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An automatic method for the determination of ammonia in sea water
~!,'ater Re~earch Vo[. I1. pp 63" to 638. Pergamon Press 1q77 Printed in Great Britain.
AN AUTOMATIC M E T H O D FOR THE DETERMINATION OF AMMONIA IN SEA WATER B. REt:SCH BERG and M. I. ABDULLAH Department of Marine Zoology and Marine Chemistry. Institute of Marine Biology, University of Oslo, Blindern. Oslo. Norway (Receired 14 December t976)
Alwatract--A method is described for the determination of ammonia in sea and estuarine water employing an autoanalyzer. The manifold design allows for the determination of ammonia concentration in the range of 0.2-20~g at NH.~-N.I over a salinity range of 35-t0"o,, with negligible interference from amino acids.
INTRODUCTION
The determination of ammonia in sea water has long been recognized as an important measurement in environmental and ecological studies. The procedures used for such determination fall into two categories: (1) based on the formation of bis-(3-methyl-lphenyl-5-) pyrazolone, described by Prochzizkovzi. (1964) and modified by Johnston (1966), and (2) based on the Berthelot reaction (1859) between ammonia, phenol and hypochlorite at alkaline pH to form the indophenol blue. Although the former method is more sensitive, it is generally multi stage and often involves organic solvent extraction, so that its automation is rendered less successful. Numerous procedures have been described for the production of the indophenol blue. Newell (1967) employed chloramine-T instead of hypochlorite and subsequently extracted the indophenol blue. Emmet (1968) using the original reaction, buffered the sample to pH 9.4 to obtain the colour. Attempts to increase the sensitivity and the speed of the reaction by Koroleff (1970) using a more alkaline buffer together with sodium nitroprusside as a catalyst, produced a precipitation and serious interference when polluted waters were analysed. In order to avoid the precipitation of magnesium hydroxide at alkaline pH, Soldrzano (1969) developed the indophenol blue (at pH 10.5) using citrate buffer. The simplicity of the latter methods renders these most suitable for automation. Attempts made to automate Sol6rzano's method, however, appeared to be fraught with difficulties, for example, Head (1971) encountered precipitation in the ethanol-phenol reagent (not met in the original manual method), while Grasshoffand Johannsen (1972) attributed some difficulties to the uncontrollable content of the hypochlorite reagent. Liddicoat et al. (1975) however, suggested that in their manual procedure, controlled photo oxidation generally produced a more reproducible colour. In almost all the published automatic procedures, heating was employed in the final indophenol blue colour development (70:C, Grasshoff and
Johannsen, 1972: 60°C. Head, 1971; 90~C, Slawyk and Maclsaac, 1972; 65°C. Benesch and Mangelsdorf, 1972) with the result that serious interference from amino acids was unavoidable (Grasshoff and Johannsen, 1972). Harwood and Huyser (1970) study of the indophenol blue reaction for the ammonia determination showed the effect of pH variation on the final colour and emphasized the necessity for the efficient buffering to obtain reproducible results. The optimum conditions found by the authors (pH 10.8 and using sodium nitroprusside) were similar to those used by Sol6rzano. EXPERIMENTAL
Reagents
Ammonia-free water obtained by passing distilled water through a cation-exchange resin (Dowex 50W) column, 100cm long and 3 cm diameter, in its hydrogen form, was used throughout. Phenol-ethanol solution. Dissolve 10g phenol (AR) in 100 ml of 95~o v/v ethanol and dilute to 200 ml with water. Sodium-nitroprusside. 0.25'~/osolution. Buffer. Dissolve 100g trisodium citrate (AR) and 5g sodium hydroxide pellets (AR) in I 1 of water. Oxidi-ing reagent. Mix 200ml of buffer solution and 25 ml of ca. I N sodium hypochlorite (AR). ChemLab multi-channel system, fitted with double beam filter colorimeter was used in the direct O/D mode. The sampler, which has an independently variable wash and sample cycle, was set to 60 s sample and 70 s wash thus allowing the analysis of 26 samples per h. Sample treatment. All samples and standards were biologically inactivated. Few drops of chloroform added to the stock and working standards were sufficient to maintain the concentration of ammonia for a long period of time. Samples for analysis should be filtered immediately on collection using 0.45/~m membrane filter and. with the addition of few drops of chloroform, be kept at -20°C until required for analysis. Attempts to automate the Sol6rzano method using the same concentration and proportions of reagents as recommended resulted in a rather erratic colour development and low sensitivity. Heating in the final stage of the indophenol blue colour development improved both sensitivity and reproducibility somewhat, but caused a high interference from amino acids due to the breakdown of these to ammonia in the presence of excess hypochlorite at 70°C. 63.7
638
B.
