VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 1 of 63
Revised: Spring 2007
NAPA VALLEY VINTNERS ASSOCIATION
TEACHING WINERY
NAPA VALLEY COLLEGE
WINE & MUST
ANALYSES MANUAL
Prepared by the Students
of
Laboratory Analysis of Musts and Wines
Fall 1997, 2001, 2002, 2003
Acknowledgements:
Thank you to:
The Napa Valley Vintners Association for generously donating the money to build the teaching winery.
The many wineries, suppliers and individuals who have willingly donated equipment, cash, knowledge and time.
Stacy Hitchcock for helping with some of the collation of the manual
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 2 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Winery Technology
Standard Operating Procedure
Table of Contents
Vineyard Sampling………………………………………………………….…3
Soluble Solids by Refractometry…………………..………………………….4
Soluble Solids by Hydrometry………...……………………………………... 7
Titratable Acidity (TA) by Indicator Titration “Quick & Dirty”.………..10
Titratable Acidity (TA) by pH Titration…………..………………………..11
pH …………………………………………………………………………......13
Formol Titration.…………………………………………………………......15
Nitrogen by Ammonia (NH4+) Electrode……………………………………17
Alpha Amino Acid Estimation Using the NOPA Procedure…………….…20
Clinitest / Dextrocheck…….……………………………………………….…25
Determination of Reducing Sugar by Rebelein Method…………….…..…27
Total SO2 (TS) by the Ripper Method……………………………………....29
Free SO2 (TS) by the Ripper Method ………………………………………30
Free SO2 (TS) using a Platinum Electrode…………………………………31
Free SO2 (TS) Aspiration Oxidation………………………………………..33
Malic Acid by Paper Chromatography…………………………………….35
Alcohol by Ebulliometry…………………………………………………….37
Spectral Measures for estimating Wine Color and Phenols………………40
Volatile Acidity (VA) using a Cash Still……………………………………46
Fining – Bentonite Fining……………….…………………………….…….49
Fining - Other Than Bentonite…….………………………………………..52
Tartrate Stability…………………………………………………………….60
Dissolved Oxygen by Orion 820 Dissolved Oxygen meter……………..….62
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 3 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Vineyard Sampling
Developed by Michelle Bowen
Date: December 1997
Introduction
Berry sampling can provide an accurate and economical technique with which to gauge grape maturity.
However, the following variables can affect the integrity of the sample:
1. The composition of berries can differ with their position on the rachis.
2. Variability due to microclimate and soil changes in a very large vineyard (the location of the vine in
the vineyard, irrigation practices, etc.)
3. The location of the cluster on the vine.
4. The degree of sun exposure (variation in leaf cover)
5. The natural tendency to select samples based on eye appeal.
Preparation
Sampling and measurements should be taken once or twice a week, depending on the rate of changes
occurring in the grapes’ development. Daily sampling is recommended when the grapes are almost mature.
Each sampling should be done at the same time of day in order to maintain accuracy.
While sampling, keep in mind that the distribution of sugar in a bunch of grapes or on bunches growing on the
same stem is not regular. The internal composition of the grape is also not homogeneous: the pulp from just
under the skin has the highest sugar and lowest acid; the intermediary area is more acid and sometimes more
sugary; and the pulp in the center of the berry nearest the seeds has a much lower sugar count and much more
acid. Therefore, a varied sample must be obtained in order to create a homogenous sample of the vineyard.
Materials
Ice chest, quart-size plastic zip-lock bags, grape knife or clippers.
Procedure
Collect 250-500 berries, each one from different cluster locations on the vine, lighted and shadowed, and from
both sides of the rows, and from different vines in the area.
Berries should be picked as whole as possible in order not to lose their juice. Leave berries whole until ready
to perform refractometry and titratable acidity. At that time, gently mash berries while still in the bag, taking
care not to break the skins and seeds up too much. Pour juice through a strainer or filter into a beaker and test
immediately.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 4 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Soluble Solids by Refractometry
Developed by: Martin and Rebecca Roman
Date: December 1997
Introduction
The measurement of Total Soluble Solids in grape juice gives a fairly good indication of the sugar content
(sugars represent 90-94% of Total Soluble Solids) and therefore the maturity of the grapes. The refractometer
is an optical device that tests refractive index by measuring the degree of bending of a beam of light when it
passes through a solution. On a Brix refractometer, the refractive index reads out directly in (% sugar) °Brix,
which has been calibrated against sucrose solutions.
Grape juice samples can be measured with greatest accuracy if allowed to settle, or if centrifuged or filtered in
the lab to remove pulp. The refractive index is critically dependent on the temperature of the sample
measured. Field type refractometers must have their reading temperatures corrected to 20°C (68°F). In the
absence of a temperature-compensating refractometer, a correction faction must be applied to samples that are
not at 20°C for an accurate measurement. In addition to temperature, alcohol is a major interference in
accurate measurement of the refractive index. Therefore, a refractometer should not be used in soluble solids
determination of fermenting must or wine. Standardize the refractometer using distilled water or a 20° Brix
Standard Solution. Distilled water at 20°C should read 0° Brix.
Materials
Hand held refractometer, distilled water, laboratory tissue and lens paper, and a light source.
Procedure
1. Make sure the refractometer prism and cover are clean and dry. Use lens paper to dry if necessary.
2. Open the prism box and place a drop or two of the sample on the glass surface, making sure the whole
glass surface is covered.
3. Close the prism cover and point the instrument towards a bright light.
4. Look through the eyepiece and read the graduated scale at the point where the dividing line between
light and dark fields crosses the scale.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 5 of 63
Revised: Spring 2007
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 6 of 63
Revised: Spring 2007
Calculations and Units of Reporting
For juice analysis a scale reading directly in % (w/w) sugar (°Brix) is used. Readability is normally 0.1-0.2
Brix, depending on the instrument and on the preparation of the sample.
Temperature correction (approximate) – for every °C above or below 20°C add or subtract 0.07° Brix
respectively to or from the indicated value.
Temperature Correction Table for Brix by Refractometry Above and Below 20°C
Temp
°C
°Brix
°Brix
°Brix
°Brix
°Brix
°Brix
°Brix
°Brix
Minus
12
13
14
15
16
17
18
19
0
0.42
0.37
0.33
0.27
0.22
0.17
0.12
0.06
5
0.45
0.40
0.35
0.29
0.24
0.18
0.13
0.06
10
0.48
0.42
0.37
0.31
0.25
0.19
0.13
0.06
15
0.50
0.44
0.39
0.33
0.26
0.20
0.14
0.07
20
0.52
0.46
0.40
0.34
0.27
0.21
0.14
0.07
25
0.54
0.48
0.41
0.34
0.28
0.21
0.14
0.07
30
0.56
0.49
0.42
0.35
0.28
0.21
0.14
0.07
35
0.57
0.50
0.43
0.36
0.29
0.22
0.15
0.08
Plus
21
22
23
24
25
26
27
28
0.06
0.13
0.19
0.26
0.33
0.40
0.48
0.56
0.07
0.13
0.20
0.27
0.35
0.42
0.50
0.57
0.07
0.14
0.21
0.28
0.36
0.43
0.52
0.60
0.07
0.14
0.22
0.29
0.37
0.44
0.53
0.61
0.07
0.15
0.22
0.30
0.38
0.45
0.54
0.62
0.08
0.15
0.23
0.30
0.38
0.46
0.55
0.63
0.08
0.15
0.23
0.31
0.39
0.47
0.55
0.63
0.08
0.15
0.23
0.31
0.40
0.48
0.56
0.64
References
1. Zoecklein, B.W., Fugelsang, K.C., Gump, B.H., Nury, F.S. 1995. Wine Analysis and Production.
Chapman & Hall, New York.
2. Iland, P., Ewart, A., sitters, J. 1993. Techniques for Chemical Analysis and Stability Tests of Grape
Juice and Wine. Patrick Iland Wine Promotions, Campbelltown, South Australia.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 7 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Soluble Solids by Hydrometry
Developed by: Didier Jacquet and Bill Wright
Date: December 1997
Introduction
The measurement of soluble solids by hydrometry is based on the measurement of the specific gravity of the
sample expressed as degrees Brix on the hydrometer at a defined temperature.
The presence of carbon dioxide (in fermenting samples of wine) may buoy the hydrometer upwards. For an
accurate analysis, de-gas the sample by flash-heating, then cooling sample, in preparation for the procedure.
An alternative is to place sample in the cylinder and gently invert several times, allowing gas to be given off
each time the cylinder is uprighted.
If the hydrometer is not floating freely, an inaccurate reading may result. Remove suspended solid particles
by settling or straining the sample prior to the procedure. The hydrometer cylinder must also be wide enough
so that the hydrometer can float freely without touching the sides to reduce frictional forces.
A faulty hydrometer will cause errors. Hydrometers can be checked by testing with aqueous solutions of
known sugar concentration. For example: an 18 degree Brix solution can be prepared by dissolving 18 grams
of sucrose in 82 ml of distilled water.
Failure to measure the temperature of a sample and apply the temperature correction factor may result in an
inaccurate measurement. The temperature for which the hydrometer has been calibrated.
Use a clean hydrometer. Grease and dirt can cause inaccuracies.
Materials
Appropriate size graduated cylinder (approximately 250 ml), an appropriate range Brix or Balling hydrometer
and a thermometer.
Procedure
1. Fill an appropriate size of measuring cylinder with settled juice to about 10 cm (2”) from the top
2. Insert a thermometer into the juice and measure the temperature.
3. Record temperature of sample.
4. Carefully slide the hydrometer into the cylinder. Try to keep the liquid from adhering to the scale.
5. Spin the hydrometer with your thumb and forefinger (to remove adhering air bubbles). The
hydrometer must float freely.
6. Read the indicated reading with your eye level with the bottom of the meniscus.
7. Apply the appropriate temperature correction (see below) to the indicated reading obtained in Step 6.
Calculations and Units of Reporting
°Brix (accurate reading) = °Brix reading + correction factor.
For every °C above 20°C add 0.06 °Brix to the indicated value.
For every °C below 20°C subtract 0.06 °Brix to the indicated value.
1 °Brix = 1 g. of sugar/100 g. of solution.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 8 of 63
Revised: Spring 2007
Note: to convert degrees Fahrenheit to degrees Celsius: (°F – 32) x .56 = °C
References
1.
Iland, P., Ewart, A., sitters, J. 1993. Techniques for Chemical Analysis and Stability Tests of Grape
Juice and Wine. Patrick Iland Wine Promotions, Campbelltown, South Australia.
Determination of Total Soluble Solids by Hydrometry
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 9 of 63
Revised: Spring 2007
Temperature Correction Table for Various Hydrometers
Temperature of
Measurement
(°C)
15
16
17
18
19
20
21
22
23
24
25
Indicated
Hydrometer
Reading
X
X
X
X
X
X
X
X
X
X
X
Corrected Value
(at 20°C)
°Baume
X – 0.25
X – 0.20
X – 0.15
X – 0.10
X – 0.05
X
X + 0.05
X + 0.10
X + 0.15
X + 0.20
X + 0.25
Corrected Value
(at 20°C)
°Brix
X – 0.30
X – 0.24
X – 0.18
X – 0.12
X – 0.06
X
X + 0.06
X + 0.12
X + 0.18
X + 0.24
X + 0.30
Corrected Value
(at 20°C)
°SG
X – 0.0010
X – 0.0008
X – 0.0006
X – 0.0004
X – 0.0002
X
X + 0.0002
X + 0.0004
X + 0.0006
X + 0.0008
X + 0.0010
Techniques for Chemical Analysis and Stability Tests of Grape Juice and Wine
References
1. Zoecklein, B.W., Fugelsang, K.C., Gump, B.H., Nury, F.S. 1995. Wine Analysis and Production.
Chapman & Hall, New York.
2. Iland, P., Ewart, A., sitters, J. 1993. Techniques for Chemical Analysis and Stability Tests of Grape
Juice and Wine. Patrick Iland Wine Promotions, Campbelltown, South Australia.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 10 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Titratable Acidity (TA) by Indicator Titration
“Quick and Dirty”
Developed by: Cheryl Zammataro and Lucinda Wolf
Date: December 1997, September 2003
Introduction
Titratable acidity (TA) is a measure of the organic acid of the wine or juice sample being analyzed. The test
may be used as a quick method to calculate the acidity of the sample. In this method, NaOH is used to titrate.
The end point is monitored using a phenolphthalein indicator (1%).
It is difficult to see the end point of red wines. The major interference with this method is CO2, therefore it is
important to degas the sample prior to starting test. Other interferences include using the incorrect
concentration of phenolphthalein, and not measuring the correct volume of wine.
Materials
100ml measuring cylinder, de-ionized water, 0.067N Sodium Hydroxide (NaOH), 5mL volumetric pipette,
250mL Conical flask, phenolphthalein indicator (1%), 10mL buret.
Procedure
1. Add 100ml de-ionized water to a 250ml Conical flask using a 100ml measuring cylinder
2. Pipette 5mL of wine (degassed)/juice into the flask.
3. Add 2-3 drops of 1% phenolphthalein indicator.
4. Carefully fill the 10mL buret with the standardized 0.067N NaOH using a squeezy bottle.
5. Place the tip of the buret into the Conical flask to begin titration. Record the initial buret reading.
6. Slowly add (titrate) NaOH and look for a pink color for white wine and a purple color in the red wine.
7. As you start to notice a color change, titrate drop by drop until the entire solution turns either pink or
purple and stays that color for 10 seconds (Endpoint). DO NOT OVER TITRATE.
