Anal. Chem. 1986, 58, 585-587
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Ammonia and Urea Determination in Water Samples Using Amberlite XAD-7 To Concentrate Indophenol Pilar Moreno, Elena Sbnchez, Antoni Pons, and Andreu Palou* Departament de Bioquhica, Facultat de CiZncies, Uniuersitat de les Illes Balears, 07071 Palma de Mallorca, Balears, Spain
A modlflcatlon of the Indophenol method applied to the determination of ammonla and urea in the same sample is described. Indophenol, formed proportionally to the amount of ammonia available, is selectively concentrated In a slngie purification-concentration step, using slmple and recyclable columns of Ambertlte XAD-7 resln, thus Increasing the method sensltivity. Its application to seawater samples Is described. The procedure Is appllcable to analysis of large numbers of samples running slmultaneousiy in routlne studles and ellmlnates possible artlfacts that may alter the precision and sensitlvlty of the method.
The indophenol method for ammonia estimation, first described by Berthelot ( l ) ,has been modified and adapted by several authors (2-5). It is yet in use, especially when adapted to the urea determination, in combination with urease (6-8). Water samples are treated in an alkaline medium with sodium hypochlorite and phenol, in the presence of nitroprusside, which acts as a catalyst. The blue indophenol formed with ammonia is then measured colorimetrically. Indophenol is not the only product formed in the reaction, thus the treatment of different types of samples can provide different suites of chromophores (9-12). As a consequence, several interferences in the measurement of ammonia have been found (10-12). However, the stability of the indophenol dye and reproducibility of the method, together with the general lack of simple and reproducible methods for ammonia, can explain its actual widespread use. The use of polymeric porous hydrophobic absorbing beads for the extraction of hydrophobic organic compounds dispersed in aqueous solutions is a well-known and documented procedure (14). We have used these resins for the concentration of specifically derivatized materials in single-step purification-concentrations (14-16). The application of these purification-concentration procedures to indophenol improves the sensitivity of the procedure for use in very dilute samples. Potential interferences, as they are reduced to only those trapped in the Amberlite columns, are minimized. Such a procedure is described in this paper applied to the determination of both urea and ammonia in seawater samples.
EXPERIMENTAL SECTION Standard solutions of urea (Sigma) or ammonium sulfate (Merck) were prepared in distilled deionized water (Milli-Q-quality Millipore; minimum specific resistance 10 MO cm). Surface seawater samples used for validation of the method were obtained from the Mediterranean Sea about 2-3 miles off the south coast of Majorca. Amberlite XAD-7 (Sigma, St. Louis, MO) was selected because its hydrophobicity is the lowest in the XAD series. It is able to retain even mildly hydrophilic materials with hydrophobic domains in the molecule, allowing the easy elution of the retained indophenol chromogen. All the other reagents and solvents used were of analytical grade. Amberlite columns were 5-mL plastic syringes, 10 cm long and 1cm in diameter, containing 0.5 g of the resin. They were fitted 0003-2700/86/0358-0585$01.50/0
with a 1mm i.d. polyethylene capillary tube for flow control and collection of the column eluates (14-16). A wide glass tube of buret with a volume of 25 mL was also connected to the top of each column to hold the sample prior to passage through the column. The low cost of the columns allowed their use in arrays so as to allow several samples (up to 50) to be processed in parallel. Columns could be recycled several times, provided that before their immediate use they were washed with acetone and finally equilibrated with 15% ethanol in water. Urease (Sigma) was purified as previously described (8)except that 2-mercaptoethanolwas used instead of dithiothreitol. Urease incubation of the samples was done as previously described (8). The standard 20-min incubation time was selected from the time course of the reaction shown in Figure 1. For both urea and ammonia determinations, volumes of sample in the range of 2-200 mL were tested. In this paper we mainly refer to 10-mL samples for easier explanations. The results of the use of this procedure with larger sample volumes (from 2 to 200 mL) are also shown (Figure 5) to provide a better idea of the volume and other limitationsof the method. The general reaction conditions for indophenol formation were those described by Solorzano (4). For the determination of urea two aliquots of the sample were treated in parallel: one was hydrolyzed with urease and the other was not. The amount of urea in the samples was calculated from the difference between the two samples. A reagent blank for urea determination was obtained by means of reversed addition of reqgents, Le., urease addition after incubation and immediately prior to the addition of ammonia color-developing reagents. However, it was found that under the experimental conditions described, no significant differences have been observed in absorbance when these blanh or the reagent blanks (without urease) are used. This is probably a consequence of using previously dialized urease (8). From the different fractions described below (A, B, C, and D) aliquots were taken to evaluate recoveries from the procedure and to validate the method. Absorbance was measured at room temperature with a 220s Hitachi double-beam spectrophotometer with 1-cm cells. Procedure. Sample volumes of 10 mL were introduced into 25-mL Erlenmeyer flasks. First, 0.4 mL of phenol solution (100 g/L in 95% ethanol in water) was added to the sample. Then 0.4 mL of sodium nitroprusside solution (5 g/L in water) and 1 mL of oxidizing solution (prepared by mixing 4 volumes of 1M trisodium citrate in 0.25 N NaOH with 1volume of 1.5 N sodium hypochlorite) were added, mixing thoroughly after each addition. The flasks were allowed to stand at room temperature for at least 45 min (fraction A). Then the samples were passed through the Amberlite columna, and the unretained materials were collected as fraction B. Then 2 mL of acetone was used to elute the trapped chromophore. The acetone was evaporated under vacuum or under a gentle dry nitrogen stream at not more than 45 O C . The residue was then redissolved in either 2 mL (fraction D) or 10 mL (fraction C) of distilled water.
