% Alcohol
Because the federal Bureau of Alcohol, Tobacco and Firearms imposes a tax of $1.07
per gallon for wines less than or equal to 14% alcohol, while the tax on wines over 14% is
$1.57 per gallon, for financial reasons alone it is important to know a wine’s alcohol
concentration. But the ethanol concentration of a wine is also important for organoleptic
and for microbiological reasons. Second to water, ethanol is the largest component of a
wine. As such, it contributes significantly to a wine’s flavor profile. The ethanol
concentration of a wine is highly correlated with the initial sugar concentration of the juice
from which it is made. A simplified description of a yeast’s metabolism of sugar to produce
ethanol and carbon dioxide is given by the equation:
C6H12O6  2CH3CH2OH + 2CO2
glucose
ethanol
carbon dioxide
A rough estimate of a wine’s final ethanol percentage can be calculated by
multiplying the degrees Brix of a juice by 0.58 (or 3/5, to simplify the math even more).
Because only 3/5 of the carbon from the sugar ends up as ethanol, a water addition to a
juice should be made prior to alcoholic fermentation in order to minimize the amount of
water required to achieve the desired final alcohol concentration.
The alcohol content is important to a wine’s flavor. The flavor of ethanol tends to
impart a subtle spiciness or heat to the wine; in fact, there has been unpublished work at the
University of California, Davis attempting to replace this spiciness in de-alcoholized wines
with capsicum, the compound that gives the heat to peppers of the jalapeño family. In
addition, higher alcohol wines have sometimes been found to have a more viscous
mouthfeel than wines of a lower alcohol concentration.
The ethanol concentration of a wine is also important from a microbial standpoint.
Combined with a wine’s relatively low pH (see section on pH below), its alcohol makes it
impossible for most yeast and bacteria to survive. Many yeast genera are present in a freshly
crushed juice, but most will have died off once the ethanol concentration reaches 10%. At
14% ethanol, many strains of Saccharomyces, the genus of yeast responsible for performing the
alcoholic fermentation, start to have difficulty as well. Lower wine pH’s tend to amplify this
problem. Although certain species of Saccharomyces have been selected for their high alcohol
tolerance, most cannot survive concentrations greater than 16%. Similarly, the malo-lactic
bacteria Oenococcus oeni will have difficulty completing a malo-lactic fermentation at this
alcohol concentration.
Ebulliometry
There are quite a few methods for measuring the alcohol concentration in a wine,
some simple, others involving sophisticated equipment and technology. A reliable method
based on boiling point depression was developed in the early 19th century. In this lab course,
we make use of the Dujardin-Salleron ebulliometer, similar to the device the company
developed back in 1881. Boiling point depression refers to the fact that an alcohol and water
1
mixture will have a lower boiling point than that of the water alone. The higher the alcohol
content, the lower the boiling point. In this procedure, the boiling point of the wine sample
is compared to that of distilled water.
Equipment
Dujardin-Salleron ebulliometer kit ($595 from Scott labs)
Reagents
Distilled water
Denatured alcohol or Everclear
0.01M sodium hydroxide (NaOH), for periodic cleaning
Procedure
1. To the clean boiling chamber, add 50mL of distilled water, ensuring that the draining
valve is closed. Also fill up the condenser chamber with cold, distilled water.
2. Close the boiling chamber with the rubber stopper and thermometer. Light the alcohol
lamp and position in its place under the ebulliometer.
3. Bring contents of the chamber to a boil (this can be heard). The boiling point of the
water is the point at which the temperature has stopped rising.
4. Record the boiling point of the water. Then, extinguish the alcohol lamp and carefully
remove the thermometer. WARNING: DO NOT TOUCH THE OUTSIDE OF THE
BOILING CHAMBER UNTIL IT HAS COOLED.
5. Grab the CONDENSER chamber and place the ebulliometer in the sink. Open the
drain valve to allow the hot water to escape. Run cold water through the chamber until the
device is cool enough to handle.
6. Turn off the water and completely empty the contents of both chambers on the
ebulliometer. The ebulliometer is now ready to run a wine sample.
7. Rinse the boiling chamber two times with a splash of the wine to be analyzed to remove
any excess water. Failure to do this will compromise the accuracy of the results.
8. Repeat steps 1 through 4, except add 50mL of the wine sample to the boiling chamber in
place of the distilled water. New cold water must also be added to the condenser. Again,
the boiling point of the wine is the point at which the temperature remains constant.
2
9. On the calculator included in the ebulliometer kit, rotate the dial so that the boiling point
of the distilled water is aligned with the . The boiling point of the wine is now aligned with
the corresponding alcohol percentage.
10. Empty and rinse the contents of the ebulliometer as described above. After 20 or so
determinations, the ebulliometer should be thoroughly cleaned by boiling 0.005M NaOH in
the boiling chamber. After treatment with the NaOH, it may be necessary to boil distilled
water in the chamber a couple of times to remove any excess.
Notes on the method
1. The thermometer in the Dujardin-Salleron kit contains mercury, a toxin. Care must be
taken to ensure that it is not broken.
2. As the alcohol measurement in this method is entirely dependent on the boiling point of
water, it is recommended that two identical readings for the boiling point of water be
obtained before any wine is analyzed. Ions in the tap water tend to increase its boiling point.