8. To determine the TA, look at the buret and read the mLs of NaOH it took to reach the endpoint.
Calculations and Units of Reporting for Titratable Acidity
At the concentration level of 0.067N, the quantity of the base (NaOH) used to reach the endpoint yields the
total acidity in the appropriate units. Therefore, the total volume of 5.0mL of base needed to reach that
endpoint, corresponds directly to a TA of 5.00 g/L.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 11 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Titratable Acidity (TA) by pH Titration
Developed by: Lucinda Wolf, Cheryl Zammataro & Gerry Ritchie
Date: December 1997, Fall 2001
Introduction
Titratable acidity is a measure of the easily dissociable organic acid content of the juice, must, or wine being
tested. Organic acid content is important in the flavor, stability, and shelf life of the wine. This procedure
determines the amount of organic acids that can be titrated with a dilute alkali solution to a pH of 8.2.
Titratable acidity is reported as grams per liter of tartaric acid.
Wine and juice samples must be degassed and centrifuged clean. Carbon Dioxide is a major interference.
Other interferences include: incorrect concentration of NaOH, incorrect volume of wine, and not allowing
sufficient time for mixing and reacting after each addition of NaOH.
Equipment
Vacuum Pump
PH meter
Magnetic Stirrer
Magnetic stirrer bar (1”)
5ml Pipette
50ml beaker
10ml Burette
Chemicals
0.067N NaOH
De-ionized / distilled water
Preparation
1.
2.
3.
4.
5.
6.
7.
8.
Standardize pH meter per operator’s manual.
Degas about 20-30ml wine for 4min using vacuum pump.
Shake occasionally during de-gassing
Pipette 5ml into a 100ml beaker, add stirrer bar.
Add 50ml DI water.
Begin stirring.
Put 0.067N NaOH in burette using squeezy bottle. Record the initial burette reading.
Place burette over beaker with tip 1/3” above level of liquid.
Procedure
The following describes how to add the alkali from the burette to achieve a pH of 8.2 in the beaker.
1. Add NaOH from burette until pH is about 5 and then close tap.
2. Wait until the pH has stabilized or starts to decrease.
3. Add more NaOH until the pH is 5.5 (or 6.0 if the pH was >5.5 after waiting for it to stabilize or to start
to decrease).
4. Wait until the pH has stabilized.
5. Add more NaOH until the pH is 6.0 (or 6.5 if the pH was >6.0 after waiting for it to stabilize or start to
decrease.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 12 of 63
Revised: Spring 2007
6. Wait until the pH has stabilized.
7. Repeat this process until pH is 7.00.
8. Add NaOH more slowly by rotating the tap through a complete circle once and waiting for pH to
stabilize / decrease.
9. Continue until the pH is 8.2 +/- 0.3.
10. Record volume of 0.067N NaOH added.
NB
It takes less than one drop of alkali to increase the pH of the de-ionized water to 8.2 and therefore it is
considered a negligible amount.
Calculations and Units of Reporting Titratable Acidity in Musts and Wines
TA (g/L Tartaric Acid) = (ml NaOH) (N NaOH) (0.075) (1,000)
ml wine sample
In this case:
TA (g/L) = volume (mls) of 0.067N NaOH added.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 13 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
pH
Developed by: Jonathan Grant and Janet McDonald
Date: October 1997
Introduction
The pH is important in winemaking as it directly affects microbial stability (bacterial growth), wine oxidation,
wine color, SO2 activity, flavor, aroma and chemical stability. pH is a measure of the concentration of
hydrogen ions in a solution. pH = -log [H+].
Errors may occur due to incorrect calibration of the pH meter or worn or insensitive electrode. If adjustment
of the sensitivity control in setting the pH 4.00 cannot achieve the correct setting or is less than 90% accurate,
this indicates that the electrode has lost sensitivity.
Another common cause of error in pH measurement is temperature. Some electrodes have automatic
temperature correction or some pH meters have temperature compensation probes that correct for
temperature. On the other hand, some set ups have neither and correction needs to be achieved by measuring
the temperature and using tables of correction factors. The temperature of the sample and standard buffers
needs to be the same, even if the electrode assembly has a temperature compensation probe.
To ensure a quick response and free-flowing liquid junction, the sensing bulb on the electrode must not be
allowed to dry out. Always store in the pH electrode storage solution.
Materials
pH Meter & electrode, 50ml beakers for each sample, pH 7.00 buffer solution, pH 4.00 buffer solution,
juice/wine sample, Pasteur pipette, distilled water, magnetic mixer and stir bar.
Procedure
1. The buffers are stored in containers that the electrode can be immersed in directly.
2. Turn on the meter, slide the rubber sleeve off of the vent hold in the electrode (if need be), check the
level of internal filling KCl solution (to ¼ inch below the hole), add if needed.
Calibration of the meter: You must first calibrate the meter with two buffers before proceeding with the
sample measurement. This must be done every time the meter is switched on. Once calibrated, do not
turn the machine off until finished for the day. If you are measuring a lot of pHs, recalibrate after 4hrs.
3. Place the pH 7.00 buffer solution container (yellow solution) on a magnetic stirrer. Immerse electrode
in the container (there is a magnetic stirrer bar already in the container). Switch on the stirrer carefully
and stir slowly, making sure that the stirrer bar does not hit the glass bulb.
4. Follow the instructions for the calibration of the pH meter that are on the wall above the pH meter.
5. When moving from the the pH7 buffer to the pH 4 buffer, rinse the electrode with a few milliliters of
deionized water from a squeezy bottle, touch the tip with a tissue to remove excess water. and then
place the electrode in the pH 4.00 buffer (red solution) Stir slowly as before and complete the
calibrations as described in the instructions.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 14 of 63
Revised: Spring 2007
Measurement of samples:
6. Place approximately 50 mL of sample in a small beaker with a stir bar and set on magnetic stirrer. If
necessary, wine and juice samples should be clarified prior to measurement either by natural settling
or centrifuging.
7. Remove electrode from buffer and rinse with deionized water. Place electrode into the beaker with the
50 mL sample and start stirring, making sure that stirring bar does not contact the tip of the electrode.
8. .Follow the instructions for pH measurement on the wall above the pH meter
9. After recording the pH value, rinse the electrode with distilled water and return the electrode to the
storage solution.
VERY IMPORTANT: When not in use, store electrode immersed in storage solution. Do not leave
electrode immersed in grape juice or wine longer than is necessary.
Storage Solution: Either the pH 4.00 buffer solution, a saturated solution of potassium hydrogen tartrate
or as per manufacturer’s recommendations.
Calculations and Units of Reporting for pH Readings
Temperature Correction Table: Variation of pH value with temperature
pH Value
pH Value
pH value
pH value
Temperature (°)
Phosphate Buffer Phosphate Buffer Phthalate Buffer
Saturated
(commercially
(lab prepared)
Solution of KHT
prepared)
10
7.12
6.92
4.00
-15
7.06
6.90
4.00
-20
7.02
6.88
4.00
3.56
25
7.00
6.86
4.01
3.56
30
6.99
6.85
4.02
3.55
Electrode Storage
Short-term Storage (up to one week): Soak electrode in pH electrode storage solution. If there is none
available use 200 ml. pH 7 buffer to which about 1 gram of KCl has been added, as a temporary substitute.
Long-term Storage: The reference chamber should be filled and the filling hold securely covered. Cover the
sensing element and reference junction with its protective cap containing a few drops of storage solution.
Before returning the electrode to use, prepare it as a new electrode (See manual for preparing new electrode).
Electrodes should be periodically cleaned to ensure proper operation. Immerse them in a solution of 75%
Methanol followed by several rinses in Distilled Water.
References
Orion Laboratory Products Division, 720A pH/ISE Meter, Part No. 216616-001 Rev. A reference manual.
Techniques for Chemical Analysis and Stability Tests of Grape Juice & Wine, Determination of pH, page 2225.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 15 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Wine Technology
Standard Operating Procedures
Formol Titration
Developed by Keith Behlmer, Roger Low and Ralph White
December 2002
Introduction
Yeast available nitrogen (YAN) is required for yeast to complete the fermentation of sugars to alcohol. It is
needed for cell division, growth and synthesis of cell parts. YAN is made up of two forms of Nitrogen:
1. Ammonium ion – NH4
2. Free alpha amino acids (yeasts will only utilize some of the amino acids)
The Formol titration is a method used to estimate the YAN in musts. Formaldehyde is used to react with free
amino groups and the released hydrogen is titrated with an alkali (sodium hydroxide). Ammonium ions are
also titrated by the sodium hydroxide
Errors
 pH adjustment is not done correctly
 Titration should be done slowly due to high buffering of the solution in the presence of formaldehyde.
Equipment
Centrifuge
Filtration system for 5 micron filter papers
pH meter
magnetic stirrer and stirring bars
10ml pipette
10ml burette and stand
100ml beakers
Chemicals
pH 4 and pH 7 buffer
1N NaOH
0.067N NaOH
Formaldehyde (37%) at pH 8
De-ionized water
Preparation
Clarify the must using a centrifuge and filtering equipment
1. Centrifuge for 10 minutes at 4000 rpm
2. Filter through a 5 micron swinnex cartridge.
Procedure
1. Pipette 10ml sample of clarified and filtered must into a 100ml beaker containing a stirring bar.
2. Add 50ml of distilled water and immerse pH electrode in the solution.
3. Start stirring the solution.
4. Adjust pH to 7.0 by adding 1 N NaOH from a Pasteur pipette.
5. Adjust pH to 8 by adding 0.067N NaOH from a Pasteur pipette.
6. Add 5 ml of 37% formaldehyde (also adjusted to pH 8) and wait 5 minutes
7. Titrate to a pH of 8.0 using 0.067 N NaOH
8. Repeat with water blank if necessary.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 16 of 63
Revised: Spring 2007
Calculation:
mg YAN /L =
(mls NaOH for sample – mls for blank) x (Normality of NaOH) x Dilution factor x (1000/sample
volume) x At. Wt. Of N
=(mls NaOH for sample-mls NaOH for blank) x 93.8
Notes on the Calculation
 1 mole of OH- in the alkali reacts with 1 mole of hydrogen released from an amino acid of an
ammonium ion
 1 mole of OH- added in titration = 1 mole of nitrogen
 Dilution factor = final volume/initial volume (NB dilution occurs before pipetting the 10 ml sample)
 Grams of Nitrogen in 1 mole or equivalent = Atomic weight of N = 14
 mg/L = ppm
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 17 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Winery Technology
Standard Operating Procedure
Nitrogen by Ammonia [NH3] Electrode
Developed by Val Zuber, Carl Bowker , Raul Perez and Gerry Ritchie
December 2002
Introduction
Yeasts require adequate nitrogen (N) to ferment sugar to alcohol. Fermentation yeasts need nitrogen
specifically for cell division, growth and synthesis of cell parts, particularly for sugar transportation across the
cell wall. Determining the amount of nitrogen in a must is therefore essential when converting said must to
wine through fermentation. The Ammonia Electrode procedure is used to determine the concentration of
nitrogen present in the must or juice of grapes in the ammonium form. It assists with decisions regarding
additions of nitrogen supplements to maintain and complete alcoholic fermentation adequately.
Equipment, Materials and Chemicals
pH Meter
Conditioned Ammonia [NH3] Electrode
Ammonium chloride
De-ionized water
50, 200 and 500ppm NH3 standard solutions prepared from an ammonium chloride stock solution (see below)
100 mls of 10N NaOH
10 ml pipette
5ml micro-pipette
4 x 100ml beakers
Magnetic stirrer
Stirring bar
Preparation of 10g/L Ammonia (NH3) Stock Solution
Dissolve 15.71g of ammonium chloride in a 500 ml flask of de-ionized water and add 0.1 ml of 1N HCl.
Preparation of Standard Ammonia Solutions
Prepare the following standard solutions by pipetting the stated volume into a 500ml volumetric flask and
adding DI water up to the mark. Mix thoroughly before using.
Concentration of
Standard Solution
(mg NH3 /L)
50
200
500
mls of Stock solution
required
Final Volume with
DI water (mls)
2.5
10
25
500
500
500
Preparation of Juice / Must sample
1. Centrifuge four 30ml centrifuge tubes of each sample for 10mins at 4000rpm
Procedure
A calibration curve is constructed from standard solutions of NH3 which is then used to estimate the
concentration of ammonium N in musts or juice.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 18 of 63
Revised: Spring 2007
Determination of NH3 in standard solutions of known concentration
1. Using a measuring cylinder, add 40mls of the 500ppm NH3 standard solution to a 50ml beaker.
2. Rinse the ammonia electrode with DI water and submerge into beaker.
3. Using the 5ml micro pipette, add 2mls of 10N NaOH.
4. Place stirring bar in beaker and place on magnetic stirrer.
5. Record the mV reading after the electrode has stabilized.
6. Repeat steps 1-5 using 40mls of 200ppm NH3.
7. Repeat steps 1-5 using 40mls of 50ppm NH3.
Determination of NH3 in juice / must
1. Repeat steps 1-5 above using 40mls of must or juice.
2. .If the mV reading of must sample falls outside the range covered by the known standard solutions, the
sample needs to be diluted. Pipette 20mls of must sample into a 100ml volumetric flask and fill to the
mark with DI water. You now have a sample with a dilution factor of 5 (100/20=5). Record the mV of
the diluted sample as described in steps 1-5 above.
Calculation
Determine the concentration of NH3 in the must sample using the following steps:
1. Draw the calibration graph. Plot a graph of mV reading on the vertical (y) axis vs. concentration of
standard solution on the horizontal (x) axis. If doing this by hand, use semi log paper. If using a
computer, use the log of the concentration on the horizontal axis
2. Fit a straight line through the points. You can either
a. use regression analysis to fit a straight line through the three points given by the standard
solutions
b. draw a straight line through the points by eye
3. Estimate the concentration:
a. By regression analysis:
mg/L of NH3 = mV-(intercept/slope)
b. By eye: find the mV equivalent to that for the unknown must sample on the vertical (y) axis
of the calibration curve. Draw a horizontal line from that point across to the calibration
curve. At the point that this horizontal line touches the calibration curve, draw a vertical
line down to the horizontal (x) axis. The value on the x axis is the log of the concentration
of NH3 in the must sample.
