Determination of Ammonium, Amide; Nitrite, and Nitrate Nitrogen in Plant Extracts J. E. VARNER, W. A. BULEN, STEVE VANECKO, AND R . C. BURRELL Agricultural Biochemistry D e p a r t m e n t , Ohio S t a t e University, Columbus 10, Ohio
A method is described whereby the ammonium, amide, nitrite, and nitrate nitrogen present in plant extracts can be determined quantitatively using a single portion of extract. In addition, each kind of nitrogen can be recovered as ammonium sulfate in aqueous solution convenient for mass spectrometric determination of isotopic ratios. The plant extract, freed of proteins, is buffered at pH 10 with borate and placed in a modified semimicro-Kjeldahl distillation unit, and the ammonium nitrogen is removed by vacuum distillation at 40" C. Concentrated alkali is added to the distillation flask and the amide nitrogen is removed by steam distilIation at 100' C. After the addition of ferrous sulfate as a reducing agent, the nitrite nitrogen is removed as ammonia. The nitrate nitrogen is reduced to ammonia by ferrous sulfate after addition of silver sulfate as a catalyst. The entire procedurerequires about 20minutes. Interferencesof various substancesand means of overcoming these interferences are discussed.
A
REAGENTS
Borate buffer, saturated solution of sodium tetraborate adjusted to p H 10 Ferrous sulfate, 20% solution Sodium hydroxide, 40% solution Silver sulfate, saturated solution Copper sulfate, 25% solution Tartaric acid, 15y0 solution Dowex-1, 200- to 400-mesh Dowex-50,200- to 400-mesh Sulfuric acid, 0.01 N , standardized Boric acid, 4% solution BASIC PROCEDURE
The following procedure gives accurate results when mixtures of ammonium salts, asparagine and/or glutamine, nitrite salts, and nitrate salts, containing no interfering substances, are analyzed. The sample in 1 to 20 ml. of water or ethyl alcohol is added to the distillation apparatus and washed in with 5 ml. of borate buffer. The receiver, containing an appropriate amount of boric acid solution, is put in place and the water pump is turned on. A small water bath a t 50" to 55" C. is brought up around the sample chamber of the distillation unit. Air is allowed to leak in slowly through the steam generator to prevent serious bumping. In 5 t o 10 minutes all of the ammonia from the ammonium salts is collected in the receiver. The receiver is removed and the contents are titrated with 0.01 N sulfuric acid. This is essentially the procedure of Archibald (1). A new receiver containing boric acid is placed under the condenser, 15 ml of 40% sodium hydroxide added to the sample chamber, and steam distillation a t 100' C. started. Under these conditions the hydrolysis of asparagine and glutamine is complete and rapid and the theoretical quantity of ammonia distills over in 2 to 4 minutes. The receiver is changed again, 5 ml. of 20% ferrous sulfate added to the sample chamber, and the distillation continued. In 2 to 4 minutes all of the nitrite nitrogen is reduced and distills over (8). After the receiver is changed, 5 ml. of saturated silver Eulfate is added and in 2 to 4 minutes the nitrate nitrogen is reduced to ammonia and distills over ( d , 6, 8).
