Experiment 5: Investigation of the Components of Wine by Chemical Methods
The process of wine making begins with growing and harvesting the fruit (usually grapes). Next the
juice, called must, is extracted from the grapes. The must is the reagent for the fermentation process.
Fermentation is an oxidation carried out in an anaerobic (no oxygen) environment by microorganisms
that grow and thrive by breaking down the sugars. Yeasts are the most common microorganisms used
to carry out the fermentation process, but there are many other species, primarily bacteria, which can
also cause fermentation.
Yeast + C6H12O6 → 2 C2H5OH + 2 CO2
C6H12O6 is the chemical for fructose, the fruit sugar that ferments. (C6H12O6 is also the formula for
glucose as well as many other sugars that have the same chemical formula but a different structure,
called isomers)
The wine making process is continually refined to optimize different characteristics of the wine. How
long should the sugars ferment? What should the fermentation temperature be? How long should the
wine age after fermentation? What should the final alcohol and/or content be? In what kind of
container should the wine be allowed to age?
Certain wines like champagne, ports, and sauternes (sweet dessert wine) are even more specialized.
Champagnes are carbonated by undergoing a second fermentation and trapping the CO2. Ports are
higher alcohol content wines because they have been spiked with brandy. And sauternes are made from
grapes that have begun to rot on the vine after being subjected to a fungus (Botrytis cinerea) that
reduces the liquid content of the grape, but leaves the sugar content unchanged.
In this experiment you will analyze three components of wine: alcohol content, acidity, and
sulfur dioxide content.
Part A: Determination of Alcohol Content
Ethanol and certain other alcohols react with ammonium hexanitrate cerium(IV), (NH4)2Ce(NO3)6, to
yield a corresponding carboxylic acid and a cerium(III) complex (compound). This reaction is depicted
below.
alcohol + (NH4)2Ce(NO3)6
→ red complex
Since the reaction stoichiometry is 1:1 between alcohol and the red complex, the red complex
concentration will be equal to the initial alcohol concentration of the solution. It is important to note that
the cerium(III) complex is not stable for long periods of time, therefore timely measurements are
important.
The colored species obeys Beer’s Law, meaning that the absorbance of light by the colored substance
is directly proportional to the concentration of the substance in solution:
A = εbC
where A = absorbance (no units)
ε = molar absorptivity constant (Concentration –1cm –1)
b = path length of cell (cm)
C = concentration (mol/L usually, but in this experiment we are measuring % concentration)
This direct proportionality will be used to determine the percent of alcohol in the wine. A Beer’s Law
plot of absorbance vs. concentration of the red complex allows direct determination of the quantity of
alcohol oxidized by ammonium hexanitrate cerium (IV). By measuring the absorbance of the complex
formed by the wine, the percent alcohol and concentration (%) can be determined from a computergenerated calibration graph.
Part B: Determination of Acid Content
The acidity of the wine will be determined by titration. The acidity in wine is primarily due to tartaric
acid, a dicarboxylic acid with the chemical formula:
HOOCCHOHCHOHCOOH
Tartaric acid, H2C4H4O6, is a weak diprotic acid with two acidic protons (highlighted in the chemical
formula above). Because tartaric acid has two acidic protons, neutralization of each mole of this acid will
require two moles of NaOH base, with the neutralization reaction:
H2C4H4O6
+ 2NaOH → Na2C4H4O6
+ 2H2O
Part C: Determination of Sulfur Dioxide
Sulfur dioxide, SO2, is used commercially as a preservative in wines to protect the wine from
bacteriological growth and subsequent infection. Sulfur dioxide is generated by the addition of
potassium metabisulfite, K2S2O5 to the slightly acidic must:
K2S2O5 + H2O → 2KHSO3
KHSO3 + H3O+ → SO2 + 2H2O + K+
The preservative concentration is strictly regulated because the added sulfites and the SO2 produced
from the reaction can be harmful to certain people. Legal limits vary from country to country, from
200-450 mg/L. Much of the added SO2 reacts with other components of wine to form bisulfite
complexes leaving only about 10% (20-40 mg/L) free in solution to protect the product from spoilage.
