Polarographic Determination of Nitrates in Blood and Urine EUGENE W. SCOTT AND KARL BAMBACH Kettering Laboratory of Applied Physiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio
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by Cholak and Bambach ( I ) . The step height in the case of urine samples was slightly higher (3 to 5 per cent) for a given amount of nitrate. Separate standard curves were therefore used for urine and for blood. The urine filtrate is prepared by mixing 0.5 ml. of fresh urine with 0.5 ml. of mercuric chloride solution (5 grams per 100 ml.) and 5.0 ml. of saturated barium hydroxide solution, diluting to 10 ml. with water, and filterin . One milliliter of this filtrate is used in the electrolysis cell unjer the conditions described above for the final blood filtrate. The lanthanum chloride solution is prepared by dissolving lanthanum oxide (obtained by ignition of the acetate) in a slight excess of hydrochloric acid. A large excess of acid is avoided, since it has to be neutralized by the filtrate used. The nitrate step appears only in neutral or alkaline solutions. The half-wave potential of the nitrate step is 1.33 to 1.39 volts in the case of blood filtrates, and 1.47 to 1.53 volts in the case of urine filtrates, when referred to the standard calomel electrode. The difference in half-wave potential was probably due to a difference in supporting electrolytes.
OLORIMETRIC methods have been used successfully
for the determination of small amounts of nitrate in water and food extracts, but in the authors' experience the application of these methods to biological materials such as blood and urine has not been satisfactory. Whelan's method (4) was also tried, but the difficulty of preventing atmospheric oxidation of the reagent, diphenylbenzidine, rendered this procedure unsuitable. The use of the polarograph for the determination of nitrates has been reported by Tokuoka (3) and has been recently discussed by Kolthoff and Lingane (2). After a suitable procedure was developed for the removal of proteins and interfering substances, this method was used for blood and urine filtrates, with only slight modifications. 130
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When the higher sensitivity of the galvanometer is used, i t is possible to determine as little as 0.5 microgram of nitrate in 1 ml. of the electrolysis mixture, which is equivalent to 40 micrograms of nitrate per milliliter of blood. The full sensitivity of the instrument cannot be employed because of the salts and other substances present in the filtrates. With pure solutions of nitrate it should be possible to determine as little as 0.1 microgram in 1ml.
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MICROGRAMS NO,
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The apparatus employed is a Leeds & Northrup ElectroChemograph, equipped with a Micromax recorder and the dropping mercury electrode and electrolysis cell which were supplied with the instrument. Galvanometer sensitivity ranges of 0 to 80 and 0 t o 8 microamperes are used. The estimations are made graphically by comparin the step height obtained with the samples to those obtainecf by adding known amounts of nitrate t o blood and urine filtrates which previously showed no nitrate step. The standard charts prepared in this way are shown in - Figures 1 and 2. The blood filtrates are prepared by mixing 0.5 ml. qf fresh blood with 3.5 ml. of water, and adding 0.5 ml. of mercuric chloride solution (5 grams per 100 ml.) and 6.5ml. of sodium carbonate solution (1 ram per 100 ml.). After filtering, 2 ml. of the filtrate are acidifiefwith 1 ml. of 0.4 N hydrochloric acid, heated t o boiling, cooled, and made alkaline with 1 ml. of 1 N sodium hydroxide. One milliliter of this solution, which is equivalent t o 0.05 ml. of blood, is placed in the electrolysis cell and mixed with 1 ml. of Ivater and 2 ml. of 0.1 N lanthanum chloride, and nitrogen is bubbled through the solution for 5 minutes. The current set up by electrolyzing this solution at 30" over a range of 1.0 to 1.8 volts is recorded and the height of the nitrate step is measured from this chart. The method of measurement was that employed
MICROGRAMS NO3
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Methods other than those described above for the preparation of the blood and urine filtrates were studied before these procedures were adopted. Most of the common protein precipitants were tried, but in every case the final solutions contained residual amounts of these precipitants, which interfered in the electrolysis. Urea does not cause interference but uric acid, which is present in human urine, does, unless it is removed. 136
February 15, 1942
ANALYTICAL EDITION
The method of polarographic determination here reported has so far been Only to rabbit and urine* The appearance and increased concentration of nitrate in both fluids after the oral or intravenous ingestion of certain aliphatic nitro compounds were successfully followed with this procedure. Results obtained will be reported elsewhere.
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Literature Cited (1) Cholak, J., and Bambach, K., IND. ENG.CHEM.,ANAL.ED.13, 583 (1941). (2) Kolthoff, I. hf., and Lingane, J . J., “Polarography”, P. 314,New York, Interscience Publishers, 1941. (3) Tokuoka, M., Collection Czechoslov. Chem. Commun., 4, 444 (1932). (4) Whelan, I f . , J . Biol. Chem., 86, 189 (1930).
Determination of Combined Nitrogen in Steel A Rapid Method H. F. BEEGHLY Research and Development Division, Jones 81 Laughlin Steel Corporation, Pittsburgh, Penna.
A rapid method for determining combined nitrogen in steel utilizes a microKjeldahl steam-distillation unit and a photoelectric spectrophotometer in conjunction with the well-known Nessler color reaction. The method is accurate and can be used for micro-, semimicro-, or macrosamples. The procedure is suitable for
N
ITROGEK is believed by many investigators to exercise a considerable influence on the physical properties of steel. The large number of determinations necessary for control purposes and to provide data for statistical studies make it essential to have an analytical method which, without sacrifice of accuracy, will reduce to the minimum the time and labor of a single nitrogen determination. The Allen and the vacuum fusion methods are the most commonly used for determining nitrogen in steel. Seither is sufficiently rapid to use for control purposes nor to care adequately for the large numbers of routine determinations made necessary by present-day quality requirements. The experience of many industrial and research laboratories has indicated the value of micro and semimicro analytical methods where rapid accurate determinations are required. Using a micro-Kjeldahl unit designed t o utilize steam-distillation in removing ammonia from alkaline solution, Klinger and Koch (5) developed a micromethod for determining the nitrogen content of steel surfaces. The method was sensitive and gave accurate results but, because of the large volume of reagents used, substantially the same time was required for a single determination as in the regular macroprocedure. Most of the micro-Kjeldahl units available commercially are not suitable for determining the nitrogen content of a series of samples in rapid sequence, The purpose of this paper is t o describe a rapid and accurate method developed in this laboratory for determining combined nitrogen in carbon steel. The method utilizes a standard micro-Kjeldahl unit which is suitable for rapid determinations either singly or in series. The ammonia in the distillate is determined by means of the color reaction introduced by Kessler. A photoelectric spectrophotometer is used t o measure the intensity of the ammonia-Xessler color complex. The procedure may be applied t o steels for which the usual macromethods are suitable and is also snitable for
investigational work and has been in use for routine determinations for approximately one year with appreciable economy of labor and time. It is applicable to all steels containing acid- or alkali-soluble nitrogen compounds and may be modified to accommodate materials containing nitrogen in a difficultly soluble form.
small quantities of material for which macroprocedures are not applicable. Modifications of the method are necessary for pig irons and alloy steels containing nitrogen compounds not readily soluble in acid or alkali. The chemical reactions for the microprocedure are essentially the same as for the macroprocedure, except that the
OF MICRO-KJELDAHL UNIT FIGURE 1. DIAGRAM
