Conductometric Determination of Ammonia Application to Nitrogen Distribution Studies R. H. HENDRICKS AND M. D. THOMAS, American Smelting and Refining Company, Salt Lake City, Utah AND
MYRON STOUT
AND
BION TOLMAN, Bureau of Plant Industry, United States Department of Agriculture, Salt Lake City, Utah
I
N T H E course of a sulfur dioxide fumigation experiment in 1935 it was suspected that some ammonia was being evolved, presumably from decaying organic matter under the crop. The inference was based on the fact that a sulfur dioxide recorder (10) showed a slight decrease in conductivity when analyzing the air from a "check" plot. Evidently the dilute acid in the absorber was being neutralized. The inference was confirmed by the Nessler test, both qualitatively and as to order of magnitude of concentration, though the amount of ammonia in the absorbing solution represented the extreme limit of sensitivity of nesslerization. This experience suggested that the conductivity technique might be applied to the determination of ammonia, particularly in the range of microquantities, which are difficult to titrate accurately, though larger amounts might also be determined. For this purpose it would generally be preferable to employ a weak acid which would increase in conductivity, rather than a strong acid which would decrease in conductivity on conversion to the salt. Boric acid with a dissociation constant of 6.4 X should be ideal for this purpose because its conductivity is very small and practically independent of its concentration, whereas ammonium borate has the conductivity of a well-ionized salt. Though boric acid has been proposed many times (3, 6, 16, 17) as an ammonia absorbent, so far as the writers are aware no one has previously suggested a conductometric measurement of the system. If a strong acid were employed it would be necessary to know beforehand the approximate amount of ammonia to be absorbed, so that the proper quantity of acid could be taken and satisfactory results obtained. I n this paper Hendricks and Thomas are responsible for the data on air analysis and the distillation method; Stout and Tolman are responsible for the aspiration method.
would depend to a large extent on the quality of the distilled water used in the absorbing solution. I n the analysis of 50 p. p. m. of ammonia, 140 liters of air were aspirated, raising the pH of the boric acid solution from 5.8 to 8.1. The efficiency of the absorbing solution remained unimpaired. With larger amounts of ammonia it would be necessary to use a stronger boric acid solution, as indicated below. TABLEI. AUTOMATIC DETERMINATION OF KNOWNMIXTURE^ OF AMMONIAA N D AIR BY MEANBOF A RECORDINQ ANALYZER
14.3 14.3 Av 14.3
Concentration of NHa in Air Found Calculated P. p . m . P . p . m. 52 3.5 49 3.5 50 Av. 3.5 A\.. 50.3 2.34 13.5 2.34 13.2 Av. 2.34 13.8 A r . 13.5 1.31 1.31 13.4 A v . 1.31 13.7 Av. 13.6 0.42
5.9 5.9 A v . 5.9
5.9 5.7 AY. 5.8
Calculated P . p . m. 51 52 50 Av. 51.0
13.5 13.5 13.5 A v . 13.6
0.30
Found P. p . m.
3.4 3.5 Av. 3.45 2.54 2.56 Av. 2.55 1.26 1.28
Av. 1.27 0.49
0.30
Vacuum Distillation and Conductometric Determination of Ammonia In the analysis of biological material it has been found (4, 6, 14) that the ordinary distillation of ammonia, even with a mild base like magnesia, causes partial hydrolysis of glutamine and asparagine. Vacuum distillation has therefore been proposed and it has been established that the rate of distillation is rapid, particularly when the distillation is aided by more or less aspiration of air through the system. For example, the vacuum distillation by Stanford (8) was completed at 35' C . in 10 minutes, during half of which time air was aspirated through the system; though Watchorn and Holmes (16) preferred to heat the distillation flask to 60' C. without any aeration. Pucher, Vickery, and Leavenworth (4) suggested a 15-minute distillation a t 40" C. with the aspiration of 2 to 3 bubbles of air per minute. In the foregoing distillations the ammonia was received in sulfuric or hydrochloric acid. It was necessary to study the behavior of boric acid under these conditions, since Kober and Graves (9)reported that boric acid was not so efficient as sulfuric acid for absorbing ammonia.
