Protein Determination

Proteins are amino acid polymers. In proteins twenty different types of amino acids occur naturally. Proteins are important constituents of foods for a number of different reasons. They are a major source of energy, it contains essential amino-acids which are essential to human health, but which the body cannot synthesize. Proteins are also the major structural components of many natural foods, often determining their overall texture. Typically, proteins are used as gelling agents, emulsifiers, foaming agents and thickeners. Many food proteins are enzymes which are capable of enhancing the rate of certain biochemical reactions. Therefore food analysts are interested in knowing the total concentration, type, molecular structure and functional properties of the proteins in foods (people.umass.edu, n.d., para. 1 ).


Nitrogen is one of the major element found in organic materials such as protein. This fact was recognized by a Danish chemist, Johan Kjeldahl, who used it as a method of determining the amount of protein in samples. In 1883 Kjeldahl presented to the Danish Chemical Society a method for determining the amount of nitrogen in mixtures of substances containing ammonium salts, nitrate, or organic nitrogen compounds. The central basis used in this procedure is the oxidation of the organic compound using strong H2SO4.

As the organic material starts to being oxidized, the carbon starts to being converted into CO₂ and the hydrogen starts to being converted into water. The nitrogen, from the amine is converted to NH₄⁺, which dissolves in the oxidizing solution, and can later be converted to NH₃. It has a balance and equilibrium point. 

The worldwide standard for calculating protein content in a wide variety of materials such as human and animal food, fertilizer, waste water and fossil fules is the Kjeldahl method.

The Kjeldahl method consists of three steps, which have to be carried out in sequence:

1.       The sample is first digested in strong sulfuric acid in the presence of a catalyst, which helps in the conversion of the amine nitrogen to ammonium ions,

N_(food) →  (NH₄)₂SO₄     

2.       This is the most time-consuming step in the analysis. The purpose of this step is to break down the bonds that hold the polypeptides together, and convert them to simpler chemicals such as water, carbon dioxide and, of course, ammonia. Therefore a transparent solution is resulted. Such reactions can be considerably speeded up by the presence of a catalyst and by a neutral substance, such as potassium sulfate (K₂SO₄), which raises the boiling point of the digesting acid and thus the temperature of the reaction.Catalysts are also used to help in the digestion process; many different one have been tried including selenium, mercury, copper, or ions of mercury or copper. The ammonium ions are then converted into ammonia gas, heated and distilled. The ammonia gas is led into a trapping solution where it dissolves and becomes an ammonium ion once again.

The purpose of the next step, distillation, is to separate the ammonia (that is, the nitrogen) from the digestion mixture. 

      (NH₄)₂SO₄ + 2 NaOH → 2NH₃ + 2H₂O + Na₂SO₄       

3.       Finally the amount of the ammonia that has been trapped is determined by titration with a standard solution, and a calculation made.

      NH₃ + HCl → NH₄Cl

      HCl(aq) + NaOH(aq) → NaCl(aq) + H₂O(l)

As the ammonia dissolves in the acid trapping solution, it neutralizes some of the HCl in the solution. Acid, that is left can be "back titrated" with a standard, solution of base (usually NaOH). By this way the amount of ammonia distilled off from the digestive solution can be calculated, and hence the amount of nitrogen in the protein determined (brooklyn.cuny.edu, n.d.).

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 (FAO, n.d.):

Food				Factor
Animal origin
	Eggs			6.25
	Meat			6.25
	Milk			6.38
Vegetable origin
	Corn (maize)			6.25
	Rice			5.95
	Rye			5.83
	Wheat: Whole kernel			5.83
		Bran		6.31
	Beans: Castor			5.30
			Soybean	5.71
	Peanuts			5.46

Nitrogen content determination by Kjeldahl method can be used for nearly all analytical areas:

·         Foodstuff and beverage

·         Feed

·         Pharma and cosmetics

·         Environment (Applications,n.d.)

References

1.       Analysis of proteins. (n.d.). Retrieved April 9, 2013 from

 http://people.umass.edu/~mcclemen/581Proteins.html

2.       Kjeldahl method. (n.d.). Retrieved April 9, 2013 from

http://www.brooklyn.cuny.edu/bc/ahp/SDKC/Chem/SD_KjeldahlMethod.html

3.       Methods of food analysis. (n.d.). Retrieved April 9, 2013 from

http://www.fao.org/DOCREP/006/Y5022E/y5022e03.htm

4.       Wise, W. (1991).  Protein Determination. Retrieved April 7, 2013 from

http://www.fmmaseattle.com/lab/protein.htm