July, 19531 MIXTURES OF NATURALLY OCCUHRIKG URANIUM ISOTOPES 405 The Amperometric Titration of Traces of Ammonia with Hypobromite at the Rotated Platinum Wire Electrode Application to the Deterininatioii of Nitrogen in Organic Coinpouiids BY I. M. KOLTHOFF, W. STRICKS ASD L. MORREN Current - voltage curves of sodium hypobromite solutions at the rotating platinum electrode as indicator electrode have been determined in buffer solutions of pH values between 8.3 and 13.0, both in the absence and presence of oxygen. In air-saturated sodium bicarbonate solutioii the current nieasured a t +Om2 volt with respect to the saturated calomel electrode is proportional to the concentration of hypobromite. Use of this is made in the amperometric titration of arsenic trioxide and ammonia. Procedures are given for the rapid titration of arsenite, of ammonia and of nitrogen in organic compounds after a Kjeldahl digestion.Ammonia can be determined at con- centrations between G s and 4 x A4 with an accuracy and precision better than 2 per cent. At high dilutions the amperometric titration methods with hypobromite are simpler, more rapid and more accurate than previous methods. The methods described in this paper should find application to the ainperometric titration of many substances that react stoicheiometrically with hypobroniite. SODIUM hypobroinite is a well-known \701umetric reagent, but it has never been used in amperometric titrations. In this paper the voltamrnetry of hypobromite a t the rotated platinum electrode is described and conditions are established under which traces of ammonia can be titrated rapidly, simply and accurately with hypobromite by the amperometric technique.Analytical use has been made of the oxidation of ammonia by hypobromite for almost 30 years. Artman and Skraball and, independently, Rupp and Rossler2 showed that ammonia and urea can be titrated with hypobromite. ,4 critical study of these titrations has been reported by Kolthoff and Ti trations with hypobromite found wide application to the determination of nitrogen in biological materials, so obviating a distillation of the Kjeldahl digest (for examples see references 4 to 10). The determinations were carried out by adding an excess of hypobromite and titrating with tliiosulphate after addition of iodide or by potentiometric titration. From current - voltage curves for hypobroinite solutions at the rotating platinum electrode it ~7as inferred that it should be possible to carry out titrations with hypobromite amperometrically.In practice it was found that, with the rotated platinum electrode as406 KOLTHOFF, STRICKS AND hIORRES THE XMPERO.IIETRIT [Val. 78 indicator electrode, traces of ammonia can be titrated more simply, rapidly and accurately than by any other titration method. The end-point in amperometric titrations with hypo- bromite can be detected very sharply. The method has been successfully applied in this laboratory to routine determinations of protein nitrogen in blood sera and their albumin and globulin fractions. EXPERIMESTAL MATERIALS USED- Water was made ammonia-free by redistillation from dilute sulphuric acid medium in an all-Pyrex glass apparatus, air being excluded.Glutathione in the reduced state was a Pfanstiehl product. The purity of this product was 99 per cent. as determined by titration with cupric copper.ll The stock solution used was 0.024 M in glutathione, which corresponded to 1 mg of nitrogen per 1 ml of solution. The water content of this product was found to be 6.0 per cent. by heating a t 110" C to constant weight. A 10-3 M stock solution of albumin was prepared, containing 1.02 mg of nitrogen in 0.1 ml. Stock solutions of ammonium chloride and ammonium sulphate were 0.003 M . Stock solutions of sodium hypobromite (0.06 M in hypobromite and 0.1 M in sodium hydroxide) and of arsenious oxide (0.025 M) were prepared according to conventional procedures.l2S13 The stock solution of hypobromite was stored in a dark bottle in a refrigerator a t 4" C.Under these conditions the titre of this solution was found to decrease by about 10 per cent. within three months. Stock solutions of sodium bicarbonate and of borax were 0.6 !M and 0.1 Jf, respectively. The titrations were carried out with solutions prepared from stock solutions by Crystallised bovine plasma albumin was an Armour product. appropriate dilution with ammonia-free water. EXPERIMESTAL METHODS- Current - voltage curves were prepared with a Heyrovskgj self-recording polarograph and with a manual apparatus and circuit,14 which was also used for amperometric titrations. Potentials were measured against the saturated calomel electrode. Amperometric titrations were also carried out with the simplified apparatus and circuit described by Kolthoff and Harris.15 In the present work with this apparatus a reference electrode was used that has a potential of +O.lS volt against the saturated calomel electrode.The electrolyte of the reference half-cell is a saturated mercurous chromate solution in M potassium chromate. Mercurous chromate was prepared as described by Gmelin and Krautle by precipitation from an excess of a slightly acid mercurous nitrate solution with potassium dichromate. The product was filtered, washed with water and suspended in a M potassium chromate solution. In order to protect the titration mixture from contamination with chromate, a vessel filled with saturated potassium nitrate was interposed between each half-cell and the titration vessel, the solutions in the two half-cells being connected by means of an agar - potassium chromate and an agar - potassium nitrate bridge, respectively.An unused platinum electrode must be cleaned with concentrated nitric acid and rinsed with water. If not in use, the electrode is kept in distilled water. One- arid two-miliilitre semi-micro burettes graduatcd at each 0.01 ml were used in the t iti-at ions. The pH was measured with a Beckman pH meter, Laboratory Model G. Oxygen was removed from hypobromite solutions by a stream of pure nitrogen, which was passed through two wash-bottles containing hypobromite solutions of the same com- position as that in the test mixture.A layer of mercury serves as the electrode of the half-cell. A motor provided rotation a t 1800r.p.m. for the platinum electrode. CURREST - VOLTAGE C ~ R V E S AT ROTATISG PLATINUX WIRE ELECTRODE- Current - voltage curves were prepared for hypobromite solutions of various concen- trations of sodium hypobromite and pH, both in the absence and presence of oxygen. Hypobromite is reduced a t the rotating platinum electrode and gives a reduction wave which, under proper conditions, exhibits a well-defined diffusion current region in the absence of oxygen. Current - voltage curves of hypobromite a t different concentrations in air-freeJuly, 19531 TITRATIOS OF TRACES OF AMMONIA 407 medium at pH 10.3 are shown in Fig. 1. Current -voltage curves for each concentration were plotted from measurements of the current while the potential was varied first from positive to negative values and then in the reverse direction.It is seen from Fig. 1 that the curves plotted in these two ways are different in shape, which indicates that the electrode 28 24 20 Potential, V Fig. 1. Current - voltage graphs of hypobromite in the absence of oxygen, a t different concentrations in a carbonate - bicarbonate buffer solution (pH 10.35). Curve A, 1.45 x 10-4 M sodium hypobromite; curve B, 0.65 x 10-4 M sodium hypobromite Potential, V Fig. 2. Current - voltage graphs of 0.65 x lO-'M hypobromite in air-free solutions a t various pH values. Curve A, 0.24 M bi- carbonate solution (pH 8.3) ; curve B, 0.05 M borax solution (pH 9.2) ; curve C, 0.15 M