4. If the samples was diluted, multiply the concentration estimated from the calibration curve by the
dilution factor (DF = Final Volume / Initial volume)
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 19 of 63
Revised: Spring 2007
Chart Example
Ammonium Electrode Maintenance and Storage
To ensure accurate electrode readings, refer to the electrode owner’s handbook and follow proper
procedures for short-term and long-term electrode storage.
Common Reasons for Errors
If inconsistent readings are being observed:
1. Ensure there are no bubbles of air trapped on the membrane when taking a measurement
2. Ensure that the pH is between 11 and 14.
3. If the above to possibilities have been discounted, an unclean or damaged membrane may be the
cause erroneous readings. Follow the electrode owner’s handbook instructions for membrane
replacement.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 20 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Alpha Amino Acid Estimation Using the NOPA Procedure
Developed by Ted Bechard, Cheryl Lauritsen, John Ruch, Ben Dolphin and Gerry Ritchie
December 2002
INTRODUCTION
At harvest, the winemaker needs to assess the availability of nutrients present in the musts to execute healthy
and successful fermentations. The most important macro-nutrient used by wine yeasts to conduct and
complete alcoholic fermentation is fermentable nitrogen. These yeasts require adequate nitrogen for the
structural development of multiple generations of cells and the transport of sugar across cell walls.
The forms of nitrogen that are available to, or assimilable by, wine yeasts most free alpha amino acids, and
ammonium ions (NH4+).
This assay concerns the estimation of free alpha amino acids using the NOPA procedure. The NOPA
procedure is based on estimating the availability of free amino acids using a reaction with the Ophthaldialdeyde / N-acetyl-L-cysteine (OPA/NAC) reagent to form compounds called iso-indoles. The
absorption of light at a wavelength of 335 nm by the iso-indoles is then measured using a spectrophotometer.
A juice blank is also prepared to account for absorbance by non iso-indole compounds, particularly phenolics.
A calibration curve of light absorbance versus concentration of amino acids is established from prepared
standards. It is then used to determine the net absorbance of nitrogen in the must measured in units of mg/L of
amino nitrogen.
Based on the results of this assay and those performed for estimating ammonium (NH4+) nitrogen, along with
taking into account viticultural factors and winemaking choices, the winemaker is able to determine the
amount of nitrogen supplements (if any) necessary to complete alcoholic fermentation. These supplements
may include Di-ammonium phosphate (DAP), blends of DAP and amino acids, or inactivated yeast. The
timing of any supplement additions is at the discretion of the winemaker but should be consistent with the
maintenance of a healthy yeast population, particularly in the presence of higher alcohol levels to avoid
sluggish or stuck fermentations.
MATERIALS AND EQUIPMENT NEEDED FOR NOPA ANALYSIS
Equipment
 Spectrophotometer capable of detection at 335nm
 4.5mL methyl acrylate cuvettes
 100uL pipette tips
 1000uL pipette tips
 3mL dispenser
 100 – 1000uL micro pipette
 10 – 100uL micro pipette
 2 X 1L vol. flasks
 Weighing Scale or analytical balance to 0.000g
 Syringe & 5um swinnex cartridges for clarification
 Tissues
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 21 of 63
Revised: Spring 2007
 Beakers for waste
Chemicals
 N-acetyl-L-cysteine (NAC)
 Ortho-phthaldialdehyde (OPA)
 L-isoleucine
 Boric Acid
 Sodium Hydroxide
 Denatured ethanol
 De-ionized water
Solution Preparation
OPA/NAC Reagent Preparation
Reagent Solution A (1000mLs)
1. Dissolve 0.671g of OPA in 100mL of 95% ethanol.
2. Dissolve: 3.837 g NAOH (s), 8.468 g ortho boric acid (s), and
0.816g NAC (s) in approximately 500mL de-ionized (DI)water in a
1000mL volumetric flask.
3. Add the alcohol/OPA brew to the flask and mix well.
4. Add de-ionized water to make 1000mL of solution
Reagent Solution B (1000mLs)
1. Dissolve 3.837 g NaOH (s), 8.468 g ortho-boric acid, and 0.816 NAC
in Approximately 500mL of de-ionized (DI) water in a 1000mL volumetric
flask.
2. Add 100mL 95% ethanol to the flask and mix well.
3. Add de-ionized (DI) water to make 1000mL of solution.
Note: Reagents can be stored in the refrigerator for 2 weeks and should be
used at room temperature.
Amino Acid Stock Solution Preparation
1.
Dissolve 0.328 g of isoleucine (s) in de-ionized water in a 250mL volumetric flask.
2.
2 mL aliquots of this solution can be frozen for use later.
Juice / Must Preparation
1.
Clarify juice sample by centrifuging for 10min at 4000rpm
2.
Filter through a 5um swinnex cartridge.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 22 of 63
Revised: Spring 2007
Procedure
A calibration curve is constructed by measuring the absorbance, at 335nm, of four different standards whose
mg N/L is known. Once the curve is plotted, the absorbance of the juice sample is measured and the
corresponding mg N/L is estimated using the calibration curve.
Calibration Curve
1. Prepare the following standard solutions (i.e. solutions that contain a known concentration of amino acids)
by pipetting the volumes of stock solution, reagents and DI water given in the table into a 4.5ml cuvette.
ml = milliliters (one thousandth of a liter)
uL = µL = microliters (one millionth of a liter)
1
2
3
4
Cuvette Code
Blank
Amino Acid
Concentration
(mg/L)
0
28
56
84
112
140
uL of Stock
Solution
0
10
20
30
40
50
uL of DI water
50
40
30
20
10
0
uL of OPA
(Reagent A)
3000
3000
3000
3000
3000
3000
2.
3.
4.
5.
5
Mix thoroughly and wait 10mins.
Zero the spectrophotometer using the blank cuvette.
Measure the absorbance of each cuvette containing a standard solution.
Construct a calibration curve by plotting the absorbance on the vertical axis (y axis) versus the known
mg/L of amino acids in each standard on the horizontal axis (x axis)
Determination of Amino Acid N in Unknown Sample
A. Sample
1. Pipette 50uL of clarified must or juice into a cuvette. Juice may need to be diluted if its nitrogen
concentration greatly exceeds the range of the calibration curve (>200Nmg/L).
2. Using pipette, add 3000µl of Reagent A.
3. Mix thoroughly and wait 10mins.
4. Measure the absorbance of light at a wavelength of 335nm.
B. Juice Blank
1.
2.
3.
4.
5.
Pipette 50uL of the clarified must or juice (diluted if necessary) into cuvette.
Using pipette, add 3000uL of Reagent B to the cuvette.
Mix thoroughly and wait 10mins
Measure the absorbance of light at a wavelength of 335nm.
Calculate Net Absorbance = “sample absorbance” –“juice blank absorbance”
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 23 of 63
Revised: Spring 2007
Calculation
Determine the concentration of amino acids in the juice / must sample using the following steps:
1. Draw the calibration graph. Plot a graph of Absorbance reading on the vertical (y) axis vs. concentration
of each standard solution on the horizontal (x) axis.
2. Fit a straight line through the points. You can either
a. use regression analysis to fit a straight line through the three points given by the standard
solutions (Excel has this capability)
b. draw a straight line through the points by eye.
3. If you are using regression analysis, you will need to know the slope and intercept of the fitted line.
Regression analysis should do this for you automatically. If not:
c. The intercept is the value on the vertical axis where the fitted line passes through that axis.
d. The slope is calculated from:
slope = y/x
select any y value on the vertical axis and find its corresponding x value on the
horizontal axis and divide the former number by the latter
4. Estimate the concentration:
net absorbance of N in must = sample abs-juice blank abs
a. By regression analysis:
mg/L of amino N = (net abs-intercept)/slope
b. By eye:
find the point on the vertical axis that is equivalent to the net absorbance for the unknown
sample. Draw a horizontal line from that point across to the calibration curve. At the point that
this horizontal line touches the calibration curve, draw a vertical line down to the horizontal (x)
axis. The value on the x axis is the concentration of amino acids in the unknown sample.
5. If the samples was diluted, multiply the concentration estimated from the calibration curve by the dilution
factor (DF = Final Volume / Initial volume)
Chart Example
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 24 of 63
Revised: Spring 2007
POTENTIAL ERRORS
Very small amounts (in the magnitude of micro liters) of juice samples, standards and reagents are used.
Micro pipetting correct amounts of reagent and samples is critical and care should be taken when using digital
micro pipettes.








Potential for miss-tracking the cuvettes (i.e.-keep track of the samples). Each cuvette will contain the
same total amount of solution, with varying amounts of stock solution and de-ionized water. When
filed, all the cuvettes will look the same!
Spectrophotometer needs to be zeroed with blank sample.
Sample may need to be diluted if nitrogen concentration exceeds 140 mg Nitrogen/L.
Regression equation needs to be correctly calculated.
Juice needs to be clarified by centrifugation or cold settling.
Care must be taken when preparing the reagent solutions. Solutions need to be brought to room
temperature before using.
Calibration curve should be plotted each time a set of new sample is measured.
Duplicate determinations are suggested.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 25 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Clinitest/Dextrocheck
Developed by Pat Collins, Mona Huisman and Gerry Ritchie
Date: November 2002 & 2003
Introduction
The Clinitest tablet is a standardized self heating method for quantitative determination of sugar by copper
reduction. The Clinitest Reagent Tablets contain copper sulfate which reacts with reducing substances in
wine converting cupric sulfate to cuprous oxide. This results in a color change which varies with the amount
of reducing substances present. The Clinitest Reagent Tablet reacts with all reducing sugars and is not
specific to glucose. Clinitest is a useful tool for monitoring fermentation progress but will react with phenolic
content in wine from red pigmentation to barrel phenolics. Fermentable sugars, glucose and fructose,
determined by enzymatic analysis will confirm dryness. Clinitest Reagent Tablets are the identical tablets
previously sold as Dextrocheck for testing reducing sugar.
Red Wines must be de-colorized before carrying out the Clinitest once the sugar concentration reaches
0.4%.
Equipment
De-colorizing:
Filter Funnel
250ml conical flask
15cm diameter qualitative filter paper
Teaspoon
Activated Carbon
Clinitest:
1 mL pipette
Test Tube
Clinitest Tablet
Color Chart (supplied with test kit)
Procedure
De-colorizing of red wines:
1. Place the filter funnel in the neck of the 250ml conical flask
2. Flute the filter paper and put it in the filter funnel
3. Put ¼ teaspoon of Activated Carbon in the filter paper.
4. Pour sufficient wine into the filter paper to fill it completely
5. Wait until about 2-5mls has filtered through the paper and then discard the rest.
Clinitest:
1. Pipette 0.5 mL of (decolorized)sample into test tube
2. Drop a sugar tablet (Clinitest) into the test tube. Note: Clinitest tablets are sensitive to moisture, so
packets should be kept sealed until used
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 26 of 63
Revised: Spring 2007
3. Watch the test tube as a boiling reaction proceeds. Caution: make sure you are not holding the test
tube as the boiling reaction occurs
4. Wait 15 seconds after boiling stops, then compare the color in the tube with color chart included with
the test kit
5. Estimate % residual sugar from chart (% approx. = g/100 mL; for g/L multiply by 10)
6. If color goes past orange to brown the sample is too sweet and must be diluted before retesting
7. For samples of 1 to 5% sugar, use 0.1 mL (2 drops) of wine and 0.4 mL (8 drops) of water. Multiply
% sugar reading from color chart x5 to compensate for dilution
Interpretation
Use color chart for 2 Drop Method with following % reducing sugar scale:
0
Blue
0.05%
Blue/Green
0.10%
Green
0.20%
Olive Green
Accuracy +/- 0.05% to 0.1% if <0.6% residual sugar
0.40%
Brown
0.60%
Brown/
Orange
1.00%
Orange
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 27 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Determination of Reducing Sugar by Rebelein Method
Developed by: JC Pijanowski, Anthony Guerrera and Erica Schubert
Date: Fall 2002
Introduction
The Rebelein method is used for determining the concentration of reducing sugars in grape juice or wine.
Wine samples are mainly analyzed by this method. Juice samples must be diluted before analysis. Copper
concentration is determined by reducing the copper with excess iodine and estimating the remaining iodine
with a standard thiosulfate solution. Starch is used as an indicator. When there is excess iodine present, the
iodine binds with the starch giving it a blue/green/black color. When all the iodine has reacted, the
blue/green/black color disappears and the solution takes on a cream appearance. The color change signals the
end point of the titration.
Equipment
 Burette
 5 mL and 1 mL Pipettes
 125 mL Conical flask
 Hot plate
 Oven mitt
 Plastic bowl and ice
 Distilled water
 Wine/juice sample
Chemicals
Reagents:
Chemical Description
Z1
Z2
Z3
Z4
Z5
Z6
Wine
Juice
Blank
Copper sulfate(CuSO4)
NaK Tartrate
Sodium Iodide (KI)
Sulfuric Acid (H2SO4)
Starch
Na Thiosulfate
Distilled water
Amount
required
5mL
2.5mL
5mL
5mL
5mL
Fill burette
1mL
1mL
1mL
Reagent Preparation
Accurate
Accurate
Accurate
Accurate
Accurate
Accurate
Decolorized for reds
Diluted
Accurate
Reagent
Addition
Accurate
Approximate
Approximate
Approximate
Approximate
Accurate
Accurate
Accurate
Accurate
Preparation
Before analysis of the wine sample, the wine sample must be prepared. If the wine sample is red it must be
decolorized (procedure below).
