Crude Protein Analysis
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2 | C r u d e P r o t e i n A n a l y s i s  Abstract The reported protein content of foods depends on the analytical method used for determination, making a direct comparison between studies difficult. The aim of this study was to calculate the crude protein content of animal feedstuff using the Kjeldahl method and appropriate chemicals. The feed sample was digested with a strong acid so that it released nitrogen which was determined by a suitable titration technique. Kjeldahl nitrogen determinations are performed on a variety of substances such as meat, feed, grain, waste water, soil, and many other samples. Various scientific associations approve and have refined the Kjeldahl method, including the AOAC International (formerly the Association of Official Analytical Chemists). Even with such recommendation, an alternative method for varying samples is suggested for the sake of reproducibility and comparison. Keywords: proteins; amino acids; Kjeldahl; Dumas I. Introduction Proteins are polymers of amino acids. Twenty different types of amino acids occur naturally in proteins. Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. As a result, they have different molecular structures, nutritional attributes and physiochemical properties. Proteins are important constituents of foods for a number of different reasons. They are a major source of energy, as well as containing essential amino-acids, such as lysine, tryptophan, methionine, leucine, isoleucine and valine, 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, e.g., tenderness of meat or fish products. Isolated proteins are often used in foods as ingredients because of their unique functional properties, i.e., their ability to provide desirable appearance, texture or stability. 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. These reactions can have either a favorable or detrimental effect on the overall properties of foods. Food analysts are interested in knowing the total concentration, type, molecular structure and functional properties of the proteins in foods. In this experiment, the Kjeldahl method was utilized. The Kjeldahl method was developed in 1883 by a brewer called Johann Kjeldahl. A food sample is digested with a strong acid so that it releases nitrogen which can be determined by a suitable titration technique. The amount of protein present is then calculated from the nitrogen concentration of the food. The same basic approach is still used today, although a number of improvements have been made to speed up the process and to obtain more accurate measurements. It is usually considered to be the standard method of determining protein concentration. Because the Kjeldahl method does not measure the protein content directly a conversion factor (F) is needed to convert the measured nitrogen concentration to a protein concentration. A conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram of protein) is used for many applications, however, this is only an average value, and 3 | C r u d e P r o t e i n A n a l y s i s
each protein has a different conversion factor depending on its amino acid composition. The Kjeldahl method can conveniently be divided into three steps: digestion, neutralization and titration. II. Methodology 50 mg of feed sample and 0.5 g catalyst were weighed and mixed well. 2.0 mL of concentrated HCl was added to the sample-catalyst mixture, making sure all of its particles were covered by the acid. This mixture was then digested in a microkjeldahl digester (setting at 6) for approximately 20 minutes, after which it was cooled; covered loosely with rubber stoppers and diluted with distilled water. The contents of the flask were transferred into a distillation apparatus, rinsed 3-4 times. 10 mL NaOH solution was added to the distillation still. The mixture was distilled using 10 mL of 4% boric acid with 4 drops of mixed indicator as the receiver. About 40 mL of the distillate was collected and cooled. The distillate was titrated with standardized HCl solution until end point (note color change of green to pink indicating end point of titration). The blank which contained all the reagents except the sample was distilled and titrated the same way as the sample. III. Results and Discussion In principle, digestion involves the food sample to be analyzed to be weighed into a digestion flask and then digested by heating it in the presence of sulfuric acid (an oxidizing agent which digests the food), anhydrous sodium sulfate (to speed up the reaction by raising the boiling point) and a catalyst, such as copper, selenium, titanium, or mercury (to speed up the reaction). Digestion converts any nitrogen in the food (other than that which is in the form of nitrates or nitrites) into ammonia, and other organic matter to CO2 and H2O. Ammonia gas is not liberated in an acid solution because the ammonia is in the form of the ammonium ion (NH4+) which binds to the sulfate ion (SO42-) and thus remains in solution: N(food) → (NH4)2SO4 (1) After the digestion has been completed the digestion flask is connected to a receiving flask by a tube. Neutralization involves the solution in the digestion flask to be made alkaline by addition of sodium hydroxide, which converts the ammonium sulfate into ammonia gas: (NH4)2SO4 + 2 NaOH → 2 NH3 + 2 H2O + Na2SO4 (2) The ammonia gas that is formed is liberated from the solution and moves out of the digestion flask and into the receiving flask – which contains an excess of boric acid. The 4 | C r u d e P r o t e i n A n a l y s i s