Polarographic Determination of Nitrate WALTER A. LAWRANCE
AND
RICHARD M. BRIGGS
Hedge Laboratory, Bates College, Lewiston, Maine
Most of the nitrate determinations in this investigation were conducted on water which had a p H range of 6.0 to 6.4.
wo methods of determining nitrate ( 1 ) have been employed Tin this laboratory for 5 years, but are somewhat unsatisfactory, when applied to Androscoggin River water. The phenoldisulfonic acid method gives brownish yellows which are not sufficiently reproducible to be of much value. The reduction method is more satisfactory, but the procedure is slow and subject to considerable error when applied to water containing very little nitrate ion. The polarographic determination of nitrate ( 3 )was extensively studied during the summer of 1952 as a control method for the application of sodium nitrate to the Androscoggin Pool ( 2 ) . About 850 determinations were made, using a Model 12 polarograph manufactured by E. H. Sargent & Co. This method was found to be rapid and to furnish reasonably reproducible results when certain modifications and precautions were made.
METHOD OF ANALYSIS
The zirconyl chloride electrolyte was prepared as directed liy Rand and Heukelekian ( 3 ) . To a carefully cleaned electrolysis vessel mercury was added to cover the platinum wire sealed into the side and 5.0 ml. of sample was pipetted into the cell. S e x t 0.5 ml. of zirconyl chloride reagent was added from a pipet, the dropping mercury electrode was rinsed with distilled water and inserted, and finally nitrogen was passed through the solution for 5 minutes. After the nitrogen was shut off, the polarogram was recorded of the current read a t an applied potential of 1.20 volts. This potential is very critical. More reproducible results were obtained, due to the better voltage control, by setting the meter a t 3.0 volts and the ring scale a t 0.4. Next 0.20 ml. of the ferrous ammonium sulfate reagent was added and nitrogen was again passed through the solution for 5 minutes. During this period a dense white precipitate formed. Finally the polarogram was recorded on the same axes as before or the current was again read with 1.20 volts applied across the cell. A shunt ratio of 10 was employed.
The use of ferrous sulfate for the reduction of nitrate and nitrite did not give consistent results and a t times appeared to be highly erratic, probably because of the amount of ferric ion pres ent in the solution. hddition of iron wire and acid to the ferrous sulfate solution was also unsatisfactory. Ferrous chloride was not investigated. Ferrous ammonium sulfate was found to be much more stable and gave more consistent results; 19.6 grams of ferrous ammonium sulfate hexahydrate and 0.5 ml. of concentrated sulfuric acid were dissolved in enough water to make 100 ml. of solution, a piece of standard iron wire which had been cleaned with sandpaper and rinsed with dilute sulfuric acid and water was added, and the flask was stoppered lightly. Commercial water-washed cylinder nitrogen required purification by passing through 5 % pyrogallol in 25% potassium hydroxide and through two bottles containing deoxygenated distilled water. Triple distilled mercury was employed. Bfter use mercury was washed with water. passed four or five times through a column of 10% nitric acid, and then washed several times JT ith distilled water. Originally, after each determination, the mercury was washed and then distilled under reduced pressure in a rapid current of air. Such frequent distillation was found to be unnecessary and so was not made part of the usual routine. However, it is essential that the mercury be freed of all traces of the whitish zirconyl film. Occasional distillation is necessaIy. An electrolysis vessel (Heyrovsk9, Erlenmeyer style) made of borosilicate glass, was used. During the early part of this work results a t times suddenly became very erratic. After considerable experimentation the difficulty was found to be due to the presence of a whitish film, frequently invisible to the unaided eye, which adhered tenaciously to the glass cell and capillary, and was very difficult to remove. The film is believed to be related to the white precipitate that forms during the second half of the analysis. The capillary was cleaned by placing it in concentrated nitric acid for about 0.5 hour each day. The electrolysis vessel was cleaned by adding concentrated nitric acid and allowing it to stand overnight. When many continuous determinations were made, the cells were used for about 3 hours and then cleaned. Slime and zoogleal suspensions must be removed prior to analysis, as they completely suppress the nitrate wave. A similar suppression may be produced by addition of a few drops of 0.01 % gelatin. In this laboratory the standard flocculation procedure employs ferric chloride as a coagulant. This, however, is impractical for polarographic nitrate determination, because the amount of ferric ion added is extremely critical if flocculation is to be obtained without leaving in solution an amount of ferric ion that seriously interferes. Aluminum chloride was adopted as a coagulating agent, as this was found to be nearly as effective as ferric chloride and a slight excess is not objectionable. At certain locations Androscoggin River water may contain sulfide, disulfide, and mercaptans. When these are present in sufficient quantities, they contaminate the mercury and thus render the analysis unreliable. The concentrations of these interfering compounds, other than sulfide, were not determined. Hydrogen sulfide (0.1 p.p.m.) in the water was found to invalidate the analysis. Owing to the high hydrogen ion concentration in the reagents, this method should be applicable to both acid and alkaline waters.
