FDA Manual of Compliance Policy Guidelines

Standards for the Identification of Acetic Acid in Vinegar

Journal Editor: Charlotte Horn

Keywords: Vinegar, FDA, titrations, validation, reference materials, replicate analyses, statistical
analysis

Introduction
Vinegar is a liquid made by the fermentation of any dilute alcoholic liquids into a solution that contains
acetic acid.1 Vinegar can be made from many different types of food, such as apples, grapes, grains, and
potatoes. 2 The percentage of acetic acid in vinegar can vary depending on the type of vinegar. 2 However,
the average acetic acid content of vinegar ranges from 4-8%. 2 The FDA helps to monitor the food supply
for chemical contaminants and takes action when a contaminant causes a portion of food to be unsafe. 3
The FDA does not have any established procedures to identify vinegar, but instead, definitions have been
developed for different types of vinegar.4 The FDA states that natural vinegar must contain 4 grams ±.05
of acetic acid per 100mL(0.666M).4 The FDA states that when vinegar is diluted with water, the label
must state “diluted with water to ____ percent acid strength”, with the blank filled with the actual percent
of acetic acids- in no case should it be less than 4% percent.4

In this paper, we will develop a standard operating procedure (SOP), to describe FDA- level set of
procedures to test vinegar samples for the amount of acetic acid contained within. 5 The SOP created
explains the hazards as well as the general steps of an experimental process. 6 SOPs should thoroughly
describe the hazards and safety protocols associated with the chemicals involved. 1 Our SOP meets a
number of FDA guidelines for creating high-quality methodology.6
Three of these guidelines are as follows:
1. Reference materials and certified reference materials: The use of known reference materials (a
substance whose property of interest is known) should be incorporated to assess the accuracy or
bias of the method.
2. Replicate Analyses: The precision of the analytical process can be evaluated using replicate
analyses. The originating laboratory should ensure that adequate sample replicates are performed
and that results from replicate measurements of each analyte are compared. Minimally, the
method repeatability should be evaluated.
3. Statistics: Statistical techniques are employed to evaluate accuracy and measurement uncertainty.
Through our laboratory, we have identified that the FDA has failed to regulate the concentration of acetic
acid in vinegar. We suggest that the FDA should follow our outlined SOP to regulate the concentrations
of acetic acid (CH3COOH). Through our laboratory, we have learned that the FDA has allowed vinegar
with various concentrations of acetic acid to be sold to the consumer.

Results
Table 1 shows the Concentrations of [HCl] calculated in the titration of NaOH
Table 1. Concentrations of [HCl] calculated from moles of HCl and Volume of HCl
Trials Volume of Volume of Concentration Moles of Moles of HCl Concentration
HCl Pipetted NaOH Used to of NaOH (M) NaOH (mol) (mol) of HCl (M)
(L) Titrate (L)

1 0.01 0.0081 0.560 0.00454 0.00454 0.454

2 0.01 0.0085 0.560 0.00476 0.00476 0.476

3 0.01 0.0086 0.560 0.00482 0.00482 0.482

A titration was performed with NaOH and HCl to confirm the concentrations of our “standard” of HCl.
The standard HCl was projected to be ~0.5M. The average concentration of the HCl solution was found to
be 0.471M (SD 0.015). Refer to Appendix A for calculations for the average concentration and standard
deviation of HCl. Each of our found concentrations was close in comparison. The greatest difference
between our three trials was between the first and last trials (difference in 0.028M).
The concentration of HCl of the solution was found using the equation:

moles (mol)
Concentration=
Volume ( L)
1 Moles of HCl (mol)
Concentration= ×
Volume of HCl pipetted (L) 1

We can use this equation because through stoichiometry we know that the moles of NaOH and moles of
HCl have a 1:1 ratio.7

A two-sample t-test was performed to compare our found concentration to the known concentration of
HCl. There was a significant difference in our found concentration (M=0.471M, SD =0.015 and the
known concentration (0.591M); (t(2)= 2.920, p>0.05). We found our confidence interval = 0.471±0.025.

Table 2 shows the Concentrations of Un-Diluted Vinegar calculated from moles of CH 3COOH and the
Volume of Vinegar pipetted

Table 2. Concentrations of CH3COOH in Un-Diluted Vinegar

Trials Volume of Volume of Concentratio Moles of Moles of Concentration Concentration
Diluted NaOH n of NaOH NaOH CH3COOH of Diluted of Un-Diluted
Vinegar Used to (M) (mol) (mol) Vinegar (M) Vinegar (M)
Pipetted (L) Titrate (L)

1 0.01 0.0004 0.112 0.000044 0.0000448 0.00448 0.0448

8
2 0.01 0.00069 0.112 0.000077 0.00007728 0.007728 0.07728
28

3 0.01 0.00041 0.112 0.000045 0.0000459 0.00459 0.0459

9

A second titration was performed to calculate the concentration of CH3COOH contained in vinegar. The
Average concentration of acetic vinegar was found to be 0.0560M (SD 0.0185). Each of our found
concentrations was close in comparison. The greatest difference between our three trials was between the
first and second trials (difference in 0.03248M).

