Water Research Vol. 12. pp. 399 to 402 Pergamon Press Ltd. Printed in Great Britain.

0043-1354/78/0601-0399 $02.00/0

AMMONIA DETERMINATION BASED ON INDOPHENOL FORMATION WITH SODIUM SALICYLATE

H. VERDOUW, C. J. A. VAN ECHTELD and E. M..l. DEKKERS Limnologisch lnstituut, Nicuwersluis, The Netherlands
(Received 29 March 1977; receivedfor publication 8 December 1977)

Abstract--An ammonia determination based on formation of a substituted indophenol with sodium salicylate as phenolic reagent has been developed and compared with other methods. Sensitivity and reproducibility are comparable with results obtained in a method where phenol was used, while a number of the disadvantages inherent to the use of phenol are avoideat. The salicylatemethod is specific for NH3-N and interferences are generallyabsent in samples from natural fresh waters. The method can be easilyapplied for seawater analysis.

INTRODUCTION The determination of ammonia based on a reaction with phenol and hypochlorite, in which a bluecolourcd indophcnol compound is formed, has found a wide acceptance in recent years Solorzano (1969); Harwood & Kiihn (1970); Nimura (1973); Liddicoat et aL (1974); Scheiner (1976). Although the details of the mechanism are unknown, the following overall reaction is generally accepted for the indophcnol formation:

resulting in a very unsatisfactory sensitivity compared to the phenol method. In the present study the optimal reaction conditions for the salicylate method have been established and compared to those for the phenol method.
METHODS AND REAGENTS

Water samples were fdtered through 0.45/~m membrane filters, Interstitial water was obtained by centrifugation of sediment samples at 23,000g. Extinction values were

2 (~}'-O-+ NH3 + 3CtO" " - O ' - ~ N : X ~ O + 2H:,O+OH-+3CtCatalysts of different nature are used for the reaction, especially sodiumnitroprusside and potassiumferrocyanide. Citrate is generally used to prevent the precipitation of ions that would form insoluble hydroxides at the high pH of the reaction mixture. The reaction is dependent on the light conditions. The method is very sensitive and can be applied to many types of samples. However, the hazards associated with the use of phenol (poisonous, volatile) and the formation of a very unpleasantly smelling compound during the reaction, led us to investigate for an alternative phenolic reagent. A compound with the very pvnetrant smell was isolated after a large-scale ammonia determination and indications were found from infrared spectroscopy that ortho-chlorophenol was present. To prevent the formation of this very poisonous and volatile compound, phenol should be replaced by an ortho-substituted analogue. The introduction of a protecting substituent should, however, affect the reactivity of the phenolic para-position as little as possible. This condition, together with the necessity of good solubility in water led to the choice of the sodium salt of salicylic acid. This compound has been used by others, Bcnesch & Mangelsdorf (1972), measured with a Zciss spectrophotometer type PMQ II. Determination of NH3-N by distillation/Nessler procedure was pcrformegl according to Golterman (1971); enzymatic determination according to Verdouw (1973). pK, values of indophenols formed in the phenol and salicylate methods were determined by titration with HC1 of the reaction mixtures of standard NH3-N determinations. After full colour development pH and extinction at 635 nm (phenol method) and 660 nm (salicylate method) were measured after each HCi addition. The formula used for calculation of pK, was: pK, = pH - log (indophenol dissociated) (indophenol) As the blue colour of the indophenol is due to the dissociated form, the quotient indophenold~/indophenol can b estimated by E/(Eo -//),where E o is extinction after full colour development and before addition of HCI, i.e. when pH is wry high and well above pK,, so all indophenol is dissociated, and E is extinction after addition of HCI. At the point where E = 0.5 Eo the 10g term in the formula equals zero, so pK a = pH. Corrections were made for changes of volume by the addition of HC1. The determinations were carried out in triplicate. All reagents were analytical grade chemicals. SELECTION OF REACTION CONDITIONS Reagent concentrations Optimal reagent concentrations were selected by varying 399