Air Analysis Mixtures of ammonia and air have been analyzed by means of the sulfur dioxide autometer (IO), which was used without modification. The mixtures were prepared by sending gas from a tank of liquid ammonia through a calibrated capillary flowmeter into a large measured air stream. The ammonia concentration ranged from 0.3 to 50 p. p. m. by volume in the air. The analytical machine automatically drew a measured sample of the gas through an absorber and recorded the resistance of the solution throughout the absorption period. The absorbing solution was 100 ml. of 0.04 M reagent grade boric acid in redistilled water. Absorption of the gas was practically complete in one absorber at an aspiration rate of 10 liters per minute. With two absorbers in series the second absorber collected only 0.5 per cent of the amount of gas absorbed by the first absorber at both 50 and 14 p. p. m. At lower concentrations the loss from the first absorber was hardly detectable. The conductivity recorder had a range from 40,000 to 125 ohms. The electrodes were a pair of platinum plates, 18 mm. square, spaced about 2 mm. apart, with a cell constant of about 0.069. The recorder was calibrated by adding ammonia to the boric acid solution in the range from 10-6 N to 3 x 10-3 N . I n Table I are given the results of a group of experiments in which the analytical concentrations are compared with values calculated for the synthetic mixtures. The close concordance of the results indicates the reliability of the method. There is no suggestion that the range of concentrations in Table I approaches either the upper or lower limits, which can be conveniently handled by this technique, though the lower limit
The following apparatus, resembling somewhat that of Pucher et al. (4), was used: A 150-ml. distillation flask was attached by means of a ground-glass joint to the still head, which carried an intake tube (with stopcock), that reached to the bottom of the flask. The receiver was a 250-ml. Erlenmeyerflask with a sealedin intake tube extendingto the bottom at one side and endin with a 15-mm. porous glass disk. A 10-mm. outlet tube was seafed on the opposite side of the flask near the top. A 12-cm. condenser connected the still head with the receiver through two standard taper joints. In this assembly ground-glass joints are convenient, but not essential. Good results have been obtained with rubber connections. The carbon dioxide absorber already described (11) has also been satisfactory. 23
INDUSTRIAL AND ENGINEERING CHEMISTRY
24
01 AMMONIUM BORATESOLUTIONS TABLE11. STABILITY Subjected to vacuum distillation and aspiration for 20 minutes a t 13- t o 15mm. pressure) Mixture before Distillation 7 hlolalityS H3 Excess Distilled Tenip. pII HaBOa SH3 Ratio HaB03 S N R
-
7 -
c.
.Ilg.
Mg.
0.1 0.0047 21 0,095 1.32 0.00 0 7.63 0.1 1.32 0.015 1.2 0.1 1.31 0 . 3 3 5 2 5 . 6 0.1 0.0094 10.5 0.091 2.62 0.02 0.8 7.94 0.1 2.60 0.085 3.3 0.1 2 . 5 1 0.47 18.7 0.4 0.0188 21 0.381 5 , 2 4 0.00 0 6.90 0.4 5.24 0.00 0 0.4 5 . 2 4 0.056 1.1 0.4 0,047 8.5 0.353 13.18 0 , 0 2 5 0 . 2 0.4 13.16 0 . 1 4 5 1.1 7.55 0.4 13.01 0.695 5.3 0.4 0.047 8.5 0.353 13.18 0.06 0.5 40" 0.4 13.12 1.66 12.7 a Snlution containing butyl alcohol distilled from flask having porous glass bubbler. 15 25 40 15 25 40 15 25 40 15 25 10 250
~
TABLE 111.
~~
VACUUM DISTILLATION O F AXMONI.4 AT 40" TO
50"
c.
(Conductometric measurement in boric acid solution) Amide SHa from Asparagine KHa from ?;Hac1 Hydrolysis S Taken N Recovered Time Temp. N Take11 N IZecuvered .Mu. My. Hours C. Mg. MMg. 96 0.466 0.400 3 0.466 0.465 0.010 16 98 0 01 0.029 1 0.466 0.435 0.09 115 0.466 0.465 0.05 0.051 1.5 115 0.470 0.099 16 0.466 115 0.10 0.932 0.956 115 0.50 0.505 3 115 1.40 1.40 0.983 3 1.00 115 1.86 1.75 0.483 1 .oo 3 1.00 1.015b .. ... 1.000 1.00 .. .. ... ... ... "00 1.99 .. B . 73 3.70 .. .. ... ... .. 5.22 5.17 .. ... ... a Solution contained phosphate, made alkaline with MgO. b Solution contained phosphate, made alkaline with 0.1 N NaOH.