Therefore, distilled water must be used for this purpose. Tap water may be used in the
condenser, but it may leave deposits.
3. As water’s boiling point is also affected by atmospheric pressure, and as atmospheric
pressure will change daily, the boiling point of water must be determined daily. If a storm is
moving in, atmospheric pressure may be changing enough to warrant rechecking water’s
boiling point after only a few hours.
4. Any substance present in appreciable amounts in a liquid may affect its boiling point.
Because of this, wines over 2% residual sugar should be diluted by a measured amount with
distilled water to a concentration below 2% sugar. The diluted wine is then analyzed as
described above, and then the alcohol concentration given on the calculator is multiplied by
the dilution factor to get actual concentration of the wine. However, great care must be
taken here to ensure an accurate dilution as any inaccuracies will be amplified with the
multiplication by the dilution factor.
3
The Malo-Lactic Fermentation
There are two main organic acids in grape: tartaric acid and malic acid. Tartaric acid
is unique in that it is found in grape and not much else other than petunia. In addition, most
microbes cannot metabolize tartaric acid, a big reason why wine can be stored for years
without microbial spoilage.
Malic acid, however, is not so unique, and is common to many fruits including
apples, cherries, peaches, plums and pears. In addition, malic acid can be metabolized by a
vast number of organisms, including bacteria that can survive the low pH, high alcohol
environment of wine. The malo-lactic fermentation, secondary to the alcoholic fermentation
performed by yeast, refers to the bacterial conversion of malic acid to lactic acid (an organic
acid commonly found in yogurt and cheese) and carbon dioxide, as described by the
following equation:
C4H6O5  C3H6O3 + CO2
malic acid
lactic acid
carbon dioxide
The malo-lactic fermentation can be desirable for two main reasons: first, especially in high
acid wines, the process has been found to result in a decrease in wine sourness; second, the
bacteria that perform this conversion also scavenge the remaining nutrients in the wine,
rendering the wine less susceptible to microbial spoilage later in its life. However, in some
wines, notably fruity white wines, the malo-lactic fermentation may be an undesirable
process, and measures will be taken to prevent its occurrence.
Many wine bacteria convert the carbohydrates they consume to lactic acid.
However, only a species of bacteria called Oenococcus oeni can perform the conversion of malic
acid to lactic acid in a single metabolic step. What is important to know here is that, because
other bacteria (and yeast to some extent) will produce lactic acid as a metabolic end product,
the presence of lactic acid alone is not proof of malo-lactic fermentation. In addition, it is a
common misconception that Oenococcus uses malic acid as a source of carbohydrate, which it
does not. Actually, the bacteria’s ability to convert malic acid to lactic acid in a single step
allows it to more easily maintain intracellular pH while it is growing on other carbon sources
in the wine. The distinction is an important one because, as Oenococcus is growing on food
sources other than malic acid, the depletion of the malic acid in your wine does not
necessarily mean that the fermentation is complete.
Oenococcus oeni is greatly inhibited by SO2, so a post-malo-lactic fermentation sulfur
dioxide addition should be timed as to not prematurely kill off the malo-lactic bacteria
present in, or added to, the wine. The growth of Oenococcus is also largely affected by wine
pH; wines with a pH of 4.0 can go through the MLF in two weeks, whereas with a pH of 3.0
the fermentation may take 3 months. Temperatures lower than 60F also tend to slow
microbial growth, as do high alcohol contents.
4
Malo-lactic determination by paper chromatography
We all know that oil and water do not mix. The reason for this is that water is a
polar compound, whereas oil is non-polar. In nature, the polarity of a given compound is
usually somewhere in between that of water and that of oil. In all chromatographic
methods, we take a mixture, such as wine, and separate it into its various constituents
according to their relative polarities. In this method, we use paper chromatography to
separate out the organic acids present in the wine, allowing us to check for the presence of
lactic acid and malic acid. With this information, we can determine whether or not the wine
has undergone a malo-lactic fermentation.
Equipment
Glass capillary tubes
Chromatography jar
Chromatography paper
Pencil, ruler and stapler
Reagents
Malic acid and lactic acid standards (~1g/L), prepared fresh weekly
Chromatography solvent, pre-made (The Wine Lab, Vinquiry) or prepared with:
 100mL reagent grade n-butanol
 100mL distilled water
 10.7mL reagent grade formic acid
 15mL bromocresol green indicator
 Separatory funnel (a bulb-shaped jar with a draining valve on the bottom)
Procedure
1. To prepare the solvent, mix the above-listed reagents together in the separatory funnel.
When you do this, the mixture will separate (according to relative polarities!) into a clear
aqueous layer, and an orange organic layer. Discard the clear aqueous layer.
2. Gather representative wine samples.
3. Take chromatography paper and, using ruler and pencil, draw a line ~2cm from the
bottom of the sheet. Then, mark with a tiny x the locations upon which you will spot the
samples. The x's should be at a minimum 1/2'' apart. Also, label the x's with the
appropriate wine sample i.d.
4. Using a separate capillary tube for each sample, carefully spot each of the two organic
acid standards and the wine samples on its corresponding x. Try to keep the spots as small
as possible.
