CHEMICAL WINE ANALYSES

A Step-by-Step Laboratory Manual for Wine & Juice Analyses with Photos,
Using Laboratory Glassware and Other Chemistry Techniques

By:

Aaron Essary

February 2022
Volume II
 TABLE OF CONTENTS
Pg.
Yeast Assimilable Nitrogen (YAN) – FORMOL TITRATION …………………………………………………………. 2

Malolactic Paper Chromatography ………………………………………………………………………………………… 13

Malic Acid ……………………………………………………………………………………………………………………………… 28

Residual Sugar ……………………………………………………………………………………………………………………….. 34

Formula for Potassium Metabisulfite Additions …………………………………………………………………….. 40

Formula for Tartaric Acid Additions ……………………………………………………………………………………….. 41

Link to CHEMICAL WINE ANALYSES – VOLUME I ……………………………………………………………………. 42

Special Thanks ……………………………………………………………………………………………………………………….. 43

About the Author …………………………………………………………………………………………………………………… 44

Contact ………………………………………………………………………………………………………………………………….. 44

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 YEAST ASSIMILABLE NITROGEN (YAN) – FORMOL TITRATION

Yeast Assimilable Nitrogen (YAN) is the combination of Free Amino Nitrogen (FAN),
Ammonia, and Ammonium that are available for wine yeast to consume during fermentation.
The Formol Titration yields an estimation of FAN (Free Amino Nitrogen) and Ammonia in a
grape juice sample. The Formol Titration gives a good estimation of fermentable nitrogen in a
sample but does not yield an exact YAN measurement.

Recommended YAN ranges for grape juice/must:

150mg/L – 350mg/L

Equipment Needed:
• 100mL Beaker
• 125mL Erlenmeyer Flask
• 100mL Graduated Cylinder
• 5mL Wide-Orifice Pipette
• 5mL Pipette
• Pipette Pump
• 25mL Burette
• pH Meter
• Small Sample Beaker with Lid (For Formaldehyde)
• Disposable Dropper(s)
• Ring Stand
• Stand Clamp
• Latex Gloves
• Goggles (For Formaldehyde Fumes)
• Face Mask (For Formaldehyde Fumes)

Reagents Needed:
• 37% Formaldehyde (Formalin) - FORMALDEHYDE IS A CARCINOGEN! USE CAUTION.
• 1N Sodium Hydroxide (NaOH)
• 0.1N Sodium Hydroxide (NaOH)

• Grape Juice Sample (20mL)

• Distilled Water

Calculation:
• (Final burette volume – Initial burette volume) x 35

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 How To Measure Yeast Assimilable Nitrogen (YAN) – FORMOL TITRATION

SAMPLE PREPARATION:
1.) Collect a sample of fresh grapes in a baggie and smash them into a liquid pulp.
2.) Pipette 20mL of clean juice into a 125mL Erlenmeyer Flask
3.) Add 30mL of distilled water to the same Erlenmeyer Flask in order to bring the total sample
volume to 50mL.
- Thoroughly mix the diluted grape juice sample.

4.) Pipette 20mL of the diluted grape juice sample into a 100mL Beaker
- Thoroughly mix the sample.

5.) Place a pH meter into the beaker containing the 20mL diluted juice sample and add drops of
1N Sodium Hydroxide (NaOH) until the pH of the sample reads 8.0
- Please note that the closer the pH climbs to 8.0 the less NaOH is required.
- Thoroughly mix the beaker and continue to add 1N NaOH until the sample pH reads
8.0

FORMOL PREPARATION:
FORMALDEHYDE IS A CARCINOGEN. DO NOT HARM YOURSELF PERFORMING THIS EXPERIMENT.
PLEASE NOTE! Formaldehyde is a CARCINOGEN and a BRONCHIAL IRRITANT! Please use
appropriate PPE and preform this experiment in a well-ventilated area!
PLACE THE LID BACK ONTO ANY VESSEL CONTAINING FORMALDEHYDE WHEN NOT IN USE.
Formaldehyde fumes are dangerous!
6.) Take a small sample beaker and label it “F” for Formaldehyde.
7.) Pour a small amount (approx. 20mL) of Formaldehyde into the small sample beaker. (There
is no specific amount to pour. 20mL – 30mL is fine)
8.) Place a pH meter into the sample of Formaldehyde and add only 1 or 2 drops of 1N Sodium
Hydroxide until the pH reads 8.0 (Literally only 1 or 1.5 drops of 1N NaOH should do)
- Mix the solution until the pH reads around 8.0 (It’s difficult to get exactly 8.0).
- If the Formaldehyde is way over-titrated then toss it out and try again.