carbonate - 0.05 M bicarbonate solution (pH 10.35) ; curve D, approximately 0.1 M sodium hydroxide solution (pH 13.0) has become polarised during the electrolysis.The curves plotted in the usual way (from positive to negative potentials) show diffusion currents that are proportional to the concen- tration of hypobromite. M solutions, the diffusion currents are 12.5 and 28.2 PA, respectively, which corresponds to a current of about 19.3 PA for a 10-4 M hypobromite solution. Fig. 2 shows current - voltage curves for hypobromite in air-free solutions at various pH values. It is seen that the hypobromite wave is shifted to more negative potentials as the pH of the medium is increased. It can be seen from Fig. 1 that, for 0.65 x lo-* and 1-46 X408 KOLTHOFF, STRICKS AND MORREN : THE AMPEROXETRIC [Vol.78 Hence a t pH 8.3 (bicarbonate), 9.2 (borax), 10.35 (carbonate -bicarbonate), 13 (0.1 31 sodium hydroxide) the waves start a t f0.72, +060, +0.50 and +0.20 volt, respectively. As liypobromite in bicarbonate medium is reduced at the rotating platinum electrode a t more positive potentials than is oxygen, it should be possible to detect the reduction wave of sodium hypobromite in air-containing media also. This is demonstrated in Fig. 3, which shows the cathodic waves of hypobromite in a 0.24 -1I sodium bicarbonate solution (pH 8.3) in the absence and presence of air, and the current - voltage curve of the air-saturated Potential, V Fig. 3. Current - voltage graphs of 0.55 x M hypo- bromite solution in 0.24 M sodium bicarbonate solution (pH 8.3).Curve A, in presence of air; curve B, in absence of air; cur\.e C, vith supporting electrolyte (0.21 M sodium bicarbonate soluticin, no lixrpobromite, air saturated); curve D, current a t V against amount of 3.1 x 10-3 M sodium hypobromite soliltion added to 30 in1 of 0.24 ;M sodinm bicarbonate solution (pH 8.3) supporting electrolyte. It is of interest that, in the presence of oxygen, the hypobromite wave (Fig. 3, curve -4) starts a t a more positive potential and is steeper than that in the absence of oxygen. This efiect has not been subjected to a detailed study because of the poor reproducibility of the liypobromite wave a t the platinum electrode, the ascending part of the wave being greatly affected by the pre-treatment of the electrode. The diffusion current in the presence of oxygen is not well defined.From the point of intersection of the steep and slowly rising part of the current - voltage curve it is estimated that the current between +0.25 and 0.20 volt corresponds to the diffusion current. In the absence of air, a well-defined diffusion current is found and is equal to the current a t +0.2 volt in the presence of air (compare curves A and B, Fig. 3). The straight line in Fig. 3 (curve D), which was constructed by plotting the current ac +O+ volt against the volume of a 3.1 x &' hypo- bromite solution added to 30 ml of an air-saturated bicarbonate solution, is evidence that the current for sodium hypobromite at +0.2 volt is strictly proportional to the hypobromite concentration. Use of these observations was niadc in the development of amperometric titrations with hypobromite. AMPERONETRIC TITRATION IVITII IITPORROMITT: Amperometric titrations with liypobromite were carried out in carbonate - bicarbonate and borax buffers a t pH values varying from 8.2 to 10.0.The end-point was found to be best defined in a 0.2 M sodium bicarboink solution (pH 8.2)), and this buffer was used in most of the titrations described belon.July, 19531 TITRATION OF TRACES OF AMMOSIA 409 TITRATION OF ARSEXIOUS OXIDE- Arsenious oxide reacts rapidly with hypobromite according to the equation- This reaction can be used for the standardisation of the hypobromite solution. The titration of 3 ml of a 5 x lo-* M arsenite solution in 30 ml of bicarbonate with hypobromite is shown