Determination of Nitrogen and Calculation of Crude Protein – Kjeldahl

1. Principle

For determination of nitrogen the sample is digested using sulphuric acid in the presence

Of a catalyst to convert sample nitrogen to ammonium sulphate. The acid solution is made

Alkaline with sodium hydroxide solution. The ammonia is distilled and collected in an excess

of boric acid solution, followed by titration with sulphuric acid solution. For determination

of crude protein nitrogen is multiplied by a factor, 6.25 (or an appropriate factor,

2. Scope

The method described is applicable for determination of nitrogen in feeds.

3. Responsibilities

Laboratory Analysts shall perform the analysis as per this method. It is the responsibility

of the Laboratory Analyst to ensure that all conditions laid down in the method are met

and strictly adhered to. Any deviations from the prescribed method shall be recorded and

Supervisor notified.

4. Equipment

4.1 Analytical balance, accurate to 0.1 mg.

4.2 Digestion tubes fitted for the Kjeldahl digestion unit.

4.3 Kjeldahl digestion unit with fume removal manifold.

4.4 Kjeldahl distillation apparatus.

4.5 Titration unit (in sophisticated equipment 4.4 is combined with 4.5).

5. Reagents

5.1 Sulphuric acid, concentrated, 95–98% (w/v), reagent grade.

5.2 Kjeldahl catalyst tablets (see remark 9.5).

5.3 Boric acid, 10 g/litter.

5.4 NaOH solution, 40% (w/v).

5.5 Indicator solution: Methyl red indicator, dissolve 1 g methyl red (sodium salt) in 100

ml methanol or ethanol.

5.6 Hydrochloric acid standard volumetric solution, c = 0.1 M (accurate to 0.1000 M).

6. Procedure

6.1 Digestion

6.1.1 Weigh approximately 1 g sample recording to the nearest 0.1 mg (W) and transfer

to the digestion tube (4.2). In each batch use a tube without sample as blank

test.

6.1.2 Add two Kjeldahl tablets (5.2) and 20 ml sulphuric acid (5.1). If fuming is a

problem, add a few drops of anti-foaming agent.

6.1.3 Place the tubes in a digestion unit (4.3) and connect to the fume removal manifold.

6.1.4 Digest the sample at least 1 hour at 420 ± 20 °C.

6.1.5 Turn the digestion off, remove the tubes and allow to cool for 10–20 minutes.

6.1.6 Add distilled water to each tube to a total volume of approximately 80 ml.

6.2 Distillation and titration

The following procedure describes the manual method of distillation and titration. If an

automatic distillation and titration unit is used, follow instructions of the manufacturer.

6.2.1 Place a conical flask containing 25–30 ml of the concentrated boric acid (5.3)

under the outlet of the condenser of the distillation unit (4.4) in such a way

that the delivery tube is below the surface of the boric acid solution.

6.2.2 Add 50 ml NaOH (5.4) and distill the ammonium by following the instructions

of the manufacturer.

6.2.3 Titrate the content of the conical flask with hydrochloric acid standard solution

(5.6) (see remark 9.4) after adding a few droplets of indicator solution (5.5)

using a titration unit (4.5) and read the amount of titrant used. The endpoint

is reached at the first trace of pink color in the contents.

6.2.4 Record the amount of acid used to the nearest 0.05 ml for the blank test (Vb)

and for each sample (Vs).

7. Calculation

Percent Nitrogen (% N)

% N = (Vs – Vb) x M–(HCl) x 1 x 14.007 / (W x 10)

where,

Vs = ml HCl needed to titrate sample,

Vb = ml HCl needed for the blank test,

M(HCl) = molarity of HCl,

1 = the acid factor,

14.007 = molecular weight of N,

10 = conversion from mg/g to %, and

W = weight of the sample (g).

Calculation percent Crude Protein (% CP):

% CP = % N x F

where,

F = 6.25 for all forages, feeds and mixed feeds,

F = 5.70 for wheat grains, and

F = 6.38 for milk and milk products.