh 78.9 + 99.8 = 178.7 g — it’s all there, but it takes up less space. In contrast, if you mix 100 g (= 126 mL) of ethanol with 100 g (= 100.18 mL) of water, you’ll end up with 200 g of solution. However, since the decrease in volume of various ethanol-water mixtures varies with the amounts of each included, there’s no easy way to predict what the final volume of that mixture will be.
However, in spite of how complicated all that seems to be, a lot of work has been done by analytical chemists studying the relationship between the specific gravity of ethanol solutions and their concentrations, expressed as either %w/w or %v/v, and these relationships have been published in various forms in a number of chemistry handbooks. Thus, similar to the Sugar in Soft Drinks lab you did, here also, we can weigh 100 mL of an ethanol solution and 100 mL of dH2O, then calculate the specific gravity of that solution, and look that number up on a chart to determine either %w/w or %v/v of that solution. Since you may be more familiar with the %v/v used on alcoholic beverages, we will use a chart that correlates specific gravity with those numbers. Thus, the table for percentage of alcohol in a solution (excerpted from a larger one in one of the chemistry handbooks) which is included here and in your protocol is based on percentage by volume, in other words, how many milliliters of ethanol per 100 mL of solution.
Background on Distillation
The technique of distillation as a method of separating ethanol from the ferment will be demonstrated in this lab exercise. Distillation is the process of heating a solution to its boiling point, passing the vapors through a cooling device called a condenser, and collecting the liquid which condenses. Because the boiling point of ethanol is 78.5° C, so considerably lower than that of water (100° C), almost all of the alcohol will boil off first, followed by the water. Thus if you start with 100 mL of solution, by the time just under 100 mL has distilled, you will have collected all of the alcohol and almost all of the water. If you then q. s. the resulting distillate to exactly 100 mL with dH2O, you will restore it to its original volume and concentration. Because of the alcohol it contains, the distillate will have a specific gravity lower than that of distilled water. By the use of the table provided, the percentage of alcohol in a solution can be determined by knowing the specific gravity of the solution, assuming that no interfering substances co-distill with ethanol which would affect the specific gravity of the distillate.
Interestingly, since the boiling point of ethanol is so far below the boiling point of water, the ethanol can be concentrated by the process of fractional distillation (collecting the distillate in aliquots rather than the whole solution). This is the source of liquors with higher alcohol contents, but is not what we will be doing in this lab.
A liquid must be brought just slightly above its boiling point before bubble initiation can begin to start it boiling. As a bubble of vapor appears within the liquid, it may do one of two things: if it is below a minimum size, it will collapse because of the surface tension of the liquid, or if it is larger than the critical size, grow and rise to the surface of the liquid. If a liquid, which is free of solid impurities or dissolved gases, is heated slowly, a temperature much above the boiling point can be reached without any boiling actually taking place. This superheating occurs because extra energy is required before bubble formation is initiated. If a bubble should start to form in such a superheated solution, it might suddenly grow with almost explosive violence enough to shatter the container. This problem, called bumping, can be overcome by adding a boiling chip, a piece of porous material, to the liquid before it is heated to the boiling point. The pores act as built-in bubbles so that a liquid cannot superheat. As the distillation proceeds, the air in these pores is replaced by vapor of the distilling material, but this vapor cannot condense because the temperature of the liquid is just slightly above its boiling point. However, whenever a distillation is stopped and then started again, a new boiling chip will be needed because the vapor in the pores will have condensed and filled the pores with liquid.
(This, by the way, has direct practical application. There have been numerous cases in which someone tried to heat water to boiling in a clean glass or china container placed in a microwave. When the person checked on the water, it wasn’t boiling, yet, so the person “nuked” it for a longer time, and then again, and again, without it ever starting to boil. Then, in frustration, the person removed the container from the microwave, jostling the water in the process, and simultaneously looked into it. However, the process of jostling the water initiated bubble formation in this superheated water, causing it to bump and explode out of the container, severely scalding the person’s face. The “moral of the story” is, if you need to heat water in a microwave, always make sure to include some porous substance such as a toothpick, teabag, or loose tea leaves, etc. that will serve as a site of bubble initiation, thereby allowing the water to boil without bumping.)
Distillation of Ethanol Solutions
|wt distillate @ 20° C||× sp gr of distillate|
|wt dH2O @ 20° C|
Table of Specific Gravity and Percent Alcohol
|Sp Gr & % by Vol 20/20||Ref
|% by V
@ 20° C
|Sp Gr||% by V||Sp Gr||% by V||Sp Gr||% by V||Sp Gr||% by V|
Other Things to Include in Your Notebook
Make sure you have all of the following in your lab notebook: