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Water Research Pergamon Press 1970. Vol. 4, pp. 805-811.

Printed in Great Britain

A COLORIMETRIC METHOD FOR IN NATURAL WATERS

AMMONIA

J. E. HARWOODand A. L. Kf3HN National Institute for Water Research of the South African Council for Scientific and Industrial Research, P.O. Box 395, Pretoria, Republic of South Africa (Received 18 May 1970)
Absa'act--An improved phenol-hypochloritc method for determining ammonia in water using nitroprusside as catalysthas been investigated. Unlike other recently published methods, the method issimple (and sufficientlysensitivefor low-levelanalyses) and does not require accurate time intervalsbetween reagents,nor expensive equipment. The method is well suited for routine
USe.

Beer's L a w was obeyed over the whole ammonia range investigated. Reproducibility was 1.3 per cent with 10 fzg N, and 5.7 per cent with I p.gN.

INTRODUCTION AMMONIA Occurs in many waters both as a trace component (less than I mg N 1-1) and at higher concentrations, when it is often used as a chemical indicator of sanitary pollution (STANDARDMETHODS, 1965). At high concentrations, distillation and subsequent absorption of the released ammonia in dilute acid followed by titration is a generally accepted method (STANDARD Mm'HODS, 1965). Distillation is generally unsatisfactory due to the possible hydrolysis of nitrogenous material to ammonia (Strl'CLIFFE and JONES, 1968). For samples containing trace concentrations of ammonia, distillation methods of analysis become tedious because of the large volumes being handled and long distillation times. Because of this, colorimetric methods are often resorted to, a standard procedure being that of Nesslerization (STANDARDMETHODS, 1965). The Nessler method is sensitive to numerous variables (THOMSONand MORRISON, 1951) including the age of the reagent (SUTCLIFFEand JONES, 1968) so it is not suited for direct routine application. JENKINS(1967) found distillation and subsequent Nesslerization to be satisfactory. It is also not sufficiently sensitive for low-level analyses (TETLOW and WILSON, 1964). Another colorimetric reaction with sufficient sensitivity is the phenol-hypochlorite reaction (TETLOW and WILSON, 1964), which is more specific and less subject to interferences than the Nessler reaction (SuTCLIFFE and JONES, 1968). The phenol-hypochlorite method consists basically of reacting a sample containing ammonia with phenol (or rather phenate) and hypochlorite. In the presence of a catalyst (Mn 2+, acetone, sodium nitroprusside) colour development proceeds rapidly at room-temperature to give a blue-coloured solution, the intensity of which is proportional to the ammonia content of the sample. The chromogenic species is believed to be an indophenol moeity. Many variations of this reaction are possible, but since the phenol-hypochlorite reaction has been shown to be less subject to interference than the hypochlorite-phenol reaction (S~ARcY et al., 1965) we have developed a method based on the former reaction. The work described here is based on the use of nitroprusside catalyst for the phenolhypochlorite reaction. Nitroprusside was selected since it has recently been shown wAczx4/12--c 805

806

J.E. HARWOODand A. L. K0mq

(HARWOOD and HUYSER, 1970a) to be better than acetone. The method does not require critical timing between addition of reagents (WEAraERBURN, 1967). The reagents are easily made up, and since the colour develops rapidly and is stable, the method appears ideal for routine analyses. EXPERIMENTAL

Reagents
All reagents should be A.R. where possible, in order to reduce the blank value. Distilled water passed through a mixed-bed deionizing unit (Elgastat) was used throughout. Buffer: 5 ~ (w/v) Na3PO, solution. Phenol stock: 500 g phenol were dissolved in methanol and diluted to 800 ml with methanol. Store at 2C. 27 ~ NaOH: 270 g NaOH pellets were dissolved in water, cooled and diluted to 1 litre. Phenate reagent A: 15 ml of the phenol stock and 0.02 g sodium nitroprusside were diluted to I00 ml with water. Reagent B: Equal volumes (15 ml) of commercial bleach (3"5~o C1) and 2 7 ~ NaOH solution were mixed, and diluted to 50 ml. Store both Reagents A and B in amber bottles in a fridge, but allow to reach room temperature before using.

Method
A suitable aliquot of sample (containing less than say 50 ~g N) is placed in a 25 ml volumetric flask and 2.0 ml buffer, added. All the flasks are then diluted to nearly the same volume (about 10 ml). Add 5 ml Reagent A, with swirling, to all the flasks, then add 2.5 ml Reagent B, also whileswirling. Dilute to the mark and mix thoroughly. Prepare both blanks and standards. After about 25 min, read the optical density at 630 m/~ against the blank, using appropriate cells for the colour observed.