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FINAL SAMLE PREPARATION:
9.) Using a clean pipette, transfer 4mL of 8.0pH Formaldehyde solution into the beaker
containing the 8.0pH grape juice solution.
- Mix this new solution thoroughly.
- Place the lid back onto the formaldehyde sample beaker and remove it from work
station.

10.) Affix a 25mL Burette onto a ring stand and fill the burette with 0.1N Sodium Hydroxide
(NaOH).
- Make sure the stopcock is closed. Once filled, open the stopcock to release any
trapped air. Top off burette if desired.

11.) Record the initial burette volume of NaOH.

TITRATION:
12.) Place the beaker containing the grape juice/formaldehyde solution underneath the 25mL
burette.
13.) Place the pH meter into the beaker.
14.) Titrate the solution until the pH reads 8.0
- Thoroughly mix the solution while titrating.
- The closer the pH climbs to 8.0, the less NaOH is required.

15.) Record the final burette volume of NaOH.

16.) CALCULATIONS:
YAN (mg/L) formula below:
(Final burette volume – Initial burette volume) x 35 = YAN (mg/L)
(2.4 – 0.7) x 35 = 60(mg/L) of YAN Using a FORMOL Titration

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Equipment and Reagents:

5
 Step 1.)

Smash grapes into a liquid pulp.

Step 2.)

Squeeze the bag so that juice pools into a corner.

Transfer 20mL of juice into an Erlenmeyer Flask.

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 Step 3.)

Add 30mL of distilled water to the 20mL of juice.

Thoroughly mix the diluted juice sample.

Step 4.)

Pipette 20mL of diluted juice into a 100mL beaker.

Mix thoroughly.

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 Step 5.)

Add drops of 1N NaOH to the diluted juice sample until the pH is 8.0
Thoroughly mix the sample during this step.
The pH will climb faster as it approaches 8.0

Step 6.) PLEASE USE EXTREME CAUTION WHEN HANDLING FORMALDEHYDE

Label a small sample beaker “F” for Formaldehyde.

Please use a beaker with a lid.

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 Step 7.) WARNING FORMALDEHYDE

Pour approximately 20mL of Formaldehyde into the sample beaker.

Immediately cap both lids when finished.

Step 8.) WARNING FORMALDEHYDE

Place a pH meter into the Formaldehyde sample and add only 1 or 2 drops of 1N NaOH until the
pH reads near 8.0, mix sample. Do not over-titrate.

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 Step 9.) WARNING FORMALDEHYDE

Pipette 4mL of 8.0pH Formaldehyde into the beaker containing the 8.0pH grape juice sample.
Once complete, cap the Formaldehyde sample and remove it from the work area.
Mix solution thoroughly.

Step 10.)

Affix a 25mL burette onto a ring stand and fill with 0.1N NaOH.
Make sure the stopcock is closed when filling, open the stopcock to release any trapped air.

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 Step 11.)

Record the initial burette volume (0.7mL shown above)

Step 12.) & Step 13.)

Place the grape juice/formaldehyde solution beaker underneath the burette and place a pH
meter into the solution.

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 Step 14.) TITRATE

Titrate the solution until the pH reads 8.0

Thoroughly mix the solution during this step.

Step 15.)

Record the final burette volume (2.4mL shown above)

Congratulations! Proceed to Step 16.) CALCULATIONS

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 MALOLACTIC PAPER CHROMATOGRAPHY
Paper Chromatography is a qualitative analytical laboratory technique used to separate
and identify different chemicals that are dissolved together in the same solution. Paper
chromatography can be applied to monitor the progress, or completion, of Malolactic
fermentation in wine by separating the Malic acid from any Lactic acid that may be present. The
separated acids are then compared against a standard to see which acids are present in the
wine.
The presence of Lactic acid and the absence of Malic acid indicates that Malolactic
fermentation is complete. The presence of both Malic and Lactic acids indicates that Malolactic
fermentation is still going. The presence of Malic acid and the absence of Lactic acid indicates
that Malolactic fermentation has not even begun.

