ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
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Determination of Urinary Ammonia by Osmometry Man S. Oh," Kenneth R. Phelps, Ruth L. Lieberman, and Hugh J. Carroll Department of Medicine, State University of New York, Downstate Medical Center, Brooklyn, New York 11203
Osmometry replaces the cumbersome titration technique in this new, accurate, and simple method for measuring urinary ammonia. Ammonia liberated from urine by addition of saturated K2CO3 is trapped as (NH4)2S04 in 0.3 N H2S04 through an aeration train. One milliliter of 0.4 N NaOH is added to 1 mL of the trapping solution and the osmolality of the mixture is measured. Sufficient NaOH Is present In the mixture to titrate completely H2S04and ("4)$04 to Na2S04and NH,. Since NH3 remains in solution unless it is agitated vigorously, the osmolality of this solution is greater, by the quantity of ammonia trapped, than the osmolality of a blank consisting of a mixture of the H2SO4 and NaOH.
Table I. Recovery of NH, from Urine sample expected, recovery, no. mmol/L mmol/L 1 58.3 57.3 57.6 2 60.7 3 85.5 85.9 4 122.5 125.0 70.3 5 70.8 6 70.8 73.1 7 70.0 74.6 8 70.0 73.1 73.6 9 70.0
T h e technique of osmometry lends itself t o the measurement of the concentration of any chemical substance which can be caused to alter the osmolality of a given solution. We have previously described a simple and accurate method for the measurement of total C 0 2 concentration in biological fluids. In that system COz is trapped in a solution of NaOH, the osmolality of which is lowered in p r o p o r t i d to the quantity of C02 trapped ( I ) . Among the methods cqnmonly used for measurement of urinary NH, (2-6) are titration technics, which require alkalinization of the sample, trapping of NH3 in acid either by aeration ( 2 ) or by microdiffusion (3), and finally back-titration of the trapping solution. T h e principle of the technique described here is that NH3 trapped by a n acid solution as NH4+ and converted back t o NH3 by addition of strong alkali, remains in solution and is readily measured as a n osmotically active substance.
NH,, and unless the tube is vigorously agitated, the loss of NH3 between mixing of the solution and measurement of osmolality is negligible. The difference in osmolality between tubes a and b is equal to the concentration of NH3 in tube a. Since osmolality of the tube b is relatively constant over a few days, one value can be used for the measurement of NH3 in multiple urine samples. With time, however, exposure of the stock NaOH solution to the atmosphere leads to trapping of C02 as NaZCO3and lowering of the osmolality, so that tube b should be prepared and its osmolality determined every few days. The concentration of NH3 in the original sample of urine is calculated as:
EXPERIMENTAL An aeration train driven by vacuum draws air in series through a solution of 1.0 N HzSO4, the urine sample, and the trapping
solution of 0.3 N HzSO4. Multiple sets of sample tubes and tubes containing trapping solution can be placed in series between the vacuum line and the first trapping solution (Figure 1). The 1.0 N HzS04 traps NH3 contained in atmospheric air before the air passes through the urine sample. A drop of antifoam is added to the urine sample before the sample tube is closed with its rubber stopper. Saturated KZCO3 is then added to the urine sample through a needle piercing the stopper, and the vacuum line is opened. The NH3 formed in the alkalinized urine is driven off by vigorous aeration and is carried into the tube containing 0.3 N HzSO4, H2S04, where it is trapped as (NH4)&304. When liberation and trapping of ammonia are complete, after 1@15 min of vigorous airflow, 1 mL of trapping solution is withdrawn and mixed with 1 mL of 0.4 N NaOH in an osmometer tube (a). In a separate osmometer tube (b), 1 mL of the same NaOH solution is mixed with 1 mL of the blank trapping solution (0.3 N H2S04). The osmolalities of tubes a and b are determined. The reaction in tube a is: H2S04+ (NH4I2SO4+ excess NaOH The reaction in tube b is: HzSO4 + excess NaOH
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Na2S04+ NaOH
+ NH3
NaZSO4+ NaOH
The excess NaOH is sufficient for complete titration of NH4+to 0003-2700/79/0351-2247$01.00/0
Samples % recovered
98.3 94.9 100.5 102.0 99.3 103.2 106.4 104.4 105.1 101.5 mean 1.2 SEM
VZ
NH3(mmol/L) = - X 2 x AOsm (mOsm/L) VI
where VI and V z are the volumes of the urine sample and the trapping solution, and AOsm is the difference in osmolality between tubes a and b. The factor 2 reflects dilution of NH3 concentration upon mixing of HzS04trapping solution with NH, solution. The technique was applied to the measurement of standard solutions made from dried (NH4)zS04,and also to measurement of recovery of standard solutions from different urine samples. To determine the specificity of the technique, the results by osmometric measurement of NH3 concentration of a known standard and 5 urine samples were compared to those measured by titration. In the latter method, HzS04 solution containing trapped NH4+was titrated, using a pH meter, with a standard solution of NaOH to an end point of pH 6.5. NH3 concentration then was calculated from the normality of the H$04 solution before the trapping of NH3 and the quantity of NttOH required to titrate HzSO4 after trapping NH3. Osmolality was measured with an Advanced Osmometer (Advanced Instrument, Inc., Newton Highlands, Mass. 02161) and titration was carried out with a Beckman Expandomatic SS-2 pH meter (Beckman Instruments, Inc., Fullerton, Calif. 92634).