REUSCHBERG and SAMPLER
0.065 Air SMC
5MC
$MC
~ee~i0 8', Sompt, 0 ~'03~ Pheno[ reogent O 0"035
N i tr o p r u ssi d e
~0.0S6 Oxidizing reogent 00-065
I COLORIMETFR
RECOROER
t$ mm f/c
M. I.
ABDLLLAH CO\CLLSION
The interference from amino acids was investigated and found to be negligible as reported by Soldrzano 11969) and H a r w o o d and Huvser [1970) who employed no heating for the indophenol blue colour development. Solutions containing 50.ug-at. N l - t ot" urea. histidine, l.vcine, gbcine and alanine were analysed. The NH.~-N detected ranged between 0,4°,, (for urea) and 2.2°0 (for alanine) of the nitrogen added.
Acknowledgemeplt--We wish to acknowledge the support of the Norwegian Research Council tbr Science and The Humanities.
625 am filter
Fig. 1. Schematic flo~-diagram for the automatic analysis for ammonia.
REFERENCES
No significant increase in the sensitivity was observed when heating to above 50°C. but improvement in the reproducibility continued up to 70=C. This together with the rather variable blank, suggested that variation in the pH due to incomplete mixing along the analytical stream, (as often encountered when joining a slow stream of concentrated reagent, as the case here, with a faster sample or main stream) may be the cause. A modification of the manil'old was then made (Fig. II where the reagent volumes were doubled keeping the final concentration the same as the original manual method, This manifold was found to give maximum colour development when heating to 35-40"C. low blank and high degree of reproducibility. The effect of ambient light on the colour development, if any, would be eliminated since the reaction is completed in the light-tight oil heating bath. Salt solution (3~o NaCI) was used for blank/wash and calibration. The amount of ammonia in this (less than 0.1/~g-at.NH,t-Nl -*) was determined and taken into account in the computation. For the analysis of samples containing less than 0.l.ug-at. NH.rNI -t, the ammonia in this salt solution should be reduced to a much lower level. Ultra-violet oxidation of the stock solution in the presence of 1 ml of H:O2 was found suitable to reduce the ammonia level to less than 0.02 ug-at. I - t. The reproducibility of the method was assessed by repeated (hourly) analysis of a known standard solution over a period of 12 h. The percentage standard deviation was found to be 1.4°; (standard deviation = 0.07). Calibration in 3~ salt solution was found linear in the region of 0.2-20ugat.NH,,-NI-*. Replicate analysis of standard made up in different salinities (35, 30, 25, 20, 15 and 107£,,) showed little or no salt effect (the ~%i;standard deviation for all replicates combined was was less than 2°0).
Benesch R. & Mangelsdorf P. 11972) Eine Methode zur colorimetrischen Bestimmung yon Ammoniak in Meerwasser. Helgoliindcr wiss. Meeresunters. 23. 365-375. Emmet R. T. 11968) Direct spectrophotometric analysis of ammonia in natural water by the phenol-hypochlorite reaction..Vac. Ship. Res. Decelop. CeJtt. Rep. 2570. Grasshoff K. & Johannsen H. (1972) A new sensitive and direct method for the automatic determination o1" ammonia in sea water. J. Copes. int. Explor. Mer. 34. 5t6-52l. Harwood J. E. & Huyser D. J. (19701 Automated analysis of ammonia in water. Water Res. 4. 695-704. Head P. C. 11971) An automated phenolhypochlorite method for the determination of ammonia in sea water. Deep-Sea Res. 18, 531-532. Johnston R. (1966) Determination of ammonia in sea water as rubazoic acid. hm Corn Explor. Sea. C.M. 1966/N: 11. Koroleff F. (1970) Direct determination of ammonia in natural waters as indophenot blue. lnj'ormation on techniques aml methods/or sea water amdysis, lnterlah. Rep. (3) 19-22. Liddicoat M. I.. Tibbits S. & Butler E. I. [1975) The determination of ammonia in sea water. Li,moL Oct'altogr. 20. 131-132. Newell B. S. (1967) The determination of ammonia in sea water. J. mar. biol. Ass. U.K. 47. 271-2g0. Prochfizkovfi L. (19641 Spectrophotometric determination of ammonia as rubrazoic acid with bispyrazolone reagent. Analyr. Chem. 36. 865-871. Slawyk G. and Maclsaac J. J. [1972) Comparison of two automated methods in a region of coastal upwelling. Deep-Sea Res. 19. 521-524. Soldrzano L. (1969) Determination of ammonia in natural waters by the phenolhypochlorite method. Limnol. Oceanogr. 14. 799-801.
AN AUTOMATIC M E T H O D FOR THE DETERMINATION OF AMMONIA IN SEA WATER B. REt:SCH BERG and M. I. ABDULLAH Department of Marine Zoology and Marine Chemistry. Institute of Marine Biology, University of Oslo, Blindern. Oslo. Norway (Receired 14 December t976)
Alwatract--A method is described for the determination of ammonia in sea and estuarine water employing an autoanalyzer. The manifold design allows for the determination of ammonia concentration in the range of 0.2-20~g at NH.~-N.I over a salinity range of 35-t0"o,, with negligible interference from amino acids.