Recommended wines for Malolactic Fermentation:

At the discretion of the winemaker

Equipment Needed:
• Chromatography Paper
• Chromatography Solvent
• Capillary tubes
• A large jar
• Pencil (DO NOT USE INK)
• Stapler

Reagents Needed:
• 0.3% Malic acid standard
• 0.3% Lactic acid standard
• 0.3% Tartaric acid standard (optional)

• Wine Sample

Calculations:
• Linearly match the positions of the acid standards with the positions of the acids derived
from the wine samples on the chromatogram (photos shown).

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 How To Perform Malolactic Paper Chromatography
NOTE: Please allow for a full 24 hours to complete this test from start to finish.
1.) Draw a line, in PENCIL, one inch from and parallel to the bottom of the chromatography
paper. Make sure the paper is landscape and not portrait.
2.) On the left side of the paper, mark “x” on the line drawn. Each “x” should be one inch apart.
Label each “x” underneath as “M”, “L”, or “T” to represent the Malic, Lactic, and Tartaric acid
standards.
3.) On the right side of the paper, mark “x” on the line drawn for each sample that will be
tested. Each “x” should be at least one inch apart. Label each “x” underneath to represent each
wine sample.
4.) Using a capillary tube, draw up about ¼” of Malic acid standard.
- Use a paper towel to expel some of the acid if too much is drawn up.

5.) Press the capillary tube onto the “x” marked “M” in order to release the malic acid standard
onto the paper.

NOTE: It is important to only use one capillary tube per wine sample/acid standard.

6.) Repeat steps 4 & 5 for the Lactic and Tartaric acid standards, placing each acid standard on
its respective “x”.
- Use a different capillary tube for each standard.

7.) Using a capillary tube, draw up about ¼” of wine sample that will be tested.
- Use a paper towel to expel some wine if too much is drawn up.

8.) Press the capillary tube on the “x” marked for that wine sample.
- Place as many wine samples that will safely fit onto the paper.
- Each sample needs to be at least one inch apart.
- Use a different capillary tube for each sample.

9.) Pour about ¼” – ½” of chromatography solvent into the large jar.

- Do this in a well-ventilated area.

10.) Delicately bend the chromatogram into a cylinder and staple the ends together.
- The edges of the paper should not overlap or be misaligned.
- All pencil markings, acid standards, and wine samples should now be facing outward.

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11.) Place the chromatogram into the solvent with the samples at the bottom and tightly seal
the lid.
- Avoid contact between the paper and the inside walls of the jar.

12.) Let the chromatogram set in the solvent undisturbed for 6+ hours.
- The solvent needs to reach to top of the chromatogram before it can be analyzed.
- The chromatogram can be left overnight if needed.

13.) After 6+ hours, unseal the lid and carefully remove the chromatogram form the jar.

14.) Hang the chromatogram in a well-ventilated area to dry.

- Drying time can take up to 12+ hours.

NOTE: The chromatography solvent can be poured back into the original container and reused.

15.) Once dry, the chromatogram should appear blue-green in color.

16.) Unstaple the chromatogram and lay it flat on a table.

INTERPRETING THE RESULTS:

17.) Acids should appear as yellow blots on the chromatogram.

18.) View the chromatogram and note where each acid standard lies, then move horizontally
across the paper to see if any wine samples produced the same acid. Any yellow blots that are
in the same linear horizontal plane are matching acids.

19.) (In the photos provided) Malic acid is underlined and Lactic acid is circled.
- Wine samples 2, 3, & 4, all have lactic acid but no malic acid.
- Wine sample 1 has malic acid with no to very little lactic acid.
- All wine samples have tartaric acid present.

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Equipment & Reagents:

16
 Step 1.)

Use a straight edge to draw a parallel line one inch from the bottom of the paper.
Make sure the paper is landscape and not portrait.

Step 2.)

Mark “x”s on the left side of the line and label them “M”, “L”, & “T” (T is optional).
Space them one inch apart.

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 Step 3.)

On the right side of the paper mark “x”s for each wine samples that will be tested and label
them accordingly. Spacing should be one inch apart.
Shown above is chromatography paper marked for four wine samples and three acid standards.

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 Step 4.)

Use a capillary tube to draw up about ¼” of Malic acid standard.

Use a paper towel to expel some acid if too much is draw up.

Step 5.)

Press the capillary tube onto the “x” to release the acid standard.

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 Step 6.)

Repeat the process for the Lactic & Tartaric acid standards. (Tartaric is optional)

Step 7.)