graphically in Fig.4, curve A. It is seen that arsenite gives a small anodic current at the rotated platinum electrode a t +0.2 volt against the saturated calomel electrode. This current decreases upon addition of hypobromite and is zero before the end- point is reached. On further addition of hypobromite a cathodic current is observed, and this increases along a curved line at first and then linearly with the volume of hypobromite .. * * (1) As,O, + 2NaOBr = As,O, + 2NaBr . . .. . . Amount of hypobromite added, mi M As,O, solution in 30 ml of 0.2 14" sodium bicarbonate solution with an unknown hypobromite solution a t + 0.2 V (S.C.E.) a t the rotating platinum wire electrode; curve B, blank titration of 30 ml of 0.2 M sodium bicarbonate solution with the same hypobromite solution Fig.4. Curve A, titration of 3 ml of 5 x added. The end-point is at the intersection of the straight reagent line with the zero current line, which corresponds to the residual current measured at +O-2 volt with the supporting electrolyte in the absence of arsenite. The curved part of the reagent line, which is observed after the zero current is reached, does not correspond to the reaction between arsenite and hypobromite but to a slow reaction between hypobromite and impurities (most likely ammonia) in the bicarbonate solution. This is substantiated by Fig. 4, curve B, which represents a blank titration of the supporting electrolyte (in the absence of arsenite). The blank value is equal to the amount of hypobromite that corresponds to the curved part of Fig. 4, curve A.In a mixture of arsenite and ammonia, the hypobromite apparently reacts first with arsenite and then slowly with ammonia. TITRATION OF AMMOSIA- Ammonia reacts with hypobromite according to the equation- 2NH, + 3NaOBr = N, + 3NaBr + 3H,O . . .. .. . * ( 2 ) This reaction is slow and therefore, in the classical titration method, an excess of hypobromite must be added and titrated back. Amperometric titrations can be carried out directly and rapidly in spite of the sluggishness of reaction ( 2 ) . From Fig. 5 , curve A, which represents an amperometric titration of ammonium chloride in a 0.2 M bicarbonate solution, it is seen that the first increments of hypobromite added give rise to a considerable cathodic current, which decreases on standing and so indicates a slow reaction.On further addition of reagent the current first increases slowly and then decreases. The reaction is complete after a slight excess of hypobromite has been added, when further addition of hypobromite gives a current that is stable and increases linearly with the volume of reagent added. The intersection410 KOLTHOFF, STRICKS ASD MORRES : THE AXPEROMETRIC [Vol. 78 of this line with the zero current line or original residual current gives the end-point. For the performance of a titration it is, therefore, only necessary to measure the current before addition of hypobromite and to record this as the residual current, and then to add reagent until a stable current is observed. A few more increments are then added and the current measured.Fig. 5 , curves B and C, shows the excess reagent lines in ammonia titrations in bicarbonate - carbonate buffers of different pH values. It is seen that a pH higher than 8.2 leads to higher results and also a decrease in the slope of the reagent lines. The results were corrected for the blanks determined for supporting electrolytes of the same pH as those Amount of 3.19x IO~’M sodium hypobromite added, ml Fig. 5. Xniperometric titration at the rotating platinnm wire electrode a t +0.2 V (S.C.E.) of 30 ml of 4.43 x 10-5 M ammonium chloride in: ;1, 0.2 M sodium bicarbonate solution (pH 8.23); B, 0.2 M sodium bicarbonate solution and 0.01 M sodium hydroxide solution (pH 8.78); C, 0.2 M sodium bicarbonate solution and 0.1 M sodium hydroxide solution (pH 9.98), with 3.19 x M sodium hypobromite used in the titrations.Because the hypobromite wave is shifted to more negative potentials at higher values of pH, titrations were carried out at -0.2 as well as at +0.1 volt. At both potentials results were high at the higher pH values. A pH of 8.6 & 0.3 is recommended for the titration. The amperometric titration of ammonia has been applied to the determination of nitrogen in glutathione, bovine plasma albumin and human sera. The Kjeldahl digestion was carried out with concentrated sulphuric acid and persulphate in a way similar to that suggested by Scott and Myers,17 and by Willard and Cake.* This Kjeldahl digestion was applied to known amounts of ammonium sulphate. It was found that ammonia is not oxidised by persulphate if the procedure is carried out under proper conditions.Thus in one instance 0486mg of ammonium sulphate was subjected to the Kjeldahl digestion and the amount recovered, as found by titration with hypobromite, was 0,899 mg, which corresponds to a positive error of 1-5 per cent. METHOD REAGESTS- 10 ml of sulphuric acid (use litmus as indicator). Sodium arsenite solutioiz-Dissolve 1.2363 g of arsenious trioxide (primary standard) in sodium hydroxide and make the solution slightly acid by addition of 0.5 M Transfer the solution to a 250-ml calibrated flaskJuly, 19531 TITRhTIOS OF TRACES OF .\MVONIA 41 1 and fill it to the mark with ammonia-free water. Dilute part of this solution to one-tenth of its strength to produce a 2.5 x 10-3M standard solution of arsenious trioxide.Sodium hypobromite solutiom---Slowly add 20 g of bromine to a solution of 12 g of sodium hydroxide in 500ml of water with constant thorough shaking. Make the solution up to 2 litres. Dilute this solution, which is about 0.06 M in sodium hypobromite and 0.1 M in sodium hydroxide, to half strength and standardise it with sodium arsenite solution. STAKDARDISATIOX OF HYPOBROMITE AGAISST ARSENITE- Place 10 ml of 0.6 M sodium bicarbonate solution and 15 ml of ammonia-free water in a 100-ml beaker. Immerse the rotating platinum-wire electrode and a glass tube with a sintered-glass bottom that is covered with a potassium nikate - agar plug. Into this tube insert the tip of the potassium nitrate - agar bridge, which, by way of a saturated potassium nitrate solution, is connected to a mercurous chromate half-cell.Connect the two electrodes directly through a microammeter. A spotlight galvanometer or a direct-reading Leeds and Northrop microammeter can be used. Record the value of the current, which usually is nearly zero. M standard sodium arsenite solution and titrate with a hypobromite solution that is approximately 3 x 10-2M in sodium hypobromite. As long as the hypobromite is not present in excess, the current is negative or zero. After the end-point, the cathodic current increases rapidly on further addition of hypobromite. When the ammeter indicates that the end-point has been passed, measure the current after the addi- tion of a few more increments of hypobromite. Plot the ammeter readings against the volume of hypobromite added.Draw a horizontal line through the point corresponding to the residual current of the supporting electrolyte (before the addition of sodium arsenite). The point of intersection between this line and the line drawn through the points recorded in presence of an excess of reagent corresponds to the end-point. Carry out a blank titration with the supporting electrolyte (10 ml of 0.6 ill sodium bicarbonate plus 15 ml of ammonia-free water) under the same conditions as for the standardisation. Deduct the blank from the volume of hypobromite used in the standarisa- tion. The molarity of the sodium hypobromite is given by- Add 5 ml of 2.5 x 2 x (volume of arsenite) x (molarity of arsenite) volume of hypobromite The hypobromite solution should be