STASDARDIZATION
Solutions containing 0.1 to 20.0 p.p.m. of nitrate nitrogen were prepared by the quantitative dilution of various amounts of x standard sodium nitrate solution and analyzed as described above. A curve of concentration us. scale reading a t 1.20 volts applied across the cell was plotted and found to be linear over those portions of the curve which were made with the instrument set a t a constant sensitivity, but there were slight variations from linearity when t’he sensitivity was changed. 4 s the curve could be represented by a straight line which did not pass through the origin, it conforms to an equation of the type
P
=
(DS - C ) / F
where P is the concentration of nitrate nitrogen in parts per million F is a proportionality factor D is the difference between the two measured diffusion currents a t an applied potential of 1.20 volts S is the sensitivity setting on the polarograph C‘ is an empirical correction factor which compensates for the “ferric ion effect” and also any possible traces of nitrate in the zirconyl chloride solution When the data were subjected to the above equation to calculate the valuesof C and F , i t was found,as had been ant,icipated, that F varies only with the sensitivity factor and even this is nearly constant, while C is essentially independent of the sensitivity but varies with time and must be redetermined several times a day. The variations in C are usually small and may be safely ignored if the nitrate concentrations are high. In working with concentrations about 10 p.p.m. C becomes negligible in comparison with the other errors and may be dropped from the calculations, provided the reagents are fresh. Because both ferrous and ferric ions, as well as many other substances, interfere in this analysis, determinations were made on river water containing known amounts of nitrate nitrogen. Usually there were not enough interfering substances present to cause difficulty and the experimentally determined constants for solutions in distilled water were found to fit the data on river water adequately. Because it is necessary to “fix” the river water with chloroform a t the time of sampling, to ensure no change in nitrate nitrogen due to microbial action during variable periods of time before analysis, river water and distilled water solutions containing known
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ANALYTICAL CHEMISTRY
amounts of nitrate were saturated with chloroform and analyzed. No interference due to chloroform could be detected even after standing a t room temperature for several days. For other work it was necessary to determine nitrate in the presence of widely varying amounts of phosphate ion, to learn the effect, if any, of such variations on the polarographic nitrate analysis. It was found that the phosphate ion (KH,POI) does not interfere in concentrations up to 25 p.p.m. Higher concentrations were not studied. Accuracy of Polarographic Method. Although concentraions of 0.10 p.p.m. or less nitrate nitrogen may be determined by the polarographic method, more consistent results were obtained by adding to the sample sufficient nitrate to bring the concentration to about 2.0 p.p.m. when testing samples known to contain 0.5 p,p.m. of nitrate nitrogen or less.
Within the range of 0.1 to 10 p.p.m. determination of known nitrate concentrations in distilled water gave results which averaged less than 2% error. Results in filtered Androscoggin River water were in error by less than 3% of a known nitrate increment. Water samples which required flocculation treatment averaged less than 5% error. LITERATURE CITED
(1) Am. Public Health Assoc., New York, “Standard Methods for
the Examination of Water and Sewage,” 9th ed., pp. 69-71, 1946. (2) Lawrance,W. A , , Sewage andInd. Wustes, 22,820 (1950). (3) Rand, AI. C., and Heukelekian, H., ANAL.CHEM.,25, 878 (1953). RECEIVED for review liovember 1 3 , lQ52. Accepted March 12, 1953. This investigation was made possible by a grant from the National Council for Stream Improvement.
Titration of Calcium and Magnesium in Milk and Milk Fractions with Ethylenediamine Tetraacetate ROBERT JENNESS Department of Agricultural Biochemistry, Unicersity of Minnesota, S t . Paul, Minn.