5
5. Allow the first round of spots to dry. This can be hastened by the use of a hair dryer.
6. Repeat steps 4 and 5 at least two more times.
7. If using a round chromatography jar, staple the chromatography paper into a selfstanding cylinder.
8. Add solvent to the jar so that the solvent's depth is about 1".
9. Place paper into solvent so that it is not touching the sides of the container, and then
close the container. Let stand for 4 to 5 hours, or until the solvent has run up to just below
the top of the paper.
10. Remove the paper from the jar and allow it to dry. This should be accomplished in a
fume hood.
11. Read the spots.
Notes on the method
1. The chromatography solvent is toxic and should be prepared and handled in a fume
hood. Do not allow the paper to dry in a poorly ventilated space.
2. The solvent is sensitive to UV light and will last longer if kept in the dark.
3. Pencil must be used on the chromatography paper. The ink of a pen or marker will
interact with the solvent.
4. Keep the paper as clean as possible. Do not allow it to come into contact with dirty
hands or counter tops as any wine/juice residues will appear on your chromatogram.
5. The organic acid standards, the malic acid standard in particular, are susceptible to
microbial spoilage and should be prepared fresh and refrigerated. I have found that spritzing
the inside of their containers with a 1% SO2 solution can help extend their shelf life.
6. Carbon dioxide, which is ubiquitous in a winery during harvest, tends to make the
chromatogram difficult to read. If the paper fails to turn green after it dries, do not despair.
Simply get a jar of household ammonia, open its lid, and pass the chromatography paper
over the top of the jar. The ammonia fumes will restore the green color to the paper,
allowing it to be read.
7. The use of a citric acid standard in addition to the malic and lactic standards is insightful,
as Oenococcus will scavenge the very small amount of citric present in a wine toward the end
of fermentation. Some winemakers also use a tartaric acid standard. But because tartaric is
always present, I don’t bother to check for it. In any case, the use of these additional
standards informative, but unnecessary.
6
Wine pH
Wine pH has an enormous effect on the sensory properties of a wine, as well as on
its microbial ecology. Low pH wines are generally perceived as more sour, and a low pH
also has a tendency to make wine tannins more astringent. As previously mentioned, a wine
with a pH of 4.0 can undergo the malo-lactic fermentation in 2 weeks, while a wine with a
pH of 3.0 may take as long as 3 months to finish the MLF. Similarly, most microbes, those
that survive the high alcohol content of wine included, will have a difficult time surviving a
pH of 3.0. On the other hand, a wine with a pH of 4.0 is much more susceptible to spoilage
and should be treated with more care. Moreover, not only does a higher pH in and of itself
provide a more favorable environment for spoilage bacteria, a higher pH also reduces the
efficacy of sulfur dioxide additions (see the sections on SO2 below).
What is pH? pH is a measure of the acidity/alkalinity of an aqueous (water based)
solution. Organic acids, such as the malic and tartaric acid in grape juice, to some extent will
dissociate, giving off a positively charged hydrogen ion and leaving the acid molecule with a
negative charge. This can be described by the following chemical equation:
HAcid  H+ + AcidThe pH is equal to the negative logarithm of the concentration of these dissociated hydrogen
ions, expressed in moles H+ per liter. All this is saying is that a pH of 3.0 means that there
are 10-3 (or 0.001) moles H+/L, while a liquid with a pH of 4.0 has 10-4 (or 0.0001) moles of
H+/L. It is important to notice that, because pH values are based on a logarithmic scale, a
pH difference of 1.0 means that there are 10 times as many acidic hydrogen ions in the liquid
with the lower pH value. The scale of pH values runs from 0 to 14, with 0 being most
acidic, 7 being neutral, and 14 being the most basic. Wines generally have a pH between 3
and 4. To give you an idea of the relative acidity of wine, lemon juice has a pH of just
under 3.0, most beers are around 5.5, and human blood is slightly alkaline at about 7.4.
Measuring pH
Placing an electrode into a wine sample and checking the readout on the display is
obviously an easy task to perform. However, it is important to ensure that we are obtaining
an accurate reading from the pH meter. This is accomplished by regularly (at least daily
during periods of heavy use) calibrating the electrode against standardized solutions. These
buffer solutions come with many pH values, 4.0, 7.0, and 10.0 being the most common, and
are often color coded to minimize confusion. Once the electrode is calibrated, we are ready
to check the pH of our juices/wines.
7
Equipment
pH meter, with proper electrode
25 and/or 50mL beakers
Larger electrode rinse beaker/container
Stir bar and magnetic stir plate (optional)
Plastic squirt bottle
Reagents
pH buffers, 4.0, 7.0, 10.0
Electrode filling solution
Electrode storage solution
De-ionized or distilled water
Procedure
1. Place aliquots (enough to completely submerge the electrode) of the three pH buffers
into three separate, clean and dry beakers.
2. Remove pH electrode from the electrode storage solution, and rinse with distilled water.
3. Follow calibration instructions for the pH meter you are using. These will vary from
meter to meter, but are generally very easy to follow. There are two things to remember: the
first is to completely rinse the electrode with distilled water before placing it in the next
buffer, and the second is to try to keep the solution gently moving when the electrode is in
it. The latter can be accomplished by a swirling action, or by making use of the magnetic stir
rod and plate.
