Determination of Protein Content Using Kjedahl and Titration
Proteins are polymers. They are the source of dietary amino acids and are used for growth and maintenance of living systems. They are costlier sources of energy compared to carbohydrates and fats and hence the human body utilizes proteins mainly for biosynthesis rather than as an energy source, though the energy yield is 5 kcal/g of protein. 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. 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 proteins in foods.
In this experiment, the methods that are used to determined the protein content which is Kjedahl and titration methods. The Kjeldahl method of nitrogen analysis is the worldwide standard for calculating the protein content in a variety of materials from human and animal food, fertilizer, waste water and fossil fuels. This method is an analytical method to quantitatively determine the nitrogen in certain organic compounds. A food is digested with 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. In the Kjedahl method, there are three principles involved which are digestion, neutralization and titration.
The first step is digestion. The sample to be analyzed is weighed into a digestion flask then digested by heating it in the presence of sulphuric acid (an oxidizing agent which digests the food), anhydrous sodium sulfate (to speed up the reaction by raising the boiling point) and a catalyst (for example copper, selenium, titanium or mercury that used to speed up the reaction). This step also converts any nitrogen in the food into ammonia, and other organic matter to carbon dioxide and water. 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-). The remains in the solution is N(food) (NH4)2SO4
The second step is neutralization. After the digestion has been completed the digestion flask is connected to a receiving flask by a tube. The solution in the digestion flask is then made alkaline by addition of sodium hydroxide, which converts the ammonium sulfate into ammonia gas as in the equation: (NH4)2SO4 + 2 NaOH 2NH3 + 2H2O + Na2SO4
The ammonia gas that is formed is liberated from the solution after distillation and moves out of the digestion flask and into the receiving flask which contains an excess of boric acid. The low pH of the solution in the receiving flask converts the ammonia gas into the ammonium ion, and simultaneously converts the boric acid to the borate ion as in this equation: NH3 + H3BO3 (boric acid) NH4+ + H2BO3- (borate ion)
The last step is titration. In this step, the nitrogen content is then estimated by titration of the ammonium borate formed with standard sulphuric or hydrochloric acid, using a suitable indicator to determine the end point of the reaction (H2BO3- + H+ H3BO3). Then, the concentration of hydrogen ions required to reach the end point must be equivalent to the concentration of nitrogen that was in the original food. To determine the nitrogen concentration of a sample that weighs m grams using a xM HCl acid solution for the titration is based on the following equation:...