Draw up about ¼” of each wine sample that will be tested. Use a paper towel to expel any
excess wine.

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 Step 8.)

Place the capillary tube onto the “x” to expel the sample.
Place as many samples that will safely fit onto the paper. (Four wine samples are shown above)

Step 9.)

Pour enough chromatography solvent into the large jar to fill ¼” – ½”

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 Step 10.)

Bend the chromatogram and staple the ends together flush.

Do not overlap the ends. Acid standards and wine samples should be facing outwards.

Step 11.)

Place the chromatogram into the jar of solvent and tightly seal the lid.

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 Step 12.)

Leave the chromatogram undisturbed for 6+ hours. It’s okay to leave it overnight if needed.

Step 13.)

After 6+ hours, remove the chromatogram from the jar.

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 Step 14.)

Hang the chromatogram in a well-ventilated area to dry.

Step 15.)

Once dry, the chromatogram should appear blue-green in color.

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 Step 16.) & Step 17.) RESULTS

Unstaple the chromatogram and lay it flat on a table.

The acid standards and any acids derived from the wine samples should appear as yellow blots.

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 Step 18.) & Step 19.) RESULTS

Notice where the acid standards lie. Linearly match the acid standards to any acids derived
from the wine sample. Malic acid is underlined and Lactic acid is circled in the photos above.

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 Step 18.) & Step 19.) Continued

Changing the angle of the chromatogram will help to view the acids

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 MALIC ACID
Malic Acid is one of the main organic acids found in grape juice and wine. Malic acid is
developed in grapes in the vineyard and is gradually decreasing as the grapes ripen. Malic acid
is required for malolactic fermentation to occur. Low to zero amounts of malic acid in grape
juice or wine will result in low to zero lactic acid production during malolactic fermentation. The
test shown here is an enzymatic-color test for the estimation of malic acid in grape juice or
wine.

Note: This test maxes out at 500mg/L and is mostly used to monitor the completion of MLF.

Recommended ranges for Malic Acid at harvest:

2.0g/L – 4.0g/L; Depending upon the stylistic direction of the wine

Equipment Needed:
• Malic Acid Test Kit
• Wine Sample

Calculations:
• Use the color chart provided with the test kit to determine Malic Acid levels in mg/L
(milligrams per liter).

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 How To Measure Malic Acid

1.) Observe the test strips and notice that one side is padded and one side has a circular dot.

2.) Take the dropper provided and draw up enough wine to fill the tube. No need to fill the bulb
as this is used to catch any overflow.

3.) Press the dropper firmly onto the PADDED side of the test strip and expel the wine sample.

4.) Wait 5 minutes.

5.) After 5 minutes, flip the strip over and match the color of the circular dot to the color chart
provided. The matching colors with reveal the amount of Malic Acid present in mg/L.

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 Equipment:

Malic Acid Test Kit

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 Step 1.)

Notice the square pad or circular dot on each side of the test strip.

Step 2.)

Draw up enough wine sample to fill the tube of the dropper. The bulb of the dropper is
designed to catch any overflow so there is no need to fill the bulb.

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 Step 3.)

Press the dropper firmly onto the padded side of the test strip and expel the wine sample.

Step 4.)
WAIT 5 MINUTES

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 Step 5.)

Match the color of the circular dot to the color chart to reveal malic acid in mg/L.

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 RESIDUAL SUGAR
Residual Sugar (RS) can appear in wine a number of different ways. One way is by
incomplete fermentation, another way is by intentionally halting fermentation early before
dryness is achieved, and a more controversially way is by adding sugar back into wine later on.
Glucose and Fructose are 6-Carbon (hexose) sugars that are the two main sugars found in wine.
Glucose is the preferred consumable sugar by Saccharomyces yeasts; while Fructose tastes
twice as sweet as Glucose to humans. Other sugars that can be found in wine, often at
negligible amounts, include 5-Carbon unfermentable sugars (pentoses), Galactose, and
Cellobiose.