4. After calibration, you are ready to measure the pH of your clarified (settling will do) juice
or wine sample. Again, rinse the electrode and keep the sample in motion.
5. Rinse off the electrode and return to the storage solution.
Notes on the method
1. The electrode is the most sensitive piece of equipment in this method, and great care
should be taken when handling it. The following rules should help extend the life of your
electrode:
o Do leave it sitting in the open air for more than a few seconds.
o Avoid wiping off the electrode; instead use a stream of distilled water to clean it.
o Do not store the electrode in distilled water. Tap water is better in this instance.
o Make sure the electrode is completely full of filling solution before using.
o Regularly check the storage solution for crystallization, and replace as needed.
2. Unstable pH readings can often be remedied by changing out the electrode filling
solution. If this does not rectify the situation, it may be necessary to change the electrode.
8
Titratable Acidity
The second measurement of a wine's acidity is called the titratable acidity, or TA. A
wine's acid content will greatly affect its sensory properties as well as its longevity. In
general, the later in the growing season the fruit is harvested, the lower the acid content of
the juice will be. Low pH values are often correlated with high titratable acidities, and vice
versa; however, young vineyards have been known to produce fruit with both relatively high
pH values and high titratable acidities. Winemakers often adjust the acid content of a juice
or wine depending on the wine style and where the grapes were grown. The range of TA
values for finished wines is about 3 to 10g/L, or 0.3 to 1.0g/100mL. Because tartaric acid is
the predominant acid in grapes, especially in finished wines that have undergone malo-lactic
fermentation, these values are expressed as grams of tartaric acid per liter.
Measuring the TA
Recall the generic equation for the dissociation of an acid:
HAcid  H+ + AcidWhereas in the pH measurement an electrode was used to measure the concentration of the
dissociated H+ present in the wine, the titratable acidity measures the sum of both the
dissociated H+ and the H+ still attached to the HAcid. In this analysis, a measured amount
of a sodium hydroxide (NaOH) solution of known concentration is added to a measured
volume of wine. When mixed together, the hydroxide part of the NaOH will react with the
H+ ions in the wine, as well as with the H+s still attached to their acid molecule, to form a
molecule of water. This acid-base reaction is described by the equation:
H+ + OH-  H2O
As we are dealing with tartaric acid, and as there are actually two H+s per each molecule of
tartaric, the acid-base neutralization reaction can be modified to:
H2Tartrate + 2OH-  2H2O + Tartrate-2
The point at which all of the acid in the wine sample has been thus neutralized can be
detected with either a pH meter, or with a colorimetric indicator. This is called the endpoint,
at which the number of molecules of hydroxide added to the wine sample will be equal to
1/2 the number of molecules or tartaric acid. And because the concentration of the sodium
hydroxide solution is known, we can then calculate how much acid there is in the wine
sample.
9
Equipment
25 or 50mL burette with stand and clamps
pH meter (optional, but recommended for red juices and wines)
Squirt bottle
250mL beakers or Ehrlenmeyer flasks
10.0mL volumetric pipets
Pipet bulb
Vacuum flask, rubber stopper, and tubing
Air pump, or split flow valve attached to sink faucet
Magnetic stir/hot plate with stir rod (optional)
Reagents
0.10M sodium hydroxide (NaOH)
Distilled water
pH buffers 4.0, 7.0, and 10.0 (for calibrating the pH meter)
1% phenolphthalein solution (optional colorimetric indicator)
Procedure
1. Calibrate the pH meter if necessary.
2. Fill clean burette with 0.10M NaOH solution. Excess water can be removed by rinsing
burette with the NaOH solution itself.
2. If analyzing a juice/berry sample, proceed to step 3. If analyzing a wine or fermenting
must, pour ~50mL of the sample into the vacuum flask. Attach tubing from the pump or
faucet and cover flask. Run pump or water for 1 to 2 minutes to create a vacuum and
remove any dissolved carbon dioxide.
3. Pipet 10.0mL of the de-gassed juice/wine sample into a 250mL beaker.
4. Add about 50 to 100mL of distilled water to the beaker, or enough that the electrode of
the pH meter is completely submerged.
5. If using the phenolphthalein indicator instead of the pH meter, add 3 drops.
6. Note start point of the burette, and add the NaOH solution incrementally until the
endpoint is reached. Be sure to constantly but gently mix the contents of the beaker. This
can be accomplished manually or with the aid of the magnetic stir rod and plate. With a pH
meter, the endpoint of the titration is at a pH of 8.2. With the phenolphthalein, the
endpoint is indicated by a color change from clear to pink.
10
7. Using the volume of NaOH added during the titration, the TA of the wine sample, in
grams of tartaric acid per liter, can be calculated with the following equation:
TA (in g/L Tartaric) = (mL NaOH)(conc. of NaOH)(75)
(mL wine)
Notes on the method
1. Throughout the wine industry, the TA is often expressed as grams of tartaric acid per
100mL. For these units, simply substitute 7.5 for 75 in the above equation.
2. 0.10M NaOH can be purchased from a chemical supply company. It can also be
prepared by dissolving 4.0g of solid NaOH in distilled water, bringing the final volume to
1.0L.
