Urea Test in Milk Procedure

In this urea test in milk procedure post we have briefly explained about DMAB test’s principle, requirements, urea test in milk procedure and results.

Urea Test in Milk

Ammonia is produced when protein is digested in the rumen. If there is too much ammonia in the rumen, it gets taken into the bloodstream and transformed to urea in the liver. The majority of the urea is expelled in the urine of the cow, but some makes its way into the milk.

Within a herd, the concentration of urea in milk varies. Natural milk contains anywhere from 20 mg/100 ml to 70 mg/100 ml of urea. Urea level in milk beyond 70 mg/100 ml, on the other hand, suggests milk with ‘added urea.’

Paradimethylaminobenzaldehyde can be used to detect the addition of urea to milk (DMAB). This approach is based on the fact that in a low acidic solution at room temperature, urea forms a yellow complex with DMAB.

Principle

Urea test in milk based on the use of Paradimethylaminobenzaldehyde (DMAB) is performed. Urea test in milk is based on the principle that urea forms a Yellow complex with DMAB in a low acidic solution at room temperature.

Requirements

Equipment's

1. Dry Test tubes

2. Spectrophotometer

3. Whatman filter paper G-42

4. Funnels

Chemicals

1. DMAB reagent (1.6%, w/v)

2. Milk Sample

3. Distilled Water

4. Ethyl alcohol

5. Phosphate Buffer pH 7.0

6. Trichloroacetic acid

7. Urea Standard Solution

Reagents

1. DMAB reagent (1.6%, w/v): Dissolve 1.6 g DMAB in 100 ml ethyl alcohol and add 10 ml concentrate HCl.

2. Phosphate Buffer pH 7.0: Dissolve 3.403 g anhydrous potassium dihydrogen orthophosphate (KH2PO4) and 4.355 g anhydrous dipotassium monohydrogen orthophosphate (K2HPO4) separately in 100 ml of distilled water. Combine solutions and dilute to 1 litre with water.

3. Trichloroacetic acid (TCA) 24%, w/v: Freshly prepared. 24.0 g TCA is dissolved in distilled water and volume made up to 100 ml.

4. Diluting Reagent: Equal volumes of 24% TCA and phosphate buffer (pH 7.0) are mixed to make the diluting reagent.

5. Urea Standard Solution: (a) Stock solution: 5 mg / ml. Dissolve 5 ± 0.001 g reagent grade urea in water and dilute to 1 litre with water. (b) Working solution: Pipette 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 ml stock solution into 250 ml volumetric flask and dilute to volume with phosphate buffer. (c) Reference solution -Use standard solution containing 1.0 mg urea / 5 ml as reference standard. Store at less than 24°C. The reagent is stable for 1 week.

Procedure

Qualitative

1. Mix 1 ml of milk with 1 ml of 1.6% DMAB reagent. Distinct yellow colour is observed in milk containing added urea.

2. The control Urea test in milk (normal milk) shows a slight yellow colour due to presence of natural urea. The limit of detection of method is 0.2% in Urea test in milk.

Quantitative

1. Pipette 5 ml aliquots of working standard solutions into 20 x150 mm (25 ml) test tubes and add 5 ml DMAB solution to each.

2. Prepare reagent blank of 5 ml buffer and 5 ml DMAB solution. Shake tubes thoroughly and let stand for 10 minutes.

3. Read absorbance in 1 cm cell at 420 nm with reagent blank at zero A. Plot A against concentration urea Plot should be straight line.

4. 10 ml of milk sample is mixed with 10 ml of TCA to precipitate the proteins and filtered using Whatman 42 filter paper.

5. 5 ml of filtrate is then treated with 5 ml of DMAB reagent to develop the colour. Blank is prepared by taking 5 ml of diluting reagent and treating with 5 ml of DMAB reagent.

6. The optical density of the yellow colour is measured at 420 nm. From standard curve the amount of Urea test in milk is calculated.

Result

Milk with more than 70 mg/100 ml urea reveals distinct yellow colour. The control or normal milk shows a slight yellow colour due to presence of natural urea.

Further Readings

Reference