...
t
.
I . .
The stability of ammonium borate solutions was studied b,y distillin mixtures of boric acid and ammonia in vacuum a t 15 , 25", anf40' C. for 20 minutes according to a definite procedure. The pressure was maintained at 13to 15 mm. and air was allowed to bubble through the flask a t 50 cc. (90 bubbles) per minute (650mm. pressure). The temperature was kept constant by immersing the flask in a large beaker of water. Condensation of moisture in the still head above the water bath was prevented by occasionally playin a stream of hot water on that part of the apparatus. Two a%sorbers, each containing 10 ml. of 0.4 M or 0.2 -11boric acid, were usually employed but only traces of am: rnonia ever reached the second absorber.
Vol. 14, No. 1
quantitative absorption has been obtained with boric acid under conditions which permitted 10 to 30 per cent of the ammonia to escape through the Van Slyke-Cullen absorber (IS), though absorption was satisfactory with the latter apparatus if sulfuric acid was the absorbent. I n most of the analyses reported in this paper (except the air analyses), the absorbing solution contained about 1 per cent of butyl alcohol, which caused some foaming and increased the time of contact between the gas and liquid. Early work with 0.04 to 0.1 M boric acid solutions showed that the alcohol was definitely helpful in raising the efficiency of the absorption. Later work with stronger solutions of boric acid indicated that the alcohol was unnecessary. Typical data, showing the recovery of ammonia after vacuum distillation and conductivity measurement, are presented in Table 111. The distillations were conducted in the apparatus described above. The volume of the liquid in the flask was 15 to 25 ml. and magnesia was used in all cases, except one. The observation of Pucher et al. ( d ) , that low results were obtained when phosphate and magnesia were present together, was confirmed. Distillations were complete in 15 to 20 minutes at 40" or 50" C. if water was prevented from condensing in the still head. Eighty-eight to 97 per cent of the ammonia was distilled in 5 minutes even when no air w w allowed to bubble through the system. In other experiments the rate of distillation from a volume of 100 ml. was found t o be about half the rate from 20 ml. Z-Asparagine was not completely hydrolyzed by normal sulfuric acid in 3 hours a t 96" C. in the boiling water bath, but the amide group was split off in 1.5hours in the autoclave at 115" C. Longer heating had no further effect. The absorbing solution, containing a definite amount of boric acid-(e, g., 10 ml. of 0.2 M or 0.4 M)-wm diluted in a volumetric flask t o 25 to 250 ml. according to the amount of ammonia present, and its conductivity determined using glass-petticoated dip electrodes of 0.069 cell constant, similar to the one described above, A conductivity recorder or a Leeds & Northrup alternating current bridge was em loyed to measure the conductivity. Temperature correction coul! be applied using the compensator of the instrument, though it was preferable to bring the solution to a definite temperature before placing it in the cell. An empirical calibration curve was prepared for each boric acid concentration, by adding known amounts of standardized ammonia solution to the boric acid, diluting to volume, and reading the resistance. The reciprocal of the resistance (or conductance) was then plotted against the concentration of ammonia, because the relation between conductance and concentration is nearly linear. The cell constant was checked occasionally, using 0.001 N potassium chloride, and analyses were run to determine the reagent blanks.
