Acetone test in Urine using Lange method | Observation + Discussion

Acetone test in urine Principle (Lange method)

For acetone test, urine sample is mixed with glacial acetic acid and sodium nitroprusside along with ammonium hydroxide forms a colored complex with acetone.

CH₃COCH₃ + OH^– = CH₃COCH₂^– + H₂O

[Fe(CN)₅NO]^2- + CH₃COCH₂^– = [Fe(CN)₅NO.CH₃COCH₂]^3-  (purple colored ring)

Beta-hydroxybutyric acid cannot be detected by this method. For acetoacetic acid Gerhardt’s test is performed.

Requirements

• Urine sample
• Glacial acetic acid
• 10% Sodium nitroprusside solution
• Ammonium hydroxide
• Test tube

Procedure for acetone test in urine

• Two test tubes were taken for two samples.
• 5 mL of different urine samples were collected in each tubes.
• Then 0.5 mL of glacial acetic acid and 0.5 mL of 10% sodium nitroprusside solution was added in both tubes.
• The tubes were kept in an inclined state for some minutes.
• After that 2 mL of ammonium hydroxide (NH₄OH) was added to the tubes and a layer was formed in both samples.
• Purple ring formation was seen between the two liquids which indicated the presence of acetone (CH₃)₂CO in both tubes.
• The test tubes were then placed in front of a white background to get better view.

Observation of acetone test

Upon adding ammonium hydroxide, two layers were seen. Lighter layer was above and slightly darker layer was below. Between the two layers there was a purple ring formation. It indicated that the urine sample contained acetone. The concentration of acetone present in the sample is directly proportional to the color intensity of the ring. In our experiment, the ring color was lighter in sample 1. On the other hand, the ring color was quite deeper in sample 2. That means urine sample 1 contained lower amount of acetone whereas sample 2 contained higher amount of acetone.

Result

Lower level of acetone(normal) was present in sample 1 and higher amount of acetone was present in sample 2.

Discussion of acetone test

Acetone is a water-soluble organic compound. Acetone is the simplest and smallest ketone. It is a colorless compound with flammable characteristic and has pungent odor. Acetone is produced in human body through normal metabolic processes. People with diabetic ketoacidosis (DKA) and those who follow keto diets produce it in larger amounts. Acetone is found in blood and urine.

Urine contains 2% acetone, 20% acetoacetic acid, 78% beta-hydroxybutyric acid during ketosis. In a normal individual, acetone is producing consistently and are off disposing via urine. During starvation, uncontrolled diabetes or prolonged intense exercise, the level of acetone in the blood goes above a certain level, some hormones such as glucagon, epinephrine and growth hormone causes the release of fatty acids from the adipose tissue into the blood. This test determines the presence acetone in the urine.

In this experiment, two samples showed different levels of acetone. The color intensity of sample 1 was lower than in the sample 2. As color intensity is directly proportional to acetone concentration, sample 2 contained higher level of acetone and that patient should see a doctor for further instructions.

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