RESULTS AND DISCUSSION Figure 1 shows the time course of ammonia production (measured with the indophenol reaction) following urease addition to standard urea solutions in seawater. From these results an optimum incubation time of 20 min can be assumed in accordance with Strickland and Parsons (8). Figure 2 shows the relation between indophenol absorbance and urea concentrations, both for the concentrated fraction (D) and the @ 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
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Figure 1. Time course of urease action upon solution of urea, measuring the ammonia evolved wlth the indophenol reaction. Each point is the mean, f standard error of the mean of four to five different determinations in 10-mL sample volumes,
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Flgure 3. Absorbance at 640 nm of reaction products (A) and convs. ammonia concentration (nmoVtube)comparing centrated eluates (0) seawater standards (solid lines) with distilled water standards (dashed lines). Fractions A and D are those described in Table I. Seawater standards are corrected for the NH, content of each. Results refer to an initial sample volume of 10 mL
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Figure 2. Absorbance at 640 nm of reaction products (A) and concentrated eluates (B) against urea content of the tubes (nmol). Fractions A and B are described in Table I. Results refer to an initial sample volume of 10 mL.
initial one (A). The absorbances in D were higher, by an average factor of 3, than those of A while their ratio of volumes was actually 5 (from 10 to 2 mL). Despite the loss of dye, this situation is compatible with indophenol being quantitatively trapped and recovered, while additional colored products, also formed in the reaction (10-12),were not trapped in the column (see below). Figure 3 shows the relationship between absorbance and ammonia concentration, in both distilled water and seawater. The slopes of the lines obtained were lower when distilled water was used. This is in accordance with a lower rate of the Berthelot reaction in distilled water vs. seawater (4). The values shown for seawater are corrected for-its own ammonia content. In Figure 4 the absorbance spectra of the different (A, B, C, and D) fractions are shown. The Amberlite column procedure resolves the products f the reaction-which, in accordance with Solorzano (4), show a maximum a t 640 nm (A)-into a fraction (C or D) with a maximum at 625 nm, the same as pure indophenol (22),and a fraction (not retained B) with a maximum a t 650 nm, corresponding to materials with lower hydrophobicity than indophenol, probably quinonechloramine derivatives or other intermediary products formed in the Berthelot reaction (9). The above observations explain why the absorbance multiplicative factor is lower than the volume concentrative factor: only indophenol, among the colored products of the reaction, is selectively retained by the Amberlite column. I
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Flgure 4. Light absorption spectra of the different fractions studied
(see Table I). Table I. Slopes Obtained Relating the 640-nm Absorbance in the Different Amberlite Isolated Fractions (A, B, C, and D) of Ammonia Estimation with the Indophenol Method in Both Distilled Water and Seawater
sample fraction A" Bb
C' Dd (B + C/A) X 100 (B/A) X 100
slope, A/kmol distilled seawater water 1.48 0.66 0.83 4.13
100.7 44.6
linear corr coeff ( r 2 )(N= 5 ) distilled seawater water
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100 39.8
OInitial. b N o t retained by the column. 'Retained by the column. dRetained (C) eluted with 2 mL of acetone.
Table I shows the slopes of the linear regresion lines obtained from the 640-nm absorbance vs. nanomoles of ammonia, for fractions from both seawater and distilled water standards. A higher variability of the data (lower correlation coefficient) was found when distilled water, was used. It can be seen that
ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
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Figure 5. Relation between absorbances at 640 nm and concentration of ammonia is distilled water, for initial sample volumes of 2-200 mL concentrated to 2 mL with columns containing 0.5 g of Amberlite XAD-7. Each point Is the mean of two to three determinations.