The data are summarized in Table 11. Ammonium borate solutions are unstable unless a large excess of boric acid is I n dealing with microquantities of ammonia it is essential present and room temperature is not exceeded. The table to employ ammonia-free water with a specific resistance of at indicates that for a given boric acid-ammonia ratio the least 3 X IO6 ohms. Water of this quality can readily be obstronger solutions are the more stable ones. The presence of tained by a single redistillation, in glass, of ordinary distilled butyl alcohol affected the stability only at elevated temperawater, discarding the first fractions until the desired specific tures. It may be expected that boric acid will absorb amresistance is reached. The water can be handled freely and monia without appreciable loss a t the rate of about 0.1 mg. of even stored for a long time in Pyrex without appreciable nitrogen per ml. for the 0.1 M solution, or about 0.7 mg. of change, because it is evidently in equilibrium with the carbon nitrogen per ml. for the 0.4 M solution. The absorbing solution tends to be cooled by evaporation during " the distillation. and this circumstance favors TABLE Iv. CHANGES I N CONDUCTIVITY O F SULFURIC AND BORICACIDS DUETO absorption. ADDITIONOF AMMOXIA I n accord with these observa(Dilution 100 ml ) tions that ammonium borate Conductirity Recorder H&Oi H3BO3 Shift solutions are not entirely Concentration + + With With stable, it has been necessary HzSO6 NHdOH "%ON>fif' HzSOa SHiOH Diff. HaBOa S H i O H Diff. H3BOz . HzSO4 Din. Diu. to employ a sintered-glass disk ,Y x 205 MQ. M h o s . X 10' of medium porosity to produce 36G.5 .5,0 ... ... ... 0.55 45.5 45.0 , . 81.8 90 71.5 1.0 40 5 24.0 25.5 0.55 10.65 10.1 21.7 I(-I 5 small bubbles in all absorbers, 10 7,l5 0.10 5 03 3 10 2.53 0.55 1.61 LOR 11.0 4 3 1 0.71 0,010 1'2: 1.00 0.25 0.55 0.66 0.11 0.Y 0.5 except those used for air analysis. Using the glass disk,
-
7
January 15, 1942
ANALYTICAL EDITION
dioxide of the atmosphere. Recrystallization of reagent grade boric acid resulted in only a slight improvement. There is uncertainty of about 10 per cent in the conductance value of 10 micrograms of ammonia. The distillate was diluted to 25 ml. in this case. It would be practicable to enlarge the electrodes and to modify the absorber and cell so that a final dilution of 5 or 10 ml. mould suffice, in which case still smaller amounts of ammonia could doubtless be accurately determined. I n this connection the distillation technique of Conway and Byrne ( I ) would be particularly effective. Some additional sensitivity could also be obtained by using sulfuric acid as the absorbent, as indicated below. With larger amounts of ammonia the error of the conductance reading approached 1 per cent without taking special precautions to secure greater accuracy. I n this connection the speed and ease with which conductivity measurements can be made should be emphasized. Boric acid was selected as the ammonia absorbent because it supplied the convenience of a direct conductance-concentration relationship, and avoided the necessity for a number of accurately standardized solutions of a strong acid-the proper one to be selected according to the amount of ammonia to be determined. On the other hand, the strong acids are more efficient absorbents of ammonia than boric acid and they also show a greater change of conductance. It is probable, therefore, that they will be more suitable than boric acid for some purposes-for example, in the analysis of microquantities of ammonia. The data in Table IV indicate that the relative change in the conductance of sulfuric acid due to the absorption of ammonia is 2 5 times that of boric acid. It is evident that the molecular conductivity of ammonium sulfate is nearly constant over the range considered, whereas the values for ammonium borate fall off appreciably a t higher concentrations. The relative sensitivity of the recorder with the two acids is
2s
also indicated in Table IV. When measurements were made with microquantities in the same conductance range, the recorder sensitivity was greater with sulfuric than with boric acid. The reverse was true in dealing with 1 mg. of ammonia nitrogen per 100 ml., because the measurements were made in the high conductance range with the former acid and in the lorn range with the latter. The range of the recorder (minimum resistance, 125 ohms) did not permit measurement of more than 1.5 mg. of ammonia nitrogen per 100 ml. of sulfuric acid solution without additional dilution or decrease in the size of electrodes. Eight milligrams could be measured in boric acid under comparable conditions. Finally, the method has been applied to nitrogen distribution studies on alfalfa leaves and on sugar beet seeds (9),using the procedure of Vickery and co-workers ( 4 , l Q ) . Reproducible results mere obtained and there was no indication that any other volatile conducting material besides ammonia interfered with the method. It is of course possible that in certain plant material appreciable amounts of volatile organic bases other than ammonia may be encountered, and these may interfere with the method.
