Rapid Determination of Nitrates and Nitrites 1. L. NELSON, L. T. KURTZ, and R.
H.BRAY
Department of Agronomy, University o f Illinois, Urbana,
T
HE purpose of thiq papei is to present a rapid and sensitive method for the quantitative determination of either nitrite nitrogen or nitrogen in a mixture of nitrate and nitrite ions, as is oftcn desired in plant and -oil analysis. The procedure utilizes a reaction which was originally suggested by Griess ( 3 ) for the estimation of nitrous acid and was subsequently modified and improved by Ilosvay ( 5 ) , Holbourn (41, Morpeth ( 7 ) , Rider and Xellon ( 8 ) , and others. The reaction, still widely used for the determination of nitrites in waters and many other diverse materials, consists of diazotization of sulfanilic acid by the nitrite ion and subsequent coupling with 1naphthylamine to form a rrsd dye If the nitrate ion is to be determined, it is first reduced to nitrite by powdered zinc and mangancir( 11)sulfate, as proposed by Bray (1). Bray also combined the rc.agcnts into a singlr dry powder to simplify the procedure and to increase greatly the etahility of the reagent. This powder reagent has been usrd e~tc~nqivcly as a qualitative test for nitrate in plant tissue. RE4GEYTS
Standard Nitrate Solution. Dissolve 0.607 gram of sodium nitrate in 1 liter of water for a stock solution of 100 p.p.m. of nitrogen. Acetic Acid Solution. Dilute 20 ml. of glacial acetic acid to 100 nil. Powder Mixture [ A s prepared by Bray ( I ) ] . One-hundred grams of barium sulfate (dried at 110" C.), 10 grams of manganese(11) sulfate monohydrate, 2 grams of finely powdered zinc, 75 grams of powdered citric acid, 4 grams of sulfanilic acid, and 2 grams of 1-naphthylamine. Grind any coarse materials to a fine powder. Mix the manganese(I1) sulfate, powdered zinc, sulfanilic acid, and l-naphthylamine separately with portions of barium sulfate. Then mix thoroughly all ingredients including the barium sulfate and citric acid. Use extreme care t o have room, table top, and equipment free of nitrate and nitrite. Store the pmvder in a blackened bottle, since light affects the I-naphthvlnmine. The reagent is stable for many months in a bottl? painted on the outside with black paint. If the determination of nitrite in the presence of nitrate is desired, omit the powdered zinr and manganese(I1) sulfate. The citric acid, besides contributing to the acidity of the solution, complexes iron which interferes when present in large quantities. The barium sulfate acts as a carrier, zinc and manganese(11) sulfate reduce the nitrate, and the sulfanilic acid and 1naphthylamine are used directly in forming the dye. For best analytical results the ponder should be made up several days before it is to be used
111. Effect of Temperature. N o effect over the iange of normal laboratory temperatures was found. Excessively high or low temperatures are not recommended ( 8 ) . Effect of Light. The powder should be kept in a black bottle until ready for use. During the color development, ordinary artificial laboratory lighting appears to have no effect, but direct sunlight should be avoided. Stability of the Powder Reagent. One major advantage of the powder reagent is its great comparative stability. If kept in a blackened, air-tight bottle, the dry, properly compounded powder i p good for at least 2 t o 3 years. Time of Contact of Powder Reagent with Solution. The zinc must be separatedfrom the solution after color development because the reducing action of the zinc will cause the color to fade. Equal color development was found for shaking periods ranging from 20 to 90 seconds; therefore, a constant 1-minute shaking time (before beginning of centrifugation) is recommended. Filter paper may be uqed instead of the centrifuge to remove the powder. Howevcr, filter paper partially adsorbs the dye, decreasing greatly the sensitivity and accuracy of the test. Effect of Amount of Powder Reagent Added. The powder should be in excess, but too much excess is also harmful. Constant color development was obtained when the amount of powder used was between 0.1 and 0.8 gram; therefore, careful measurement of the powder in a 0.4gram scoop is recommended. Interfering Ions. Several ions interfere with the reaction. An excellent table and a discussion of these interferences are given by Rider and Mellon (8).Iron is complexed by the citric acid and copper does not interfere appreciably up to 107. This would cover the range usually encountered in soil and plant material extracts. It has becn found that aluminum does not interfere up to at least 500 times the concentration of the nitrate.
Table I. Extract Soil 1
Soil 2
EXPERIXIEYTAL
Effect of Order of Addition of Reagents. Several workers have advised t h a t the sulfanilic acid be added several minutes before the 1-naphthylamine, ostensibly because any unreacted nitrite still present is partially destroyed by reaction with the amino group when the coupling agent is added. However, since a proportionate amount of nitrite is destroyed in the standard as well as in the unknown, simultaneous addition results only in a slight loss of sensitivity. Effect of pH. As shown by Rider and Mellon (8),the reaction is fairly sensitive to pH changes. The pH curve given by those workers was found to hold generally for the conditions of this determination-that is, maximum color development is obtained between the approximate pH values of 1.7 and 3.0. Since the dye is more stable and the over-all reaction (with simultaneous addition of reagents) is faster a t the lower pH values, a pH level 3f about 2.0 is recommended. For this purpose a 20% acetic acid solution gives approximately the correct p H and is a medium from which the powder is easily centrifuged.