In the laboratory, you will need to perform titrations in order to determine sulfur dioxide concentration
of the wine. The concentration of free SO2 can be determined by the following reaction:
SO2(aq) + I2(aq) + 2H2O(1) → 2I–(aq) + SO42–(aq) + 4H+(aq)
An aqueous solution of iodine is reddish-brown, whereas the products of the reaction are colorless. As
iodine is added, the color of the wine will remain unchanged until all the sulfur dioxide is consumed.
As soon as iodine is present in excess, the following reaction occurs:
I2
+ I–
→ I3 –
The triiodide ion is slightly yellow. However, if starch is added, a dark blue-black starch-triiodide
complex forms and serves as an endpoint indicator:
I3 –
+ starch → blue complex
To measure the total concentration of SO2, sodium hydroxide is added to the wine to break down
bisulfite complexes. Then it is acidified and titrated using the same technique and the same titration
reaction as in the determination of the free SO2. You can then compare the free and total
concentrations of SO2 in your wine sample.
In this experiment, you will use titration to determine free SO2.
You will calculate total SO2 using a concentration of K2S2O5, provided by the instructor. (The given
will be g/L K2S2O5, you will need to convert this to mg/L of SO2 using the stoichiometry.)
You can calculate the % free SO2 from your titration data and calculated total SO2.
Procedures
The procedures in this experiment revisit many of the techniques you have already learned in
Chemistry 161. Accordingly, you will be expected to already know how to use much of the equipment.
Part A
In this part of the experiment you will (1) make ethanol standards (2) react the standards with the
cerium complex (to generate a color) (3) measure the absorbance of standards (4) create a calibration
curve (with Logger Pro) (5) measure the absorbance of your unknown (after diluting and reacting with
the cerium to create the colored complex (6) calculate the % ethanol in your unknown using your
calibration graph. (Step (6) to be completed at home and/or after completion of the experiment).
Make ethanol standards and wine unknown.
The stock ethanol provided is 10% ethanol. Make 6 ethanol standards that have concentration of 1.0%,
2.0%, 3.0%, 4.0%, 5.0%, 6.0%. For example a 1.0% by volume ethanol solution contains 1.00 mL of
10% ethanol that is brought to a total volume of 10.00 mL. This is dilution by 1/10. Make the other
standards the same way. The standards should be made quantitatively.
Questions to consider: How much stock ethanol do you need? What type of glassware/equipment do
you need?
Make a table to show your work including amounts of each solution component and final
concentration.
Example:
1.0% final
1.00 mL 10% ethanol
9.00mL H2O 1.0% final concentration
Finally make your wine unknown by using 2.00 mL of wine in a total of 10.00 mL of H2O.
React standards and wine unknown with cerium complex.
You will need about 45 mL of the cerium reagent. For each of the standards and for the wine unknown
from above, pipet 1.00 mL into another test tube (you can use the smaller test tubes for this). Then add
5 mL of cerium reagent. Allow the color to develop for a few minutes as you ready the spectrometer.
Measure the absorbance of the standards.
Set up the spectrometer and the logger pro equipment (if you don’t remember how to do this, ask).
Then open the logger pro program on the computer. Open Beer’s Law 11B in the Chemistry with
Vernier folder. If you don’t know where this is, ask. Set the spectrometer to measure absorbance at
565nm. Calibrate the spectrometer to 0.0 Absorbance with a cuvette filled with 10% ethanol (from the
stock).
Press start and measure the absorbance of each standard, remembering to press keep and to enter the
known concentration of each standard. You should see a linear pattern develop. If you don’t remember
how to do this, ask. (You should remember a little bit though!)
Press stop after you have measured all 6 standards.
Create a calibration line by pressing the linear regression button in the toolbar.
Print the graph.