Aspiration Method The chief advantage of the aspiration method for the separation of ammonia from plant extracts and solutions is its adaptability to the routine analysis of large numbers of samples. The principal objection t o the method has been the length of time necessary for complete recovery of the ammonia (12). Incomplete absorption has been reported with boric acid ( 2 ) . A satisfactory aspiration apparatus, shown in Figure 1, consisted of 12 units connected in series. The evolution tubes were supported in a compact water bath, the temperature of which was controlled by the rate of feeding water into an "instantaneous" heating coil. The rate of aspiration (200 to 300 cc. per minute)
J A C K L T ED COPPER COIL
__b
FROM
caNsrAHr
T-
TO P U M P
L E V E L FELD
vw r
n
-8 - c m .
G A L V A N I Z E D DQAIN
PIPE
(LENGTH
125 cm.)
I
DQAIN
1
FIGURE 1. ABSORPTION APPARATUS WITH ACCESSORY EQUIPMENT .%bsorbersshould be supported in cold bath for rapid separations at increased temperature and reduced pressure (Table 11).
INDUSTRIAL AND ENGINEERING CHEMISTRY
26
OF TEMPERATURE AND ALKALINEREAGENT TO TIME OF ASPIRATION TABLEV. RELATIONSHIP
--
(Time necessary t o effect complete recovery of ammonia from ammonium sulfate solution containing 2.00 mg. of nitrogen in 10 ml. of sample.) Nitrogen Recovery with Different BasesTemperature Average NaOH, KzCOs, NaOH-Sa2B407. Aspiration of Aspirated hspiration I\.lgo, Period Solution Rate 50 mg. 1 ml. 3 N 1 ml. 50% buffera
c. Cc./min. Mg. Mg. 65 1.93 1.97 1.32 35 1.33 1.99 65 1.99 1.80 35 1.86 1.99 2.00 65 1.95 1.97 35 2.01 2.00 65 2.00 1.98 35 ... 65 1.9s 2.00 35 1.99 2.00 35 Av. 65 9.50 11.15 PH 36 9.80 11.35 3 ml., 50 grams of NazBdO, made t o 1 liter with 0.5 N NaOH.
Hours
O
...
a
Mg.
M Q.
1.99 1.31 2.00 1.81 2.00 1.96 2.00 1.97
1.97 1.32 2.00 1.80 2.01 1.95 2.01 1.99 2.01 2.00 2.01 10.15 10.10
...
2.00 2.00 10.40 10.10
NITROGEN DISTRIBUTION IN A SEED-BALL EXTRACT
TABLE VI.
(With and without the addition of ammonium sulfate) Solution Volume 20 MI.
(NHI)zSOL Extract Extract corrected Extract (NH4)zSOd Extract C (NHd:SO4 corrected
+
Ammonia
1st aspiration, 2nd aspiration, 4 hours 3.5 hours Mg. Mg. 2.01 1.78 1.74b 3.77 3.728
0.005 0.043
: ,..
0 Os7
Nitrogen Recovered Amide aspiration, Residual by 4.0 hours Kjeldahl 0 . oos=
s:iSs
Sum
Total By analysis
M ' U.
2.023
1,052 1.126 1.012
8 :223
.... 13.062
1.110
...
....
Reagent blank. b Subtract ammonia obtained in second aspiration assuming 5 Add decomposed amide value less reagent blank. a
MQ.
MU.
11.041
(was derived from amide.
MQ.
....
10.990
....
....
....
