Beverage analysis in forensic and liquor samples

The Bureau of Indian Standards (BIS) and the Prevention of Food Adulteration (PFA) Act
provide guidelines for the composition and analysis of alcoholic beverages in India. Here is a brief
overview of the guidelines:

1. Alcoholic beverages are defined as drinks containing more than 0.5% alcohol by volume.
2. The composition of alcoholic beverages is governed by the type of beverage. For example,
beer must contain malted barley or wheat, hops, water, and yeast. Whiskey must be made
from grain, distilled to a maximum of 94.8% alcohol by volume, and aged for a minimum of
three years in oak casks.
3. The BIS and PFA Act mandate that alcoholic beverages must not contain any harmful
substances. The presence of impurities, such as methanol, is strictly prohibited.
4. The labeling of alcoholic beverages must be accurate and in compliance with the BIS and
PFA Act. The label must indicate the name of the product, the type of beverage, the alcohol
content, the name and address of the manufacturer, and the batch or lot number.
5. Alcoholic beverages must be tested and analyzed for quality control purposes. Tests include
measurement of alcohol content, pH, and specific gravity. The presence of harmful substances
is also tested, such as methanol and other impurities.
6. The storage and transportation of alcoholic beverages must be done in compliance with the
BIS and PFA Act. Alcoholic beverages must be stored at appropriate temperatures.

Adulteration of liquors and alcohols refers to the addition of substances that are not meant to be
present in the beverage, either for economic gain or to mask the presence of low-quality ingredients.
Some possible adulterants in liquors and alcohols include:

Methanol: Methanol is a toxic alcohol that can be produced during the distillation process, and can
cause serious health effects if consumed in large quantities.

Sugars: The addition of sugars such as sucrose or high-fructose corn syrup can increase the sweetness
of the beverage, but can also lead to a higher calorie count and potentially harmful effects on blood
sugar levels.

Coloring agents: Artificial coloring agents may be added to make the beverage appear more
appealing, or to mask the presence of low-quality ingredients.

Flavoring agents: Flavoring agents such as essential oils or artificial flavors may be added to mask
the taste of low-quality ingredients or to enhance the flavor of the beverage.

Furfural: Furfural is a chemical compound that can be produced when certain sugars are heated or
fermented, and has a bitter almond-like taste. It is not usually added as an adulterant, but can be
present in low levels in some alcoholic beverages, especially those that have been aged or stored for a
long time. Furfural can be harmful if consumed in large quantities, and has been linked to stomach
irritation, liver damage, and other health effects.

Chloral hydrate: Chloral hydrate is a sedative medication that was historically used to treat insomnia
and other sleep disorders. It has a bitter taste and can be added to alcoholic beverages to increase their
sedative effects or to produce a "knockout" drink. However, chloral hydrate is highly toxic and can
cause serious health effects, including respiratory depression, coma, and death.

Barbiturates: Barbiturates are a class of sedative medications that are sometimes added to alcoholic
beverages to produce a stronger sedative effect or to produce a "knockout" drink. However, like
chloral hydrate, barbiturates are highly toxic and can cause serious health effects, including
respiratory depression, coma, and death.

It is important to note that adulteration of liquors and alcohols is illegal and can have serious health
effects. Consumers should purchase products from reputable sources and be aware of the signs of
counterfeit or adulterated products, such as unusually low prices or unusual tastes or odors.

On the basis of preparation there are two kinds of alcohols. They are (i) distilled alcohols and (ii)
undistilled alcohols.

Distilled alcohol forms from a process of heating the fermented plant matter at high
temperatures to produce steam that is collected, condensed and bottled. Liquor production by this
involves extra steps of distillation, which reduces the original water content and significantly
increases the alcoholic strength. Distilled alcoholic drinks, also known as spirits or hard liquor, vary
in fermentation processes, ingredients, and alcohol by volume (ABV). Eg- Gin, Brandy, Whiskey,
Vodka, Rum.

In contrast, undistilled alcohol is made by fermenting sugar or plant matter and yeast with water
to produce ethanol as a byproduct. It is less pure and lower in strength. Eg; wine, beer, hard ciders.

There are different types of alcohols that people consume. Various types of alcoholic beverages,
including commercial, licit non- commercial, illicit home brewed and sometimes adulterated alcoholic
beverages that diverge state by state and area, are consumed.

Colour test for ethanol.

Ethanol and other volatile reducing agents in urine/blood - dichromate test.

About 1 ml or appropriate amount of sample (distilled or as such depending upon the nature of
samples and concentration of ethanol) is added with about 0.2 ml of 2% Potassium Dichromate
solution followed by about 1 ml of concentration Sulphuric Acid. The yellow colour of the
dichromate changes to green or blue indicates the presence of ethanol.

Test for Ethyl Alcohol in suspicious samples.

Iodoform Test:

Appropriate amount of sample (distilled or as such depending upon the nature of sample and
concentration of ethanol) is taken and about 1 ml of 5% Sodium Hydroxide solution added to it
and then iodine solution is added drop-wise with shaking until the liquid becomes persistent dark
brown in colour. It is kept for 2-3 minutes. If the iodine colour disappears more drops of iodine
solution is added until persistent brown colour of iodine appears. Few drops of dilute Sodium
Hydroxide solution are added to remove extra iodine. Equal volume of water is added and left for ten
minutes. Yellow crystalline precipitate indicates the positive test for the presence of ethanol.