standardised daily.PROCEDURES- Tifration of ammonia-Place bicarbonate solution and water in a 100-ml beaker and measure the current through the supporting electrolyte in the cell as described in the standardisation. to G x 10-4M in ammonia. Titrate with a hypobromite solution of suitable concentration (3 x As long as the hypobromite is not present in excess, the current increases a t first and then decreases in the last stage of the titration before the end-point. As soon as the end-point is reached, the current increases regularly on the addition of reagent. Determine the end-point as described for the standardisation and subtract the blank determination. Add a volume of the sample that will make the solution 4 x to 3 x 10-2M in sodium hypobromite). 1 ml of 3 x 10-2 M hypobrornite solution = 0.34 mg of ammonia (NH,) or 0.28 mg of nitrogen Determination of nitrogen in organic materials-Heat 0.1 to 1 ml of the sample, containing approximately 1 mg of nitrogen, with 1 ml of concentrated sulphuric acid in a 100-ml Kjeldahl flask until the escaping vapour is transparent.Continue heating for 15 minutes and then allow the flask to cool. To the cold solution add 150mg of dry potassium persulphate through a long-stemmed thistle funnel, taking care to prevent the powder from sticking to the neck of the Kjeldahl flask. The solution as well as the persulphate must be water- free, otherwise some ammonia will be oxidised. Heat the flask gently for about 1 minute, at the end of which time the mixture should be colourless.If not, add more persulphate. Generally the amount of persulphate required is not more than 10 times the weight of organic matter in the sample. Boil the colourless solution for 5 minutes to destroy the excess of persulphate. Boil the solution again to remove sulphur dioxide and then transfer it completely to a 25-1111 calibrated flask and allow it to cool. Neutralise it carefully with sodium hydroxide, a t first partly Allow the solution to cool and add 5 ml of ammonia-free water.412 KOLTHOFF, STRICKS AND MORKES : THE AMPEROMETRIC [Vol. 78 with 10 &' sodium hydroxide and then with a 0.1 A4 solution, using a drop of bromocresol green as indicator. Make up the cold neutral solution to the mark with ammonia-free water. Titrate 5 ml of this solution amperometrically with approximately 3 x 10-2 M hypobromite solution as described for the titration of ammonia.Make a blank Kjeldahl digestion and titration with ammonia-free water in place of the sample. RESULTS The results of standardisation of hypobromite solutions of different concentrations It is seen that the results are more reproducible against arsenite are summarised in Table I. TABLE 1 STA?;DARDISATIOli OF HYI'OBROMITE AGAISST ARSEYITE Supporting electrolyte: 30 ml of 0.2 -11 sodium bicarbonate if not otherwise stated Results are corrected for blank Total volume of Hypobromite Concentration Concentration hypobromite used in o f hypobromite -w ml ml M of arseriite, used, blank, found, Remarks Fresh hypohromite solution 3.32 x 0.670 0.043 3.32 x 10-5 (a) 0.695 0.060 3.32 x 10-5 0.688 0.060 3.18 x 10-3 3.32 x 10-6 5.00 x 10-5 5.00 x 5.00 x 10-3 5.00 x 10-6 (a) 2.05 Y 10-4 4.18 x 10-4 4.18 x 10-4 4.18 x 10-4(b) 4-18 x 10-4(4 8.25 x 10-4 6.00 x 10-5 2.Oi x lo-' 0.692 1.005 ...I .005 0.986 0.984 1.000 0.837 0,837 0.853 0.952 0.953 0.970 1.607 0.060 0.060 0.060 0.053 0.053 0.055 0.008 0.008 0.005 0.006 0.005 0.011 0.005 Hypobromite solution one day old 3.17 x 10-3 3.17 x 10-3 3.21 x 1.51 x 1.51 Y lo-? 