Vol. 14, No. 1
atilize conducting substances other than ammonia. It is improbable that other conducting materials would show the same conductivity as the estimated amount of ammonia in the system. This was verified by nesslerizing the solutions after the conductivity measurements. It was found that the conductivity and Nessler values agreed within about 1 per cent. The results also show complete recovery of the added ammonium salt. Slight hydrolysis of other nitrogen compounds, most of which are presumably amides, is suggested by the second aspiration rather than incomplete removal of ammonia in the first. Assuming that the ammonia obtained in the second aspiration was derived from amides, the values for ammonium and amide nitrogen have been corrected in accordance with the continuous amide decomposition observed by Schlenker (6) I
through the ap aratus was controlled by a stopcock on the intake tube or by reg3ation of the filter pump. The sample volume was 25 ml. or less and the absorbing solution was 15 ml. of 0.4 A4 boric acid containing 2 drops of butyl alcohol. The delivery tube in the absorber ended with a 6-mm. sintered-glass disk of "coarse" porosity. The method of connecting and disconnecting the apy t u s was similar t o the procedure of Sessions and Shive (7'). he ammonium borate solutions were measured conductometrically, as described above. Two dip cells, one having a constant of 0.05 and the other 0.15, seryed conveniently to cover the range from 0.01 to 10 mg. of ammonia when the absorbing solution was made up to 100 ml. Experiments summarized in Table V indicate that 2 mg. of ammonia were completely volatilized in 2 hours or less a t 60" C. At 35" C., 4 to 5 hours were required. (At 25' C. the process may require 24 hours.) The different bases gave nearly identical results, and illustrate the reliability of the procedure. The final values were within 0.5 per cent of the amount of ammonia taken. In other experiments, slightly low results were obtained when phosphate and magnesia were present together. An extended investigation (9) was carried out by the aspiration method of certain nitrogen fractions in aqueous extracts of sugar beet seed balls, before and after the extracts had been in contact with germinating seeds. In this study the ammonium salts were first determined; then the amide nitrogen was hydrolyzed in the autoclave at 15 pounds' pressure for 3 hours, using 10 per cent sulfuric acid, and the hydrolyzed ammonia was removed by another aspiration after making the solution slightly alkaline with sodium hydroxide. The nitrogen fractions of a fresh seed-ball extract are considered in Table VI. To one aliquot of the original solution was added an ammonium sulfate solution containing 2.02 mg. of ammonia nitrogen. Another aliquot was analyzed for total nitrogen by the Kjeldahl-Gunning method, as were also the solutions after the amide determinations. The results show that the ammonia volatilized by aspiration, plus the residual nitrogen, corresponds closely with the total nitrogen value, thus suggesting that the aspiration proceduredid not vol-
Summary The absorption of ammonia by boric acid was satisfactorily accomplished after vacuum distillation or volatilization by aspiration, if sintered-glass bubblers and sufficient excess of boric acid were employed. It may be necessary to use butyl alcohol in the absorbing solution in some cases. The change in the conductance of the solution provides a simple and accurate method of measuring the amount of ammonia absorbed. Sulfuric acid is a better ammonia absorbent than boric acid and gives a larger change of conductance. Boric acid is preferred in general use because of its convenience. Sulfuric acid may be more suitable in some cases, particularly in microanalyses. The method has been applied to nitrogen distribution studies of plant materials and to automatic analysis of traces of ammonia in air.
Literature Cited (1) Conway, E. J., and Byrne, A,, Biochem. J., 27, 419 (1933). (2) Kober, P. A,, and Graves, S. S.,J . Am. Chem. Soc., 35, 1594 (19 13). (3) Meeker, E. W., and Wagner, E. C., IND.ESG. CHEN.,Aiv.4~. ED..5. 396 (1933). (4) Pucher, G. W., Vickery, H. B., and Leavenworth, C. S.,Ibid., 7, 152 (1935). (5) Scales, F. M., and Harrison, A. P., J. IND.ENG.CHEM.,12, 350 (1920). (6) Schlenker, F. S., Plant Physiol., 15, 701 (1940). (7) Sessions, A. C., and Shive, J. W., Ibid.,3, 499 (1928). (8) Stanford, R. V., Biochem. J., 17, 847 (1923). (9) Stout, M., and Tolman, B., J . Agr. Research, 63,687-713 (1941). (10) Thomas, M. D., IND. ENQ.CHEU.,ANAL.ED.,4,253 (1932). (11) Ibid.,5, 193 (1933). (12) Tiedjens, V. A., and Robbins, W. R., N. J. Agr. Expt. Sta., Bull. 526 (1931). (13) Van Slyke, D. D., and Cullen, G. E., J . Biol. Chem., 19, 218 (1914). (14) Vickery, H. B., Pucher, G. W., Clark, H. E., Chibnall, A. C., and Westall, R. G., Biochem. J., 29,2710 (1935). (15) Wagner, E. C., IND.ENQ.CHEM.,ANAL.ED., 12, 771 (1940). (16) Watchorn, E., and Holmes, B. E., Biochem. J.,21, 1391 (1927). (17) Winkler, L. W., Z . angew. Chem., 26, 231 (1913).