RESULTS

The above method was tested on river samples containing 0.1 to 10 gg NH3-N ml- i. The samples containing from 2 to 10 gg N ml- 1 were analysed with 1 cm cells, and gave a correlation coefficient of r = 0.996 (n = 11) between samples analysed according to this procedure and using the Autoanalyser (HARwOOD and HUYSER, 1970b). With samples covering the range 0.1 to 1 gg N ml-1, the correlation coefficient was r = 0.998 (n = 18), using 4 cm cells. These results are shown in TABLEI. The method was reproducible, giving a coefficient of variation of 1-3 per cent with 10~g N. DISCUSSION Calibration graphs for both 4 cm and I cm cells are shown in FXG. 1. The regression lines shown, had correlation coefficients of r -----0-9993 and r = 0.9989, respectively. Beer's Law was obeyed over the whole range investigated in both cases.

A Colorimetric Method for Ammonia in Natural Waters


TABLE 1. AMMONIA ANALYSES ON RIVER WATERS, USING THE MANUAL METHOD REPORTED IN THIS PAPER AND THE AUTOANALYSER~ WITH 4 c m CELLS

807

Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Ammonia (rag N 1- l) Manual* Autoanalyser~1"17 0-94 1"13 N.D. 0"50 0'46 0.47 0"46 0'48 0'23 0"25 0'21 0"23 0'19 0-11 0"13 0"15 0.11 0'11 1'14 0"93 1"12 N.D. 0-45 0"44 0"44 0-44 0"45 0'20 0"26 0"23 0'25 0"22 0.10 0"t0 0-12 0.08 0"I1

* Average of triplicates. I"Average of duplicates.

The indophenol-blue reaction is widely used in the clinical field, but has not found the same popularity in water analyses. The reason for the popularity arose from the use of catalysts that gave rapid, room-temperature colour development, so eliminating the need for heating. Since hydrolysis of other nitrogenous materials was reduced, the method became more or less specific for ammonia. Of the catalysts, the use of acetone (CROwTH~ and LARG~, 1956) resulted in increased sensitivity over the use o f manganous ions (RuSSEL, 1944). Acetone was subsequently used in a method for trace analysis (TZTLOW and WIUSON, 1964). Nitroprusside (LtmocHI~S~tY and ZALTA, 1954) resulted in further improvements in sensitivity. HARWOOD and HuYsF_~ (1970a) showed that nitroprussidc was better than acetone as a catalyst. Major advantages were that the rate of colour development exceeded that obtained with acetone; the colour was stable, while that from acetone was not; and there was a considerable enhancement in sensitivity. In the light of these findings we decided to develop a manual method based on nitroprusside as catalyst, since it appeared far superior to acetone. The study also showed that at a final p H of about 12, the colour would be stable, develop rapidly, and be sensitive. O f the methods using nitroprusside, the early results were not reproducible. FAWCETT and SCOTT (1960) observed decreasing final intensities when there were increasing delays between addition of the reagents. CHANEY and MAmaACH (1962) attributed this to alkaline decomposition of the nitroprusside reagent. On addition of the nitroprusside in the phenol, and the caustic in the hypochlorite, the method became far more reliable, with no accurate time intervals required between addition

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J.E. HARWOOD A. L. KOHN and


22 2-1 2,0

1.9 t,8 1.7 1.6 1.5


1.4 1.3 1.2

I.I

{-0

09
0.8 0 0-7 0.6

0"5
04 0.3 0-2 O.l 0 0 ;, 3 ,* ~ 6 30 7 e 4o 9 ~o l(3~gN, 4era

io

zo

50Fg

N,

I cm

FIG. I. Calibrationgraphfor the determinationof NH3-N by the phenol-hypochloritemethod.

of the reagents. WEATHERBURN(1967) has confirmed these findings. HORN and SQUIRE (1967), and WEICI-ISELBAUMet al. (1969) reported that the nitroprusside should be reacted with caustic prior to use. We have found decreased sensitivity in the automated method (I-IAxWOODand HLrYSER, 1970b) if the nitroprussid was added prior to the hypochlorite, i.e. the nitroprusside was apparently adversely affected by the alkaline solution. We decided rather to use the reagent combinations of CHANEYand MARBACH (1962) and WEATHERBURN(1967). To avoid weighing out the phenol frequently, we chose to dissolve it in methanol, using a modification of the reagent of CROWTHER and LARGE (1956). The use of methanol also resulted in increased sensitivity over the phenol in water solution only. This last solution resulted in a slightly turbid solution, so that in order to take large volumes of sample it was necessary to make the phenol up in methanol. In an initial study of the phenol hypochlorite reaction (HARWOOD and HUYSER, 1970a) suitable final concentrations for the reagents were phenol (2 per cent), hypochlorite (0.05 to 0-10) and nitroprusside (0.02 per cent). This concentration of nitroprusside proved unsuitable for manual work due to the intense yellow colour of the blank. The final nitroprusside concentration was reduced, to bring it more into line with the concentrations used by CHANEY and MARBACH (1962) and WEATHERBURN (1967) (0"0025 per cent). The sensitivity remained unaltered, and the blank value was reduced. This final concentration (0.004 per cent) was adopted in the method.