2.95 x 10-2 3.64 x 10-2 2.61 x 10-2 2.92 x 10-2 (a) Supporting electrolyte: 0.05 M borax (pH 9.2). ( b ) Supporting electrolyte : 0.2 M sodium bicarbonate, approximately 0.01 A t in sodium hydroxide (c) Supporting electrolyte : 0.2 M sodium bicarbonate. approximately 0.1 M in sodium hydroxide (pH 8.68). (pH 9.87).TAELE I1 AMPEROMETRIC TITRATIOSS OF AMBIOSIA WITH HYPOBROMITE Supporting electrolyte : 30 ml of 0.2 M sodium bicarbonate Results are corrected for blank Approximate Titre of molarity in solution, salt added mixture, ammonia, added, found, Error, hypobromite Ammonium ammonia of Blank of Ammonia Ammonia ;M M mg mg "fi % 3.19 x iYH4C1 4.43 x 10-5 0.0016 0.0227 0.023 + 1.3 3.19 x 10-3 (NH4)$04 4.46 x 10-5 0.0016 0.0228 0.023 + 0.9 1.52 x lo-* NH4C1 2.77 x 10-4 0.0017 0.1419 0.147 + 3.4 1.51 x 10-2 (NH4)2S04 2.22 x 10-4 0.0014 0.1140 0.116 + 1.8 1.51 x (NH4)ZSOd 3.22 x 10-4 030014 0.1140 0.117 + 2.9 2.97 x 10-2 NH,Cl 5.60 x 10-4 0.001 7 0.2840 0.286 + 0.5 0.2840 0.287 + 1.0 2.99 x (NH,),SO, 5.80 x 10-4 0.0017 0.2855 0.288 + 0.9 2.99 x 10-2 (NH,)ZSO, 5.60 x 10-4 0.0017 0.2855 0.290 + 1.4 2.64 x NH4C1 5.60 x 10-4 0.0018 0.2840 0.287 + 1.0 2.97 x 10-2 NH4C1 (a) 5.60 x 1.0-4 0.0017 0.2855 0.291 + 1.7 2.97 x 10-2 NH,Cl ( b ) 5.60 x 10-4 0.0135 0.2855 0.300 + 4.9 2.64 x XH4CI (b) 5.60 x 10-4 0,0033 0.2840 0.300 + 5.5 2.97 x 10-2 XH4C1 5.60 x 10-4 0.0017 (a) Supporting electrolyte: 0.2 M sodium bicarbonate, 0.01 M in sodium hydroxide (pH 8.78).( b ) Supporting electrolyte: 0.2 M sodium bicarbonate, 0.1 M in sodium hydroxide (pH 9.98).July, 19531 TITRATION OF TRACES OF 9MMOKI.4 413 in titrations with more concentrated solutions to 3 x 10-2M sodium hypobromite). At these concentrations the blank correction is less than 1 per cent. of the total volume of hypobromite used. From Table I it is also seen that the results of titrations in a borax buffer (pH 9.2) and in a carbonate buffer of about pH 9.9 are from 1 to 2 per cent.lower than those found with bicarbonate as supporting electrolyte. Hypobromite solutions kept for one or two days in a refrigerator show hardly any change in titre. Table I1 shows results for amperometric titrations of ammonia. It is seen that 23 pg of ammonia at a concentration of about 4 x M can be determined with an error of less than 1 per cent. The accuracy and precision of the ammonia titrations is better than 2 per cent. All errors are positive. This must be attributed to formation during the titration of nitrogen oxides, as found by Kolthoff and L a ~ r . ~ At any pH markedly higher than 8.6 the result is high, as is seen from the last two experiments shown in Table 11.Table I11 gives results for the nitrogen content of reduced glutathione and albumin as found after Kjeldahl digestion and titration of the digest with hypobromite. The nitrogen content of glutathione is found to be 13.72 per cent., which compares favourably with the theoretical value of 13.68 per cent. The nitrogen content of albumin is generally assumed to be 16 per cent.,la a value which is in fair agreement with our average result of 16.22 per cent. It is also seen from Table I11 that the blank corrections are not larger than 3 per cent. of the titration results, a factor that is of importance for the accuracy of the method. TABLE I11 AMPEROMETRIC DETERMISATION OF NITROGEN IX REDUCED GLUTATHIONE AND IN BOVINE PLASMA ALBUMIN AFTER KJELDAHL DIGESTION Supporting electrolyte: 30 ml of 0.2 M sodium bicarbonate Concentration of hypobromite used: approximately 3 x 10-2 M Results are corrected for blanks Theoretical amount of Quantity of material nitrogen in used for Kjeldahl Blank the titration Sitrogen Average No.of deter- digestion, of nitrogen, mixture, found, error, minations mg mg mg mg 70 7.37 mg of glutathione 0.0058 0.2016 0.2022 + 0.3 4 6.98 mg of albumin . . 0.0035 0.2232 ( a ) 0.2264 + 1.4 2 (a) This value corresponds to a nitrogen content of 16 per cent. in albumin. Xitrogen determinations in normal and pathological blood sera and in their albumin and globulin fractions are being carried out in this laboratory by the Kjeldahl digestion and titration method described in this paper. 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