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H. VERDOUW,C. J. A. VAN ECHTELDand E. M. J. DEKKERS Table I. Reaction conditions and typical results for the phenol and salicylate method. Concentrations given for final reaction mixture. Final volume 50ml. Room temperature. "Laboratory light". 20/~g of NH3-N present, except in blank determinations. Equivalent optical density = extinction caused by 1/zg of NH3-N in final volume, measured in 1 cm caret. Sequence of reagent additions as described in standard procedure for salicylate method Phenol method Phenol Sodium salicylate NaOCI NaOH K4Fe(CN)6.3H20 pH Reaction time Measuring wavelength Equivalent optical density Blank extinction Colour stability pK, ofindophenol 0.5% = 0.053 M -0.15% = 0.021 M 0.1% = 0.025 M 0.02% = 4.7 x 10 -4 M 10.8 40 min 635 nm 0.030/zg- 1 N H 3-N cm- 1 0.022 cm- t At least 12 h 7.91 + 0.20 Salicylate method -4 % = 0-24 M 0.077% = 0.01 M 0.04% = 0.01 M 0.2% = 4.7 x 10 -3 M 12.0 60 min 660 nm 0.029#g- 1 NH 3-N cm 0.030 cm- t At least 12 h 10.44 + 0.23

phenol or salicylate, NaOH, NaOCI and catalyst concentrations over a wide range, covering most of the values mentioned in the literature. Every concentration of phenol or salicylate was combined with different NaOH concentrations; to each of these combinations varying amounts of NaOCI were added, etc. This laborious procedure was the only way to find out the optimal reaction conditions, as the data from the literature are extremely variable. The selected reaction conditions and other data for the phenol and salicylate method are summarized in Table 1.

Influence of li#ht
Data from the literature concerning influence of light show the necessity of further research in this area. Liddicoat et al. (1975) found good results with the phenol method when u.v. light was applied during the reaction period; potassiumferrocyanide was used as a catalyst because better blanks were obtained than with sodiumnitroprusside. Gravitz & Gleye (1975) preferred the reaction to proceed in the dark, due to light-induced interferences; they used sodiumnitroprusside as a catalyst. Using the phenol method we also found, that potassiumferrocyanide should be preferred to sodiumnitroprussid for better colour stability in light and dark. In our work with the salicylate method, "laboratory light" did not interfere with the results. On the other hand, light appears to be an even more important factor than for the phenol method: colour development in the dark was very poor (see Table 2). This Table gives a

comparison of colour development/stability in the dark and in normal light conditions. Though the light effects were not quantified, we got reproducible results under varying light conditions in the laboratory, i.e. daylight plus T.L. light. Only in the case of very dark weather, it proved to be necessary, to put the reaction flasks closer to the windows, in order to maintain the reaction time of 60 min. The analytical results were not affected by this procedure, which was checked by running standard samples in each series of determinations. We prefer this procedure, as putting the flasks very close to T.L. light sources resulted in higher blanks. Further investigations in this regard, which are in progress, certainly will lead to more information on the details of the reaction mechanism of indophenol formation, especially about the role of the catalyst. THE SALICYLATE METHOD

Standard procedure for the salicylate method Stock solutions of reagents. (A) sodium sahcylate solution
40 % in distilled water, i.e. 40 g dissolved in water and filled up to 100ml. Prepare directly before use. (B) NaOCI solution 1.93~'o in 0.1 N NaOH. This solution is stable for months if stored in a cool and dark place. (C) K4Fe(CN)~. 3H20 2 % + Na3Citrate. 2H20 10% in 0.1N NaOH. Stable for weeks if stored cool and dark. Procedure. A filtered water sample, max 38 ml and containing not more than 25 pg of NH3-N, is put in a 50ml

Table 2. Colour development in the light and in the dark for the salicylate method. Standard procedure for salicylate method. 20/~g of NHa-N present Light Ebla,k 0.030 0.031 0.031 0.031 0.032 0.036 0.041 0.044 Dark Ebj,nk 0.012 0.016 0.020 0.022 0.022 0.024 0.030 0.036