Methanol in alcohols.

Methanol is formed in very small amounts during fermentation, the process by which alcohol is made
from plant products like grape juice or cereal grains. There are small amounts in wine and beer, but
not enough to cause problems. After consumption of methylated alcohol, Methanol is converted in
the body into formic acid, during metabolism. It's the buildup of this in the blood that causes the
devastating problems. In large amounts it can lead to the following problems; kidney failure,
problems with your heart and circulation, liver damage, visual disturbances such as blurred vision,
tunnel vision, changes in colour perception, and temporary or permanent blindness, nerve and brain
damage.

Test for methanol in alcoholic sample

(1) Chromotropic Acid Test:

About 1 ml or appropriate amount of sample (distilled or as such depending upon the nature
of sample and concentration of methanol) is taken in a test tube and about 2 ml of Potassium
Permanganate solution in Phosphoric/Ortho Phosphoric Acid is taken and shaken well. Few
crystals of Sodium Bisulphate are added with shaking till disappearance of colour (Potassium
Permanganate colour) of the solution. About 1 ml of 5% aqueous chromotropic Acid and
concentrate Sulphuric Acid is added slowly with inner sidewall of the test tube. Appearance of
violet colour indicates the presence of methanol.

(2) Schiff’s reagent Test

 Take 8 mL of a sodium dichromate solution with 4 mL of sulfuric acid. Swirl gently to

mix, then add 10 drops of the mixed solution to a test tube or other small container containing the
alcoholic sample. Swirl this container gently a few times, then waft the air from the mouth of the
container towards your nose by fanning the air toward you with a hand, with the container placed
roughly 8-12 inches from your face. Take note of the scent. If it is pungent and irritating, methanol
is present in the alcohol. If the scent is dominating and fruity, only ethanol is present, and the
beverage is safe.

The whole content is left for 10 minutes. 1 ml of 10% Oxalic Acid is added followed by
1ml of concentrated Sulphuric Acid. The contents are cooled at room temperature. 5 ml of Schiff’s
reagent is now added and kept for half an hour to observe the colour. Appearance of purple colour
indicates positive test for the presence of methanol.

Test for Furfural

About 5 ml or appropriate amount of sample (distilled or as such depending upon the nature
of sample and concentration of furfural) is taken in a test tube and about 1 ml Aniline and about 0.5
ml Hydrochloric Acid is added to it and kept for 15 minutes. Appearance of red colour indicates the
presence of Furfural.

Alternative method About 2 ml or appropriate amount of the sample (distilled or as such depending
upon the nature of sample) is taken in a test tube and about 0.2 ml of Aniline and about 0.4 ml of
Glacial Acetic Acid is added to it. If the furfural is present in the sample, red colour develops in a
few seconds & reaches its maximum intensity in 5-10 minutes.

Procedure for determination of alcoholic samples by Gas Chromatography.

i) Transfer 5 ml of sample into a 10 ml stoppered test tube and add 1ml of n- pentanol internal
standard and mix well.
ii) Inject 2 μL of methanol standard solution into GC and record the chromatographic profile.
iii) Adjust the operating parameters and attenuation to obtain good resolution of the peaks.
iv) Determine the retention time of methanol and n-pentanol.
v) Inject 2 μL sample solution into GC and record the chromatogram.
 Chloral hydrate in toddy samples.
Chloral hydrate is a colourless solid with anti- convulsive properties that has use in the
manufacture of sedative and hypnotical pharmaceutical drugs. While natural toddy is prepared
from the alcoholic sap of coconut palms, the synthetic variety sees the use of chloral hydrate
among other ingredients.
Fujiwara test, which gives intense red colour by the adding Pyridine in the presence of
an alkali, is generally used to identify Chloral hydrate in toddy.

Alcohol samples were filtered through whatman No-1 filter paper and 1 ml of the filtrate was
thermostated on autosampler at 80oC for 20 minutes and all of vapour is injected onto the
chromatograph. The standard chloral hydrate sample was analyzed under the identical condition.
The presence of chloral hydrate was determined by comparing the rotation times with that of the
standards and their quantity was estimated from the areas of the respective peaks.
A thin-layer chromatographic method for selective detection of chloral hydrate in alcoholic
beverages is described. This hypnotic compound reacts with orcinol under alkaline conditions
producing a yellow fluorescent compound. Other adulterants of alcoholic beverages, for example
saccharin, diazepam, and phenobarbitone, do not interfere with the test. The detection limit for
chloral hydrate is approximately 10 µg per spot
Procedure to find out aldehyde in alcohol samples.
(i) Take 50 ml of distillate of liquor in a 250 ml Iodine flask and add 10 ml of bisulphite solution. Keep
the flask in a dark place for 30 min. with occasional shaking.
(ii) Add 25 ml of standard iodine solution and back titrate excess iodine against standard thiosulphate
solution using starch indicator to light green end point.
(iii) Run a blank taking 50 ml of distilled water in the same way.
(iv) The difference in titer value in milliliters, of sodium thiosulphate solution gives the equivalent
aldehyde content.
Questions
Explain the qualitative as well as quantitative determination of ethyl alcohol in suspicious
samples
Explain the qualitative as well as quantitative determination of (i) methyl alcohol (ii) furfural
(iv) chloral hydrate and ((v) aldehyde in a liquor samples.