3. The phenolphthalein indicator works well for many juices, especially for those of white
grapes, but its color change is difficult to see in red wines. For this reason, the use of the pH
meter is recommended.
4. As dissolved carbon dioxide acts as an acid in this titration, degassing of the wine sample
is necessary to remove any lingering CO2 that was generated during fermentation. Instead of
subjecting the wine to a vacuum and thereby forcing the CO2 out of the wine as described
above, some methods call for the gentle boiling of the wine to remove any dissolved gases.
Either method is effective.
11
Sulfur Dioxide
Sulfur dioxide has been used in wine production since the ancient Egyptians. It is a
powerful anti-microbial agent, even in small concentrations. Although toxic to humans in
large doses, (as is carbon dioxide and ethyl alcohol for that matter), when consumed in wine
the SO2 can be readily metabolized, as in the synthesis of sulfur-containing amino acids. In
addition to its anti-microbial properties, sulfur dioxide has been found to inhibit the
oxidative browning action of the plant enzyme poly-phenol oxidase, or PPO. Although not
by itself an anti-oxidant at wine pH, SO2 acts as an indirect anti-oxidant of wine tannins and
pigments by binding to these compounds, thereby inhibiting their oxidation. These
characteristics of SO2 explain its continued use.
Sulfur dioxide solutions are usually prepared by bubbling SO2 gas into distilled water,
or by dissolving solid potassium meta-bisulfite (K2S2O5) in water or the wine itself. When
dissolved in an aqueous solution such as wine, the majority of the SO2 will react with a water
molecule and be converted to the bisulfite ion, while a small percentage of this bisulfite ion
will in turn dissociate (by losing its acidic hydrogen!) into sulfite. This process is described
by the following chemical equation:
H2O + SO2  H+ + HSO3-  2H+ + SO3-2
molecular form
bisulfite ion
sulfite ion
At wine pH, more than 90% of all of the sulfite will be present as bisulfite. At a pH of 3.0,
6% will be present as molecular SO2 while at a pH of 4.0 only 0.6% will exist as the
molecular form. The laws of chemical equilibria ensure that these relative percentages are
maintained, regardless of the total amount of sulfite present. This is important from a
winemaker’s perspective because it is the molecular form that is the anti-microbial and PPOinhibitory form. Although low pH wines will more rapidly lose their SO2 due to the
volatility of the molecular form, they generally require a smaller sulfite addition to receive an
equal amount of protection.
The sulfur dioxide situation is made more complicated by the fact that the bisulfite
ion will readily bind to many compounds in the wine, notably tannins and pigments. When
this occurs, the bisulfite is considered “bound” and it loses its anti-microbial capability.
Because red wines are so much more rich in tannin and pigment than are white wines, a
good percentage of the sulfur dioxide added to a red wine will immediately be bound up. A
rule of thumb that many winemakers use is to assume that 1/3 of any sulfur dioxide added
will end up bound to tannins, pigments, and acetaldehyde. But the bound bisulfite does not
remain so permanently, as an equilibrium between free and bound exists as well.
Taking the two equilibria together, what this all means is that as the molecular form
volatilizes out of the wine over time, a corresponding amount of bisulfite will convert back
to the molecular form to maintain their relative percentages. As this happens, eventually a
corresponding amount of the bound bisulfite will be freed up, restoring the sulfite equilibria.
These equilibrium reactions are important to understand, not only for winemaking decisions,
but for also understanding how the methods of sulfur dioxide analysis work.
12
Free and total sulfur dioxide by aeration-oxidation
Sulfur dioxide is not an easy compound to measure in wine, in that many other
methods making use of more sophisticated technologies have been attempted, but with
limited success. To date, the aeration-oxidation method remains the most accurate and
precise (although it should be noted that a Fourier infrared analytical technique has recently
been successfully deployed). In this procedure, a wine sample is acidified with 25%
phosphoric acid to encourage a larger percentage of the free sulfite to be converted to the
volatile molecular form. The acidified wine sample is then aspirated by vacuum pump,
thereby removing the SO2 gas. After bubbling through the wine, the air stream then passes
through an 0.3% solution of hydrogen peroxide, whereupon the volatile sulfur dioxide is
immediately oxidized to form non-volatile sulfuric acid, as described by the following
equation:
SO2 + H2O2  H2SO4
sulfur dioxide peroxide
sulfuric acid
The sulfuric acid can then be titrated with a base, in this case, 0.010M NaOH. (Please note
that this is 1/10th as strong as the NaOH used to measure titratable acidity.) The endpoint
of this acid-base titration is indicated by a color change.
Equipment
Aeration/oxidation apparatus ($230 from R & D Glass, Berkeley, or $430 from Richmond
Glassblowing, Richmond, CA)
Vacuum pump and tubing
25 or 50mL burette
2 burette stands and clamps
20mL volumetric pipet and pipeting bulb
Timer
Repipet dispenser (optional) or two 10mL graduated cylinders, or two 10mL pipets
Dropper bottle
Bunsen burner, alcohol lamp, or heating mantle (for total SO2 only)
Sink or recirculating chiller for the AO condenser (again, total SO2 only)
Vacuum grease (if necessary)
Reagents
0.3% reagent grade hydrogen peroxide (H2O2), prepared fresh daily
0.010M sodium hydroxide (NaOH)
25% phosphoric acid (H3PO4)
SO2 indicator
13
Procedure, Free SO2
1. In the trap flask, add 10mL of the 0.3% hydrogen peroxide solution and three drops of
the SO2 indicator. Reattach flask to the apparatus.
