Kjeldahl Nitrogen Analysis

Mary Jane L. Andes, Ma. Cristina F. Areola, Melanie Anne Arlante Department of Natural Sciences, College of Science Polytechnic University of the Philippines, Sta. Mesa, Manila, Philippines

Objective: To determine the nitrogen content of pure organic compound.

Introduction:

Nitrogen occurs in a wide variety of substances of interest in research, industry and agriculture. The most common method for determining organic nitrogen is the Kjeldahl method, which is based on a neutralization titration. The Kjeldhal method is the standard method of nitrogen determination dating back of its development in the late 1800s. This method consists of three basics steps: 1) digestion of the sample in sulphuric acid with a catalyst, which results in conversion of nitrogen to ammonia; 2) distillation of the ammonia into a trapping solution; and 3) quantification of the ammonia by titration with a standard solution. Digestion is the decomposition of nitrogen in organic samples utilizing a concentrated acid solution. Distillation is the adding of excess base to the acid digestion mixture to convert NH4+ to NH3, followed by

boiling and condensation of the ammonia gas in receiving solution. Titration is to quantify the amount of ammonia in the receiving solution. The amount of nitrogen in a sample can be calculated from the quantified amount of ammonia ions in the receiving solution. The Kjeldahl method of nitrogen analysis is the worldwide standard for calculating the protein content in a wide variety of materials ranging from human and animal food, fertilizer, waste water and fossil fuels.

Technical Approach: Materials and equipment The apparatus to be used in the experiment are Kjeldahl digestion flask, condenser, adaptor, rubber stopper, iron stand, iron ring, iron clamp, buret clamp, acid and base buret, hot plate, 250-mL beaker, watch glass, spatula, and graduated cylinder. In the experiment, the standard NaOH and HCl, 10 g of Potassium Sulfate, Seleniumcoated boiling chips, alternative catalysts0.1 g of Se, 0.2 g CuSeO3, or a crystal of CuSO4 and concentrated H2SO4 will be used as reagents. For the indicators, bromocresol green and phenolphthalein solution will be used. Methodology Kjeldahl method has three basic steps, the digestion, distillation and titration. For the digestion, the unknown will be dried at 105C for 45 minutes and an amount will be weighed such that it will produce 2-3 mmol of ammonia. The sample must be placed in a dry 500- mL Kjeldahl flask, so that as little as possible sticks to the walls. Add 10 g of potassium sulphate (K2SO4) to raise the boiling temperature and add also three selenium-coated boiling

chips to help speed up digestion. Pour in 25 mL of H2SO4, washing down any solid from the walls. Then, in the fume hood, clamp the flask at 30 angle away from you. Heat gently with a burner until foaming ceases and the solution becomes homogeneous. Continue boiling gently the mixture for an additional 30 min. After that, cool the flask for 30 min in the air, and then in an ice bath for 15 min. Slowly and with constant stirring, add 50 mL of ice-cold, distilled water. Dissolve any solids that crystallize. Transfer the liquid to the 500-mL 3-neck distillation flask. Wash the Kjeldahl flask five times with 10-mL portions of distilled water, and pour the washings into the distillation flask.

Figure 1. Kjeldahl distillation setup

For the distillation, set up the apparatus for the Kjeldahl distillation process and tighten the connections well. Pipet 50.00 mL of standard 0.1 M HCl into the receiving beaker and clamp the funnel in place below the liquid level. Then, add 5-10 drops of phenolphthalein indicator to

the three-neck flask and secure the stoppers. Pour 60 mL of 50 wt% NaOH into the adding funnel and drip this into the distillation flask over a period of 1 min until the indicator turns pink. Do not let the last 1 mL through the stopcock and heat the flask gently until two-thirds of the liquid has distilled. After that, remove the funnel from the receiving beaker before removing the burner from the flask. Rinse the funnel well with distilled water and catch the rinses in the beaker. For the titration, add 6 drops of bromocresol green indicator solution to the beaker and carefully titrate to the blue end point with standard 0.1 M NaOH. Look for the first appearance of the light blue color. Calculate the wt % of nitrogen in the unknown.

Discussion: Digestion of the sample is the most time-consuming step in the analysis. The purpose of this step is to decompose and dissolve the solid sample in boiling sulfuric acid, or in the other words, to breakdown the bonds that hold the polypeptides together, and converts nitrogen into ammonium sulphate, and oxidizes other elements present: It is affected by temperature, addition of salt, reflux rate, length of digestion and catalyst addition. The addition of inorganic salts such as K2SO4 decomposes organics slowly, speeded up the reaction by raising the boiling point of the acid (338C) up to ~390C with the added salts. But too much salt can result in +400C, where volatiles lost to atmosphere. Catalysts are also used to help in the digestion process, such as mercury, copper and selenium compounds catalyze the digestion. Digestion is carried out in a long-neck Kjeldahl flask that prevents loss of sample from spattering.

After digestion is complete, the acid digestion is diluted and made alkaline using NaOH. The solution containing NH4+ is made basic, and the liberated NH3 is distilled and trapped into a receiver containing a known amount of HCl. As the ammonia dissolves in the acid trapping solution, it neutralizes some of the HCl it finds there. What acid is left can then be back titrated that is titrated with a standard NaOH. In 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. Calculations One mole of ammonia coming from the digestion mixture (and hence from the original protein) will neutralize exactly one mole of the acid in the trapping flask.

The first calculation, therefore, is to find the number of moles of ammonia that have been produced and then trapped from your sample(s). This is done by,

calculating the number of moles of acid in the trapping flask originally (before any ammonia was trapped) by multiplying the molarity of the acid solution by the volume of the trapping solution

calculating the number of moles of base (NaOH) that were added from the buret to neutralize the remaining acid (that NOT neutralized by the ammonia).

calculating the amount of ammonia.

since 1 mmol of ammonia is equal to 1 mmol of nitrogen, you can get directly the weight of nitrogen from the calculated mmol of ammonia.

For the percentage of nitrogen in the sample,

To determine the % protein in the sample, multiply % N by the corresponding factor*.

Protein Factor* 6.38 6.25 5.95 5.70

Protein sources Milk and dairy Other grains Rice Wheat flour