RESULTS The measured concentration of NH3 in standard solutions agreed well with predicted values, with a correlation coefficient of 0.99. The average recovery of NH3 from 9 urine samples was 101.5 f 1.2 (Table I). Results of osmometric determinations and of titrations were in close agreement (Table 11). This indicates t h a t negligible loss of NH3 occurred between the mixing of NaOH solutions with trapping solutions and the measurement of osmolality. It also appears that NH3 was the only substance 0 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
1.0 N HzS04
Urine sample (a)
Trapping solution (0.3 N H z S 0 4 ) (b)
Figure 1. Schematic diagram of the aeration bain. The syring attached to the sample tube contains saturated K2C03, which is to be injected into the sample
Table 11. Comparison of Titration Technique t o Osmometric Technique osmometric titration sample technique, technique, no.a mmol/L mmol/L 1 57.6 58.5 2 29.5 27.9 3 36.8 35.9 4 8.0 6.0 5 23.7 23.9 6 35.5 33.0 a No. 1 is an ammonia standard at 57.5 mmol/L. Nos. 2 to 6 are samdes of urine. extracted from urine and trapped in H z S 0 4 in significant quantities. DISCUSSION NH3 formed by the alkalinization of the trapping solution is not readily released into the environment, presumably because of its extraordinarily high solubility coefficient; 10 to 15 min of vigorous aeration are required to drive NH3 from alkalinized urine. The agreement between the osmometric method and the titration method (Table 11) proves that NH3 loss prior to the measurement of osmolality is negligible. The concentration of HzS04and NaOH need not be exact, provided t h a t NaOH is present in excess when the two solutions are mixed; NH4+is completely converted to NH3 when t h e solution p H is 12 or greater. The quantity of ammonia
that can be trapped is limited by the concentration of H2S04; for a solution of 0.3 N H2S04the maximal concentration of NH3 in the solution will be 300 mmol/L. For urine containing higher concentrations of NH3, smaller volumes of urine or larger volumes of trapping solution can be used. I t is also possible to use trapping solutions with a greater acid concentration and proportionally greater concentrations of NaOH, provided that the final osmolality of the mixture of the trapping solution and NaOH is within the range of the osmometer. If the concentration of NH3 in the urine is very low, the use of a larger volume of urine will enhance the accuracy of the technique. Vigorous aeration is very important in ensuring complete recovery of NH3 within a given time period; incomplete recovery of NH3 results from insufficient release of NH3 from the alkalinized sample rather than from incomplete trapping in the HzS04 solution. However, overly vigorous aeration may cause the H2S04solution to coat the sides of the tube, and a spuriously high NH3 concentration may be obtained. This difficulty may be obviated by the use of tubes of larger diameter for trapping and also by inversion of the tube a t the end of trapping to allow complete mixing of the HzS04solution. By using test tubes of 2.3-cm diameter, complete release and trapping of NH3 was possible in 10-15 min of aeration, whereas 20-30 min of aeration were needed with tubes of 1.5-cm diameter. Cunarro et al. concluded that the ammonia electrode provides the most accurate and rapid determination of urinary ammonia concentration (6). However, potential disadvantages of the ammonia electrode include its expense and short life span (average, 3 months). These disadvantages make the electrode method primarily suitable for laboratories doing large numbers of determinations. If an osmometer is available, ordinary laboratory supplies can be utilized to complete the aeration for the method described herein. The method is technically simple and rapid. The ease with which ammonia can be extracted and trapped and the precision of osmometry provide for accurate results. LITERATURE CITED (1) Oh, Man S.;Whang, Edmund M. S.;Carroll, Hugh J. Kidney Int. 1975, 8, 56-59. (2) Sobel, Albert E.; Hirschman, Albert; Besman, Lottie. Ind. Eng. Chem. 1947, 49, 927-929. (3) Conway, E. J.; O'Malley, E. Biochem. J . 1942, 36, 655-661. (4) Jorgensen, K. Scan. J . Ciin. Lab. Invest. 1957, 9 ,287-291. (5) Wilcox, Alan A,; Carroll, Wallace E.; Sterling, Rex E. Clin. Chem. 1986, 12, 151-157. (6) Cunarro, Julia A.; Weiner, Michael W. Kidney Int. 1974, 5 , 303-305.
RECEIVED for review July 9, 1979. Accepted September 4, 1979.
Ligand Exchange Chromatography of Alkyl Phenyl Sulfides Vaclav Horak," Mercedes D e Valle Gurman, and George Weeks Department of Chemistry, Georgetown University, Washington, D.C. 20057
Chromatographic behavior of eight alkyl phenyl sulfides (RS-Ph; R = Me, Et, Pr, /-PI, Bu, sec-Bu, i-Bu, and I-Bu) was examined using Hg2+,Ag+, Cd2+, and Pb2+ impregnated silica gel plates. Very efficient separation was achieved with Hg2+ and Ag' ions in solvents of medium polarRy (chloroform, ethyl acetate). The contribution of polar ((TI), molecular weight (MW), and steric effects E s to R, values was determined. For Hg2+ and Ag' impregnated plates, respectively, the foilowing equations were calculated: 0003-2700/79/035 1-2248$01 0010
+ 0.077 Es + 0.679 R M ( A g + ) = -2.397~*- 0.0077 MW + 0.156 Es + 1.152
RM(Hg*+) =
-1.933~*- 0.0061 MW
Recently, numerous selective separations have been achieved with compounds carrying either K- or n-electrons using ligand exchange chromatography. Chromatographic separations of organic sulfides on stationary phases with anchored Hgn (1,2),Znn (3), and Cun ( 4 ) have been reported in the literature. For some of the chromatographic systems 0 1979 American Chemical Society
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