Recommended Residual Sugar ranges for wine:

None

Equipment Needed:
• Reducing Substances Tables
• Color Chart
• Dropper
• Test Tube

• Wine Sample

Calculations:
• Use the color chart provided.
• Please note that RS is measured in g/dL (grams per Deciliter)
• Example: 1% RS = 1g/dL RS

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 How To Measure Residual Sugar
Note: Color charts usually contain instructions on how to test for RS. This manual will cover
testing procedures instructed by the color chart using both white and red wines.
1.) Draw up a wine sample with a dropper and drop precisely 10 drops of wine into a test tube.
2.) Drop one Reducing Substances tablet into the test tube.
3.) Wait 60 seconds.
- Gently agitate the test tube at this time

4.) Match the color of the solution in the test tube to a color on the color chart to reveal
percentage of Residual Sugar in g/dL (grams per Deciliter).

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Equipment:

36
 Step 1.)
WHITE WINE RED WINE

Drop precisely 10 drops of wine into a test tube.

Step 2.)
WHITE WINE RED WINE

Drop one Reducing Substances tablet into the test tube.

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 Step 3.)
WHITE WINE RED WINE

Gently agitate the test tube and wait 60 seconds.

Step 4.)
WHITE WINE RED WINE

Match the color of the solution to the color on the chart.

Percentage residual sugar is revealed in grams per deciliter (g/dL).

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39
 FORMULA FOR POTASSIUM METABISULFITE (KMBS) ADDITIONS
Potassium Metabisulfite (KMBS) is a preservative that is added to wine that inhibits the
growth of microbial organisms while also protecting the wine against oxidation. Potassium
metabisulfite is only useful in the free molecular form. Once KMBS binds, it no longer protects
against microbes or oxidation.

Formula:
(Desired ppm to add x Volume in Liters) / 0.57 = Grams of KMBS
Example:
(50ppm x 225 Liters) / 0.57
(0.05 x 225) / 0.57
11.25 / 0.57 = 19.7 grams of KMBS

NOTE: The decimal point has to be moved three places to the left in order to convert ppm into
a numerical value.
50ppm = 0.05
33ppm = 0.033
20ppm = 0.02

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 FORMULA FOR TARTARIC ACID ADDITIONS
Tartaric acid is naturally found in grapes and many other fruits. Tartaric acid is the most
commonly used acid to acidulate grape juice, must, or wine. It takes one gram of tartaric acid
dissolved in one liter of wine to lower the pH by 0.1 units.

Formula:
((Initial pH – Desired pH) x 10) x Volume in Liters = Grams of Tartaric Acid
Example:
((3.9 pH - 3.7 pH) x 10) x 2,300 Liters
((3.9 – 3.7) x 10) x 2,300
(0.2 x 10) x 2,300
2 x 2,300 = 4,600 grams of Tartaric Acid

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 LINK TO CHEMICAL WINE ANALYSES – VOLUME I
To purchase CHEMICAL WINE ANALYSES – VOLUME I please follow the link below or scan the
QR code:
CHEMICAL WINE ANALYSES (lulu.com)

https://www.lulu.com/en/us/shop/aaron-essary/chemical-wine-analyses/ebook/product-
zvz568.html?page=1&pageSize=4

CHEMICAL WINE ANALYSES – VOLUME I is a step-by-step laboratory manual with photos for
setting-up and running routine wine tests.
Content covered in Volume I include:
Potential Hydrogen (pH)
Titratable Acidity (TA)
Free Sulfur Dioxide (FSO2)
Bound Sulfur Dioxide (BSO2)
Total Sulfur Dioxide (TSO2)
Volatile Acidity (VA)
Alcohol Percentage (alc. %)

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 SPECIAL THANKS

Dr. Andreea Botezatu

Square Cloud Winery – Gunter, Tx

Inwood Estates Vineyards – Dallas, Tx / Fredericksburg, Tx

The Vintner’s Vault – Hye, Tx

REFERENCES
Nitrogen: Estimate of FAN by Formol Titration
Gump, B.H., Zoecklein, B.W. & Fugelsang, K.C. (2001). Prediction of prefermentation nutritional
status of grape juice.

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 ABOUT THE AUTHOR

Aaron was born in Tyler, Texas in 1992.

He was raised in the small town of Van, Texas and graduated from Van High School.
He attended Stephen F. Austin State University in Nacogdoches, Texas and received a
Bachelor’s of Science Degree in Plant Biology.
He later attended Texas A&M University in College Station, Texas to study Oenology and
received a Master’s of Science Degree in Horticulture.
Aaron has also completed a Certificate of Viticulture and a Certificate of Winemaking both from
Texas Tech University – Fredericksburg.
Aaron currently resides in Fredericksburg, Texas.

Contact: essarycellars@gmail.com

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