PREREQUISITE to the effective study of nitrogen metabolism is the availability of convenient and accurate methods for assaying the nitrogen compounds of primary concern. A recent review by Street (7) summarized the methods then available for the study of the nitrogen metabolism of plants. Ammonium nitrogen is probably determined and recovered most satisfactorily by the method of Archibald (1). The hydrolysis and recovery of amide nitrogen according t o Pucher and Vickery ( 9 ) , although otherwise acceptable, are rather lengthy. Colorimetric procedures for nitrite in low concentrations are fairly satisfactory ( 4 ) but a t higher concentration subject to much interference. Colorimetric determinations of nitrate are subject t o inference from nitrit,e and from amino compounds that are converted to nitrate by-oxidation procedures (3). The determinaTable I. Recoveries of Various Forms of Nitrogen from Known Solutions tion of nitrate by reduction with (Meq. X 102) Devarda's alloy is subject to interfer("c)zSO4 Glutamine Asparagine KN02 KNOi ence from nitrite. N N N N N N N N N N added recovered added recovered added recovered added recovered added recovered In view of these advantages and limi4.80 4.80 6.66 6.66 tations, the methods described in this .. 0.20 .. .. 3:46 .. .. .. .. .. .. 3 . . 1U.Yti paper were developed for use in a study IU.UL 5100 5.48" 6:66 6'iO .. .. 11'00 11',02 10.00 of nitrate and nitrite reduction by higher plants. Under ideal conditions, these a Distillation conducted a t looo C., pH 10. methods permit the determination and ~~ . _ _ ~ recovery (as ammonia) of the ammonium, amide, nitrite, and nitrate forms Table TI. Use of Ion Exchange Resins to Overcome Interference of Glucose of nitrogen in a single sample in about (Rleq. X 102) (NH4)rSO& Asparagine KNOs KNOa 20 minutes. N N N N N NN N APPARATUS
added 5.00 5.00
recovered added recovered added recovered 5.30 3.35 3.30 5.00 4.92 4.92 5.30 3.35 3.30 5.00 4.86 0.20 ... 5.00 0.00 1.22 E 0:52 E Ea E+1.00 2.22 E+0.64 1.06 E+1.00 0.92 a E is an aliquot of an alcohol extract of soybean leaves. ..I
h'lodified semimicro-Kjeldahl distillation unit (Figure 1) Microburet, 10-ml. capacity Water pump
added 5.00 6.00
zoo
E-tl.00
____
1528
recovered 5.15 5.00 5.25 0.32 1.32 ..
_
_
V O L U M E 25, NO. 10, O C T O B E R 1 9 5 3
1529
The entire procedure requires about 20 minutes and as Table I shows, the separation and recovery of these forms of nitrogen are clean-cut and quantitative. PROCEDURE IN CASE OF INTERFERENCES
When plant extracts are analyzed by the basic procedure the recovery of nitrite and nitrate is incomplete, primarily because of the presence of reducing sugars. The determination of a given quantity of nitrite in the presence of different quantities of glucose results in recoveries inversely proportional t o the total glucose present. For example, 10.2 X 10-2 meq. of ammonia was meq. of nitrite plus 36 mg. of glucose recovered from 11.0 X and 9.2 X 10-2 meq. v a s recovered when the glucose was increased to 72 mg. The determination of varying quantities of nitrite in the presence of a constant quantity of glucose results in absolute losses proportional, but not directly proportional, to the nitrite present, and percentage losses inversely proportional to meq. of ammonia the nitrite present. For example, 0.8 X was recovered from 1.0 X l o w 2meq. of nitrite and 6.4 X meq. from 7.0 X 10-2 meq. in the presence of 36 mg. of glucose. The loss of nitrate under similar conditions is comparable. By the use of ion exchange resins this interference can be overcome.
r,
Figure 1. Diagram of Apparatus
A \\ater solution of the plant extract (from 1 to 5 grams of tissue) is passed through a 2-cc. resin bed 2 cm. long consisting of an intimate mixture of Dowex-1 and Do~vex-50. (The Doweu-1 should be regenerated with sodium hydroxide solution on the day that it is used, ai: it slowly releases ammonia on standing.) There should be a slight exceqs of Dovex-1 in the mixture, so that the extract never becomes acid. The resin is then washed with 10 ml. of water. The effluent up to this point is discarded. The resin mixture is now eluted with 10 ml. of 1 M sodium fluoride and this solution is followed by 10 ml. of water. .The slightly alkaline effluent solution contains the ammonium and amide nitrogen and must be collerted in a slightly acid solution to prevent loss of ammonia. This effluent is then analyzed by the basic procedure given. The resin bed is now eluted with 15 ml. of a solution 1 M with re5pect to sodium bromide and sodium hydroxide, then with 10 ml. of water. This second effluent contains the nitrite and nitrate free of reducing sugars and ii analyzed by the basic procedure. Table I1 indicates how successfully this method can be applied. The total elapsed time for an analysis under these conditions is 1 t o 2 hours. As in many plant extracts the level of nitrite is below the limit of detection by this distillation procedure, it is usually convenient to determine the nitrite colorimetrically on a separate sample and to determine nitrite plus nitrate by the distillation method. In this case the interference of glucose can be overcome by oxidation with alkaline cupric tartrate. The ammonium and amide nitrogen are removed according to the basic procedure.