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the added B C slopes correspond well with the A slope, in both seawater and distilled water. Slightly less indophenol was formed when distilled water was used. Conversely, the percent of the unretained material was higher in distilled water solutions (44.6 vs. 39.8%). Thus, the combined B + C slopes give the lower final A slope obtained from the initial fraction of the distilled water standards vs. seawater standards. An immediate conclusion is that when ammonia is to be measured in seawater, seawater standards should be used (4) to prevent inaccuracies due to enhanced formation of indophenol in seawater. However, the absorbance values for a distilled water blank should be corrected by multiplying by the ratio of the slopes of seawater and distilled water standard solutions. The measurement of absorbances at 625 nm instead of at 640 nm as is described in the literature ( 4 ) can provide a slight, but consistent, increase in sensitivity. The spectra in Figure 4 suggest that indophenol is not actually the only dye extracted, as some other products that mainly absorb light at wavelengths lower than 600 nm are found. However, this situation does not significantly affect the validation of the procedure described. These materials can be largely eliminated by rinsing (before eluting with acetone) the column with 15% ethanol; however this was accompanied by the loss of some indophenol. Since these chromogens did not interfere in the proposed method, it was decided not to apply this prior extra purification step. When the absorbance spectra of the unretained fraction (B) was determined within a few minutes after its elution and was compared with those taken several hours after elution (results not shown), a displacement of the maximum absorbance of this fraction B toward shorter wavelengths could be observed,
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whereas the indophenol fraction (C or D) remained practically stable, Thus it can be inferred that in the classical methods, a possible artifact could appear due to this slowly forming dye. The actual relevance of this effect in most cases is probably minimal, but cannot be neglected. This possible artifact can be eliminated by using the procedure outlined in the present paper. In Figure 5 the results obtained by applying this procedure to larger initial sample volumes (from 2 to 200 m L in distilled water) are shown. It can be seen that the proportionality between absorbance and ammonia concentration was well maintained up to 50 mL, but not completely for sample volumes of 100 and 200 mL. This seems partly due to incomplete recovery of chromogen in the larger volume samples. The linearity of the response-linear correlation coefficient (r2)for 2-50-mL sample volumes higher than 0.994-allows the secure and correct use of samples up to 50 mL in our conditions; for larger sample volumes or higher ammonia concentrations more Amberlite resin as well as new adjustments of the elution volumes would be required. In conclusion, the procedure described can be applied to the simultaneous determination of urea and ammonia in water samples, improving, by means of a single step of concentration-purification, the sensitivity of the method, while providing increased accuracy and precision. Registry No. Urea, 57-13-6; ammonia, 7664-41-7; water, 7732-18-5;indophenol, 500-85-6;Amberlite XAD-7,37380-43-1.
LITERATURE CITED (1) Berthelot, M. Rep. Chim. Appl. 1859, 1 , 284. (2) Chaney, A. L.; Marbach, E. P. Clin. Chem. (Winston-Salem, N.C.) 1962, 8, 130-132. (3) McCullough, H. Ciln. Chlm. Acta 1968, 19, 101-105. (4) Solorzano, L. Limnol. Oceanogr. 1969, 14, 799. (5) Pym, R. V. E.;Milham, P. J. Anal. Chem. 1976, 48, 1413-1415. (6) Fawcett, J. K.;Scott, J. E. J . Clin. Pathol. 1960, 13, 156-159. (7) Clapp, J. R. Am. J . Physiol. 1966, 210, 1304-1308. (8) Strickland, J. D. H.; Parsons, T. R. “A Practical Handbook Of Sea Water Analysls”; Fisheries Research Board of Canada: Ottawa, 1972; pp 87-89. (9) Bolleter, W.T.; Bushman, C. J.; Tldwell, P. W. Anal. Chem. 1961, 33, 592-594. (10) O’Donovan, D. J. Clin. Chim. Acta 1971, 32, 59-61. (11) Forgan-Smith, J.; Slaughter, El. D.; Cross, R. 6. Clin. Cbim. Acta 1976, 69, 139-141. (12) Ngo, T. T.; Phan, A. P. H.; Yam, C. F.; Lenhoff, H. M. Anal. Chem. 1982, 54, 46-49. (13) Dressler, M. J . Cbromatogr. 1979, 165, 167-207. (14) Roca, P.; Palou, A.; Alemany, M. J. Biochem. Biopbys. Methods 1984, 10, 181-185. (15) Roca, P.; Glanotti, M.; Palou, A. Anal. Biochem. 1985, 148, 190-194. (16) Gianotti, M.; Roca, P.; Palou, A. J. Biochem. Biophys. Methods 1984, 10. 181-185.
RECEIVED for review June 27,1985. Accepted October 7,1985. This work was supported by a grant form the Universitat de les Illes Balears-Govern de la Comunitat Autonoma de les Balears and C.S.I.C., Spain.