Decolorization
Place a funnel in a 250 ml Erlenmeyer flask and line the funnel with filter paper (Whatman Qualitative will
work). Add ½ teaspoon of carbon into the filter paper. Add 10 ml of wine sample into carbon filled filter
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 28 of 63
Revised: Spring 2007
paper and stir gently. When 10 ml wine sample has filtered through; discard the paper and carbon and repeat
until enough wine sample has been obtained.
Dilution
Wines containing more than 20 g/L reducing sugars require dilution so that accurate volume of Na Thiosulfate
may be used as the titrate. Check that wine is less than 20 g/L by using Clinitest. If the sugar level is more
than 20 g/L the wine sample must be diluted. Dilution factor (DF) can be calculated as follows:
DF = (volume wine + volume distilled water)/volume wine
Once you have decolorized wine sample (if necessary) and determined whether or not to do a dilution factor,
two analyses must be completed: blank analysis (using distilled water) and analysis of the wine sample.
Procedure
Blank Analysis:
1.
Pipette 5ml of Z1 (copper sulfate) and add 2.5ml of Z2 (NaK Tartrate) into a conical flask. Touch
pipette to side of flask to release last drops into flask.
2.
Add 3 boiling chips
3.
Pipette 1ml of distilled water (Blank) into flask.
4.
Turn hot plate on to 10. Place flask on hot plate until a boil is observed. Pick up flask using the
oven mitt and hold (swirl) over hot plate so that a slow boil is maintained for 30 seconds. (heating
of solution speeds up copper reaction)
5.
Run the side of the flask under cool water for a few seconds and then place in an ice bath until
bottom of flask is moderately cool to the touch.
6.
Now add 5ml each of Z3 (sodium iodide), Z4 (sulfuric acid) and Z5 (starch), in this order, stirring
after each addition.
7.
Fill burette with Z6 (Na thiosulfate) and record the initial burette reading.
8.
Titrate the mixture in the conical flask with Na thiosulfate, swirling the flask throughout the
titration. Solution goes from a blue/green/black color to gray then to a cream color (end point).
Initial tinges of yellow indicate there is Cu still to titrate.
9.
Record the final burette reading and calculate the difference between the final and initial burette
readings. The Blank Titre should be in the range of 29 to 31 mL and will vary slightly for each set
of reagents prepared.
Wine/juice Analysis:
1.
Repeat the above procedure, but instead replace distilled water with the same amount of
decolorized (and if necessary, diluted) wine sample at step 3.
2.
Note a red/brown color while titrating indicates too much sugar and the wine/juice must be diluted
first. See Preparation section above.
3.
Record the final burette reading and calculate the difference between this final burette reading and
the corresponding initial reading. This is called the Sample Titre.
Calculation:
Reducing Sugars (g/L) = Dilution factor x [Blank Titre (mL) – Sample Titre (mL)]
Source of Errors
 Incorrect judgment of the end point – take as the first obvious change to cream.
 Failure to decolorize red wines.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 29 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Total Sulfur Dioxide (TS) by the Ripper Method
Developed by Fernando Montanez and John H. Wagner
Date: December 1997
Introduction
Sulfur Dioxide is added as a preservative and antioxidant in juices and wines mainly to prevent unwanted
microbial infection and oxidation (seen as browning in white musts / juice). In this method, standard iodine is
used to titrate SO2. The endpoint is monitored using starch indicator. The sample must first be treated with
sodium hydroxide to release the bound sulfur dioxide.
Note that it is extremely hard to see the end point of red wines by Ripper, and the SO2 will dissipate rapidly if
you shake the solution vigorously.
Materials
250ml Erlenmeyer flask, 10ml burette, 20ml volumetric pipette, rubber stopper for flask, high intensity light
source, 0.02N iodine, starch indicator, 1N NaOH, and 1+3 Sulfuric Acid.
Procedure
1. Measure 25 mL 1N NaOH using a measuring cylinder into a clean 250 mL Erlenmeyer flask.
2. Pipet 20 mL of wine sample into the flask, stopper, and gently swirl. Allow 10 minutes for hydrolysis
of the Sulfur Dioxide to occur.
3. Remove the stopper and add 10 mL of 1 +3 sulfuric acid, and a squirt of starch indicator. Do not over
mix at this stage, as the Sulfur dioxide will be given off rapidly.
4. Place the mouth of the flask under the burette containing the Iodine, noting the beginning reading.
Gently swirl and begin titrating.
5. Titrate to a grey / black end point that remains stable for approximately 10 seconds. The use of a highintensity light source is useful for end point detection in red wines.
6. Take the reading on the burette, and determine the milliliters used to reach the end point. Calculate
the total Sulfur Dioxide concentration (mg/L) as follows below.
Calculations and Units of Reporting for Total SO2 in Musts and Wines
SO2 (mg/l) = (ml iodine) (N iodine) (32) (1000) = (ml Iodine)(.02)(32)(1000)
ML wine sample
20
= (32)X(Mls I2)
Additional Notes
1. If necessary, you may want to dilute red wines to see the end point clearer.
2. As iodine rapidly deteriorates, standardization of the iodine with sodium thiosulfate is recommended.
3. You may want to add a small amount of baking soda bicarbonate during procedure #3 on front page to
create a gas above liquid, which keeps the SO2 from being given off too quickly.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 30 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Free SO2 (FS) by the Ripper Method
Developed by: Gerry Ritchie
Fall 2001
Introduction
This method is used mainly for white wines as it is very difficult to see the end point in a red wine sample.
Equipment
Light Source (for reds)
10ml burette
50ml pipette
250ml conical flasks
Chemicals
25% sulfuric acid
1% starch solution
0.02N Iodine
Procedure
1. Put 0.02N iodine in a 10ml burette using a squeezy bottle.
2. Pipette 50ml of wine into a 250ml conical flask.
3. Add 5ml of 25% sulfuric acid.
4. Add 1 squirt of starch.
5. For red wines, arrange light source so that it illuminates the sample when it is placed below the
burette.
6. Add iodine from the burette while slowly swirling the beaker.
7. A cloud of black will appear and then disappear at first.
8. As the end point is approached the cloud will persist for longer.
9. The end point is achieved when the black cloud persists for approx. 10 secs.
10. Record volume of Iodine added
Calculation
FS (ppm) = mls iodine x normality of iodine x 32000 / mls of wine
FS (ppm) = mls iodine x 12.8
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 31 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Free SO2 (FS) using a Platinum Electrode
Developed by: Gerry Ritchie
Date: Fall 2001
Equipment
Hanna Platinum Electrode HI3131
Hanna PH 201 pH meter
10ml burette
50ml beaker
Magnetic stirrer and stirring bar
Thermometer
Chemicals
0.0155M iodine/iodate soln
25% H2SO4
De-ionized/distilled water
Titre = volume added from the burette
Method
1. Fill burette with 0.0155M iodate. Note stating volume.
2. Pipette 50ml wine sample into 50ml beaker and stir.
3. Add 5ml 25% H2SO4.
4. Keep stirring while adding 0.0155M iodate soln. from burette.
5. After adding the iodate, the mV reading goes down at first. Eventually, the general trend will be
upwards but not in a consistent manner.
6. When an increase in mV is observed after an addition, it often decreases quite quickly.
7. When the mV starts to increase (e.g. 10-20mV) after an addition, begin recording the titre and the mV
reading after counting to 5. The aim is to record the mV reading after a consistent time period has
elapsed each time some titrant is added from the burette
8. Stop the titration when the max mV after an addition becomes constant (record at least 4 times with
subsequent additions).
9. The first titre at which this max was observed in the end point of titration.
e.g.
Starting Value
End Point
Titre
3.35
5.25
5.35
5.45
5.5
5.55
5.6
5.65
5.7
5.75
mV
250
274
300
313
322
353
360
402
400
400
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 32 of 63
Revised: Spring 2007
Volume added at endpoint = 5.65-3.35
FS = 2.3 x 9.92 = 22.8ppm
10. After each titration, clean the Platinum electrode by rubbing the platinum with cardboard (as if using
sand paper).
11. Rinse with water.
12. Store in electrode solution when finished.
Calculation
Ppm FS = titre x molarity of iodate (0.0155) x 32000 / (volume of sample (50ml))
= titre x 9.92
Example curves of the titration
mV
Redox titration
420
410
400
390
380
370
360
350
340
330
320
7.00
8.00
9.00
Volume (mls)
10.00
11.00
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 33 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Free SO2 (FS) using Aspiration Oxidation
Developed by: Gerry Ritchie and Scott Macindoe
Date: February 2007
Introduction
In this method, free SO2 (FS) is changed into the molecular form by adding excess phosphoric acid. The
molecular SO2 is then aspirated out of the wine sample using a vacuum pump and collected in another vessel
containing hydrogen peroxide. The hydrogen peroxide oxidizes the SO2 to sulfuric acid which is then titrated
against sodium hydroxide (NaOH) in a simple acid base titration.
Ascorbic acid interferes with this method.
Materials
1 – Vacuum pump
1 – 20 ml pipette
1 – Glass Aspiration-Oxidation (AO) unit (see diagram)
10ml burette and stand
Chemicals
Sodium hydroxide (0.01 N NaOH)
Hydrogen Peroxide (0.3%)
Phosphoric Acid (1 + 3)
SO2 Indicator Solution (Methylene Blue and Methyl Red)
Preparation
(Refer to Diagram for general assembly and reference numbers used in the script below)
1. Secure the round-bottom, wine flask (4) to the stand.
2. Fill the 10ml burette with 0.01 NaOH
3. Put 0.3% hydrogen peroxide solution up to the mark on the side of the impinger flask(3) and add one
drop of the SO2 indicator solution.
4. If the peroxide turns violet, add 0.01 N NaOH drop wise from the 10ml burette, until the solution just
turns from the original violet color to a green color. (note the shade of green, as it should be the same
shade as the final end point of your titrations in Step 8)
5. Attach the impinger flask (3) to the stand and insert the impinger (2) securely into the flask
Procedure
1. Pipette 20mls of wine into the round bottom flask (4) using the top opening.
2. .Take the plastic bottle containing the 1+3 Phosphoric acid and squeeze the bottle until the plastic cup
is about two thirds full. When the bottle is released, the level in the cup will self adjust to 10mls.
Carefully add this to the round bottom flask, using the top opening.
3. Insert the Stopper/Glass tubing (7) securely into the top opening of the round bottom flask.
4. Put the Bubbler /Stopper (6) securely into the side port of the round bottom flask.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 34 of 63
Revised: Spring 2007
5. Turn on vacuum pump and aspirate the sample for 10 minutes. In the presence of SO2, the peroxide in
the impinger flask should turn violet during this period.
6. After 10 minutes, turn off the vacuum pump by unplugging it.
7. Lift the impinger (2) to just above the peroxide solution and blow into the Bubbler/Stopper (6) to
remove any liquid remaining in the impinger
8. Titrate the contents of the impinger flask to the endpoint described in step 4 of the preparation, above.
Read the titration volume to the nearest 0.05 ml.
Calculations
Free SO2 (ppm) = ml NaOH x N NaOH x 32 x 1000
20 ml (sample size)
If one uses 0.01N NaOH, then the above equation becomes:
SO2 (ppm) = ml NaOH x 16
Report the Free sulfur dioxide concentration to the nearest +/- 0.5 ppm (mg/l)
Example calculation: a 1.5 ml titration with 0.01N NaOH will have a “free” SO2 content of 24.0 ppm
Examples of Aspiration-Oxidation Apparatus
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Support Stand
Impinger
Impinger flask for sample collection
Round bottom wine flask
Flask stand
Bubbler and stopper
Stopper & glass tubing adaptor
Vacuum system
Quick disconnect
Latex tubing
Tygon tubing
Flow meter
Clamps
Green joint clamp
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 35 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Malic Acid by Paper Chromatography
Developed by: Kevin Keane and Mike Watson
Date: December 1997
Introduction
Malolactic Fermentation is bacterial process that occurs in many wines. Lactic acid bacteria metabolize bitter
Malic acids to softer Lactic acid. Chromatography is used to monitor this conversion. In chromatography
organic acids separate at different rates as they travel up through a filter paper by capillary action.
Preparation and Interferences
Wash your hands before beginning as acids and oils on your skin can interfere with accuracy of the
chromatogram. Hold paper by edges and top corners. Work on a clean and dry surface. Avoid contamination
of your wine samples and standards. Store chromatography paper tightly sealed, as well as the solvent.
Materials
Whatman #20 chromatography paper, developing tank (one gallon mayonnaise jar), capillary tubes, 0.3%
standards malic and lactic or citric and tartaric acids, chromatography solvent (available at the Wine Lab) or
see directions in this packet on how to make your own. A ruler and a pencil (not a ball point pen).
Procedure
1. With a pencil draw a horizontal line two centimeters from the bottom of the chromatography paper.
2. Make dots of X’s along this line for each sample or standard and label the positions clearly (marks
should be at least 2 cm apart).
3. Collect samples and standards in capillary tubes. This is done by simply dipping the capillary tube at a
slight angle into the sample or standard. The tube will fill by capillary action. Wipe the outside with a
tissue to remove any drops.
4. Place the chromatography paper in such a way that the pencil line is not touching any surface. This
can be accomplished by placing a ruler underneath the paper near the draw-line.
5. Carefully touch the bottom end of each tube to its corresponding mark along the pencil line (you
should have a malic acid and a lactic acid standard spot, as well as your samples). It is important to
keep the spots small (less than ¼” in diameter). This may require some practice.