Recovery of Added Nitrate in Soils and Plants Added, P.P.M. 0 10 20
Found, P.P.A.I. 16 2 2.5 6 37 3
0
10 20
14.8 24 1 34 8
Alfalfa
0 6
3 4
Oat
0
2 0 3 8 5 7
2
4
I)
:i
Recovered, % 94.0 105.5 93.0 100 0 98 3
90 0 92 5
RESULTS
This method is easily sensitive to 0.05 p.p.m. of nitrogen as nitrate in solution, and the standard curve is conveniently plotted between 0.2 and 1.0 p.p.m. in steps of 0.2. More concentrated solutions should be diluted to this range. Duplicates can be reproduced in this range within &5%. Varying quantities of standard nitrate solution were added to soil and plant material extracts which were then analyzed by the recommended procedure. The results are given in Table I. RECOMMENDED PROCEDURE
Procure a representative sample of the material to be tested, and obtain a colorless extract of it. An extraction with 0.1N hydrochloric acid is recommended for soils or ground plant material. Strongly colored plant-material extracts may be decolorized by a
1081
1082
ANALYTICAL CHEMISTRY
method such as that described by Frear (8). Solutionswhiohare strongly acid or alkaline should be neutralized, and any interfering ions present should he removed or complexed. To 1 ml. of the test solution in a 12-ml. centrifuge tube, add 9 ml. of the acetic acid solution. With a mall measuring scoop add 0.3 to 0.5 gram of the powder reagent, stopper, shake far 50 to 60 seconds, and centrifuge until the supernatant liquid is elem (about 4 or 5 minutes a t 4000 r.p.m.). ,Decant the white film on top and pour the clear red solution into an absorption cell. Trausmittancy mrty be read conveniently in a filter photometer with a green filter or in a spectrophotometer at 520 m r . Convert trsnsmittmcy readings to amounts of nitrogen with it standard curve established with known solutions. The standard eume should cover the range from 0.2 to 1.0 p.p,m. in solution in the absorption cell, or 2 to 10 p.p.m. in the sample solution.
to close the tubes during shaking must he washed cmefully. As reported by Warington (9),solutions and reagents undulyexposed to laboratory air may pick up small rtmounts of nitrite. Distilled water must be checked frequently for contamination. LITERATURE CITED
(1) Bray, R. H.. Soil Sci., 60,219 (1945). (2) Frear, D.E., Plant Physiol.. 5, 359 (1930). (3) Griess, Peter, Rer. deut. chem. Ges., 12,426 (1879). (4) Holbourn, A.H. S., and Pattle. It. E., J . Lab. C i h . Med.. 28, 1025 (1943). ( 5 ) Ilosvay, M. L.. Bull. w e . ehim. Paris, 2. 347 (1889) Itt, W. G., Analyst, 63, 655 (1938)
PRECAUTIONS
All glassware, table taps, etc., must be kept SONPU~OUS~Yclean. Reagents should be nitrite- and nitrate-free. X-ray grade barium sulfate is satisfactory if thoroughly dried. Rubber stoppers used
Device for Measuring Change!i of Optical ... I ranemittanrra wttn Iamnaratsre
I
..I
I
ARTHUR FURST, Department of
Pharmacology and Therapeutics, Stanford University School of Medicine,
San Francisco, Calif., and
JUSTIN J. SHAPIRO. American
T.
instrument
CO, Silver SI
HE reladive purity i f a compound is invariably associated with a physical constant. A melting point or melting range is customarily used for solids and this value is most oft,en drtwmined by the capillary tube technique. The melting point. oht.ained by this method is not thermodynmieally defined, for it is neither the liquid-solid equilibrium temperature under constant atmospheric pressure nor the triple point, However, it is extremely convenient and universally employed. Reproducible results are obtained and no difficulties are encountered if the sample to he tested is relatively pure, of definite composition, and actually melts below 230" C. Frequent,ly samples are encountered whose melting point cannot be determined reproducibly by the capillary tube method. These solids e m be classified in one or more of the following catogories: hydrates; high melting-Le., above 250' C.; compounds which decompose before or a t their melting point; amorphous waxes, asphalts, or plastics; and vegetable oils or fats, In each of these categories special techniques must bo used. In besting these chemicals, operators may get results that differ as much &R several degrees from each othel'. Morton (8) lists the factors that affeot melting point values. Included among these are rate of heating, thickness of t,hc capillary wall, size of the sample, and stem corrections. If capillary tube technique is employed for t.hese special samples, different manipulative techniques BPP necessary. For compounds which decompose or melt above 250" C. the capillary tube must be either submerged when the temperature of the bath is just a few degrees below t,he melting value, or a6 an slternative, the capillary must he heated at a mtc of 10' to 20" C. per minute, constantly, rather than slowing to thc usually prescribed 1' to 2" C. per minute near the melting point. A ball and ring test ( I ) is often employed to te8t asphalts. Fats present specid problems; an allotropic form may result aftcr the sample is melted and drawn up into the eitpillmtry tube (6); hence the filled tube must be left in the icebox over night. Variations to avoid the capillary tube method were cmployed by Dennis and Shelton (e), who introduced the copper bar, and by Johns (4),who used an aluminum block upon which the sample, placed between two watch glasses, was heated. .4ttcmpts have been made to avoid the human equation by
making this determination automatic. Perhaps the first of thcec w m made by Dubosc (3) and a lstcr one by Wick and Barchfdd (11). At this time true automatic equipment was not available. Kardos ( 6 ) used a Kofler (7) apparatus and measured the melting point by attaching a recordor to a photoelectric cell and noting the change in current in milliitmporees. More recently U.liillcr and Zenchelsky (9) made a fully automatic instrument based upon the sudden increase of rcflceted light from the surface of t.he melted sample to a photoclnetrie eel!. This instrument has becn :able t o attain a precision of +0.3" C. The meltomoter ( I O ) here described reduces to fined and reproducihle values the vnrinhles contributing to the malting point
Figure 1. Meltometer
