The amount of nitrogen in a sample can be calculated from the quantified amount of ammonia ion in the receiving solution. Both methods indicate the ammonia present in the distillate with a color change. In back titration commonly used in macro Kjeldahl , the ammonia is captured by a carefully measured excess of a standardized acid solution in the receiving flask.
The excess of acid in the receiving solution keeps the pH low, and the indicator does not change until the solution is "back titrated" with base. In direct titration, if boric acid is used as the receiving solution instead of a standardized mineral acid, the chemical reaction is:.
The boric acid captures the ammonia gas, forming an ammonium-borate complex. As the ammonia collects, the color of the receiving solutions changes. The boric acid method has the advantages that only one standard solution is necessary for the determination and that the solution has a long shelf life.
Whitaker, JR. An absolute method for protein determination based on difference in absorbance at and nm. Anal Biochem; 1 Harris, DA.
CL, Bashford. Spectrophotometric assays. In, eds, Spectrophotometry and Spectrofluorimetry: a Practical Approach. Measurement of protein using bicinchoninic acid. Related Articles. Comprehensive Pipette Guide As an Amazon Associate Conductscience Inc earns revenue from qualifying purchases The modern pipette has had a colorful history as a standard tool in the.
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Such reactions can be considerably speeded up by the presence of a catalyst and by a neutral substance, such as potassium sulfate K 2 SO 4 , 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.
Weighing out approximately 1 gm of the sample containing protein, making a note of the weight, and placing the sample into a digestion flask, along with ml of concentrated sulfuric acid H 2 SO 4. The purpose of the next step, distillation, is to separate the ammonia that is, the nitrogen from the digestion mixture. This is done by,. 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, known solution of base usually 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. This dye should turn a strong color, indicating that a significant amount of the original trapping acid is still present.
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.
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