SOP: Determination of Protein Content in Milk Powder (Kjeldahl)

GIFSQ — Global Institute for Food Safety & Quality

Scope

This method is applicable to milk and milk products.

1. Principle

Protein in milk and milk products is digested with concentrated sulphuric acid in the presence of a catalyst containing a mixture of sodium or potassium sulphate and copper sulphate, in a digestion chamber. During this process, nitrogen of protein is converted into ammonium sulphate, which under treatment with NaOH liberates NH₃. Thus liberated NH₃ is distilled and collected in boric acid solution. Finally, the NH₃ solution absorbed in boric acid solution is titrated with standard H₂SO₄ acid solution to get the percentage of nitrogen value, which is converted to protein by multiplying by the factor 6.38.

2. Apparatus

a. Kjeldahl digestion set

b. Distillation set

c. Pipette, burette, measuring cylinder

d. Conical flask

3. Reagents

a. Digestion mixture: CuSO₄·5H₂O and anhydrous K₂SO₄ or Na₂SO₄ (20:100)

b. 4% boric acid solution: dissolve 40 g of boric acid (AR) in a 1000 mL volumetric flask with 400 mL of very hot distilled water. Mix and add more hot water to a volume of 900 mL. Cool the solution to room temperature and add 12 mL of bromocresol green and 8 mL of methyl red indicator. Add distilled water to make the volume up to the mark.

c. Concentrated H₂SO₄ (analytical grade)

d. 40% NaOH

e. 0.1 N H₂SO₄ solution: dilute about 2.7 mL of AR grade H₂SO₄ (sp. gr. 1.84) to 1 litre with distilled water. Shake well and standardize with standard sodium carbonate (CRM grade), with a mixture of methyl red and bromocresol green indicator.

f. Methyl red indicator (0.1%): dissolve 0.1 g of methyl red indicator in 100 mL rectified spirit (95% alcohol).

g. Bromocresol green indicator (0.1%): dissolve 0.1 g of bromocresol green in 100 mL rectified spirit (95% alcohol).

4. Standardization of sulphuric acid

Accurately weigh 50–60 mg of anhydrous sodium carbonate (CRM grade), previously dried at 110°C in an oven for 1 hour, transfer to a 250 mL Erlenmeyer flask and dissolve in 20 mL distilled water. Add 2–4 drops of each of methyl red and bromocresol green indicator. Titrate to pink with the acid to be standardized and note the amount in mL used (A1). Boil the solution gently for 2 minutes — the solution will turn green — and cool at room temperature under tap water. Continue the titration until the next pink color change occurs; note this volume (A2). Boil the solution again for a few minutes, cool rapidly to room temperature under tap water, and continue the titration until the next pink color occurs; note this volume (A3).

Normality = (Weight of Na₂CO₃ × 1000) ÷ (mL of acid consumed (A1 + A2 + A3) × 52.994)

Note: The concentration of the H₂SO₄ solution must be accurate to five digits.

5. Sampling

The representative sample of the product is taken. The sample is thoroughly ground, mixed and drawn with a clean spatula from different parts (top, middle and bottom) of the container, until about 300 g of the material is drawn; it is divided into three equal parts. Then 0.5–1.0 g of sample is drawn for the determination.

6. Procedure — Digestion

Take 0.5–1.0 g of finely ground sample and transfer quantitatively into the digestion tube

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Add about 2.5 g of digestion mixture and add 12 mL of concentrated H₂SO₄ slowly, and mix

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Place the tube in the tube rack and put into the digestion well of the digestor. Put the exhaust manifold of the scrubber on top

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Press start the program in the digestor for temperature and time (400°C for 1 hr 30 min, until the solution becomes clear light blue)

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After completion of digestion (the solution will turn into green/light bluish color), remove the whole system from the digestor with the exhaust still in place and put it in the stand to cool for 10–20 minutes

7. Procedure — Distillation and titration

Set up the automatic distillation set

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Place the digestion tube with the digested sample in the distillation unit

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Take 25 mL of 4% boric acid in a 250 mL conical flask and place it in the receiver tube of the distillation unit

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Set the distillation program to add 50 mL of 40% NaOH and 80 mL water. Set SAFE (Steam Addition For Equilibrium) mode steam capacity to 80%, YES to tube drain, and running distillation time for 5 minutes

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After completion of distillation, the pink color of the receiver boric acid solution turns into green. Titrate the distillate with standardized H₂SO₄ solution until the faint pink color appears

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Note the volume of acid consumed in the titration carried out in the blank distillation without sample

8. Calculation

Total % Nitrogen = 1.4 × (V − V₁) × N ÷ W

% Protein = % Nitrogen × 6.38*

Total % Nitrogen for milk and milk products (db) = % Nitrogen × 6.38 × [100 ÷ (100 − M)]

Crude protein is defined as N × 6.38.

Where, * Nitrogen factor for milk and milk products
V = volume of standard acid used to neutralize the distillate
V₁ = volume of standard acid used to neutralize the blank
N = normality of standard acid
X = total Nitrogen percent
W = weight of sample taken for digestion
M = moisture percentage
100/(100 − M) = factor for dry basis (db)

9. Limit of detection

This method is suitable for food containing more than 0.1% of nitrogen content.

References

Official Methods of Analysis, AOAC, 21st Edition (2019); 930.29.