M.M. Rahman*
Department of Chemistry, Shah Jalal University of Science and Technology, Sylhet 3114, Bangladesh
M. Alamgir,
Department of Chemistry, Shah Jalal University of Science and Technology, Sylhet 3114, Bangladesh
A. Alam and
Department of Chemistry, Shah Jalal University of Science and Technology, Sylhet 3114, Bangladesh
S.A. Begum
Department of Chemistry, Shah Jalal University of Science and Technology, Sylhet 3114, Bangladesh
Protein Content, Vegetable, Foodstuffs, Sylhet, The Kjeldahl Method
Sylhet, Comilla and Bhola
Resource Development and Management
Vegetables, Protein
There are numerous methods that are employed for estimation of proteins such as Dumas method (Sternanglanz et al. 1962), Biuret method (Riegler 1914), phenol-reagent method (Wu 1922), Ninhydrin determination (McGRATH 1972), Graves method ( Graves et al. 1968), Lowry and modified Lowry method (Peterson 1877; Hartee 1972; Fryer et al. 1986, and spectrophotometric method (Warburg et al. 1941) etc but most of them have some limitations.
A calorimetric protein assay technique for the determination of protein concentration was reviewed by Sapan et al. (1999). Kjeldahl method nitrogen determination is performed on a variety of substances such as meat, feed, grain and many other samples. Kjeldahl method is widely used for the determination of nitrogen and consequently estimation of protein. It is a well-established method for the determination of protein content in biological food-stuffs and it gives reproducible results. Kjeldahl method has a number of advantages over the classical Dumas method, namely because of its speed and simplicity. Thus considering the advantages and availability of the reagents and micro Kjeldahl distillation apparatus the well-established Kjeldahl method have used for the estimation of protein in food samples. Proteinious nitrogen is not only the source of nitrogen in the sample. There are many other sources of non-protein nitrogen. At first the percent of total nitrogen (%TN) and then the percent of non-protein (%NPN) were determined. The percent of protein nitrogen (%PN) was estimated by difference between % NPN and % TN. All nitrogen determinations were estimated by the Kjeldahl method. The true protein was calculated as (total N- non-protein N) x 6.25. Reagent required: Hydrochloric acid, Sulfuric acid, Trichloroacetic acid. Sodium Sulphate (Na2SO4), Copper Sulphate (CuSO4).Sodium hydroxide (NaOH), Sodium carbonate.Methyl Orange etc.
Determination of total nitrogen (TN): Procedure: The procedure for total nitrogen (TN) determination can be conveniently divided into two parts: (i) Digestion of the sample (ii) Distillation of the digested product.
(i) Digestion of the sample: In a typical procedure 0.5 g sample, 5.0 g sodium sulphate (Na2SO4) and 0.5 g copper sulphate (CuSO4) with 11 mL of concentrated sulfuric acid (H2SO4) was taken in a digestion flask (also called iodine flask). This mixture was heated for about 2 hours until the colour of the digestion mixture changed to colourless. Then the digestion mixture was cooled and about 10 ml of water was added to dissolve the digestion product and to make it ready for the distillation.
(ii) Distillation of the digested product: Digested product obtained in earlier step was poured into the Kjeldahl apparatus with care and approximately 35 ml of sodium hydroxide (40%) was added to that digested product. 20 mL of 1 M hydrochloric acid was taken in a another large capillary tube. After the addition of 35 ml of 40% NaOH solution ammonia gas was evolved and it was absorbed in hydrochloric acid of known concentration. The steam distillation was continued until no ammonia gas is evolved which was tested with red litmus paper and phenolphthalein indicator. After absorption of the ammonia gas, the change of the strength of hydrochloric acid was determined with standard sodium carbonate solution using methyl orange as indicator.
Determination of Non-protein nitrogen (NPN): Procedure: Approximately 0.2 g sample was taken and was dissolved in 20 ml of water and 30 ml of 20% trichloroacetic acid was added with it. This solution was filtered on a filter paper and the filtrate was subjected to for digestion process. Approximately 5.0 g of sodium sulfate (Na2SO4), 0.5 g copper sulfate (CuSO4) and 8.0 ml concentrated sulfuric acid (H2SO4) was taken with the filtrate in a digestion flask. The mixture was digested and distilled in the same way as mentioned earlier and the quantity of ammonia, as well as the NPN, was determined from the change of concentration of HCl solution as mentioned for total nitrogen determination.
Journal of Science and Technology 12(June 2014): 94-96
Journal