334
ANALYTICAL CHEMISTRY
metric and T.B.P.P.I. methods is greatly reduced. It is suggested therefore that substances causing high results in the T.A.P.P.I. method are removed by dialysis and are consequently of fairly low molecular weight. The effect in both methods of a large concentration of free sulfurous acid is shown in results for the “synthetic” sulfite waste liquor in which the concentration of free sulfurous acid present was much greater than normal for sulfite waste liquors. The magnesium-base sulfite waste liquor n-as obtained from the Keyerhaeuser Timber Company, Longviem, Wash. The ammonium-base liquor was prepared in an excerimental digester at the University of Kashington. The calcium-base sulfit’e waste liquor was obtained from the Puget Sound Pulp and Timber Company, Bellingham, Kash.
LITERATURE CITED
“Barlow’s Tables” (L.J. Comrie), 4th ed.. Sew York, Chemical Publishing Co., 1944. Britton, H. T. S., “Conductometric Analysis”, London, Chapman & Hall, (1934). Doering, H., Papier-Fabr.. 39,159 (19111. Jander., G., and Pfundt, O., “Leitfiihigkeitstitrationen und Leitfihigkeitsmessungen”, 2nd ed., Stuttgart, F. Enke, 1934.
Jones. G., arid Bollinger, D. AI., J . A m . Chem. Soc., 57, 280 (1936). Kolthoff, I. XI.. “Konduktiometrische Titrationen”, Dresden and Leipzig. T. Steinkopf, 1923. Partansky. .I. >I., and Benson, H. K., T.A.P.P.I. Standard 0-403-SM40(1940). Pfuiidt, 0 . .Z.angeu.. Chem., 46,200 (1943).
Rapid Turbidimetric Determination of Inorganic Nitrogen in Soil and Plant Extracts B E S J i3IIN F O L F , T h e G.L.F.-Seabrook Farms Raw Products Research Dicision, Seabrook Farms, Bridgeton, S. J . ..irapid method for determining inorganic nitrogen fractions in soil and plant extracts is presented. Kitrate and nitrite nitrogen are reduced quickly to ammonia nitrogen without heating by the use of a titanous chloride solution. The reduced nitrogen along with the original ammonia nitrogen is determined photometrically with a precision of *lo% by use of modified Graves’ reagent. Since nitrites are not fully recovered, a separate determination and correction for nitrite nitrogen must be made, when the method is applied to the few soil or plant extracts containing large quantities of nitrite nitrogen.
T
HE determination of inorganic nitrogen fractions (ammonia, nitrite, and nitrate) is useful to investigators dealing with soils or plants. The amount present in a soil extract is an indication of nitrogen available for plant use; the amount present in a plant extract can give much information as to the nutrition of the plant. Within, certain concentrations, varying with the ion and conditions of the substrate, all three forms are used by plants. It is, therefore, necessary in many cases to determine only the total nitrogen without distinguishing the amounts in each form. I n routine rapid soil and plant analyses, the concentration of nitrate nitrogen alone is often used as an index of the amount of available nitrogen present. The concentration of ammonia nitrogen or the total nitrogen in nitrate and ammonia forms is also used. Kitrite nitrogen is seldom determined because of its usually low concentration and its rapid change-over to other forms. Ascertaining the total amount of inorganic nitrogen present, or what is present RS ammonia and nitrate, usually involves three or a t least two separate determinations. Emmert ( 3 ) has devised a method based on the oxidation of soluble nitrogen to nitrate which determines the various forms of inorganic and soluble organic nitrogen. The method, although fairly rapid and useful, has the disadvantage of using strong acids in what is a rather active reaction. Several procedures designed t o reduce nitrates t o ammonia looked promising as a possible means of rapidly determining soluble inorganic nitrogen. The nitrates and nitrites could be reduced to ammonia nitrogen and this in addition t o ammonia nitrogen originally present could be determined by Nessler’s or Graves’ reagents. The reduction of nitrogen in an alkaline medium would be an advantage, because this is necessary for the actual determination of ammonia nitrogen. Three procedures for the reduction of nitrogen to ammonia were investigated: the reduction by means of aluminum foil as used in water analyses ( I ) , the reduction by Devarda’s alloy ( d ) , and the use of titanous chloride solution (6).
