Nitrogen analysis by micro-Kjeldahl method

Nitrogen is the major element next to carbon, hydrogen and oxygen found in living things. Nitrogen occurs in amino acids, purine and pyrimidine bases, vitamins, aminosugars, alkaloids, compound lipids etc. however, the major nitrogen source is proteins. In most proteins, nitrogen constitutes 16% of the total make-up and hence, the total nitrogen content of a sample is multiplied by 6.25 to arrive at the value of the crude protein. By and large micro-kjeldahl technique is adopted to estimate the total nitrogen content in a variety of samples ranging from microbial cells to meat. The procedure described here is highly suitable for food samples such as cereals and pulses flour. In addition, procedures for non-protein nitrogen, protein nitrogen and amino nitrogen are also called.

Principle

The nitrogen in protein or any other organic material is converted to ammonium sulphate by H₂SO₄ during digestion. This salt, on steam-distillation, liberates ammonia which is collected in boric acid solution and titrated against standard acid. Since 1mL of 0.1N acid is equivalent to 1.401mg N, calculation is made to arrive at the nitrogen content of the sample.


Material
» Kjeldahl Flasks : 30mL hard glass flasks (10mL size for micro samples)
» Digestion Rack : Commercial heating apparatus
» Distillation  Apparatus : Glass distillation apparatus assembly
» Sulphuric Acid Sp. Gr. 1.84
» Mercuric Oxide
» Potassium Sulphate
» Sodium Hydroxide – Sodium thiosulphate solution: Dissolve 600g NaOH and 50g Na₂S₂O₃.5H₂O in distilled water and make to one liter.
» Indicator Solution: Methyl red 0.2g/100mL ethanol, methylene blue 0.2g/100mL ethanol. For mixed indicator, two parts of methyl red solution are added to one part of methylene blue solution.
» Boric Acid 4% solution
» Standard HCl or H₂SO₄, 0.02 N
» Boiling Chips and/or Glass Beads

Procedure

1. Weigh 100mg of the sample (containing 1 to 3mg nitrogen) and transfer to a 30mL digestion flask.
2. Add 1.9 ±0.1g potassium sulphate and 80 ±10mg mercuric oxide and 2mL conc. H₂SO₄ to the digestion flask. If sample size is larger than 20mg dry weight, 0.1mL H₂SO₄, should be added for each 10mg dry material.
3. Add boiling chips and digest the sample till the solution becomes colorless (the time of digestion will vary with regard to the size of the sample, temperature, and the mode of digestion).
4. After cooling the digest, dilute it with a small quantity of distilled ammonia-free water and transfer to the distillation apparatus (when the nitrogen content of the sample is high, the digest can be made up to a known volume and an aliquot may be transferred to the distillation flask). The kjeldahl flask should be rinsed with successive small quantities of water.
5. Place a 10mL conical flask containing 5mL of boric acid solution with few drops of mixed indicator with the tip of the condenser dipping below the surface of the solution.
6. Add 10mL of sodium hydroxide-sodium thiosulphate solution to the test solution in the apparatus.
7. Distill and collect the ammonia on boric acid (at least 15-20mL of distilled should be collected).
8. Rinse the tip of the condenser, and titrate the solution against the standard acid until the first appearance of violet color, the end point.
9. Run a reagent bank with an equal volume of distilled water and subtract the titration volume from that of the sample titre volume.

Calculation

The nitrogen content of the sample can be calculated based on any one of the following formulae as the case may be

Ng/kg =	(mL HCl – mL blank) x normality x 14.01
	Weight (g)

Ng/kg =	(mL HCl – mL blank) x normality x 14.01 x final volume
	Weight (g) x aliquot volume

Non-Protein Nitrogen
1. Extract a known quantity of powdered material (100mg) with ice cold 10% TCA (10mL). Proteins are precipitated while non-protein nitrogen gets extracted).
2. Centrifuge, wash the precipitate with TCA, pool all the supernatants and make up to a known volume (25 or 50mL)
3. Take an aliquot and distill as described earlier.
4. Titrate against the standard acid , and calculate the nitrogen content. This gives the percentage of non-protein nitrogen.

Protein Nitrogen
Multiplying total nitrogen value with 6.25 will give the crude protein content which also includes non protein nitrogen. To get true protein content, deduct the non-protein nitrogen from the total nitrogen and then multiply with the factor.

Amino Nitrogen
1. Estimate the total free amino acid content by clicking here.
2. Multiply the percentage equivalent of leucine with 14/131 to get the percentage of amino nitrogen. If any amino acid other than leucine is used as standard, introduce the molecular weight of that amino acid in the denominator.

Notes

1. Care must be taken so as to get a representative and homogeneous sample.
2. When greater quantity of sample (500mg) is used and the digest is diluted before an aliquot is transferred to the distillation set, care should be taken that the actual quantity of sulphuric acid so transferred does not exceed the capacity of the 10mL of NaOH- Na₂S₂O₃ solution. The solution being distilled out should always be strongly alkaline.
3. The method described is meant for the conventionally operated distillation apparatus and does not attempt to give the methodology for automatic distillation sets that are commercially available. In such cases, the research worker has to follow the supplier’s manual for specific procedures.
4. If the test solution is still yellow-colored even after prolonged digestion, addition of few drops of perchloric acid or hydrogen peroxide will assure complete oxidation and to get a colorless solution.
5. Appropriate factors should be calculated for the acid normalities prepared in the laboratory based on the following
   1mL 0.1N acid = 1.401mg N
6. A known concentration of ammonium sulphate solution can be distilled as a standard check, often.
7. In general, the nitrogen content is multiplied by the factor 6.25 to arrive at the percentage of crude protein which is based on the assumption that nitrogen constitutes to 16% of a protein. However, the nitrogen percent varies with the amino acid composition of the proteins. For more refined expression of protein percentage in samples, different factors are used. These factors were arrived at by amino acid composition. Some such factors are given below:

Item	Conversion factor
Wheat (whole)	5.83
Wheat flour	5.70
Wheat bran	6.31
Rice	5.95
Rye, barley and oats	5.83
Groundnut	5.46
Soya	5.71
Sesame, safflower and sunflower	5.30
Milk and cheese	6.38
Other foods	6.25

References

1. FAO Nutritional Studies No. 24 (1970) Amino Acid Content of Foods and Biological Data on Proteins FAO Rome.
2. Pellett, L P and Young, V R (1980) Nutritional Evaluation of Protein Foods UN Univ Publ.
3. Theymoli Balasubramanian and Sadasivam, S (1987) Plant Foods Hum Nutr 37 41.