6. Allow spots to dry and reapply two times, drying spots between applications using the hair dryer.
7. Allow spots to dry and put just enough solvent into developing tank to cover the bottom, so as not to
dilute the standards and samples.
8. Arrange chromatography paper into cylindrical shape. Staple the edges together, but do not overlap
the paper.
9. Place the paper cylinder in the developing tank (spotted end down) and secure the lid. Keep out of
direct sunlight.
10. When the solvent has reached near the top of the paper (about ¾ the way up) remove and hang to dry.
Reporting
The developed chromatogram will appear as yellow spots on a blue green background. Compare your wine
samples with the malic acid and lactic acid standards. Absence of a malic spot and the presence of a bright
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 36 of 63
Revised: Spring 2007
lactic spot should indicate completion of the malolactic fermentation. If there is an indication of any malic
spot the fermentation is not complete.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 37 of 63
Revised: Spring 2007
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedures
Alcohol by Ebulliometry
Developed by: Martin Garcia, Matt Gardner, and Paul Jenkins
December 1997
Introduction
Ebulliometry is a quantitative analysis for measuring the alcohol content of a given wine sample.
Ebulliometric analysis is a quick and easy method for general purposes, and is accurate to + 0.5% v/v. The
ebulliometric procedure is based on the fact that wine has a lower boiling point relative to the boiling point of
pure water at 1 atmosphere.
Alcohol (Ethanol) is a byproduct of fermentation, and should be tested for tax/legal purposes as well as
winemaking practices. A wine containing alcohol of 14% (by volume) and greater is taxed at a higher rate
than wine containing less than 14% alcohol. In addition, alcohol plays an important role in the structure of a
wine; high alcohol can cause “hot” characteristics within a wine, and relatively low alcohol content can cause
a wine to be “flabby” and unbalanced.
The major interference involved in this test is residual sugar. Ebulliometric analysis will not give an accurate
measurement of alcohol content for wines containing more than 2% residual sugar. Ebulliometric results for
samples that contain residual sugar between 0.2% and 2.0% must be corrected for in accordance with the
formula given in the Calculations section of this analysis. In testing, ice-cold water must be placed in the
condenser to prevent premature alcohol evaporation; this water should be emptied and refilled before each
new sample is placed in the ebulliometer. Due to changes in barometric pressure, the boiling point of water
should be determined at least once every two hours. If a gas burner will be used instead of the alcohol lamp,
the ebulliometer should be elevated away from the flame to prevent overheating which would lead to
improper boiling point readings (“bumping”). Record the temperature when it reaches the first stable reading
on the thermometer. Make sure to inspect the thermometer to ensure that the mercury tube is not broken
(separated). Debris may coat the boiling chamber with a boiling solution of 1% NaOH.
Materials
1. DuJardin-Salleron Ebulliometer
2. Alcohol or gas fueled burner
3. “Magic Dial” (supplied with most Ebulliometers)/alcohol scale
4. Ice water
5. Deionized/distilled water
6. 50 or 100 mL graduated cylinder
7. A chart summarizing the Churward formula is needed if measuring samples of alcohol content greater
than 16%. The chart is shown in figure 2.
Procedure
See diagram for ebulliometer components (fig.1).
A. Determine the boiling point of water:
1. Rinse boiling chamber twice with approximately 20 mL of deionized water.
2. Add 20 mL of deionized water to the boiling chamber. Do not place water in condenser.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 38 of 63
Revised: Spring 2007
3. Insert thermometer into its respective position as shown in diagram (fig. 1).
4. Place burner under the projecting tube and ignite burner.
5. Watch the mercury column rise, and note when it reaches a stable point for 30 seconds.
Record this temperature reading (T1).
6. Set the “magic dial” so that T1 is opposite the 0% alcohol mark.
7. Drain the ebulliometer.
B. Determine the boiling point of the sample:
1. Flush the boiling chamber with 2 rinses of approximately 25 mL each of the wine sample.
Close the outlet tap.
2. Measure 50 mL of the wine sample into the boiling chamber.
3. Fill the condenser with ice water.
4. Insert the thermometer.
5. Place burner under the projecting tube and ignite burner.
6. Watch mercury column rise. Note when the temperature remains constant for 30 seconds.
Record this temperature (T2).
7. Find the % alcohol that corresponds to T2 on the “magic dial”. This is the % alcohol of the
sample.
Calculations and Units of Reporting
1. The % alcohol should be calculated as follows for samples greater than 16% alcohol:
a. Find the difference between T1 and T2 (T1 – T2), this is the ‘ebulliometer degree;.
b. Enter this number into the table (fig. 2) to find corresponding % alcohol.
2. The % alcohol should be calculated as follows for samples with Baume > 0.5.
a. True Alcohol % (v/v) = Apparent Alcohol [%(v/v)] x [1 – °Baume level x 0.015]
b. Note, Correct Baume to 20°C.
3. Alcohol is measured and reported on a percent by volume basis.
Dujardin & Salleron Ebulliometer
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 39 of 63
Revised: Spring 2007
Table 1: Ebulliometer Degree (Wine) Table
Ebull
Deg.
% v/v
Alcohol
6.15
6.20
6.25
6.30
6.35
6.40
6.45
6.50
6.55
6.60
6.65
6.70
6.75
6.80
6.85
6.90
6.95
7.00
7.05
7.10
7.15
7.20
7.25
7.30
7.35
7.40
7.45
7.50
7.55
7.60
7.65
7.70
7.75
7.80
7.85
7.90
7.95
8.00
8.05
8.10
8.15
8.20
8.25
8.30
8.35
8.40
8.45
7.4
7.5
7.6
7.6
7.7
7.8
7.9
7.9
8.0
8.1
8.2
8.3
8.3
8.4
8.5
8.6
8.7
8.8
8.8
8.9
9.0
9.1
9.2
9.3
9.3
9.4
9.5
9.6
9.7
9.8
9.8
10.0
10.0
10.1
10.2
10.2
10.4
10.5
10.6
10.7
10.8
10.9
10.9
11.0
11.1
11.2
11.3
Ebulliometer Degree (Wine) Table
By Churchward. ACJ Jrnl & Proc. Jan. 1940)
Ebull
% v/v
Ebull
% v/v
Ebull
% v/v
Deg.
Alcohol
Deg
Alcohol
Deg
Alcohol
8.50
8.55
8.60
8.65
8.70
8.75
8.80
8.85
8.90
8.95
9.00
9.05
9.10
9.15
9.20
9.25
9.30
9.35
9.40
9.45
9.50
9.55
9.60
9.65
9.70
9.75
9.80
9.85
9.90
9.95
10.00
10.05
10.10
10.15
10.20
10.25
10.30
10.35
10.40
10.45
10.50
10.55
10.60
10.65
10.70
10.75
10.60
11.4
11.5
11.6
11.7
11.8
11.9
12.0
12.1
12.2
12.3
12.4
12.5
12.6
12.7
12.8
12.9
13.0
13.1
13.2
13.3
13.4
13.5
13.6
13.8
13.9
13.9
14.0
14.2
14.3
14.4
14.5
14.6
14.7
14.8
14.9
15.1
15.2
15.3
15.4
15.5
15.6
15.8
15.9
16.0
16.1
16.3
16.4
10.85
10.90
10.92
10.94
10.96
10.98
11.00
11.02
11.04
11.06
11.08
11.10
11.12
11.14
11.16
11.18
11.20
11.22
11.24
11.26
11.28
11.30
11.32
11.34
11.36
11.38
11.40
11.42
11.44
11.46
11.48
11.50
11.52
11.54
11.56
11.58
11.60
11.62
11.64
11.66
11.68
11.70
11.72
11.74
11.76
11.78
11.80
16.5
16.6
16.7
16.7
16.8
16.8
16.9
16.9
17.0
17.1
17.1
17.2
17.2
17.3
17.3
17.3
17.4
17.4
17.5
17.6
17.6
17.7
17.7
17.7
17.8
17.9
17.9
18.0
18.0
18.1
18.1
18.2
18.3
18.3
18.4
18.4
18.5
18.5
18.6
18.7
18.8
18.8
18.8
18.9
19.0
19.0
19.1
11.82
11.84
11.86
11.88
11.90
11.92
11.94
11.96
11.98
12.00
12.02
12.04
12.06
12.08
12.10
12.12
12.14
12.16
12.18
12.20
12.22
12.24
12.26
12.28
12.30
12.32
12.34
12.36
12.38
12.40
12.42
12.44
12.46
12.48
12.50
12.52
12.54
12.56
12.58
12.60
12.62
12.64
12.66
12.68
12.70
12.72
12.74
19.1
19.2
19.2
19.3
19.4
19.4
19.5
19.5
19.6
19.6
19.7
19.8
19.8
19.9
19.9
20.0
20.1
20.1
20.2
20.2
20.3
20.3
20.4
20.5
20.5
20.6
20.6
20.7
20.8
20.8
20.9
20.9
21.0
21.1
21.2
21.2
21.3
21.3
21.3
21.4
21.5
21.6
21.7
21.7
21.8
21.8
21.9
Ebull
Deg
% v/v
Alcohol
12.76
12.78
12.80
12.82
12.84
12.86
12.88
12.90
12.92
12.94
12.96
12.98
13.00
13.02
13.04
13.06
13.08
13.10
13.12
13.14
13.16
13.18
13.20
13.22
13.24
13.26
13.28
13.30
13.32
13.34
13.36
13.38
13.40
13.44
13.46
13.48
13.50
13.52
13.54
13.56
13.58
13.60
13.62
13.64
13.66
13.68
22.0
22.0
22.1
22.2
22.2
22.3
22.4
22.4
22.5
22.5
22.6
22.7
22.8
22.8
22.9
22.9
23.0
23.1
23.2
23.2
23.3
23.3
23.4
23.5
23.6
23.6
23.7
23.8
23.8
23.9
23.9
24.1
24.2
24.2
24.3
24.4
24.5
24.5
24.6
24.7
24.7
24.8
24.9
25.0
25.0
25.1
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 40 of 63
Revised: Spring 2007
Napa Valley College Viticulture and Enology Department
Standard Operating Procedure
Spectral Measures for estimating Wine Color and Phenolics
Developed by: Denise Eldridge and John Liles
December 1997
Revised December 2002 by: Andrea Bechard and Gerry Ritchie
Introduction
As wine ages, color changes form, intensity shade and brilliance. The ability for quantitative measures are
important for color and phenols due to the fact that color has become an indicator of quality for grapes in
specific viticultural regions. Phenols contribute to wine quality by providing structure, balance & body.
Oxidation of phenolic compounds will change the color of white wines from green-yellow to golden-yellow
and red to red-brown in red wines. These aspects of color depend on grape variety, viticultural practices and
winemaking techniques which in turn affect pH, SO2, co-pigment-type and concentration. A method of
measuring these changes is done using color spectrometric tests in order to measure the hue and intensity of
wine color as well as the phenolic concentration in the wine. Values for juice or wine can be assigned by
measuring the absorbance readings at different wavelengths. Samples of white wines are measured at 420nm
and red wines at 420nm and 520nm. Samples of natural wine are modified to compensate for the influence of
pH and SO2 in order to standardize the results.
Use a high quality spectrophotometer for absorbance measurements. Zero the instrument on distilled water in
the appropriate size cell. All particulate matter must be removed from the sample before analysis. This is
done by filtration through a .45 micron membrane filter.
Errors
 Incorrect volume additions
 Inadequate mixing
 Neglecting to recalibrate the spectrophotometer when changing from one wavelength to another or
changing cell sizes
 Remember to correct for cell size prior to carrying out calculations.
Equipment
UV-VIS Spectrophotometer (for 280620nm wavelengths)
pH Meter,
6 test tubes
0.45 micron membrane filters
10mm methyl acrylic cuvettes
1mm matched quartz cuvettes
10 ml burette
25 micro liter pipette
100 micro liter pipette
Chemicals
1 N NaOH
1 N HCl
10% w/v acetaldehyde (CH3CHO)
5% SO2
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 41 of 63
Revised: Spring 2003
Preparation of Buffer Solution
Add 24ml of pure ethanol to 176ml of de-ionized water. Dissolve 0.5g of KHT into the solution.
Adjust pH to 3.6 with HCl or NaOH.
Procedure
Measure the pH of the wine.
Sample Preparation
1. Take 50ml of wine and filter through <0.45micron using a swinnex cartridge
2. Adjust pH to 3.6 by adding dropwise and mixing with either 1 M NaOH or 1 M HCl
depending on the initial pH of the wine..
Use this wine on all the measurements below.
3. For each wine sample, set up five, 10mm cuvettes labeled as shown in the first column of the
table below
4. Add the samples / solutions given in Table 1 and leave for the designated time period.
Table 1
Cuvette Measurement
#
Code
Contents
Quantities
Reaction Time
1
Wine at
pH 3.6
Wine
Fill
cuvette
NA
2
AHCl
Wine
HCl (Hydrochloric
Acid)
20μL
3 hours
Wine
10% CH3CHO
(Acetaldehyde)
2ml
3
4
5
AAcet
ASO2
A20
2ml
45mins
20μL
wine
SO2
160μL
2ml
Wine
Buffer
100μL
1900μL
NA
5 min
5. Make a blank of de-ionized water in both a 10mm and a 1mm cuvette cuvette.
6. Run blank in the Spectrophotometer. Run blank every time you change cuvette size.
7. Following the instructions in the table below, transfer (if necessary) an aliquot of the
prepared sample to the correct cuvette size (see column 3 in Table 2) and measure the
absorbance of light at the wavelengths indicated in the fourth column of Table 2.