Then 2 ml. of 15% t?rtaric acid and 0.5 ml. of 25% cupric sulfate are added and heating is continued for 3 to 5 minutes, 5 ml. of 20% ferrous sulfate is added, and the nitrite plus nitrate is reduced and distilled off. Because the copper ions catalyze the reduction of nitrate, it is not possible to recover nitrite nitrogen and nitrate nitrogen separately by this procedure. DISCUSSION
The basic procedure for the distillation of ammonia from ammonium salts without concurrent hydrolysis of glutamine ( 1 ) is generally accepted and requires no explanation. If ammonia is distilled a t 100' C. in a buffer a t pH 9.0, 5 to 10% of the glutamine present will be hydrolyzed during the distillation. Under the same conditions no asparagine is hydrolyzed. The hydrolysis of glutamine and aspara ine by strong alkali is, of course, subject to the interference o f other nitrogen-containing compounds which may yield ammonia under these severe conditions. In plant extracts protein appears t o be the substance most likely to cause interference. A hot water extract of plant tissue will give a higher value for amide, nitrite, and nitrate nitrogen than a 70% alcohol extract. This is due to the presence of some protein in the water extract, which hydrolyzes fairly rapidly during the determination. It has been shown (6) that i o % alcohol is one of the most effective precipitants of plant proteins. Although one might anticipate interference from those amino acids unstable in alkali (arginine, cystine, serine, threonine), the rate of release of ammonia from the quantities of these amino acids that occur in the leaf tissues studied is so slow as to cause little or no trouble. Several ways were tried t o overcome the interference of glucose in the reduction and recovery of nitrite and nitrate salts. Oxidation with hydrogen peroxide or with alkaline potassium permanganate failed to prevent the interference. This suggested that the compound or compounds interfering were products of the alkaline degradation of glucose resistant to oxidation by these reagents-for example, acetone was found to interfere much more than glucose. Alkaline hypochlorite removed the interference of glucose entirely but could not be used, because it releases ammonia from amino acids. The use of copper tartrate as described in the procedure was satisfactory for overcoming the interference of glucose. Saabo and Bartha (8) describe the use of silver catalysts in the reduction of nitrates by ferrous hydroxide. The reduction of nitrite to ammonia is very rapid with ferrous hydroxide alone, while the reduction of nitrate to nitrite is very slow in the absence of any catalyst. Precision and Accuracy. In the absence of interfering substances the precision and- accuracy are the same as in ordinary semimicro-Kjeldahl procedures. Even in the presence of interfering substances such as reducing sugars the precision is good. Only the addition of known increments of the various forms of nitrogen or analysis by an independent method can establish whether one has successfully removed these interferences and is obtaining accurate analyses. Applications. These methods are in use in the authors' laboratory in a study of the reduction of nitrate and nitrite by green leaves, and have also been found useful for the recovery of the various forms of nitrogen for isotope ratio analyses in tracer experiments. The residue remaining in the distillation unit can be recovered and subjected to the regular Kjeldahl digestion to recover the nitrogen from the other soluble nitrogen compounds, while the residue from the iOyo ethyl alcohol extraction can be analyzed to determine total insoluble nitrogen. LITERATURE CITED
(1) Archibald, R. XI., J . Biol. Chem., 151, 141 (1943). ( 2 ) Cotte, J., and Kahane, E., Bull. soc. chim., 1946, 542. (3) Johnson, C . XI., and Ulrich, d.,ANAL.CHEhf., 22, 1526 (1950). (4) Rider, B. F., and XIellon, AI. G., TND. ENG.CHEM., ANAL. ED., 18, 96 (1946).
( 5 ) Sandonnini, C., and Beesi, S., Gam. chim. ital., 60, 693 (1930). (6) Steward, F. C., and Street, H. E., Plant Physiol., 21, 155 (1946). (7) Street, H. E., iYew Phytol., 48, 84 (1949). (8) Seabo, G.. and Bartha, L., Acta Chim. Hung., 1, 116 (1951). (9) Vickery, H. B., Pucher, G. W., and Leavenworth, C. S., IND. ENG.C H E M , d N . 4 L . ED., 7, 152 (1936). RECEIVED for review May 11. 1953. Accepted July 23, 1953. Presented before the Division of Agricultural a n d Food Chemistry a t the 123rd Meeting of the AMERICANC A ~ M I C ASOCIETY, L Los Angeles. Calif. Work supported in part by a contract between the Charles F. Kettering Foundation and The Ohio State University Research Foundation,