The concentration of HCl of the solution was found using the equation:
moles (mol)
Concentration=
Volume ( L)
1 Moles of CH 3 COOH (mol)
Concentration= ×
Volume of Vinegar pipetted (L) 1
We can use this equation because through stoichiometry we know that the moles of NaOH and moles of
CH3COOH have a 1:1 ratio.7

A two-sample t-test was performed to compare the experimental average of acetic acid in vinegar
(0.0560M, SD 0.0185) to the manufacturer’s reported value (0.666M). Refer to Appendix B for
calculations to convert the known [CH3COOH] from 0.04g/mL to M. The experimental value was
significantly different than the manufacturer’s reported value; t(2)= 2.920, p>0.05. Our confidence
interval = 0.0560± 0.0312M.

Discussion
Through our laboratory we were unable to verify that our HCl concentration was statistically the same as
our known concentration of HCl. Our average concentration of HCl did not fall within a 95% confidence
interval, therefore we can accept our null hypothesis. Thus we are 95% confident our [HCl] is not
statistically the same as our true value. An error may have occurred during our titrations, however, we
had limited amounts of NaOH, so we had to calculate our t-test with our first 3 trials. All three of our
calculated concentrations of HCl were below the known concentrations. Leading us to believe that either
we didn’t add enough NaOH or the known concentration of NaOH(M) was inaccurate. Not adding
enough NaOH would cause our calculated concentrations of HCl to be lower than the known. Similarly,
an inaccurate concentration of NaOH could have altered our final concentration value for HCl.

Finally, we titrated NaOH and vinegar to calculate the concentration of acetic acid in vinegar. This was
done to validate the definition set by the FDA (4.0 g ± 0.05 CH3COOH per 100mLor 0.666M). After we
performed our t-test, we found that we are 95% confident that the true value falls within 0.0.560 ± 0.0312
M. These results indicate that we are 95% confident that our average value of the concentration of acetic
acid (0.0560M) is statistically different from the reported value(0.666M). However, because we were
unable to prove that we can accurately find the concentration of our standard, we cannot conclude that the
concentration of acetic acid in vinegar is inaccurate. Thus, I suggest that we continue to perform titrations
and trials so that we can conclude that the FDA should create standard operating procedures to regulate
vinegar sold to the consumer. This way we can discover that the procedures/scientists are not the issue,
but the FDA-reported value is incorrect/not regulated.