8. Use Table 3 to record your results.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 42 of 63
Revised: Spring 2003
Table 2
Cuvette Measurement
#
Code
Cuvette size
for
Measurement
Wavelengths
of
measurement
420
520
620
Number to
multiply readings
by:
1
Wine at
pH 3.6
1mm
10
2
AHCl
10mm
520
101
3
AAcet
1mm
520
10
4
ASO2
1mm
520
10.8
5
A20
10mm
280
520
365
20
Table 3
Cuvette
#
Measurement
Code
Wavelengths
of
measurement
Actual
Reading
(Absorbance
units):
Number
to
multiply
readings
by:
1
Wine pH 3.6
420
10
1
Wine pH 3.6
520
10
1
Wine pH 3.6
620
10
2
AHCl
520
101
3
AAcet
520
10
4
ASO2
520
10.8
5
A20
280
20
5
A20
520
20
5
A20
365
20
True
Value
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 43 of 63
Revised: Spring 2003
Calculations
1.
Use Tables 3 and 4 to calculate and record the spectral measures. Explanations of the
Spectral Measures are given after the table.
Table 4
Cuvette # and
Wavelength
Measurement
Code
Spectral
measure
1 /420
A420
Yellow /
brown
1 / 520
A520
Red
1 / 620
A620
Blue
A420 + A520
Color
Density
A420/ A520
Color Hue
2 / 520
AHCl
TA
3 / 520
AAcet
TC
4 / 520
ASO2
Polymerized
Color (P)
5 / 280
A20280 - 4
Total
Phenols
5 / 520
A20520
A+P
5 / 365
A20365
FlavonePhenols
Free
Colored
Anthocyani
ns (A)
A20- ASO2
AAcet- A20
(AAcet / AHCl) x 100
Co-pigment
Color (C)
Degree of
Coloration
Value
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 44 of 63
Revised: Spring 2003
Interpretation of the Results
Assumptions
 Diluting the wine x 20 breaks down all the co-pigments to non colored anthocyanins and
leaving behind colored, free anthocyanins and colored, polymerized anthocyanins
 Adding SO2 bleaches away free anthocyanins and co-pigments leaving behind the colored,
polymerized anthocyanins
 Adding acetaldehyde reacts with SO2 preventing it from bleaching any color compounds and
hence gives an estimate of the total color possible at a particular pH.
Measures of Color
Wine Color Density relates to the color intensity of the red pigments (A520) plus the yellow/brown
pigments (A420):
= (A420 + A520)
Wine Color Hue relates to the color yellow/brown pigments (A420 ) divided by the red pigments
(A520):
= (A420/ A520)
Brilliance of red is determined by the shape of the spectrum from 350nm – 700nm. Wine is more
brilliant when the peak of the curve is at 520nm.
Free Colored Anthocyanins (A) at pH 3.6:
A = A 20 –ASO2
Co-Pigmented color (C) at pH 3.6
C=A acet - A 20
Polymerized Color (P) at pH 3.6:
P = ASO2
Total Colored Anthocyanins at pH 3.6 gives an estimate of the concentration of free colored
anthocyanins (A) plus co-pigmented, colored anthocyanins (C) plus polymerized, colored
anthocyanins (P):
(TC) = (A + C + P) = A acet
Total Colored and Uncolored Anthocyanins gives an estimate of all the anthocyanins in the wine
irrespective of their color:
(TA) = AHCl
Degree of Coloration of Anthocyanins gives the percentage of all the anthocyanins that are in the
colored form in the natural wine:
= (TC / TA) x 100
= (AAcet / AHCl) x 100
Measures of Phenols
Total Phenolics measures the concentration of all phenolic material present in the wine. The
subtraction of the (-4) in the formula allows for absorbance of non-phenolic materials:
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 45 of 63
Revised: Spring 2003
= A20280 – 4
Flavones (co-pigments)
= A20365
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 46 of 63
Revised: Spring 2003
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Volatile Acidity (VA) Using a Cash Still
Developed by: Nick Legg, John Peck, Scott Macindoe & Gerry Ritchie
Date: December 1997, Fall 2001, Spring 2007
Introduction
Volatile acidity is used routinely as an indicator of wine spoilage. VA is generally interpreted as
acetic acid content (g/L). In this procedure, steam distillation of the sample is followed by titration
(of the distillate) with standardized sodium hydroxide. Results are reported as acetic acid (g/L).
The Cash should never be operated without water in the boiling changer. De-ionized or distilled
water should be used to fill the boiling chamber as faucet water will lead to erroneously high VA
values.
Carbon dioxide, titrated in the distillate as carbonic acid, would constitute a major source of error.
CO2 may be removed from the distillate by boiling the sample for a few seconds before collecting
the distillate.
SO2 interference can be corrected by adding hydrogen peroxide or carrying out a titration.
Sorbic acid, when used as an additive, must also be corrected for (1g sorbic acid = 0.535g acetic
acid), results should be reported as + or – 0.05 g/L. However, the standard method in the Californian
industry is to quote VA in g/100ml.
Equipment
Cash Still
10 ml pipette
10ml burette for 0.01N NaOH
250ml Erlenmeyer flasks
Distilled water squeezy bottle
Chemicals
Phenolphthalein
0.01N NaOH or 0.067N NaOH
0.3% hydrogen peroxide (kept in fridge)
De-ionized/distilled water
Procedure
Set up and cleaning
1. Using the small plastic funnel connected to the bottom of the cash still by latex tubing, fill
the boiling chamber with DI water so that the water level is approx. 1” above the heating
coil.
2. Attach condenser tubing to the sink faucet and switch on cold water so that the flow is not
too fast or too slow.
3. Empty any liquid in the sample (inner) chamber by turning Stopcock B to a horizontal
position and Stopcock A to a vertical position, arrow up (see Photo 1). If the liquid will not
flow out, slightly adjust the flow of water using the faucet.
4. After the sample chamber has emptied, rinse it by completely filling the glass sample
delivery funnel above stopcock A. with DI water. Let it drain into the sample chamber and
then evacuate. Repeat the process two more times.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
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Revised: Spring 2003
Collecting the distillate
5. Turn Stopcock B to the vertical position and stopcock A to the vertical position, arrow up
(Photo 2).
6. Pipette 10ml of wine into the sample chamber from the sample delivery funnel.
7. Add 0.5ml of 0.3% hydrogen peroxide using a Pasteur pipette.
8. Rinse the sample funnel with about 10ml distilled water using a squeezy bottle
9. Place a 250ml Erlenmeyer flask under the condenser outlet to collect the distillate
10. Switch on heater.
11. When water boils, let steam escape for ~10 seconds and then close stopcock A (horizontal
position-Photo 3).
12. Collect 100ml of distillate. This should take ~7 minutes
13. SWITCH OFF HEATER.
14. Remove Erlenmeyer flask from below the condenser.
15. Evacuate the wine sample and rinse the sample chamber by repeating steps 3 and 4 above
16. Repeat step 5-14 for other wine samples
Titrating the distillate
1. Add 2-3 drops of phenolphthalein to the distillate in the Erlenmeyer flask.
2. Titrate with NaOH to a pink endpoint that lasts 10-15 seconds.
Calculation
VA (g/L) = (mL NaOH) (N NaOH) (0.060) 1,000)
mL wine
VA (g/L) = mls NaOH x N NaOH x 6
VA is usually reported in g/100ml in the US, so the above figure needs to be divided by 10.
Using 0.01N NaOH,
VA (g/100ml) = mls NaOH x 0.006
Using 0.067N NaOH,
VA (g/100ml) = mls NaOH x 0.0402
Cleaning the Still
Weekly or as needed, mix ~10mls of 0.01N NaOH with 30mls of DI water. Put into the inner
chamber and boil for 5 minutes. Evacuate the NaOH mixture then boil DI water for 2 minutes to
flush the inner chamber.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
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Revised: Spring 2003
Alternative Sulfur dioxide correction
Governmentally regulated limits for the VA content of a wine are listed as exclusive of SO2.
When the VA approaches legal limits (1.2 g/L for reds, 1.1 g/L for whites in California) it is
necessary to correct for the contribution of SO2.
1. Immediately upon completion of the VA titration, cool the sample.
2. Add approximately 1 mL of starch indicator and 1 mL of 1 + 3 sulfuric acid.
3. Titrate with standardized iodine solution to a faint blue-green end point.
4. Calculate the free sulfurous acid equivalent according to the following equation:
F.S.A.E. (g acetic acid / L
Equivalent to SO2 present)
=
(mL I2) (N2) 32) (2) (60) (1,000)
(1,000) (sample vol) (64)
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 49 of 63
Revised: Spring 2003
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Fining – Bentonite Fining
Developed by Angela Camp, Michael Conversano, Brian Kosi, and Myles McMonigle
Date: December 2002
Introduction
In winemaking, fining is the addition of a reactive or absorptive substance in order to
remove or decrease the concentration of one or more undesirable components of a wine or must.
Fining is performed on wines for the purpose of achieving clarity and to improve color, flavor,
and/or physical stability. Fining agents are classified into numerous categories according to their
general nature: proteins (gelatin, isinglass, casein, and albumen), synthetic polymers (PVPP, nylon),
earths (bentonite, kaolin), polysaccharides (agars), carbons, silicon dioxide (kieselsols), and others
(metal chelators, enzymes, etc.)
Fining, although one of the least expensive operations in winery production, can have the
greatest impact on wine quality. Fining trials in the lab need to be performed at varying levels to
ensure that desirable results are attained using the least amount of fining agent necessary. Any
resulting alteration to wine’s color, aroma, or flavor must also be evaluated. To achieve consistent
outcomes, it is imperative that the preparation techniques, temperature, mixing, and timing in the
cellar are identical to those employed in the lab trials. Improper preparation of the fining agent can
reduce its effectiveness by up to 50%.
Fining agents often contain an electrical charge. When this charge is the opposite of the
charges present on particles suspended in a wine or must, neutralization and absorption may occur.
During the fining operation, these small particles are induced to coalesce into larger units, which will
then settle out from solution due to their relative density. Thus the fining material facilitates
clarification by forming particles of high density, which are more easily removed from the solution
through settling and/or filtration.
Bentonite is the most commonly used fining agent in the wine industry. It is primarily
utilized for clarification and protein stability in white wine and juice. It is a negatively charged clay
of volcanic origin that reacts with positively charged proteins to precipitate out compounds that
could adversely affect the sensory properties of wine. There are two forms commercially available
in the wine industry, sodium and calcium bentonite. However, the sodium form is preferred because
it hydrates best and provides more reactive surface area, thus increasing overall effectiveness.
Problems and Errors
 Too dilute a solution of bentonite will not precipitate out the targeted proteins and can dilute
the wine due to the excess water added.
 Improper rehydration of bentonite can result in clumping of the clay, resulting in ineffective
concentration of the fining agent.
 Poor compaction of bentonite lees after addition to the wine
 Improper disposal can clog drains and fill irrigation ponds
 Undesirable odors and off-flavors can result from improper use of bentonite
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
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Revised: Spring 2003
Reagents and Equipment
5 % bentonite slurry (*see preparation instructions below)
150ml beaker
Wine or juice samples
6 x 100ml-graduated cylinders
several 1ml graduated wide-bore pipettes
syringe with tubing on end
6 disposable 0.45um swinnex cartridges (or re-usable swinnex cartridge and 0.45um filters)
12 x 35ml nalgene centrifuge tubes
6 x 20ml test tubes
2 x 100ml beakers
saran wrap
marker pen
test tube and centrifuge tube racks
glass rod
stir bar
magnetic stirrer/hot plate
pin-point light source (i.e. pen light)
*5% Bentonite slurry preparation:
Weigh out 5 grams of bentonite. Measure 85ml of tap water in a graduated cylinder. Add the
water to a 150ml beaker, place beaker on a hotplate/stirrer and heat up to 60 °C. Place a stir bar
in the beaker and adjust the stir speed to a medium rate. Slowly sprinkle the bentonite until all of
the clay has rehydrated. Turn off the heat and allow to cool overnight while continuing to stir.
The bentonite will swell considerably during this period. Add more tap water until the final
volume of the slurry is 100ml and mix well.
Procedure:
Turn on hot water bath and set control about 6 to reach 80°C.
Sample Preparation
1. Label each of six 100ml graduated cylinders with the code # 1-6.
2. Mix the bentonite slurry thoroughly and add the appropriate quantity to each cylinder, using
a graduated, wide-bore 1ml pipette, according to the following table:
Cylinder #
mls of 5% Bentonite
slurry
lbs.Bentonite/1000 gal
1
0
0
2
0.125
0.5
3
0.25
1
4
0.50
2
5
0.75
3
6
1.0
4
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 51 of 63
Revised: Spring 2003
3. Add 100 ml of wine or juice to cylinder #1 , cap it immediately and immediately mix after
each addition by inverting several times before proceeding to next cylinder.
4. Let cylinders settle for about 30 minutes, until solution starts to clear. Carefully decant about
35 ml from each cylinder into 6 individual 35ml centrifuge tubes, numbering each tube the
same as the respective cylinders.
5. Place tubes in centrifuge and spin at 4000 rpm for 10 minutes.
6. Attach plastic tubing to the end of a 60ml syringe (in order to reach the bottom of the
centrifuge tubes) and carefully suck up as much wine as possible from centrifuge tube #1.
Then hold syringe vertically upward and suck the wine from the tubing into the body of the
syringe.
7. Remove the tubing and attach a swinnex filter over the syringe and filter all the wine into a
clean 35ml centrifuge tube, labeled according to the sample number.
8. Pour the filtered wine into a 20ml test tube (also labeled) as near to the top as possible.
9. Repeat this procedure for samples #2 through 6. Assess the protein stability of each trial
sample utilizing the heat stability test (see below.)
Heat Stability Test:
1. Cover each 20ml test tube with saran wrap and place tubes upright in 100ml beakers, 3 tubes
per beaker.