2. To the other flask, add 20.0mL of wine sample by pipet, and 10mL of the 25%
phosphoric acid. Reattach flask.
3. Ensure that tubing is properly connected to the AO apparatus and to the air pump, and
turn on the pump. Run for 10 minutes, at a flow rate of 1000 to 1500 cm3/min.
4. After 10 minutes, turn off the pump and unclamp the trap flask.
5. Titrate to colorimetric end point the contents of the trap flask with 0.010M NaOH. The
concentration of sulfur dioxide (in parts per million) is calculated with the following
equation:
ppm SO2 = (mL NaOH)(concentration of NaOH)(32,000)
(mL wine)
Procedure, total SO2
1. Run tubing from the water source/recirculating chiller to the inlet and outlet on the
condenser of the AO apparatus. The cold water should enter on the bottom (closer to the
wine sample) and exit from the top of the condenser.
2. As in steps 1 and 2 above, add 10mL of 0.3% peroxide and three drops of indicator to
the trap flask, and 20.0mL of wine and 10mL of 25% phosphoric acid to the sample flask.
3. After reattaching both flasks, apply heat to the contents of the sample flask with either a
Bunsen burner, alcohol lamp, or heating mantle. Turn on the air pump and run it for 10
minutes.
4. After 10 minutes, remove the trap flask and titrate contents with the NaOH as in step 5
above. The calculation for the total SO2 is the same as for the free.
Notes on the method
1. Be sure to use reagent grade hydrogen peroxide for this method as the store-bought
peroxide is unreliable. Even the solutions purchased from chemical supply companies can
sometimes have degraded enough to no longer be effective in oxidizing the SO2 to SO4-2.
When the peroxide has gone bad, you will be hard pressed to detect any sulfite in your wine.
Also, the stronger the stock peroxide solution, the longer it will last. I usually purchase a
14
30% solution and keep it in the refrigerator. However, protective gloves and goggles should
be worn when handling a solution of this strength.
2. In addition to a degraded peroxide solution, another main source of error is an improper
rate of air flow through the wine and peroxide trap. Too weak a flow rate will not be
sufficient to remove the molecular SO2 from the wine sample. Conversely, an aspiration rate
too vigorous can cause the SO2 to bubble through the peroxide before it has a chance to be
oxidized. Flow rates are affected mainly by the pump, but also by leaks at the connections
in the AO apparatus. A leak can sometimes be stopped with a small amount of vacuum
grease.
3. To ensure accurate SO2 values, it is highly recommended that the aeration/oxidation
apparatus be calibrated before use, after being taken apart, or after extended periods of
disuse. Calibration can be accomplished through the use of standard SO2 solution. To
determine how many grams of potassium metabisulfite to add per liter of distilled water, use
the following equation:
g of K2S2O5 = (desired ppm SO2)(1.73)
(1000)
After the standards are prepared, measure the amount of SO2 in them with the AO
apparatus as described above. The numbers generated by the apparatus should be same as
the calculated concentrations of the SO2 solutions. Please note that because of the volatility
of sulfur dioxide, the standards must be analyzed immediately after their preparation or the
numbers obtained from the AO apparatus will not be valid.
4. So too will sulfur dioxide volatilize over time out of a wine sample. Any time a volatile
compound is to be analyzed, care must be taken to ensure that it is not inadvertently
removed from the sample by poor sample handling. If a wine cannot be analyzed
immediately, it should be kept at a low temperature in a container with no head space. In
addition, just as racking a wine will remove a lot of the SO2, transfer of the sample from one
container to another should be avoided to prevent additional analyte loss.
5. One may have noticed that the amount of potassium metabisulfite required for 1 liter of
distilled water is very small. For example, 1 liter of a 25 ppm SO2 standard calls for a mere
43mg, or 0.043g of metabisulfite. As many scales do not have this level of precision, another
method of making the model wine is to prepare a stock solution of, 1.0g potassium
metabisulfite in 1.0L of distilled water, which gives an SO2 concentration of 1743ppm.
Aliquots of this stock solution can be added to the model wine to achieve a particular
concentration of sulfur dioxide. In this example, by taking 10.0mL of stock metabisulfite
solution and bringing it to a final volume of 1.0L with the model wine will make a solution
with 17.4ppm SO2. 20.0mL of this stock solution in 1.0L of wine will make a 34.8ppm
solution, etc. However, the volatility of SO2 is again a problem, even more so at higher
concentrations. At the same time, successful recovery of the SO2 out of you standard
solutions will help you to conduct the AO analysis with confidence.
15
Free SO2 by Ripper titration
Although generally not as precise, the Ripper titration is quick method for
determining the SO2 content of a wine. Because the method makes use of a colorimetric
indicator, the endpoint of the titration of red wines and juices is difficult to see. However,
the rapidity of the method makes it a useful in wine analysis, particularly of whites.