A Colorirnetric Method for Ammoniain Natural Waters

809

The rate of colour development was also checked, and found that a constant value was reached after 20 rain, remaining stable for another 4 h thereafter. Since the rate of development is temperature dependant (WEATHm~UgN, 1967) these criteria may alter slightly from one laboratory to another, and from time to time. A routine method should have certain characteristics, in addition to the usual properties of accuracy and precision. The reagents should be stable to avoid the necessity of the frequent preparation of reagents. There should be few restrictions on either the time intervals between reagents or the volume added (to enable a reagent to be added serially to all the samples prior to adding the next reagent), while the colour developed should develop rapidly and be stable (to avoid having to perform an operation after narrowly defined time intervals). The fewer the number of manipulations required the less likely will the method be dependant on operator skill. Applying these criteria to other published manual methods, we see that most of the published methods fail on at least one of the above criteria. It has been shown (HARWOOD and HUYSER, 1970b) that the colour developed with acetone catalyst was unstable. Methods using acetone would require that the colour be read within a fairly well defined interval so making them unsuitable for routine use. TETLOWand WIU~ON 0964) used a time interval of 60 -4- 5 rain. Lengthy procedures (NEWELL, 1967; EMMET, 1969; ROMMERSand VISSER, 1969) are considered unsuited for routine application, especially those of ROMMERS and VISSER (1969) and EMMET (1969) which required accurate time intervals between reagents. Methods requiring specialized equipment (WEICHSELBAUMet al., 1969) are also of limited use. WEICrmELBAUMet al. (1969) used a reaction rate method for ammonia and claim one analysis in 2 rain, i.e. about 25 h-1. This could also be achieved manually using automatic dispensers (zipettes etc.) since colour development was rapid. However, even doing 25 determinations on a single sample, their method resulted in coefficients of variation of 13-9 per cent and 8 per cent and does not appear very reproducible. Due to the expense of the equipment, their method does not seem suitable for routine use. Methods utilizing manganous ions (RoSSUM and VILLhR~UZ, 1963) are much more insensitive and was found to be non-linear above 10 tzg N, and less reproducible (c.v. = 5.6 per cent). The method reported in this paper on the other hand satisfies all the criteria for a suitable method, viz. the reagents were readily made, colour developed rapidly and was stable; no special equipment was required; there were no accurate time intervals between addition of reagents, and the method was short. The sensitivity of the method has been compared with that obtained by other methods (TABLE2), by utilizing the data given by the author (or obtained here using the method) to calculate an equivalent optical density (defined as the optical density expected using 1 cm cells for 10/zg N taken and a final volume of 25 ml). The nitroprusside methods are more sensitive than any other method. The superiority of nitroprusside over acetone (HARwOOD and HUYSER, 1970a) is clearly evident. The method was finally assessed by analysing other types of samples. Effluents from anaerobic digesters gave a correlation coefficient of r = 0.980 (n = 18) between results obtained manually with this method, and with the Autoanalyser. Sample volumes of 1 ml were used, and the range was 10-30 t~g N ml- 1. A series of effluents from activated sludge plants (n = 7) gave r = 0.987, for samples covering 5-15/zg N m1-1. The method appeared suitable for ammonia analyses over a wide concentration range.

810 TABLE2.

J. E. HAII.WOOD and A. L. KiJHN


COMPARISON OF THE SENSITIVITIES OBTAINED BY DIFFERENT AUTHORS

Author

Method*

Cell path length (cm) 7.62 1 5 2 1 4 1 2 1 1 1

Nitrogen taken (~, N) 10 10 5.6 10 10 12-5 10 20 10 5 10

Final volume (ml) 25 I1.1 25 50 10 50 25 25 10 11 25

Optical density Observed 0-768 0.400 0-582 0"238 0.37 0.357 0"162 0.570 1"3 0"45 0"587 Equivalent1" 0.101 0.177 0.208 0"238 0.148 0.143 0'162 0.143 0"52 0'396 0'587

RILEy (1953) ROSSUMand V ~ u z (1963) E ~ r r (1969) ROMMm~Sand VrSSER (1969) RILEy and SlmtmENI (1957) TETLOWand WmsoN (1964) StrrcLIFFE and JONES (1968) CROWTI-mRand LARGE (1956) CHANEYand MAR.BACH (1962) WEATrmRntmN(1967) H ~ w o o o and KOHN (this paper)

Mn 2+ Mn 2+ H-P H-P Acetone Acetone Acetone Acetone N.P. N.P. N.P.

