Rapid Determination of Nitrates and Nitrites

NELSON, L. T. KURTZ, and R. H. BRAY

J. L.

Department of Agronomy, University of Illinois, Urbana, III.

purpose of this paper is to present a rapid and sensitive Effect of Temperature. No effect over the range of normal
THE
method for the quantitative determination of either nitrite laboratory temperatures was found. Excessively high or low
nitrogen or nitrogen in a mixture of nitrate and nitrite ions, as is temperatures are not recommended (6).
often desired in plant and soil analysis. Effect of Light. The powder should be kept in a black bottle
The procedure utilizes a reaction which was originally suggested until ready for use. During the color development, ordinary ar-
by Griess (3) for the estimation of nitrous acid and was subse- tificial laboratory lighting appears to have no effect, but direct
quently modified and improved by Ilosvay (5), Holbourn (4), sunlight should be avoided.
Morpeth (7), Rider and Mellon (S), and others. The reaction, Stability of the Powder Reagent. One major advantage of the
still widely used for the determination of nitrites in waters and powder reagent is its great comparative stability. If kept in a
many other diverse materials, consists of diazotization of sul- blackened, air-tight bottle, the dry, properly compounded powder
fanilic acid by the nitrite ion and subsequent coupling with 1- is good for at least 2 to 3 years.
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naphthylamine to form a red dye. If the nitrate ion is to be de- Time of Contact of Powder Reagent with Solution. The zinc
termined, it is first reduced to nitrite by powdered zinc and man- must be separated from the solution after color development because
ganese(II) sulfate, as proposed by Bray (1). Bray also combined the reducing action of the zinc will cause the color to fade. Equal
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the reagents into a single dry powder to simplify the procedure color development was found for shaking periods ranging from 20
and to increase greatly the stability of the reagent. This powder to 90 seconds; therefore, a constant 1-minute shaking time (be-
reagent has been used extensively as a qualitative test for nitrate fore beginning of centrifugation) is recommended. Filter paper
in plant tissue. may be used instead of the centrifuge to remove the powder.
REAGENTS
However, filter paper partially adsorbs the dye, decreasing
Standard Nitrate Solution. Dissolve 0.607 gram of sodium greatly the sensitivity and accuracy of the test.
nitrate in 1 liter of water for a stock solution of 100 p.p.m. of Effect of Amount of Powder Reagent Added. The powder
nitrogen. should be in excess, but too much excess is also harmful. Con-
Acetic Acid Solution. Dilute 20 ml. of glacial acetic acid to stant color development was obtained when the amount of pow-
100 ml.
Powder Mixture [As prepared by Bray (1)]. One-hundred der used was between 0.1 and 0.8 gram; therefore, careful meas-
grams of barium sulfate (dried at 110° C.), 10 grams of manganese- urement of the powder in a 0.4-gram scoop is recommended.
(II) sulfate monohydrate, 2 grams of finely powdered zinc, 75 Interfering Ions. Several ions interfere with the reaction. An
grams of powdered citric acid, 4 grams of sulfanilic acid, and 2 excellent table and a discussion of these interferences are given
grams of 1-naphthylamine.
Grind any coarse materials to a fine powder. Mix the man- by Rider and Mellon (8). Iron is complexed by the citric acid and
ganese(II) sulfate, powdered zinc, sulfanilic acid, and 1-naphthyl- copper does not interfere appreciably up to IO7. This would cover
amine separately with portions of barium sulfate. Then mix the range usually encountered in soil and plant material extracts.
thoroughly all ingredients including the barium sulfate and citric It has been found that aluminum does not interfere up to at least
acid. Use extreme care to have room, table top, and equipment
free of nitrate and nitrite. Store the powder in a blackened bottle, 500 times the concentration of the nitrate.
since light affects the 1-naphthylamine. The reagent is stable
for many months in a bottle painted on the outside with black
paint.
If the determination of nitrite in the presence of nitrate is de- Table I. Recovery of Added Nitrate in Soils and Plants
sired, omit the powdered zinc and manganese(II) sulfate. Extract Added, P.P.M. Found, P.P.M. Recovered, %
The citric acid, besides contributing to the acidity of the solu- Soil 1 0 16.2
tion, complexes iron which interferes when present in large quan- 10
20
25.6
37.3
94.0
105.5
tities. The barium sulfate acts as a carrier, zinc and manganese-
(II) sulfate reduce the nitrate, and the sulfanilic acid and 1- Soil 2 0 14.8
naphthylamine are used directly in forming the dye. For best 10
20
24.1
34 8
93.0
100 0
analytical results the powder should be made up several days be-
fore it is to be used. Alfalfa 0 3 4
6 9.3 98.3
EXPERIMENTAL Oat 0 2.0
2 3.8 90.0
Effect of Order of Addition of Reagents. Several workers have 4 5.7 92.5
advised that 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 RESULTS
group when the coupling agent is added. However, since a pro- This method is easily sensitive to 0.05 p.p.m. of nitrogen as
portionate amount of nitrite is destroyed in the standard as well nitrate in solution, and the standard curve is conveniently plotted
as in the unknown, simultaneous addition results only in a slight
between 0.2 and 1.0 p.p.m. in steps of 0.2. More concentrated
loss of sensitivity. solutions should be diluted to this range. Duplicates can be re-
Effect of pH. As shown by Rider and Mellon (§), the reaction
produced in this range within ±5%.
is fairly sensitive to pH changes. The pH curve given by those
Varying quantities of standard nitrate solution were added to
workers was found to hold generally for the conditions of this soil and plant material extracts which were then analyzed by the
determination—that is, maximum color development is obtained recommended procedure. The results are given in Table I.
between the approximate pH values of 1.7 and 3.0. Since the
dye is more stable and the over-all reaction (with simultaneous RECOMMENDED PROCEDURE
addition of reagents) is faster at the lower pH values, a pH level
of about 2.0 is recommended. For this purpose a 20% acetic acid Procure a representative sample of the material to be tested,
and obtain a colorless extract of it. An extraction with O.IN hy-
solution gives approximately the correct pH and is a medium drochloric acid is recommended for soils or ground plant material.
from which the powder is easily centrifuged. Strongly colored plant-material extracts may be decolorized by a
1081
1082 ANALYTICAL CHEMISTRY