The Ripper method is different to that of the aeration-oxidation procedure, in that
instead of removing the SO2 from the wine sample and trapping it in peroxide, in this
method a wine sample is directly titrated with 0.020N iodine. The endpoint of this titration
is indicated by a 1% starch solution, which turns blue at the endpoint. The difficulty of
seeing the blue color change in a red wine is somewhat ameliorated by using yellow bug
light. Because the iodine solution is relatively unstable, it should be standardized once per
day. The procedures for the standardization of the iodine solution and for the Ripper
titration are offered below.
Equipment
25 or 50mL burette, with stand and clamps
250mL beaker or Erlenmeyer flask
50mL volumetric pipet
Repipet dispenser or 5.0mL pipets
Pipet bulb
Desk lamp equipped with yellow bug light
Analytical balance, sensitive to 0.000g
Magnetic stir plate and stir rod (optional)
Reagents
0.020N iodine solution, standardized daily
25% phosphoric acid (H3PO4)
1% starch indicator
Solid, reagent grade ascorbic acid (vitamin C)
Procedure
1. For the daily standardization of the iodine solution, accurately measure about 0.020g of
ascorbic acid and carefully place in a DRY 250mL beaker or flask. (0.020g is equal to 20mg.
This is an extremely small amount. The mass of the ascorbate does not have to be 20mg but
the exact amount needs to be recorded for our calculations.)
2. Fill the burette with the 0.020N iodine solution. The iodine solution can be purchased
pre-made, and/or diluted to the desired concentration.
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3. To the vessel holding the ascorbic acid, add ~50mL of distilled water and 5mL of the 1%
starch indicator.
4. Note the starting point on the burette. Add the iodine solution incrementally to the
ascorbic acid solution until a blue color persists for more than 30 seconds. Record the
endpoint of on the burette.
5. The normality, or N, of the iodine solution is calculated with the following equation (For
the molarity of the solution, or M, multiply the result of the calculation below by 2):
N = (mg ascorbic acid)(11.355)
(1000)(mL of I)
6. After the daily iodine standardization, to a clean 250mL beaker or flask pipet 50.0mL of
wine. Add 5mL of the 1% starch solution, and 10mL of 25% phosphoric acid.
7. Turn on the bug lamp, placing it close to the wine sample.
8. Record the starting point on the burette and add the iodine incrementally, keeping the
contents of the beaker/flask well mixed.
9. Record the endpoint on the burette and determine the number of milliliters of iodine
added during the titration. The concentration of free sulfite, in mg/L or parts per million
SO2 is calculated with the following equation:
ppm SO2 = (mL of I)(conc. of I)(32,000)
(mL of wine)
Notes on the method
1. Other methods, such as those involving the use of a sodium thiosulfate solution
(Na2S2O3), have been described for the standardization of the iodine titrant. This method is
not discussed here because the use of ascorbic acid is much simpler. However, one problem
with the method described in this manual is that it requires the use of an expensive analytical
balance to measure out the appropriate amount of ascorbic acid. The cost of the scale may
be prohibitive. In any event, the standardization of the iodine solution is important because
of its tendency to degrade over time.
2. The ascorbic acid is stable in its crystalline, solid form. However, once dissolved in water
it will rapidly oxidize. Because of this, be ready to quickly titrate the ascorbic acid once the
distilled water is added.
3. Again, the main problem with the Ripper method is the difficulty measuring the color
change in red wines. For reds, the AO method is recommended. However, for white wines
it should be noted that while an AO run will take about 15 minutes from start to finish, a
Ripper titration can be performed in about 2 to3.
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Volatile Acidity (VA)
The term volatile acidity refers primarily to a wine’s acetic acid content. While other
compounds present in a wine in much smaller amounts technically are volatile acids, it is not
incorrect to use the terms acetic acid and VA interchangeably. Acetic acid is a pungent
compound present in most wines at about 0.5g/L (or 0.05g/100mL). The sensory threshold
for acetic acid as detected by the human nose is around1.0g/L, while the legal limits for red
wines in California and the rest of the United States are 1.2 and 1.4 g/L, respectively.
There are two ways that acetic acid is produced. The first is the conversion of
ethanol to acetic acid by species of Acetobacter, a genus of aerobic bacteria.
CH3CH2OH + O2  CH3COOH + H2O
ethanol
oxygen
acetic acid
water
Certain anaerobic bacteria, including the malo-lactic bacteria Oenococcus oeni, can also produce
acetic acid, and not from ethanol but from glucose. Although the situation is more complex
than the equation suggests, the production of acetic acid from glucose can be described as
follows:
C6H12O6 + O2  CH3COOH + C3H6O3 + CO2 + H2O
glucose
oxygen
acetic acid
lactic acid
carbon dioxide
water
Notice that on the left hand side of both equations is a molecule of oxygen. Without
oxygen, the extent of these spoilage reactions is limited. The first spoilage scenario can
largely be prevented by proper barrel and tank topping practices. The second however, can
be more difficult to control as it most often occurs during a stuck alcoholic fermentation of
higher pH wines. In this situation, the higher pH favors bacterial growth, there is residual
sugar around because the fermentation has not finished, and the yeast are no longer
producing enough carbon dioxide to displace the oxygen and keep the tank or bin
environment anaerobic. It is under these circumstances where monitoring the VA can be
critical.