References
(1) Vinegar | Description, Production, Types, Uses, & Facts | Britannica.
https://www.britannica.com/topic/vinegar (accessed 2023-04-25).
(2) admin. What is the Percentage of Acetic Acid in Vinegar?. Infrared for Health.
https://infraredforhealth.com/what-is-the-percentage-of-acetic-acid-in-vinegar/ (accessed 2023-
04-25).
(3) Nutrition, C. for F. S. and A. Food Chemical Safety. FDA.
https://www.fda.gov/food/food-ingredients-packaging/food-chemical-safety (accessed
2023-04-25).
(4) Affairs, O. of R. CPG Sec 562.100 Acetic Acid - Use in Foods - Labeling of Foods in Which
Used. U.S. Food and Drug Administration.
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cpg-sec-562100-ace
tic-acid-use-foods-labeling-foods-which-used (accessed 2023-04-25).
(5) SP23 Titrations Overview Document. Google Docs.
https://docs.google.com/document/d/1Odd9t-Ei-yW8rRIwdPf_Kp7NE8R4WiP7zG_2r2JsTdY/ed
it?usp=sharing&usp=embed_facebook (accessed 2023-04-11).
(6) Neiles, K.Y.; Larsen, R.K.; Mertz, P.S.; Bowers, R.A. SP23 Call for
Papers_Titration Lab. Google Docs.
https://docs.google.com/document/d/1I7RJJUHj2zgxWECWUJbsggKaii2bcN2oq6cMfpnuulg/
edit?usp=sharing&usp=embed_facebook (accessed 2023-04-11).
(7) Bowers, G. Presented in Goodpaster Hall on Strong Acid Titrations, SMCM, April 19, 2023.
(8) Neiles, K. Bunsen Burner SOP
https://docs.google.com/document/d/1Zrk1g2bYmFHdVqCfj01L4s0RXpeVLz1N5kH8L8
C1eao/edit?usp=sharing (accessed 2023-04-26).
(9) PubChem. Sodium Hydroxide. https://pubchem.ncbi.nlm.nih.gov/compound/14798
(accessed 2023-04-26).
(10) Water SDS (Safety Data Sheet) | Flinn Scientific, July 8th, 2015. Flinn Scientific.
Accessed April 27, 2023 https://www.flinnsci.com/sds_845-water/sds_845/
(11) Vinegar 5-6% SDS Specialties, J. JMN Specialties, Inc. Accessed April 27, 2023
JMN Specialties, Inc.
(12) Acetic Acid SDS (Safety Data Sheet) | Flinn Scientific, May 5th, 2009. Flinn Scientific.
Accessed April 27, 2023 https://www.flinnsci.com/sds_845-Acetic Acid/sds_845/
(13) Hydrochloric Acid SDS (Safety Data Sheet) | Flinn Scientific, August 24th, 2009. Flinn
Scientific. Accessed April 27, 2023 https://www.flinnsci.com/sds_845-hydrochloric
acid/sds_845/
Appendix A:
Appendix B:
Appendix C:
Site-Specific Chemical Process SOP
Lab Location: St. Mary's College of Maryland, Goodpaster Hall, Rm 200
Chemical Process Name:
1. Determining the Concentration of a “Standard” HCl
2. Determining the Concentration of Vinegar using NaOH(aq) Standard for the Identification of
CH3COOH in Vinegar
Chemicals Involved:
- NaOH(aq)
- HCl
- CH3COOH
- DI Water
Chemical Process Steps:1
Determining the Concentration of a “Standard” HCl
1. Put on gloves and goggles before proceeding to the next step.
2. Obtain the required equipment volumetric flask, burette, pipette, and storage container.
3. Titrate the HCl with your concentration of NaOH solution
4. Prepare the burette for titration by rinsing the burette with the NaOH solution.
5. Prepare the HCl by measuring out HCl in a volumetric flask. The more HCl you pipette the more
NaOH is needed to titrate.
6. Add 3 drops of your indicator to the mixture.
7. Titrate quickly for the first trial. This is our “scout” trial to ballpark the amount of NaOH needed
to titrate the HCl. We will not include this value in our data.
8. Record the volume of the titrant before beginning the titration.
9. Titrate until 2 mL before the value indicated in the scout titration. Then start adding dropwise.
10. Swirl after each drop to ensure mixing, and go very, very slowly as it is easy to overshoot.
11. Titrate only to a PALE PINK. Repeat the titration for 2-4 trials, or until three “good” trials have
been completed.
12. Once all data has been collected, dispose of each of the titrated solutions and leftover NaOH
solution appropriately as directed by the lab instructor.
13. Calculate the average concentration of the HCl solution along with the standard deviation of this
calculation. Be sure to use a t-test and confidence intervals to prove the comparison. Refer to the
blank calculations below.
14. Ensure that we have a statistically accurate prediction of the known concentration before leaving
the lab.
Calculating the Standard Deviation of the Average
1. Find the average of all trials: n=number of trials
trial1❑+trial 2+trial 3
Avg .=¿
n
 2. Calculate the standard deviation
S . D .=√ ❑
Calculate the Confidence Interval
t−value× S . D .
1. Confidence Interval=avg ±
√❑
Determining the Concentration of Vinegar
1. Put on gloves and goggles, then proceed
2. Prepare the burette for titration by rinsing the burette with NaOH solution
3. Add 3 drops of indicator
4. Titrate the solution with the NaOH solution in the burette
5. Do three good trials of the above steps (as well as the first ballpark sample)
6. Titrate quickly for the first trial. This is our “scout” trial to ballpark the amount of NaOH
is needed to titrate the vinegar. We will not include this value in our data.
7. Record the volume of the titrant before beginning the titration.
8. Titrate until 2 mL before the value indicated in the scout titration. Then start adding
dropwise.
9. Swirl after each drop to ensure mixing, and go very, very slowly as it is easy to
overshoot.
10. Calculate the average concentration of the solution using (w/v) and the standard deviation
of the values collected from our three good trials.
11. This will be compared to the FDA-reported value using a t-test with confidence intervals.
Calculating the Standard Deviation of the Average
1. Find the average of all trials: n=number of trials
trial1❑+trial 2+trial 3
Avg .=¿
n

2. Calculate the standard deviation

S . D .=√ ❑
Calculate the Confidence Interval
t−value× S . D .
1. Confidence Interval=avg ±
√❑
Associated Hazards (check all that apply to this specific SOP):

❏ Cryogenic hazards
❏ Compressed gas hazards
❏ Electrical hazard
❏ High temperature
❏ Crush/Pinch hazards
❏ Radiation hazard
❏ Fire/explosion hazard
❏ Other(describe)_____________________
 Hazardous Chemical(s) used in process/experiment/equipment (check all that apply to this
specific SOP):
Hazard Class(es):
❏ Reproductive Toxin
❏ High Acute Toxicity
❏ Carcinogen
❏ Flammable
❏ Corrosive
❏ Oxidizer
❏ Peroxide Former
❏ Other:_______________________________

Other Chemical Precautions Specific to this SOP:

Always store NaOH in a dry container that can be closed tightly. NaOH absorbs water.