Considerable time was spent i n trying to adapt reduction by either aluminum foil or Devarda’s alloy to a rapid procedure. Graves’ solution ( 7 ) was used t o determine nitrogen because it is less affected by heavy metals than Sessler’s solution. I n general, these two reducing agents were unsatisfactory because of the length of time required to reduce nitrate and nitrite in the cold to ammonia nitrogen. It was also very difficult t o wash glassware in which the reaction had taken place. On the other hand, titanous chloride proved satisfactory, reducing nitrates quantitatively in an alkaline solution a t room temperatures in a few minutes. It was less satisfactory for nitrites, since it was not possible to recover nitrite nitrogen completely. Since nitrite nitrogen is present in such small quantities, it was still possible to use titanous chloride in the method given below as a means of determining inorganic nitrogen fractions in soil and plant extracts. MATERIALS AND REAGENTS
Photoelectric Colorimeter. A Fisher electrophotometer equipped with 425 mp color filter was used. The turbidities formed can be matched with standards but the photoelectric method is more accurate. Waring Blendor for preparing plant extracts. Stirring rods with flattened ends. Titanous Chloride, 20y0solution (La Motte). If not used soon after the bottle is opened, the solution is protected from oxidation by storing under an atomsphere of hydrogen. Carbon, activated, Darco grade 0-97. Extracting Solution. Morgan’s universal, which is N sodium acetate buffered a t pH 4.8 with acetic acid ( 5 ) . Modified Graves. Sodium chloride (40 grams) is dissolved in about 750 ml. of water in a 2000-ml. volumetric flask and 3.5 grams of mercuric chloride are added and dissolved. The volume is diluted to 2000 ml. with water. The solution is stored in a brown bottle. Sodium hydroxide, 15% by weight. Gum arabic, 0.25% aqueous solution by weight. Standard Kitrogen. Ammonium sulfate in extracting solution to give 50 p.p.m. of nitrogen.
335
V O L U M E 19, N O . 5, M A Y 1 9 4 7 Table I. Rapid Determination of Inorganic Nitrogen in Soil and Plant Extracts bv Titanous Chloride Method
Extract
Soil 1 Soil 2 Soil 3 Plant 1 Plant 2 Plant3
5 P.p m. of Sitrogen Added So As (SHI),SOI As NaNOa As KaSOz S N reSitrogen N N reX ?reI found covered found covered found covered Added P.p.m. P.p.m. P.p.m. P.p.m. P.p.m. P.p.m. P.p.m. 8.3 4.0 9.3 5.0 9.2 4.9 4.3 7.4 3.8 8.8 5.2 8.8 5.2 3 6 7 3 3.9 8.2 4.8 8.0 4.6 3.4 4.0 11.8 4.8 11 0 li.3 4 . 8 7.0 5.0 4.2 0 . 3 4 7 5.8 5.0 0 8 5.4 4.2 6.5 5.3 6.5 6.3 1.2
PROCEDURE
Preparation of extracts. SOIL. A 12.5-gram sample, 0.5 teaspoon of carbon, and 25 ml. of extracting solution are shaken in a small flask for 1 minute. The contents are filtered onto a tThatman KO. 1 filter paper and filtrate is collected for the test. PLANT.Five grams of fresh minced tissue or 1 gram of dry ground tissue, 200 ml. of extracting solution, and 0.5 teaspoon of charcoal are agitated in a Waring Blendor for 5 minutes. The suspension is filtered onto a Whatman No. 1 filter paper and filtrate is collected for the tests. Determination. Five milliliters of soil extract or 10 ml. of plant extract are pipetted into 50-ml. Erlenmeyer flasks; 5 ml. of extracting solution are added to soil extracts. ITater (2.25 ml.) and 2.5 ml. of 15% sodium hydroxide are added to all flasks. The contents are mixed by rotating and 0.25 ml. of titanous chloride solution is added to each. The contents are again mixed, allowed to stand 10 minutes, then filtered through a Whatman No. 1filter paper on a funnel vial. Meanwhile, 10 ml. of modified Graves’ reagent are pipetted into a series of photometer vials and 0.5 ml. of gum arabic solution is added. The contents are mixed thoroughly with a flat-bottomed rod. Ten milliliters of the above filtrate are pipetted rapidly into the photometer vial containing modified Graves’ reagent plus gum arabic. The contents are mixed thoroughly with the flat-bottomed rod and allowed to stand for 15 minutes. Readings are then taken in the photometer, using a 425 mfi blue filter and adjusting the null to give 100% transmission with a blank. The blank is prepared from 10 ml. of extracting solution and carried through the entire determination. Readings are compared to those of a series of standards (0 to 20 p.p.m. on a IO-ml. basis) treated as above. A standard curve or chart prepared from such readings forms a permanent and convenient means of determining the amounts of nitrogen in the unknowns. DISCUSSION OF THE METHOD