To measure the absorbance of your unknown put some of your unknown into a cuvette and insert into
the spectrometer and read the absorbance from the screen. You don’t need to press start, keep, stop or
anything else.
Note the type of wine. (The composition of the two wines are different.)
Note: Make sure you have everything tabulated and recorded in your notebook. This includes pasting
in any graphs etc.
Part B
In this part of the experiment you will determine the concentration of tartaric acid in wine by titration
with NaOH.
The directions here will be abbreviated because you should remember how to do a titration form the
previous experiments this quarter.
The titrant (which goes in the buret) will be 0.05M NaOH.
Pipet 10.00 mL of you analyte (wine) into a Erlenmyer flask, and 40 mL of DI water and 3-4 drops of
phenolphthalein indicator.
Titrate until a constant pale pink color. This indicates the endpoint of the titration.
Your first titration should be a “quick run” to determine about how much NaOH is necessary. Then do
two more analytical titrations, adding NaOH slowly near the equivalence point. Your two analytical
titrations should be within 1 mL, otherwise repeat again.
Be sure to sure record all your data in tables in your notebook.
Part C
In this part of the experiment you will perform titrations to determine free SO2. You will then compare
your determined free SO2 to the known total SO2.
Free SO2 titrations
Your titrant this time is the standard I2 solution. Make sure to record the concentration from the bottle.
The analyte is of course the wine. Use 20.00 mL of wine, 4 mL of 6M sulfuric acid and 3 mL of starch
solution. (you need to start your titration as soon as you add sulfuric acid. WHY?) The endpoint is a
dark blue (almost purple/black color) that persists. Again, you need two consistent trials.
Total SO2
Be sure to record the total SO2 concentration in your data table. (calculated from given K2S2O5 mg/L)
Data Tables
Make sure all of your data are neatly organized in data tables. For part A, make a table where you
record the volumes you used to make your stock solutions as well as the absorbance values for the
stocks and the unknown. For part B and C, you’ll need titrant initial and final amounts, titrant
concentration, and analyte amounts.
If you are unsure of how to make a table or what data needs to be included, refer back to the relevant
experiment this quarter and follow the examples therein. (The Beer’s Law lab was experiment 5,
titrations were done in both experiments 4 and 5.)
Analysis and Calculations
For part A, calculate the % alcohol in (a) the wine sample you used in the spectrometer and (b) your
original undiluted wine sample.
Using you calibration graph and equation of the best fit line (which need to be attached in your
notebook), you should be able to easily calculate the % ethanol in the diluted wine sample that
you analyzed. (If you don’t remember how, go back and look at Experiment 5).
The wine sample you analyzed was diluted from the wine sample that you took from the hood.
You used 2 ml of wine (from the hood) and added 8 mL of water (for 10 mL of solution total).
That means in the sample you analyzed only 2/10 of the sample is actually wine. (8/10 is water)
How many times more concentrated should the original wine be than the sample you analyzed?
Report the results.
For part B and C, these are typical titration calculations. You can determine the moles of the titrant
from the concentration of the titrant (mol/L) and the volume of the titrant (L).
Then using the stoichiometry of the reaction you can calculate moles of analyte (in part B
analyte is acid, in part C SO2).
To calculate the concentration of the analyte (mol/L), use the moles you found above and
divide by the volume of analyte used.
For part B, express the concentration of tartaric acid as a %. (grams of solute/ml solution) x 100
For part C, express the concentration of SO2 as mg/L.
Remember in Part C you are determining the free SO2 by titration and the total SO2 by calculation
(calculated from given K2S2O5 mg/L) then you can use the free and total SO2 determine a percent
free SO2.
Be sure to determine a percent error for every value where you have a known value.
Results
See the lab report format for a description of the results table.
Discussion
The discussion is a qualitative and quantitative description of the experiment. Describe what you did
(qualitative) and what the result was (quantitative – number). Indicate two sources of error (indicate if
the error was random or systematic) and indicate specifically how they would affect your data. You
should make sure the error is quantifiable and actually relates to your data.