Designated Areas:
Process/Experiment/Equipment shall be performed only in the following designated area:

Designated area within the lab: Can be done at student workbenches

Security protocol: Only faculty, laboratory assistants, laboratory coordinator, and facilities personnel have
keys to access the laboratory space. Students should not be in the lab unsupervised.

Training requirements (check all that apply to this specific SOP): The user must demonstrate
competency and familiarity regarding the safe performance of this process. Training should include the
following:
❏ Review of current MSDS
❏ Review of the OSHA Lab Standard
❏ Site-specific training (needed for specific instrumentation)
❏ Lab Safety training
❏ Respirator training

Engineering/Ventilation/Isolation Controls (check all that apply to this specific SOP): To be used to
reduce employees’ exposure to hazardous chemicals and physical hazards.

❏ Chemical Fume Hood

❏ Specialty Hood (describe)
❏ Glove box (zero Oxygen environment)
❏ Vented gas cabinet
❏ Explosion Shield
❏ Other(describe) _______

Personal protective equipment (check all that apply to this specific SOP): All personnel are required
to wear the following personal protective equipment whenever performing this process.
❏ Safety glasses
 ❏ Chemical safety goggles
❏ Face shield
❏ Lab coat
❏ Rubber apron
❏ Tyvek clothing
❏ Respirator type____________________
❏ Gloves type/use: Nitrile
❏ Incidental Contact: _________________
❏ Extended Contact: _______________
❏ Proper lab attire (avoid wearing synthetic clothing)
❏ Other (describe) __________________________________

Emergency Controls Needed Nearby (check all that apply to this specific SOP):
❏ First aid kit
❏ Spill kit
❏ Eyewash/Shower
❏ Other____________

Significant Routes of Chemical Exposures Specific to this SOP:2

Sodium Hydroxide
Skin/eye contact
Symptoms:
Causes eyes to burn. May even
cause blindness, chemical
conjunctivitis, and corneal
damage. Causes skin burns.
Ingestion
Symptoms:
This may cause severe and
permanent damage to the
digestive and gastrointestinal
tract. Causes severe pain,
nausea, vomiting, diarrhea, and
shock.
Inhalation
Symptoms:
May lead to chemical
pneumonitis and pulmonary
edema. Causes severe irritation
to the upper respiratory tract.
Causes coughing, burns,
breathing difficulty, and
possible coma.

First aid: Flush eyes with
plenty of water for at least 15
minutes at the eyewash station.
Get medical help and wash
clothing.
First aid: If swallowed, do not
induce vomiting. Get medical
aid immediately. If conscious,
drink a cupful of water.
First aid: If inhaled, move to
clean air, If not breathing, give
artificial respiration, and if
breathing give oxygen. Seek
medical attention.

CH3COOH
Skin/eye contact
Symptoms:
Contact with skin may
aggravate diseases such as
eczema and contact dermatitis
Ingestion
Symptoms:
Irritant to mucous membranes,
esophagus, and stomach.
Abdominal pain, nausea,
vomiting, and general gasto-
Inhalation
Symptoms:
May lead to lung damage and
aggravate pulmonary
conditions. High
concentrations of vapor may
 intestional upset can be cause irritation of the
expected. respiratory tract.

First aid: Flush eyes with
plenty of water for at least 15
minutes at the eyewash station.
Get medical help and wash
clothing.
First aid: If swallowed, do not
induce vomiting. Get medical
aid immediately. If conscious,
drink a cupful of water.
First aid: If inhaled, move to
clean air, If not breathing, give
artificial respiration, and if
breathing give oxygen. Seek
medical attention.

Emergency shutdown/Evacuation procedures Specific to this SOP:

Properly label all chemicals with scientific names, names of group members, as well as date created

Spills/Decontamination Specific to this SOP:

Spill cleanup:
Vacuum or sweep up material and place into a suitable disposal container. Avoid generating dusty
conditions. Provide ventilation. Do not let chemicals enter the environment. If acidic or basic solutions
are spilled, neutralize the spill as much as possible and absorb the spill. Dispose the waste in an
appropriate manner, if waste cannot be disposed of in normal trash then contact the environmental health
and safety officer for guidance in packaging and labeling cleanup residue.

Waste disposal:
The authorized person using this process is responsible for the safe collection, handling and storage of
chemical waste managed in accordance with all government regulations and SMCM policies and
procedures. Provide any process-specific details. Do not allow material to contaminate groundwater
systems. Do not flush into surface water or the sanitary sewer system. Place any liquid waste into
designated liquid waste disposal beakers.