Time 1h 2h 3h 4h 8h 12 h 24 h 48 h

E 0.607 0.609 0.608 0.610 0.610 0.612 0.609 0.458

E - Ebla,k 0.577 0.578 0.578 0.579 0.578 0.576 0.568 0.414

E 0.182 0.267 0.392 0.512 0.517 0.532 0.540 0.546

E - Ebla.k 0.170 0.251 0.372 0.490 0.495 0.508 0.510 0.510

Ammonia determination based on indophenol formation with sodium salicylate measuring flask. Care should be taken with vacuum filtering of samples with pH of 9 and higher, as considerable amounts of NH3-N can be lost, because a big portion of the ammonia is in the volatile NH 3 form in that case (Golterman, personal communicatiofi). Distilled water is used to fill up to 35 ml if a smaller sample is used. Neutralize acid samples before proceeding further. Add 5 mi of solution A, 5 ml of solution C and 2 ml of solution B, mixing the contents of the flask after each addition. If necessary, fall up to 50 ml with distilled water. After 60 rain, measure the extinction at 660 nm in a l cm curet. Calibration curve. A calibration curve was prepared according to the procedure given above, using different volumes of an (NH4)2SO 4 solution containing 2#g of NH3-N per ml. See Fig. I.

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Application in the determination of Kjeldahl nitrogen

Checking the salicylate method for the determination of NHs-N formed after destruction of organic nitrogen (Kjeldahl nitrogen), we used the destruction technique of Scheiner (1976). Before NH3-N determination the strongly acid samples were neutralized. Interestingly, a greater sensitivity was obtained when samples for NH3-N calibration curve were prepared using the destruction procedure. In that case the equivalent optical density was 0.034 #g-1 NHs-N cm-t. If only the destruction reagents were added and NH3-N was determined without the destruction procedure, the results were not different from those in the normal standard procedure, i.e. equivalent optical density was 0.029 pg -1 NH3-Ncm -I. These results were very reproducible. We are using this method for the routine determination of Kjeldahl nitrogen for some time now, although the effect of the destruction procedure on the indophenol colour is not understood.

Possible interferences
E66o The following ions have been tested separately for interference in the salicylate method (standard procedure, 20/~g of NH3-N present). Ca 2+ up to Mg 2 + up to S 2- up to Fe 3+ up to Cu 2+ up to Fup to SiO~--Si up to 5 mg in final volume 1 mg in final volume 150/zg in final volume 25/zg in final volume 100/~g in final volume 20/zg in final volume 100/zg in final volume

o5oo

I0

,20

H-gNHs-N
Fig. 1. Calibration curve for the salicylate method. Standard procedure for salicylate method. The amounts of NH3-N present in the final reaction mixture are indicated. Triplicate determinations.

Effect of temperature
Increase of temperature affects the velocity of indophenol formation, but maximum extinction is not influenced. At temperatures of 21, 31 and 41C the maximum colour development was reached after 50, 40 and 25 rain respectively. In spite of the possible time saving at higher temperatures, the chances of decomposition of organic N-compounds are greater. Therefore, the determinations are carried out at room temperature. Reagents stored in the cold should be brought to room temperature before use.

Neither decrease in sensitivity nor any other interference was observed, except for Fe 3+, in which case a red colour developed after addition of the salicylate, changing to yellow when the alkaline solution was added. This colour, due to complex formation of Fe 3+ with salicylate (as does Fe 2+) does not influence the measurements, as there is no absorption in the 600-750 nm region. Interference effects of organic nitrogen compounds separately, as done by Nimura (1973) with a phenol method, were not investigated. We felt, that sufficient information about possible interferences by naturally occurring organic N-compounds could become available by recovery experiments with NH~-N added to several types of natural water. Using samples from different sources, all NH3-N was recovered, indicating no significant interferences. Next to this, comparison with other methods, especially the highly specific enzymatic method, gives reliable information on possible decomposition of organic N-compounds under the conditions of the salicylate method.

Comparison with other methods

The salicylate method has been compared with other techniques for ammonia determination in our laboratory.

Table 3. Comparison of different methods for ammonia determination. All determinations performed with the same filtrate of the samples. Data given as rag. 1- i NH3_N. Mean values of triplicate determinations, with maximum deviations from these values Method Sample Lake Vechten (interstitial water) River Vecht River Rhine Salicylate Enzymatic Salicylate Nessler after distillation 10.21 (0.17) 6.78 (0.11) 2.43 (0.12) 10.75 (0.21) 6.79 (0.15) 2.51 (0.17)

8.80 (0.11)* 6.33 C0.09) 2.15 (0.09)

8.91 (0.15) 6.12 (0.11) 1.99 (0.19)

* Initial yellow colour due to iron present.

