ETHANOL IDENTITY

TEST METHODS

September 2017
Prepared by the American Herbal Products Association

This document is the property of the American Herbal Products Association (AHPA) and is for AHPA purposes
only. Unless given prior approval from AHPA, it shall not be reproduced, circulated, or quoted, in whole or in
part, outside of AHPA, its Committees, and its members. Cite as: American Herbal Products Association. August
2017. Ethanol Identity Test Methods. AHPA: Silver Spring, MD.
Ethanol Identity Test Methods

Introduction and Statement of Purpose

Dietary supplement operations are required to comply with title 21 of the Code of Federal Regulations
Part 111 (Current Good Manufacturing Practice in Manufacturing, Packaging, Labeling, or Holding
Operations for Dietary Supplements) specifically to confirm the identity of non-dietary components used
in their products and determine whether other applicable components’ specifications are met. To fulfil
these requirements, manufacturers need to conduct appropriate tests or examinations; or rely on
certificates of analysis from suppliers of these components provided that the criteria listed in the rule
are satisfied. 1

This document describes test methods that can be used to identify ethanol.

This document is not an exhaustive collection of test methods for ethanol, other methods not discussed
here may be equally suitable.

1 21 CFR 111.75(a)(2).

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Ethanol Identity Test Methods

Ethanol
Description
Ethanol is an alcohol. It is a clear, volatile liquid with a characteristic pleasant odor, and a burning taste.
It is completely miscible with water in all ratios and with organic solvents, and is very hygroscopic. 2

Physical properties and other descriptive information2,3,4

CAS number: 64-17-5

Molecular formula: C2H6O

Structural formula: CH3CH2OH

Molecular weight: 46.07

Melting point: -117 °C

Boiling point: 78 °C

Specific gravity: 0.7893 at 20°/20°C (Absolute) and 0.8096 at 25°/25°C (94.9% by

volume)

Other names: ethyl alcohol; alcohol; ethyl hydrate; ethyl hydroxide; C2H5OH; fermentation alcohol;
sometimes ethanol is named after the source material such as grain alcohol, molasses alcohol, and
potato alcohol.

2 Ethyl Alcohol. https://www.osha.gov/dts/sltc/methods/organic/org100/org100.html (accessed February 13, 2017)

3 NIST Chemistry WebBook listing for Ethanol. http://webbook.nist.gov/cgi/cbook.cgi?ID=C64175&Mask=80 (accessed
February 13, 2017)
4 The United States Pharmacopeial Convention Food Chemicals Codex (FCC) 10th Edition

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 Ethanol Identity Test Methods

Identification Tests for Ethanol

• Organolepsis: It is a clear, volatile liquid with a characteristic pleasant odor, and a burning taste.

• Miscibility: Ethanol is completely miscible with water at all ratios.

• Boiling point: 78 °C

• pH: Ethanol is a neutral compound and does not change the color of litmus paper.

• Specific gravity: 0.7893 at 20°/20°C (Absolute) and 0.8096 at 25°/25°C (94.9% by volume)

• Combustion test5
Transfer about 5 mL ethanol to a large test tube, add a boiling chip, hold with a test tube holder and
heat until the liquid is boiling. Hold the open end of the test tube to the flame and ignite the ethanol
vapors. Ethanol burns with a pale blue flame with no smoke.

• Ceric ammonium nitrate test5

Reagent: 0.4 g ceric ammonium nitrate in 2M nitric acid. Dilute 1:2 in water.

Procedure: To 1 mL of the ceric ammonium nitrate reagent, add 5 drops of ethanol and mix thoroughly.
The yellow color of the reagent changes to red.

Note: This test gives positive results with primary, secondary, and tertiary alcohols (up to 10 carbons),
poly hydroxylated compounds such as carbohydrates, and hydroxylated carboxylic acids, aldehydes and
ketones.