* H-P refers to reactions where the hypochlorite was added first, then the phenol. Acetone and N.P. refers to methods using acetone or nitroprusside as catalysts. ~"The equivalent optical density = optical density expected for I0/zg N with 1 cm cell and 25 ml final volume = Observed o.d. Final volume 10 Cell path length 25 ml /~g N taken

SUMMARY The i n d o p h e n o l - b l u e reaction has been used for developing a m e t h o d for a m m o n i a analyses in water samples. The c o n d i t i o n s were selected f r o m a previous study of the p h e n o l - h y p o c h l o r i t e reaction. T h e m e t h o d was s h o w n to be precise, a n d was more sensitive t h a n other m a n u a l m e t h o d s for a m m o n i a . A m a j o r a d v a n t a g e was the observance of accurate time intervals between additions of reagents or colour m e a s u r e m e n t . D u e to its simplicity, the m e t h o d appeared suitable for r o u t i n e analyses. REFERENCES CHAmmYA. L. and MARBACaE. P. (1962) Modified reagents for determination of urea and ammonia. Clin. Chem. 8, 130-132. CROWa'aERA. G. and LAROER. S. (1956) Improved conditions for the sodium phenoxide-sodium hypochlorite method for the determination of ammonia. Analyst 81, 64-65. EMr~T R. T. (1969) Spectrophotometrie determinationsof urea and ammonia in natural waters with hypochlorite and phenol. Analyt. Chem. 41, 1648-1652. FAwc~rr J. K. and SCOTTJ. E. (1960) A rapid and precise method for the determination of urea. J. olin. Path 13, 156-159. HARWOOD J. E. and HuWSEx D. J. (1970a) Some aspects of the phenol-hypochlorite reaction as applied to ammonia analyses. Water Research 4, 501-515. HARWOODJ. E. and HUYSERD. J. (1970b) Automated analysis of ammonia in water. Water Research 4, 495-704.

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HORN D. B. and SQUIREC. R. (1967) An improved method for the estimation of ammonia in blood plasma. Clin. chim. Aeta 17, 99-105. JENKINS D. (1967) Analysis of estuarine waters, d. Wat. Pollut. Control Fed. 39, 159-180. LUBOCHINSKY B. and ZALTA J. P. (1954) Colorimetric microdetermination of total nitrogen. Bull. Soc. Chim. Biol. 36, 1363-1366. NEWXLLB. S. (1967) The determination of ammonia in sea-water. J. mar. biol. Ass. U.K. 47, 271-280. RILEY J. P. (1953) The spectrophotometric determination of ammonia in natural waters with particular reference to sea-water. Analyt. chim. Acta 9, 575-589. RILEY J. P. and SINHASENIP. (1957) The determination of ammonia and total inorganic nitrogen in sea water, d. mar. biol. Ass. U.K. 36, 161-168. ROMMERSP. J. and VISSERJ. (1969) Spectrophotometric determination of micro-amounts of ammonia as indophenol. Analyst 94, 653-658. ROSSUM J. R. and VILLARRUZP. A. (1963) Determination of ammonia by the indophenol method. d. Am. Wat. Wks. Ass. 55, 657-658. RUSSELL J. A. (1944) The colorimetric estimation of small amounts of ammonia by the phenolhypochlorite reaction, d. biol. Chern. 156, 457-461. SEARCY R. L., SIMMSN. M., FOREMANJ. A. and BERGOUISTL. M. (1965) A study of the specificity of the Berthelot colour reaction. Clin. chim. Acta 12, 170-175. Standard Methods for the Examination o f Water and Waste Water. (1965) 12th edn. Issued by the APHA, New York. SUTCLIFFE R. A. and JoNEs G. A. (1968) Research into some methods of analysis of sewage. Wat. Pollut. Control 67, 209-220. TETLOW J. A. and WILSON A. L. (1964) An adsorptiometric method for determining ammonia in boiler feed-water. Analyst 89, 453-465. THOMPSON J. F. and MORmsoN G. R. (1951) Determination of organic nitrogen. Analyt. Chem. 23, 1153-1157. WEATI-mP, BURN M. W. (1967) Phenol-hypochlorite reaction for the determination of ammonia. Analyt. Chem. 39, 971-974. WEICHSELBAUMT. F_., HAGERTYJ. C. and MARK H. B. (1969) A reaction-rate method for ammonia and blood urea nitrogen utilizing a pentacyanonitrosyloferrate catalyzed Berthelot reaction. Analyt. Chem. 41,848-850.