Extraction. Morgan’s extracting solution is very efficient in extracting the inorganic forms of nitrogen. The sodium present is useful in displacing the ammonium ion from soils. The extracts obtained can be used for determination of other nutrients as well. Extracts of soil will keep indefinitely if properly stoppered. Certain plant extracts may undergo changes due to mold growth unless kept refrigerated or protected by a thin layer of toluene. Method. The method is based on reduct,ion of nitrate and nitrite forms of nit’rogen to ammonia and determination of ammonia, including nitrogen originally present as ammonia, by modified Graves’ reagent. The reduction without heat in an alkaline medium is almost instantaneous. A 10-minute period was chosen as a waiting period, however, to allow handling of a number of flLasks a t one time. The filtration after reduction yields a colorless clear solution which is ideal for turbidity formntion with Graves’ reagent. The rapid reduction is evidently limited to very simple forms of nitrogen such as nitrates and nitrites. Experiments with more complex nitrogen substances such as alloxantin, glycine, benzidine, urea, brucine, and diphenylamine failed to show any reduction. Graves’ reagent was modified to give more accurate results. The usual reagent tends to form a precipitate upon standing. Shaking the reagent prior to use tended to give more uniform results, but was not entirely satisfactory. By diluting the reagent and eliminating lithium carbonate, a more satisfactory reagent ryith great stability has resulted. Precision has also been increased by changing the order in
wl!ich Graves’ reagent is mixed with the filtrate. A much more uniform precipitate is obtained by adding the filtrate to modified Graves’ reagent containing gum arabic. The gum arabic is useful in limiting the size of particle formed and in preventing the precipitation of red or yellow oxides of mercury. Interferences. The precipitate tends to darken if exposed to strong light for any great length of time, especially when soluble nitrogen is determined in plant extracts, and this is evidently due to reduction of mercury compounds in light. This darkening effect can be best prevented by keeping the vials until readings are made in a nooden block having holes 6.25 em. (2.5 inches) deep. Other faulty tests may be brought about by insufficient or escess amounts of sodium hydroxide during reduction. Insufficient amounts will give turbid or dark extracts upon filtering. A n escess of alkali may tend to precipitate red or yellow oxides of mercury despite presence of gum arabic. Substances such as alcohol and formaldehyde interfere with reduction, and therefore should never be used for preserving plant extracts. PRECISION AND ACCURACY
The method is suitable for determining from 3.6 to 7.2 kg. (8 to 160 lb.) of inorganic nitrogen per 2,000,000 lb. of soil (based on an extraction of 1 part of soil to 2 parts of ext’racting solution). The amount of soluble nitrogen in most soils does not exceed the upper level. Where it does, as in greenhouse soils or those recently fertilized with large amounts of nitrogen fertilizers, it is necessary to use a smaller aliquot. An extraction of 1 part of plant tissue to 40 parts of extracting solution permits the determinat,ion of 80 to 800 p.p.m. of nitrogen. Again higher concentrations can be handled by using smaller aliquots. Analyses can be repeated with a precision of *lo%. Precision is enhanced by using a uniform procedure, especially in pipetting the filtrate into modified Graves’ reagent and subsequent mixing. The method is appreciably accurate for both ammonia and nitrate forms of nitrogen, but is less accurate for nitrogen originally present as nitrites. The results in Table I show fairly good recovery of nitrogen originally present in nitrate or ammonia form but less satisfactory recovery of that present as nitrite. This indicates a 92Yc or better recovery of nitrogen when present i n ammonia or nitrate forms but only about 80% recovery when present as nitrites. S o satisfactory explanation has been found of t’he poor recovery of nitrite nitrogen. I t is evidently not due to loss of nitrites in acid mediums, because adding alkali before adding nitrites has not given better result’s. Varying the time of reduction from 1 to 30 minutes has had no appreciable effect, nor has varying the time between filtration and subsequent turbidity development. These variations were tried to determine whether there \vas a loss due to reduction of nitrites to gaseous forms of nitrogen. Regardless of the cause, the failure to recover nitrogen complettly from nitrites is not serious because of the very low concentrations of nitrite nitrogen in soils and plants. Where the concentration of nitrites is high as in alkali soils (4),nitrites must be determined separately for accurat’e results. In most soil and plant extracts, the usual procedure will give satisfactory information for all practical purposes. LITERATURE CITED (1) Am. Public Health Assoc., “Standard Methods of F a t e r Analysis”, 8th ed., pp. 49, 50, New York, 1936. (2) Assoc. Official Agr. Cheni. “Official and Tentative Methods of Analysis”, 6th ed., p . 28, Washington D. C., 1945. (3) Emmert., E., Plant Physiol. 10, 356-64 (1935). (4) Fraps, G . S., and Sturgis, A . T., Texas Agr. Expt. Station, BuI1. 515 (1938). (5) Morgan, M . F., Conn. Agr. Expt. Station, BUZZ.450 (1941). (6) Reifer, I., .Vew Zealand J. Sci. Tech., 20B,341-5 (1939). (7) Yoe, J. H., “Photometric Chemical A‘nalysis”, 1st ed.. Vol. 2, p. 78, New York, John Wiley & Sons, 1929.