method of Schwarzenbach and coworkers ( 4 , for Ttitrating . calcium and magnesium with ethylenediamine 15) 16)
HE
tetraacetate and suitable indicators offers the advantages of rapidity, simplicity, and accuracy. It has found wide application in the determination of hardness in water ( 2 , 3, 8, 9 ) and in determination of calcium and magnesium in limestone and soils ( 1 , 6, 7 ) . I t has been employed successfully for titrating calcium in blood serum, urine, and spinal fluid ( 1 1 , 18, 1 7 ) . Attempts to use it for plant materials (6, 10, 1 8 ) have not been entirely suceessful because orthophosphate interferes with the end point. Obviously, milk, in which the atomic ratio of calcium to phosphorus is approximately 1 to 1, cannot be analyzed by this technique without precautions to eliminate the interference of phosphate. The present paper describes a simple anion exchange technique for overcoming this difficulty by removing phosphate. Furthermore, several alternative methods of preparing milk samples for the analysis have been studied and shown to be satisfactory. APPARATUS AND REAGENTS
Porcelain casseroles, 2-inch and 3-inch diameter Muffle furnace Electric heaters, such as Precision Scientific Co.’s type R H or Ful-Kontrol, with refractory having hole 1 inch in diameter Kjeldahl flasks, 100 ml. Volumetric flasks, 50 and 100 ml. Anion exchange columns. Place 3 grams of the resin Duolite A-4 (Chemical Process Co., Redwood City, Calif.) in a column 7 X 250 mm., with a reservoir 15 X 100 mm. a t the top and a capillary 1 X 25 mm. a t the bottom. Attach a short length of rubber tubing to the capillary with pinch clamp. Prepare the columns for use by backwashing with water to stratify the resin particles and eliminate air, passing several portions of 1 N sodium acetate, and rinsing with distilled water. Microburet, 5-ml. capacity, calibrated in 0.01-ml. divisions Hydrochloric acid, 1 N Nitric acid, concentrated, reagent grade Perchloric acid, 72%) double distilled Sodium hydroxide, 0.5 N and 1.5 N Standard titrating solution. Dissolve 10 grams of disodium dihydrogen ethylenediamine tetraacetate dihydrate (Versene from Bersworth Chemical Co., Framingham, Mass., or Sequestrene from Alrose Chemical Co., Providence, R. I.) and 2 grams of sodium hydroxide pellets in water, and make up to 1 liter. Standardize this solution, which has a titer of approximately 1.0 mg. of calcium or 0.6 mg. of magnesium per milliliter, by titrating standard calcium chloride and magnesium chloride solutions prepared by dissolving respectively calcium carbonate or metallic magnesium in hydrochloric acid and making up to the desired volume.
Calcium indicator. Prepare indicator by grinding 0.2 grams of ammonium purpurate and 100 grams of sodium chloride to an intimate mixture ( 2 ) . Calcium and magnesium indicator. Dissolve 1 gram of Eriochrome Black T in a mixture of 30 ml. of distilled water and 1 ml. of 1 A’ sodium carbonate, and make up to 100 ml. with 2propanol ( 2 ) . Buffer solution 1. Dissolve 4 grams of C.P. sodium tetraborate decahydrate in approximately 80 ml. of distilled water. Buffer solution 2. Dissolve 1 gram of C.P. sodium hydroxide and 0.5 gram of C.P. sodium sulfide in 10 ml. of distilled water. Cool. Mix the two solutions and make to 100 ml. PROCEDURE
Preparation of Samples. Milk samples are prepared for analysis by dry ashing, wet digestion, or acid precipitation of the casein. In dry ashing, a 5-ml. sample in a porcelain dish of 2-inch diameter is evaporated to dryness on the steam bath and incinerated in a muffle furnace a t 600” C. overnight. The ash is moistened with a little distilled water, dissolved with 1 ml. of 1 N hydrochloric acid, transferred quantitatively to a 50-ml. volumetric flask, and made to volume. In wet digestion a 5-ml. sample is digested in a 100-ml. Kjeldahl flask on an electric heater with 5 ml. of concentrated nitric acid until copious brown fumes cease to be evolved. The flask is cooled, 2 ml. of 72% perchloric acid is added, and the digestion is continued until heavy white fumes appear. The digest is transferred to a 50-ml. volumetric flask and made to volume. After some experimentation, the following method was developed for acid precipitation of the casein to yield a filtrate with a maximum calcium content. Ten milliliters of milk are placed in a 100-ml. volumetric flask and diluted with 20 ml. of distilled water. Two milliliters of 1 N hydrochloric acid are then added, and the sample is allowed to stand for 10 minutes, after which 2.5 ml. of 0.5 N sodium hydroxide are added. The acid dissolves colloidal calcium salts and disperses the casein on the acid side of its isoelectric point. Addition of alkali brings the pH to 4.0 to 4.1, whereupon casein is precipitated, and the calcium remains in solution. The contents of the flask are made to volume and, after thorough mixing, the precipitate is filtered off. The filtrate should be water clear. A few determinations were made on rennet whey and on milk dialyzate. Whey is prepared by treating 500-ml. of skim milk a t 35’ C. with 0.5 ml. of commercial rennet extract. After 20 minutes the firm curd is cut into small cubes, and the whey exuded by syneresis is collected and filtered. Dialyzate is prepared by equilibration of 80 ml. of distilled water enclosed in a Visking sausage casing against 4 liters of skim milk for 48 hours a t 5’ C. Anion Exchange. An aliquot (usually 10 ml.) of solution prepared from milk is passed through a column, followed by two 10-ml. portions of distilled water to rinse the column. Air is prevented from entering the column by not allowing the liquid