method such as that described by Frear (#). Solutions which are to close the tubes during shaking must be washed carefully. As
strongly acid or alkaline should be neutralized, and any interfering reported by Warington (S), solutions and reagents unduly exposed
ions present should be removed or eomplexed.
To 1 ml, of the test solution in a 12-ml, centrifuge tube, add 9 to laboratory air may pick up small amounts of nitrite. Distilled
ml. of the acetic acid solution. With a small measuring scoop water must be checked frequently for contamination.
add 0.3 to 0.5 gram of the powder reagent, stopper, shake for 50
to 60 seconds, and centrifuge until the supernatant liquid is clear
(about 4 or 5 minutes at 4000 r.p.m.). Decant the white film LITERATURE CITED
on top and pour the clear red solution into an absorption cell.
(1) Bray, It, H,, Soil Sci., 60, 219 (1945).
Transmitfancy may be read conveniently in a filter photometer (2) Frear, D, E., Plant Physiol., 5, 359 (1930).
with a green filter or in a spectrophotometer at 520 mg. Griess, Peter, Ber. deut. chem. Ges., 12, 426 (1879).
Convert transmittancy readings to amounts of nitrogen with a (3)
standard curve established with known solutions. The standard (4) Holbourn, A. H. S., and Pattle, It. E., J. Lab. Clin. Med.. 28, 1028
curve should cover the range from 0.2 to 1.0 p.p.m. in solution in
(1943).
the absorption cell, or 2 to 10 p.p.m. in the sample solution. (5) Ilosvay, M. L., Bull, soc. chim. Paris, 2, 347 (1889).
(6) Moffitt, W. G„ Analyst, 63, 655 (1938).
(7) Morpeth, J. C., Chemistry & Industry, 1946, 370.
PRECAUTIONS (8) Rider, B. F., and Mellon, M. G., Ind. Eng. Chem., Anal. Ed.,
All glassware, table tops, etc., must be kept scrupulously clean. 18, 96 (1946).
(9) Warington, Robert, J. Chem. Soc., Trans., 39, 229 (1881).
Reagents should be nitrite- and nitrate-free. X-ray grade barium
sulfate is satisfactory if thoroughly dried. Rubber stoppers used Received for review November 12, 1953. Accepted March 5, 1954.

Device for Measuring Changes of Optical

Transmittance with Temperature
ARTHUR FURST, Department of Pharmacology and Therapeutics, Stanford University School of Medicine,
San Francisco, Calif., and

JUSTIN J. SHAPIRO, American Instrument Co., Silver Spring, Md.

relative purity of a compound is invariably associated making this determination automatic. Perhaps the first of these
THE
with a physical constant. A melting point or molting range is was made by Dubose (S) and a later one by Wick and Barchfeld
customarily used for solids and this value is most often deter- (11). At this time true automatic equipment was not available.
mined by the capillary tube technique. The melting point ob- Kardos (6) used a Kofler (7) apparatus and measured the melting
tained by this method is not thermodynamically defined, for it is point by attaching a recorder to a photoelectric cell and noting
neither the liquid-solid equilibrium temperature under constant the change in current in milliampores. More recently Muller and
atmospheric pressure nor the triple point. However, it is ex- Zenchelsky (9) made a fully automatic instrument based upon
tremely convenient and universally employed. Reproducible re- the sudden increase of reflected light from the surface of the
sults are obtained and no difficulties are encountered if the sam- melted sample to a photoelectric cell. This instrument has been
ple to be tested is relatively pure, of definite composition, and able to attain a precision of ±0.3° C.
actually melts below 230° C. The meltomcter (10) here described reduces to fixed and repro-
Frequently samples are encountered whose melting point can- ducible values the variables contributing to the melting point
not be determined reproducibly by the capillary tube method.
These solids can be classified in one or more of the following cate-
gories: hydrates; high melting—i.e., above 250° C.; compounds
which decompose before or at their melting point; amorphous
waxes, asphalts, or plastics; and vegetable oils or fats. In each
of these categories special techniques must bo used. In testing
these chemicals, operators may get results that differ as much as
several degrees from each other. Morton (S) lists the factors that
affect melting point values. Included among these are rate of
heating, thickness of.the capillary wall, size of the sample, and
stem corrections. If capillary tube technique is employed for
these special samples, different manipulative techniques are
necessary. For compounds which decompose or melt above 250°
C. the capillary tube must be either submerged when the tem-
perature of the bath is just a few degrees below the melting value,
or as an alternative, the capillary must be heated at a rate of 10°
to 20° C. per minute, constantly, rather than slowing to the usu-
ally prescribed 1° to 2° C. per minute near the melting point. A
ball and ring test (I) is often employed to test asphalts. Fats
present special problems; an allotropic form may result after the
sample is melted and drawn up into the capillary tube (5); hence
the filled tube must be left in the icebox over night.
Variations to avoid the capillary tube method were employed
by Dennis and Shelton (%), who introduced the copper bar, and
by Johns (J), who used an aluminum block upon which the
sample, placed between two watch glasses, was heated.
Attempts have been made to avoid the human equation by Figure 1. Meltometer