• Jones Oxidation6
Reagent: Jones Reagent, chromium trioxide solution in sulfuric acid (available from chemical companies,
CAS number 65272-70-0).

Procedure: To 1 mL of acetone in a small test tube, add 1 drop of ethanol and 1 drop of Jones reagent.
Within 2 seconds, an opaque suspension with a green to blue color is formed.

Note: This test shows presence of primary and secondary alcohols and aldehydes. No reaction with
tertiary alcohols, ketones, carboxylic acids or esters. Phenols react with Jones reagent but do not
produce a blue to green solution.

5 Harsch G and Heimann R. Experiments for an Introduction to Organic Chemistry.

http://research.uni-leipzig.de/~pinkon/upload/chemistry_box.pdf (accessed February 13, 2017)

6 Weaver November 2011. Qualitative Analysis II Notes.

http://www.xula.edu/chemistry/documents/orgleclab/Qual%202_Notes_v4.pdf (accessed February 13, 2017)

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 Ethanol Identity Test Methods

• Triiodomethane test 7
Reagents:
1M sodium hydroxide solution in water
0.5M iodine solution, dissolved in 0.2M potassium iodide solution in water

Procedure: Transfer 10 drops of ethanol to a clean and dry test tube, add 25 drops of iodine solution and
enough sodium hydroxide solution to remove the color of the iodine and mix gently for a few minutes. If
nothing happens in the cold, it may be necessary to warm the mixture very gently. A very pale yellow
precipitate of triiodomethane (previously known as iodoform) is formed.

Note: This test gives positive results for other alcohols containing a methyl group and a hydrogen atom
attached to carbon bearing the hydroxyl group. Methanol gives negative result.

• Infrared spectroscopy
Procedure: Record the spectrum for a thin film of ethanol (neat). Compare the spectrum with that
obtained for a standard ethanol measured under the same conditions. Characteristic bands include: a
broad and intense band at about 3342 cm-1 due to O–H stretching vibration, a band at about 2980 cm-1
due to C–H stretch, 1090 cm-1 due to C–O stretch, and at 1050 cm-1 due to C–C–O asymmetric stretch. 8

• Nuclear Magnetic Resonance spectroscopy

Procedure: Acquire the spectra in deutro-chloroform. The proton nuclear magnetic resonance (NMR)
spectrum exhibits a triplet corresponding to three protons at about 1.2 ppm representing the terminal
methyl protons, a triplet corresponding to one proton at about 3.0 ppm due to the hydroxyl proton, and
a multiplet corresponding to two protons at about 3.6 ppm due to the methylene protons. The carbon
NMR spectrum shows two peaks, at about 20 ppm corresponding to the methyl carbon and at about 60
ppm corresponding to the methylene carbon. 9

• Gas Chromatography
Gas Chromatography (GC) is the most commonly used technique for qualitative and quantitative
analysis of ethanol, whether alone, in mixtures and in different matrixes. Flame ionization is the most
commonly used detection method followed by mass spectrometry. Commonly used GC columns are
based on polar stationary phases, the most common being polyethylene glycol and modified versions. 10
Suitable methods of analysis are available from national or international pharmacopoeias and
standards-setting organizations, such as the United States Pharmacopeia and AOAC International.

7The Triiodomethane (Iodoform) Reaction.

http://chemwiki.ucdavis.edu/Organic_Chemistry/Alcohols/Reactivity_of_Alcohols/The_Triiodomethane_(Iodoform)_Reaction
(accessed February 13, 2017).
8 Brian Smith. Infrared Spectral Interpretation- A systematic approach, CRC Press, 1998.
9 Martha D. Bruch, Editor. NMR Spectroscopy Techniques, Second Edition, Mercel Dekker, Inc. 1996
10 Sigma-Aldrich GC Column Selection Guide. http://www.sigmaaldrich.com/content/dam/sigma-

aldrich/docs/promo_NOT_INDEXED/General_Information/1/gc-column-selection-guide.pdf (accessed February 13, 2017).

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