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H. VERDOUW, J. A. VANECHTELDand E. M. J. DEKKERS C. high concentrations of amino acids on a phenol method. Some care must be taken with the pH of the samples, as the pK~ value of the indophenol formed in the salicylate method (10.4) is rather high compared to the optimal pH for the reaction (12.0). In other words, a pH of < 12 might easily result in a decreased sensitivity, as less indophenol is dissociated and therefore less colour develops. This is clear from our findings with the analysis of seawater, for which the method can be easily adapted by addition of extra NaOH. Several phenomena occurring in indophenol formation are not yet understood, i.e. role of catalyst, effects of light and the influence of the destruction procedure when Kjeldahl nitrogen is determined. Further investigation on these subjects is needed. In spite of these theoretical uncertainties, the salicylate method has proved to be very useful because of its ease in application and great reproducibility.

The ammonia content of samples from different sources was determined as follows: salicylate method with and without distillation, the direct enzymatic method and the Nessler method afiex distillation. The results shown in Table 3 indicate, that the salicylate method is almost as specific as the enzymatic method: very little difference between the results, which means, that also in the salicylate method only NH~N as such is determined and that the risk of decomposition of organic N-compounds is small, even in the case of interstitial water of sediments, which are particularly rich in these compounds. At the same time, the disadvantage of distillation is shown very clearly, as the ammonia content is overestimated due to decomposition of organic material.

Application in seawater analysis; effect of pH Using artificial seawater (salinity 3"43%), with (NH,)2SO4 added for making a calibration curve, we found that the time for colour development did not change, but the sensitivity was less: equivalent optical density was 0"020/ag-1 NH3-N cm- i. This effect appeared to be caused by a lowering of the pH: 10 ml of seawater caused a decrease oftbe pH to 11.2.This effect could simply be prevented by the addition of more NaOH. The amount of citrate present was sufficient to prevent precipitation of hydroxides.
DISCUSSION

Acknowledgements--We are indebted to Dr. H. L. Golterman for critical interest in this work and to Dr. R. D. Gulati and Dr. R. Soekarjo for review of the manuscript.
REFERENCES

Comparison of the salicylate method with the phenol method indicates, that salicylate is a good alternative for phenol. Although the reactivity of salicylate is less than that of phenol, due to the introduction of a carboxylgroup (see Table 1: salicylate concentration is 4 times the phenol concentration for optimal conditions), the disadvantages of phenol, i.e. volatile reagent and o-chlorophenol formation, are much reduced, whereas the decrease of sensitivity is negligible. There is no need for frequent calibration. The calibration samples run with every set of determinations for a year resulted in the same equivalent optical densities, even though new reagent solutions were prepared from time to time. Interference effects are unlikely with samples from natural waters, so pretreatment of samples (distillation) is unnecessary. The only case in which we noticed difficulties was when ammonia was determined in water in which zooplankton (Daphnia magna) had been for some time: poor colour development which was probably due to very high levels of excretion products. See Nimura (1973) for interferences of

Benesch R. & Mangelsdorf P. (1972) Eine Methode zur coiorimetriscben Bestimmung von Ammoniak in Meerwasser. Helooltinder wiss. Meeresunters. 23, 365-375. Golterman H. L. with Clymo R. S. (1971) Methods for Chemical Analysis of Fresh Water. IBP Handbook No. 8, Blackwell, Oxford. Gravitz N. & Gleye L. (1975) A photochemical side reaction that interferes with the phenolhypochlorite assay for ammonia. Limnol. Oceanoor. 20, 1015-I017. Harwood J. E. & Kfihn A. L. (1970) A colorimetric method for ammonia in natural waters. Water Res. 4, 805-811. Liddicoat M. I., Tibbits S. & Butler E. I. (1974)The determination of ammonia in seawater. Limnol. Oceanoor. 19, 131-132. Nimura Y. (1973)A direct estimation of microgram amounts of ammonia in water without salt-error. Bull. Jap. scient. Fish. 39, 1315--1324. Schein~ D. (1976) Determination of ammonia in Kjeldahl nitrogen by indophenol method. Water Res. 10, 31-36. Solorzano L. (1969) Detrmination of ammonia in natural water by the phenolhypochlorite method. Limnol. Oceanogr. 14, 799-801. Verdouw H. (t973) Enzymatic NH3-N determination: a specific method for the determination of ammonia in water and sediments. Water Res. 7, 1129-1136.