2. Set beakers in the 80°C water bath and leave for about six hours.
3. At the end of this period, remove test tubes from bath and allow cooling to room temperature.
4. Mix the contents and observe each sample for clarity and haze. The presence of a haze can
be estimated by shining a strong pin-point light source diagonally through the sample. Any
internal reflection indicates the presence of a haze.
Results and Interpretations:
The solution corresponding to the lowest fining level which produces no haze after the heat
stability test is the bentonite addition rate at which the wine is considered heat stable.
References:
1. Zoecklein, B.W., Fugelsang, K.C., Gump, B.H., Nury, F.S. 1995. Wine Analysis and
Production. Chapman & Hall, New York.
2. Illand, P., Ewart, A., Sitters, J. 1993. Techniques for Chemical Analysis and Stability
Tests of Grape Juice and Wine. Patrick Iland Wine Promotions, Campbelltown, South
Australia.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
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Revised: Spring 2003
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Fining – Other Than Bentonite
Developed by Jeff Ames, Robert Lloyd, John Oesleby, Toshiaki Wakayama
Date: December 2002
INTRODUCTION
Winemakers must consider the expectations of their customers in producing their wines. Consumers
typically expect wine to be clear and to be stable for a reasonable period of time in conditions to
which they are commonly subjected. For example, white wines are often refrigerated, which can
cause the formation of potassium bitartrate crystals. Although perfectly safe, these crystals are
easily mistaken for broken glass which can needlessly alarm the consumer. Similarly, other
substances in wine can cause visible or potential hazes and undesirable aromas, flavors, tactile
sensations (e.g. astringency), and undesirable colors.
Wine stability is defined as the attainment of a particular state in which a wine, for some definite
period and set of conditions, will not exhibit undesirable physical or sensory changes. Clarification
is simply the removal of visible solids or colloids from juice or wine to achieve clarity.
Stabilization, on the other hand, is the removal of wine components that have the potential to cause
undesirable hazes, flavors, aromas, color, and tactile sensations.
Fining is the addition of a substance that interacts or reacts with a wine component that is causing or
may cause a haze, instability or undesirable characteristics. Fining agents can both clarify and
stabilize a wine. Fining might be employed in the production of a wine to eliminate, either
immediately or in the future, off odors or colors, astringency, oxidized colors, bitterness or
harshness. Although these problems do not always detract from the flavor or aromas of a finished
wine, they are often addressed to ensure that the product appeals to the consumer. Other times, as
with bitterness or harshness, the chemical compounds creating the undesirable characteristic must be
removed for the wine to taste the way the winemaker intended. When fining is contemplated or
undertaken the winemaker must:
 Treat every wine as an individual.
 Treat every vintage as different.
 Run lab trials to determine the appropriate process and quantities of a fining agent(s) needed.
 Always use the minimum quantity of an agent or least invasive process to achieve the desired
result.
 Not use more processes than are required, remembering, however, that more than one process
may be required to achieve the desired result.
Types of fining agents:
Bentonite: This was discussed in the previous procedure.
Polysaccharide Agents: Polysaccharide agents are positively charged, high molecular weight, long
chain alginates on a diatomaceous earth carrier which are extracted from marine brown algae. These
agents remove other substances via ionic bonding and are quite often utilized to clean up the haze
that is left by other fining agents. These tend to function best when the pH of the wine being fined is
less than 3.5 and, consequently, are used more often on white wines than red. Sparkalloid and
Klearmor are proprietary formulations used in both hot and cold forms. The hot is considered more
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
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Revised: Spring 2003
effective. A benefit of Sparkalloid is that it has minimal effects on color or flavor and will, also,
make the wine easier to filter.
Proteinaceaous Materials: Proteinaceaous materials include gelatins, casein, egg whites and
isinglass. These agents all have a positive charge when they are in a solution with a pH typical of
wine (3-4) and react with phenols via hydrogen bonding. They favor reacting with larger phenols,
such as those that cause astringency, because they have more potential bonding sites.
 Gelatins: Gelatins are created from collagen obtained from animal bones and skins. They
are often used to soften red wines and reduce the phenol and brown color level in white
wines. There is a large risk of over-fining when using gelatin. Kieselsol or tannin is
commonly added to wine with gelatins as co-fining agents to improve the
flocculation/precipitation of the gelatin.
 Casein: Casein is the principle protein in milk. Casein is nearly insoluble and must be
dissolved at pH 11. Potassium caseinate is water-soluble and is preferred for this reason.
Casein is a positively charged protein that flocculates in acidic media such as wine and
absorbs and removes suspended materials. Casein is used to remove undesirable odors and
bitterness, to bleach color and to clarify white wines. It is sometimes used as a substitute for
carbon in color modification of juice and white wine and often used to remove the cooked
character from sherries.
 Egg Whites: Albumen is a common fining agent for red wines that is found in egg whites.
Albumen is colloidal in nature and has a positive charge that attracts negatively charged
tannins. Albumen removes less fruit character and fewer phenols than gelatin. Fresh eggs
contain 3 to 4 grams of active product per white and are preferred over frozen egg whites
 Isinglass: Isinglass is a positively charged agent derived from the air bladder of a sturgeon.
Its flocculated form is easier to work with than the sheet form because it does not have to be
rinsed to get rid of fishy smells. Isinglass is used mainly in still white wine and sparking
wines to improve aroma and clarity and modify the finish.
Polymers: Polymer fining agents are high molecular weight (HMW), cross-linked polymers that
react with phenols via hydrogen bonding. Their molecules are inflexible, which tends to make them
more useful at removing smaller molecules. Polyvinyl polypyrolidone (PVPP) is the most common
type of polymer that is used as a fining agent. It is a HMW fining agent that binds with phenolic and
polyphenolic molecules in wine by absorption and also attracts low molecular weight catechins
through hydrogen bonding. It is most often used to remove bitter compounds, in both red and white
wines, and browning precursors. This is a very fast acting fining agent and no preparation is
required to use it. After fining, the wines must be filtered to remove the PVPP and these wines may,
in the end, taste more astringent after the bitter compounds are taken out.
 Polyclar: Polyclar is a highly absorbent proprietary PVPP product used to remove
polyphenolic compounds and oxidized melanoidins that, when used in a finished wine, can
help remove haze-causing proteins. It can also remove oxidized flavor and aroma compounds
and reduce tannins. Excessive amounts can strip melanoidins (color and flavor compounds)
from a wine.
Carbon: Activated carbons are nonspecific adsorptive agents made from wood. The sponge like
carbon binds with weakly polar molecules, especially those containing benzene rings. Carbon
effectively removes phenolic compounds, especially small phenolic compounds. Compounds larger
than dimers are too large to be adsorbed. Stripping of wine color and odor is often a problem with
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
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Revised: Spring 2003
carbon because of the low selectivity and great care has to be taken with its use. Carbon also
contains a large quantity of air, and oxidation sometimes follows carbon addition if the carbon is not
quickly and thoroughly removed. The addition of carbon to juice, rather than wine, helps to
diminish carbon induced oxidation.
Co-fining agents: Compounds like silica oxides, tannins and gelatin are sometimes used in addition
to proteinaceaous fining agents to either improve their performance or, as more often the case, to
facilitate the removal of the fining agents from the wine. Co-fining agents are used mainly in white
juices and wine. Tannin is used with proteinaceous fining agents, especially gelatin in white wines,
to aid in the flocculation and precipitation of the fining agent. Silica oxides and gelatin are
sometimes used together in a 7:1 ratio where the silica oxide is used as an aid in clarification of
white wines and as a substitute for tannin in facilitating the settling of solids. Silica oxide is usually
not necessary when fining red wines due to the naturally high level of tannin that they possess.
Proteinaceaous fining agents should be added first and fining trials must be done to assure proper
settling.
 Kieselsol: Kieselsol is a proprietary name for aqueous suspensions of silicon dioxide. The
primary use of Kieselsol is for clarification and as a replacement for tannin during gelatin
fining of white wines because some winemakers do not want to add any tannin to their white
wine. Kieselsols are negatively charged and electrostatically bind to and adsorb positively
charged proteins and initiate flocculation and settling. Several different Kieselsol
formulations are available at a variety of pH levels.
Fining Trials:
Fining trials are commonly conducted to determine which fining agent and concentration has the
desired effect upon the wine that is to be treated with minimum impact on flavor and aromas. Small
samples of the target wine are treated with different agents at different concentrations and then
examined and compared for clarity, aroma and taste. The fining agents used are selected by the
winemaker based upon the type of problem(s) being experienced with the wine and the known
efficacy of the agent for the problem(s). When trying to determine what would be the best fining
agent(s) to be used on a particular wine, the winemaker must keep in mind:
 The smallest quantity of agent necessary should be used.
 Use pure fining agents free of off-odors and flavors.
 Contact time should be limited to that necessary for complete reaction.
 Lab trials should simulate as closely as possible the parameters to be used in the cellar, i.e.
the agents should be prepared by the same method, and the temperature of the wine should be
the same.
 The fining agent must be completely dehydrated and mixed thoroughly into the sample.
 The wine should be low in dissolved CO2. Gas will impede settling of the agent.
 Lower pH wines require less fining agents than higher pH wines, due to charges.
 Young wines are more forgiving than older wines to the actions of the presence of greater
numbers/concentration of polymerized compounds.
 Be aware of the levels of use approved by the ATF before additions to the wines in the cellar.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 55 of 63
Revised: Spring 2003
MATERIALS
For each fining agent to be tested, the following items will be required
 Stock solution of fining agent
If filtering Wine Sample
 4 – 100ml cylinder
● Syringe with tubing on end
 Wine (600ml)
● Swinnex cartridge
 4 – wine glasses
● Filters ranging in size from 2.0µm
 Plastic food wrap
to 0.45µm
 Centrifuge
● 250 ml beaker
 Centrifuge sample containers
 Pasteur pipette
 Labels (or masking tape)
METHOD
General Methodology:
1. Prepare a stock solution of the fining agent. Instructions for mixing different stock solutions are
provided in the following section of this procedure.
2. Mix the stock solution well.
3. Using a pipette, add appropriate volumes of the stock solution to each measuring cylinder to
create samples with a range of fining agents and concentrations.
a. Group the cylinders for each fining agent together on the lab bench.
b. For each fining agent group, the cylinder furthest to your left should be the control sample (0
PPM of the agent) then add the appropriate amount of the agent so that the cylinders to the
right of the control are in increasing concentration.
c. Fill the pipette to the “0” mark and release the required volume of fining agent into the
cylinders.
4. Measure 100ml of wine into each of four (4) – 100ml measuring cylinders for every fining agent
tested.
5. Cover and invert the cylinder several times to thoroughly mix the fining agent with the wine.
6. Allow time for the fining agent to react, normally less than five minutes
7. Either cover the cylinders with plastic food wrap and allow precipitate to settle out over night,
centrifuge for 10 minutes at 4,000 rpm, or filter the samples.
8. If you choose to centrifuge the samples:
a. Carefully decant equal volumes of wine/fining agent from a single 100ml cylinder into two
centrifuge sample containers. The volume of liquid must be identical in each container, if
necessary use a Pasteur pipette to equalize the volumes. Repeat for all samples.
b. Cap the centrifuge sample containers and add labels of the same size/weight to each
container; clearly identifying its contents.
c. Place the two cylinders for each sample in the centrifuge so that they are directly opposite
one another to ensure proper balance when the samples are spun.
d. Run the centrifuge at 4000rpm for 10 minutes.
e. Remove all samples from the centrifuge.
f. Decant the wine carefully into labeled wine glasses.
9. If you choose to filter, for each sample:
a. Remove the wine from the cylinder using a syringe with tubing on the end, retaining the
liquid in the body of the syringe.
b. Attach a Swinnex cartridge with a 2µm filter to the end of the syringe.
c. Press the syringe’s plunger to push the wine through the filter into a 250ml beaker.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
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Revised: Spring 2003
d. Repeat the above steps with progressively finer filters finishing with a 0.45µm filter.
e. Pour samples into labeled wine glasses.
10. If wines are settled overnight, the following morning decant the wine carefully into wine glasses.
11. Carry out a blind sensory evaluation of clarity, aroma and flavor.
12. If the smallest concentration of fining agent tried produces satisfactory clarity, repeat above
process with smaller concentrations of the agent.
13. Determine the appropriate quantity of the fining agent (i.e. balance between adequate removal of
the problem without excessive stripping of positive attributes including taste and aroma)
14. Compare the selected samples for each fining agent with each other to determine the one that has
the best clarity with the least effect on the wines positive attributes.
PREPARING STOCK SOLUTIONS OF FINING AGENTS
The following table shows stock solutions and suggested concentration ranges for fining trials.
These are guidelines and may be adjusted based upon what works with the tested wine.
Fining Agent
Egg White (1%)
Gelatin (1%)
Isinglass (0.5%)
PVPP (1%)
Sparkalloid/Klearmor (1%)
Potassium Caseinate (1%)
Carbon (1%)
Treatment Range
(mg/liter)
60 – 600
Preparation Notes
1 egg white/59 gallon barrel is
approximately 120 mg/L
Whites: 15-120
Reds:
30 – 300
Whites: 10-100
Reds:
30 – 150
30 - 240
100-1000
50-250
Odor: 15 – 60
Color: 100-200
Procedures for Specific Fining Agents
Equipment
 100 ml volumetric flask for each fining agent stock solution prepared
 500 ml beaker (for egg white stock solution)
 Weigh scale
 Deionized water
 Magnetic stirrer with stirring bar
Egg Whites
1. Determine concentrations of egg whites to be tested. The following table contains suggested
concentrations for the first trial.