VA determination by steam distillation with a Cash still
The Cash still consists of a relatively complicated piece of glassware with a heating
element on the bottom and a condenser on the side. In this method, the ethanol and acetic
acid are distilled out of a de-gassed wine sample, and the distillate is titrated with 0.02M
NaOH to determine the amount of acetic acid present in the wine. (See section on titratable
acidity for more details on performing an acid-base titration).
18
Equipment
RD80 self-evacuating VA still ($495 from R & D Glass, Berkeley)
110V electrical outlet
Water faucet and sink
Tubing to connect still to water source
250mL Erlenmeyer flask
Pan, or large beaker for ice bath
25 or 50mL burette
10mL volumetric pipet and pipet bulb
Dropper bottle
Squirt bottle
Vacuum flask, rubber stopper and more tubing
Air pump, or split flow valve attached to faucet (for de-gassing the wine)
Reagents
0.020M sodium hydroxide (NaOH)
1% phenolphthalein indicator
Distilled water
Ice
Procedure
1. Connect all tubing from the water source to the still, making sure the exit flow from the
still ends up in the sink.
2. Add about 15mL of distilled water to the 250mL Erlenmeyer flask. Place the flask in the
ice bath, and position them to collect the distillate exiting the condenser on the still.
3. Turn on the water supply to maintain an adequate flow of water out of the inner sample
chamber, but not so much that too high a pressure builds up inside the still. Once proper
water flow is achieved, close the valve to prevent evacuation of the inner sample chamber.
4. Fill up the outer chamber of the still with distilled water so that the heating element is
completely submerged.
5. Rotate the stopcock valve 180 degrees, and add 10.0mL of a de-gassed wine sample to the
inner chamber of the still. (For instructions on de-gassing a wine, see step 2 in the titratable
acidity procedure.) Quickly rinse the sample port with a stream of distilled water to collect
all of the wine in the sample chamber. Then rotate the stopcock valve to seal-off both the
inner and outer chamber.
6. Turn on the heating element, allowing the water to boil until about 125mL of the distillate
has been collected in the Erlenmeyer flask. Remove the flask, add three drops of the 1%
phenolphthalein indicator, and quickly titrate the contents with the 0.10M NaOH.
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7. Using the volume of the NaOH added during the titration, the concentration of acetic
acid is given by the following calculation:
VA in g/L = (mL of NaOH)(conc. of NaOH)(60)
(mL of wine)
8. To empty the contents of the inner sample chamber, simply open the evacuation valve.
The vacuum created by the flow of the water will force the wine out of the still. To rinse the
chamber in preparation for another sample, leave the water on and spray a stream of distilled
water into the sample chamber until the exit flow runs clear.
Notes on the method
1. It is most important to carefully watch that the water level in the outer chamber does not
fall below that of the coils of the heating element. When this happens, the heat is not
properly distributed and may cause the still to crack. MORE IMPORTANTLY,
HOWEVER, is that if the water does fall below the coils, DO NOT ADD ANY MORE
WATER UNTIL THE CONTENTS HAVE COOLED. Simply turn off the heating
element and wait. If the chamber gets too hot because the water level is too low, adding a
stream of cool water will only ensure that the still cracks.
2. Distilled water must be used in the sample chamber as well as in the outer chamber. The
ions in regular tap water, combined with the high temperatures, will leave deposits that are
difficult to remove.
3. As with the ebulliometer, the inner chamber should be cleaned periodically with a dilute
(0.005M) sodium hydroxide solution. The NaOH can be boiled in the inner chamber to
remove any wine residue and bacteria.
20
Contact Info
Basic Lab Supplies and Reagents
Wine Analytical Services
Fisher Scientific
1-800-766-7000
www.fishersci.com
ETS Laboratories
St. Helena: 1-707-963-4806
McMinnville: 1-503-472-5149
Walla Walla: 1-509-524-5182
www.etslabs.com
Spectrum Chemical
1-800-722-8786
www.spectrumchemical.com
VWR International
1-800-932-5000
www.vwr.com
Northwest Wine Consultants
1-509-829-6751
Vinquiry, Inc. (see below)
The Wine Lab (see below)
Enological Supplies and Reagents
Supplemental Reading
Vinquiry, Inc.
1-707-838-6312
www.vinquiry.com
Boulton, R.B. et al
Principles and Practices of Winemaking
1998 Aspen Publications, Inc.
Gaithersburg, MD
The Wine Lab
1-800-224-WINE
www.thewinelab.com
Zoecklein, B.W. et al
Wine Analysis and Production
1999 Aspen Publications, Inc.
Gaithersburg, MD
Scott Laboratories
1-707-65-6666
www.scottlab.com
Margalit, Y.
Concepts in Wine Chemistry
1997 The Wine Appreciation Guild
San Francisco
R & D Glass Products and Equipment, Inc.
1-510-547-6464
http://go.to/RandD
General Glass Blowing Company
1-510-232-9172
Ough, C.S. and Amerine, M.A.
Methods for Analysis of Musts and Wines
1988 John Wiley and Sons
New York
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