Kozelka and Hine test is a method of determination of ethanol (ethyl alcohol) from blood or urine samples. The test is considered one of the reliable means of estimation of alcohol concentration in the biological sample.
However, there are some major drawbacks that make it not that reliable (discuss later in the post). So, let’s have a close look at the Kozelka and Hines method of ethanol determination.
What is the Kozelka and Hine Method?
Kozelka and Hine’s test is a macro method of ethanol determination. The test is based on the chemical oxidation of ethyl alcohol by potassium dichromate and sulphuric acid to acetic acid.
The method was first developed in 1941 by F.L. Kozelka and C.H. Hine at the University of Wisconsin.
In addition, there is also another test called the “Cavett test” which uses the same potassium dichromate and sulphuric acid as the reagent. You can check more details from our dedicated post on the Cavett test.
Comparably, the amount of sample that you needed in the Kozelka Hine test is about 10x more than you needed in the Cavett test.
Moreover, with this test, you can determine the alcohol level in urine and blood sample.
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Principle of Kozelka Hine Test Method
The test is based on the oxidation reaction of the ethanol to form acetic acid in presence of oxidizing reagents.
The amount of conversion of ethanol to acetic acid by the action of potassium dichromate will give the resultant alcohol in the blood or urine sample.
In the above reaction, potassium dichromate (orange-red color) when reacting with ethanol is the presence of sulphuric acid produces ethanoic acid along with chromium sulfate (green).
The reaction is an example of a redox chemical reaction, as:
- Dichromate get reduced to Chromium (III) sulphate
- Alcohol gets oxidized to ethanoic acid.
Procedure for Kozelka and Hine Test
- Biological Sample:
- 1ml of blood (anticoagulant sodium citrate, oxalate, or fluoride), or
- 2 ml of urine.
- Potassium dichromate solution, 0.1 N, 4.903 grams per liter
- Sulfuric acid
Step 1: Distillation
Distillation is performed to remove or precipitate proteins from the blood sample.
To the sample of blood, add 2ml 10% of Sodium tungstate and 0.5 ml of sulfuric acid.
As there is no protein in the urine, you don’t need to perform distillation for the urine samples.
Nowadays, for more efficiency and less time consumption, diffusion of alcohol under low pressure is employed.
Step 2: Removal of Acetone and Aldehyde
Potassium dichromate can oxidize primary alcohols, secondary alcohols, acetone, and aldehydes. So, it is necessary to remove them before reacting with potassium dichromate.
So, in the second step of the Kozelka and Hine test, there is the addition of 5 ml of saturated mercuric chloride and 5ml of sodium hydroxide solution to a tube on a hot water bath.
Now, the resultant distillate from the distillation is made to connect with the tube. This all removes the acetone and aldehyde.
Step 3: Reaction With Dichromate
The resultant distillate is now brought to a digestion flask and the addition of 0.1 N potassium dichromate solution with concentrated sulfuric acid is done.
This all produces the green-colored reduced chromium sulfate with acetic acid.
As each ml of dichromate solution reduces 0.575 mg of alcohol. So, the consumption of potassium dichromate to form acetic acid can be used to determine the alcohol concentration.
The color and concentration lead to the estimation of both qualitative and quantitative estimation of ethanol in the urine or blood sample.
Limitation of Using Kozelka Hine Test
The following are the limitation of using the Kozelka and Hine test for ethanol:
- Kizelka Hine test is difficult to perform and time-consuming.
- Not specific to ethanol.
- Reagents are corrosive and toxic, maybe the cause of occupational hazard, if performed regularly.
- Though Kizelka is good at reproducibility so can be performed a number of times along the way, but with very less accuracy.
- False +ve results in case a person is suffering from diabetes and ketoacidosis.
- Also gives false +ve result in decomposed bodies.
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- Kozelka and Hine, Method of Method for Determination of Ethyl Alcohol for Medicolegal Purposes, 1941 [ACS Publications]
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