Egg White Trial (Stock Solution Concentration 1%)
Final Volume (ml)
PPM of Fining Agent
Mls Stock Solution Required
of Wine
Being Assessed
100
0
0
60
0.6
120
1.2
240
2.4
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 57 of 63
Revised: Spring 2003
2. First make a 10% solution as follows: Carefully separate one egg white into a 500ml beaker,
weigh it, and add deionized water adjusted to pH 7 along with 0.5% potassium chloride (to
help dissolve it). For example, a 30 gram egg white would require 270 ml deionized water
with 15 g KCl. Stir gently until dissolved, being careful not to denature the albumin by
stirring too vigorously. Finally, in dilute this to a 1% stock solution by mixing 10 ml of the
mixture with 90 ml deionized water in a 100ml volumetric flask. The solution should be
made fresh daily.
Gelatin
1. Determine concentrations of gelatin to be tested. The following table contains suggested
concentrations for the first trial.
Gelatin Trial (Stock Solution Concentration 1%)
Final Volume (ml)
PPM of Fining Agent
Mls Stock Solution Required
of Wine
Being Assessed
100
0
0
30
0.3
60
0.6
120
1.2
2. To prepare a 1 % stock solution of gelatin, use a liquid gelatin prepared especially for
winemaking. Prepare the solution by noting the manufacturer’s figure for gelatin activity
(e.g. 25%) and diluting with the appropriate volume of deionized water.
If using powdered gelatin, add 1 gram of powder made up to 100 ml with deionized water in
a 100ml volumetric flask. Stir gently while heating (but do not exceed 40ºC, to avoid
denaturing the proteins). This solution should be made fresh every few days. Conduct trials
as described above, using the table for the first trial. Note that Kieselsol is often used in
conjunction with the gelatin fining of white wines, to help precipitate the gelatin.
Isinglass
1. Determine concentrations of isinglass to be tested. The following table contains suggested
concentrations for the first trial.
Isinglass Trial (Stock Solution Concentration 0.5%)
Final Volume (ml)
PPM of Fining Agent
Mls Stock Solution Required
of Wine
Being Assessed
100
0
0
15
0.3
30
0.6
60
1.2
2. Isinglass is available in sheet or flocculated (powdered) form. The flocculated form is by far
the easiest to work with. It is first made into a 0.5% stock solution by adding 0.5 grams to
100ml cold deionized water and stirring gently until completely dispersed.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
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Revised: Spring 2003
PVPP/ (Polyvinylpolypyrrolidone)
1. Determine concentrations of PVPP to be tested. The following table contains suggested
concentrations for the first trial.
PVPP Trial (Stock Solution Concentration 1%)
Final Volume (ml)
PPM of Fining Agent
Mls Stock Solution Required
of Wine
Being Assessed
100
0
0
30
0.3
60
0.6
120
1.2
2. To make a 1% stock solution of PVPP, add 1 gram of PVPP made up to 100ml with
deionized water in a 100ml volumetric flask. Stir to form thoroughly mixed slurry.
Sparkalloid/Klearmor
1. Determine concentrations of Sparkalloid/Klearmor to be tested. The following table contains
suggested concentrations for the first trial.
Sparkolloid / Klear-mor Trial (Stock Solution Concentration 1%)
Final Volume (ml)
PPM of Fining Agent
Mls Stock Solution Required
of Wine
Being Assessed
100
0
0
60
0.6
120
1.2
240
2.4
2. To make a 1% stock solution of Sparkalloid or Klearmor, stir 1 gram of powder into 100ml
of boiling water, and continue boiling and stirring for 20 minutes. The trials are conducted
with the hot solution, without allowing it to cool.
Potassium Caseinate
1. Determine concentrations of Potassium Caseinate to be tested. The following table contains
suggested concentrations for the first trial.
Potassium Caseinate Trial (Stock Solution Concentration 1%)
Final Volume (ml)
PPM of Fining Agent
Mls Stock Solution Required
of Wine
Being Assessed
100
0
0
20
0.2
40
0.4
80
0.8
2. Potassium caseinate is soluble in water. To make a 1% stock solution of potassium caseinate,
stir 1 gram of potassium caseinate in 100ml of deionized water. Warm the solution but do
not exceed 40ºC. Stirring may be required for several hours (up to 24) to completely mix the
powder into solution. This solution should be used within a day or two.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 59 of 63
Revised: Spring 2003
If only casein is available, the preparation needs to be alkaline. Stir 1 gram of casein in
100ml of deionized water, which has been adjusted to about pH 8 by the addition of
potassium carbonate.
Carbon
1. Determine concentrations of carbon to be tested. The following table contains suggested
concentrations for the first trial.
Carbon Trial (Stock Solution Concentration 1%)
Final Volume (ml)
PPM of Fining Agent
of Wine
Being Assessed
100
0
30
60
120
Mls Stock Solution Required
0
0.3
0.6
1.2
2. To make a 1% stock solution of carbon, add 1 gram of carbon made up to 100ml with
deionized water in a 100ml volumetric flask. Stir to prepare thoroughly mixed slurry.
Co-fining Agents
1. The following table shows suggested concentrations and treatment ranges for co-fining
agents used with proteinaceous fining agents.
Co-Fining Agent
Tannin (1%)
Gelatin
Kieselsol (15 or 30%)
Treatment Range
40 – 200 mg/L
40-200 mg/L
40-300 mg/L
2. To make a 1% stock solution of tannin, add 1 gram of tannin made up to 100ml with
deionized water in a 100ml volumetric flask and mix. The solution should be prepared fresh.
3. Kieselsol in normally supplied as a 15-30% SiO2 solution.
Calculating the Required Addition of Selected Fining Agent to Wine
General formula for calculating required volume:
Formula
Example:
Volume of
Required rate
Volume of wine (ml)
stock solution = of addition
X Conc. of stock solution of fining agent
(ml)
(mg/L)
(mg/L)
The volume (ml) of a 0.4% stock solution that must be added to a 100ml sample of wine
to achieve a desired concentration 200 mg/L of the tested fining agent is:
(Note: 0.4% solution = 0.4 grams in 100 ml = 4,000 milligrams in 1 liter)
= 200 mg/L
X (100 ml ÷ 4000 mg/L)
= 5 ml
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 60 of 63
Revised: Spring 2003
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Tartrate Stability
Developed by: Barry Jan & Rick Smith
Date: December 1997
Introduction
Wine is a supersaturated solution of potassium bitartrate (KHT). Therefore, under certain
conditions, especially at low temperatures, crystals of KHT will precipitate out. While having these
crystals on the bottom of the bottled wines might not be objectionable for home winemakers, it is
probably not desirable for commercial wines because of consumer resistance. In the case of
sparkling wines, it is imperative that no crystals be formed because it is at these points where carbon
dioxide will be released when the bottle is opened. This increased amount of released carbon
dioxide will cause gushing of the wine out of the bottle, which is highly undesirable. The cold
stability of the wine, based on the non-formation of crystals under normal storage conditions, can be
determined by the five methods below:
1. Conductivity Test. Roger Boulton of UC Davis designed this test. The test measures the
electrical conductivity of a wine sample before and after the addition of KHT. A change of
less than 5% during the test indicates a stable wine. This test is probably the most practical
one for small wineries in terms of equipment and accuracy. A photograph of the equipment
is shown in Appendix 1.
2. Concentration Product Test. This test takes into account the concentration of the
potassium, calcium, total tartrates, pH and alcohol. The potassium and calcium contents are
determined by either Atomic Absorption Spectrophotometry or Flame Photometry. The
estimated tartrates are obtained on a table based on the pH and alcohol of the wine. All these
values are plugged into a formula to determine the CP and compared to the value which is
considered stable for that particular wine type.
3. Metavanadate Spectrometric Analysis. The metavanadate method can be used to
determine the amount of tartaric acid. This method involves a colorimetric reaction between
sodium (or ammonium) metavanadate and tartaric acid in acetic acid solution. One system
uses activated carbon to decolorize the wine samples prior to the reaction with metavanadate.
Another system is to subject the wine sample to an acetate-charged resin. Both systems use
high-performance liquid chromatographic technique (HPLC) to determine results.
4. Refrigerator Test. This test subjects a wine sample to 2 to 3°C for 4 days. Absence of
crystal indicates stability.
5. Freezer Test. This test is similar to the Refrigerator Test except that temperature and time
are changed to –10 to –20°C and overnight, respectively.
The conductivity test involves the use of a cooling bath and an electrical conductivity meter. It is
recommended for wineries because of its simplicity and reliability. The Concentration Product and
Metavanadate Tests appear to be more accurate than the other three tests; however, they require
more expensive equipment such as Atomic Absorption, spectrograph, and HPLC. The Refrigerator
and Freezer Tests are simple tests and the results are rather subjective. They depend on the opinion
of the tester on what is considered to be acceptable for a particular storage condition. These two
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 61 of 63
Revised: Spring 2003
tests should be limited to home winemakers or small wineries where availability of equipment might
be a problem. The procedures for running them are given in this document.
Preparation and Interferences
Finely ground KHT must be used in these tests. If the crystals are coarse, they can be prepared by
grinding the KHT in a mortar and pestle. Some wineries run the stability tests without seeding the
wine with finely powdered KHT; however, the seeds provide nuclei for immediate crystal growth.
The size and growth of the crystal should be more pronounced by seeding.
Procedure: Refrigerator Test
1. Place approximately 90 mL of a clear (but not filtered) sample of wine separately into each
of two 100 mL clear glass screw cap bottles.
2. Add about 5mg of finely ground KHT to each bottle. Store one of the bottles in a
refrigerator, set at 2 to 3C, for 4 days and leave the other bottle at room temperature.
3. Observe both bottles for the presence of crystals. “Stability” is indicated if the refrigerated
sample does not show an appreciable increase in the amount of crystals compared to the
control.
Procedure: Freezer Test
1. Place approximately 90 mL of a clear (but not filtered) sample of wine separately into each
of two 100 mL clear glass screw cap bottles.
2. Add about 5mg of finely ground KHT to each bottle. Store one of the bottles in the freezer
compartment of a refrigerator, set at –10 to –20°C, overnight and leave the other bottle at
room temperature.
3. Remove the sample from the freezer and allow ice crystals to thaw. KHT crystals will not
thaw or dissolve back into solution during the short period of this test.
4. Observe both bottles for the presence of crystals. “Stability” is indicated if the thawed
sample does not show an appreciable increase in the amount of KHT crystals compared to
the control.
Calculations and Units of Reporting
Report any increase of KHT crystals over that of the control sample.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 62 of 63
Revised: Spring 2003
Napa Valley College, Viticulture and Enology Department
Standard Operating Procedure
Dissolved Oxygen by Orion 820 Dissolved Oxygen Meter
Developed by: Mark Bunter
Date: December 1997
Introduction
Controlling the uptake of oxygen is critical in winemaking. The molecular oxygen in air (21%) O2)
dissolves readily into juice or wine, reaching saturation at 6 to 8 mg/L (ppm). Dissolved O2 reacts
with many components in wine, combining with them to form various beneficial or unfavorable
compounds until used up.
A wine’s ability to consume oxygen is directly related to its polyphenol content. Accordingly a red
wine can consume more O2 without harm than can a white. Generally, aeration of whites should be
minimized. One of the benefits of normal levels of oxygen in red wine is increased polymerization
of phenols, reducing astringency and stabilizing pigments.
In normal practice O2 levels are measured after or during movements which expose wine to air, such
as racking, transport, filtration, and bottling. Exposure is minimized through the use of nitrogen,
argon, or carbon dioxide gas to displace air in hoses and storage vessels. One year’s aging in full 59
gallon barrels results in an O2 pickup of 3 to 7 ppm, in partially full ones up to 40 ppm. Industry
accepted levels prior to bottling are .5 and 1.5 ppm. Depending upon how careful one is, home
winemakers can expect bottling O2 pickups of .4 to 2 ppm. The dissolved oxygen probe is a twoelectrode system separated from the sample by an oxygen permeable membrane. Current flow
between the electrodes is proportional to the amount of oxygen in the sample.
The dissolved oxygen probe is a tow-electrode system separated from the sample by an oxygen
permeable membrane. Current flow between the electrodes is proportional to the amount of oxygen
in the sample.
Note: It takes 30 to 50 minutes after switching the unit on and connecting the probe for the
electrodes to polarize. Once polarized, they remain so as long as the probe is connected to the meter,
whether the unit is switched on or not. This is a battery powered, portable meter (no electrical plug).
Preparation
Collecting a sample without aerating is imperative. Immerse sample bottle, or use sample hose,
flushing bottle with four volumes of sample with hose or tubing immersed. Fill the bottle
completely and close it tightly. Measure oxygen immediately upon opening.
Procedure
The meter should be calibrated before use or daily if used frequently. There is a bulky plastic sleeve
that fits over the electrode. This is the calibration sleeve. It can also be used as a protective sleeve
when analyzing tanks directly.
VWT 172: Laboratory Analysis of Musts and Wines
Standard Operating Procedures
Page 63 of 63
Revised: Spring 2003
Calibration
1. Saturate the sponge in the calibration sleeve and gently insert the probe as far as it will go.
2. Switch on the meter.
3. Depress and hold the Mode Key Pad until the display cursor is at Cal. As long as the Mode
Key Pad is depressed, the display will cycle continuously between Cal, %, mg/L, and degrees
C°.
4. Depress quickly and release the Mode Key Pad. The display will show three dashed (---)
followed by the slope of the membrane/electrode (good slope is 0.7 to 1.2).
5. Remove the Calibration sleeve. The probe is now ready to use.
Measurement
1. Depress the Mode Key Pad to choose mg/L or %.
2. Immerse the probe in sample, making sure the stainless steel thermostat is submerged. Stir
the sample or slowly move the probe through the sample. Take a reading when the value is
stable.