ミクロキェルダール法
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概要
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The principle of this method is a direct determination of ammonia. in Kjeldahl digest by phenol-antiformine procedure without previous distillation. REAGENTS (1) Sulfuric Acid Mixture for Digestion. About 2 N sulfuric 'acid containing 1 g. of potassium sulfate, 0.025 g. of copper sulfate and 0.011 g. of selenium dioxide per 100 ml. of the solution. (2) About 2 N sodium hydroxide. Concentration of this solution should be so adjusted that 10 ml. of it neutralize just an equal volume of sulfuric acid used for preparation of the mixture mentioned above. (3) 0.001 M Ammonium sulfate. (4) 5% Phenol. (5) Saturated sodium bicarbonate. (6) Antiformine-soda solution, (see J. Biochern. 39, 207, 1952). PROCEDURE 1. Oxidation. Common reagent tubes of 1.5 cm.×16.5 cm. size, graduated at 10 ml. are used. Introduce 1 mI. of test solution into the test tube, add 1 ml. of sulfuric acid mixture. Digestion is carried out in a drying chamber regulated at 120℃. In one hour the contents of the tubes are concentrated and become reddish. Each tube .is then fixed vertically to a stand by holding the upper part of the tube with a metallic holder and heated over a microburner. Sulfuric acid creeps up soon about 2 cm. high and the contents become colorless. Remove the burner. At this moment some drops of water condense on inner wall of middle and upper parts of the tube, which should be then evaporated off by heating with burner around the side. The tube is now removed from the holder, held with fingers horizontally rotated along the axis so that the concentrated sulfuric acid wets the inner wall all around up to 10 ml. mark. Fix the tube again vertically to the stand and heat over the burner for five minutes, regulating the flame so that sulfuric acid creeping up through heating remains at the height of the 10 ml. mark. Digestion is thus completed. 2. Color Development and photometry. The tube is immersed in ice-water and the contents are dissolved by introducing 2 ml. of water. To the solution while being cooled are added successively with gentle shaking at each addition 2 ml. of 5 % phenol, 1 ml. of 2 N sodium hydroxide, 1 ml. of saturated bicarbonate and 3 ml. of antiformine-soda solution. The tube is taken out from ice-water and after leaving at room temperature for 20 minutes filled with water to the 10 ml. mark. Indophenol formed is photometrised by Pulfrich's apparatus with filter Sot. As a blank test 1 ml. of water instead of the test solution is digested and treated simultaneously in the similar manner. Besides in order to standardize the extinction coefficient,. which will be indicated by a known amount of ammonia, 1 ml. of 0,0011 ammonium sulfate and 1 ml. of sulfuric acid mixture are introduced to a test tube immersed in ice-water and indophenol formation is carried out in similar way. Another blank test for the latter is carried out with a mixture of 1 ml. of water and 1 ml. of sulfuric acid. 3. Calculation. E. of unknown-E. of its blank/E. of standard-E. of its blank×28=γ per ml. of the test solution REMARKS Indophenol formation is intensified by the presence of copper, therefore sulfuric acid mixture has been added to either ammonium sulfate solution or water as its blank before coloring the solutions. Estimation of nitrogen according to the method above mentioned with M/1000 asparagine or tryptophane, 2M/1000 tyrosine or 2M/3000 histidine gave fair results with highest deviation of 1 % from theoretical values. Nitrogen contents of casein were measured by this method with 1 ml. of 0.02 4 solution and by macromethod with 10 ml. of 2 /o solution. The results agreed with each other. In a similar way nonprotein nitrogen is measurable employing 1 ml. of deproteinised supernatant obtained by introducing 0.1 ml. of serum to 2 ml. of 5 % trichloracetic acid.
- 千葉大学の論文
- 1953-09-28