INTRODUCTION
The determination of ammonia in sea water has long been recognized as an important measurement in environmental and ecological studies. The procedures used for such determination fall into two categories: (1) based on the formation of bis-(3-methyl-lphenyl-5-) pyrazolone, described by Prochzizkovzi. (1964) and modified by Johnston (1966), and (2) based on the Berthelot reaction (1859) between ammonia, phenol and hypochlorite at alkaline pH to form the indophenol blue. Although the former method is more sensitive, it is generally multi stage and often involves organic solvent extraction, so that its automation is rendered less successful. Numerous procedures have been described for the production of the indophenol blue. Newell (1967) employed chloramine-T instead of hypochlorite and subsequently extracted the indophenol blue. Emmet (1968) using the original reaction, buffered the sample to pH 9.4 to obtain the colour. Attempts to increase the sensitivity and the speed of the reaction by Koroleff (1970) using a more alkaline buffer together with sodium nitroprusside as a catalyst, produced a precipitation and serious interference when polluted waters were analysed. In order to avoid the precipitation of magnesium hydroxide at alkaline pH, Soldrzano (1969) developed the indophenol blue (at pH 10.5) using citrate buffer. The simplicity of the latter methods renders these most suitable for automation. Attempts made to automate Sol6rzano's method, however, appeared to be fraught with difficulties, for example, Head (1971) encountered precipitation in the ethanol-phenol reagent (not met in the original manual method), while Grasshoffand Johannsen (1972) attributed some difficulties to the uncontrollable content of the hypochlorite reagent. Liddicoat et al. (1975) however, suggested that in their manual procedure, controlled photo oxidation generally produced a more reproducible colour. In almost all the published automatic procedures, heating was employed in the final indophenol blue colour development (70:C, Grasshoff and
Johannsen, 1972: 60°C. Head, 1971; 90~C, Slawyk and Maclsaac, 1972; 65°C. Benesch and Mangelsdorf, 1972) with the result that serious interference from amino acids was unavoidable (Grasshoff and Johannsen, 1972). Harwood and Huyser (1970) study of the indophenol blue reaction for the ammonia determination showed the effect of pH variation on the final colour and emphasized the necessity for the efficient buffering to obtain reproducible results. The optimum conditions found by the authors (pH 10.8 and using sodium nitroprusside) were similar to those used by Sol6rzano. EXPERIMENTAL
Reagents
Ammonia-free water obtained by passing distilled water through a cation-exchange resin (Dowex 50W) column, 100cm long and 3 cm diameter, in its hydrogen form, was used throughout. Phenol-ethanol solution. Dissolve 10g phenol (AR) in 100 ml of 95~o v/v ethanol and dilute to 200 ml with water. Sodium-nitroprusside. 0.25'~/osolution. Buffer. Dissolve 100g trisodium citrate (AR) and 5g sodium hydroxide pellets (AR) in I 1 of water. Oxidi-ing reagent. Mix 200ml of buffer solution and 25 ml of ca. I N sodium hypochlorite (AR). ChemLab multi-channel system, fitted with double beam filter colorimeter was used in the direct O/D mode. The sampler, which has an independently variable wash and sample cycle, was set to 60 s sample and 70 s wash thus allowing the analysis of 26 samples per h. Sample treatment. All samples and standards were biologically inactivated. Few drops of chloroform added to the stock and working standards were sufficient to maintain the concentration of ammonia for a long period of time. Samples for analysis should be filtered immediately on collection using 0.45/~m membrane filter and. with the addition of few drops of chloroform, be kept at -20°C until required for analysis. Attempts to automate the Sol6rzano method using the same concentration and proportions of reagents as recommended resulted in a rather erratic colour development and low sensitivity. Heating in the final stage of the indophenol blue colour development improved both sensitivity and reproducibility somewhat, but caused a high interference from amino acids due to the breakdown of these to ammonia in the presence of excess hypochlorite at 70°C. 63.7
638
B.
REUSCHBERG and SAMPLER
0.065 Air SMC
5MC
$MC
~ee~i0 8', Sompt, 0 ~'03~ Pheno[ reogent O 0"035
N i tr o p r u ssi d e
~0.0S6 Oxidizing reogent 00-065
I COLORIMETFR
RECOROER
t$ mm f/c
M. I.
ABDLLLAH CO\CLLSION
The interference from amino acids was investigated and found to be negligible as reported by Soldrzano 11969) and H a r w o o d and Huvser [1970) who employed no heating for the indophenol blue colour development. Solutions containing 50.ug-at. N l - t ot" urea. histidine, l.vcine, gbcine and alanine were analysed. The NH.~-N detected ranged between 0,4°,, (for urea) and 2.2°0 (for alanine) of the nitrogen added.
Acknowledgemeplt--We wish to acknowledge the support of the Norwegian Research Council tbr Science and The Humanities.
625 am filter
Fig. 1. Schematic flo~-diagram for the automatic analysis for ammonia.
REFERENCES
No significant increase in the sensitivity was observed when heating to above 50°C. but improvement in the reproducibility continued up to 70=C. This together with the rather variable blank, suggested that variation in the pH due to incomplete mixing along the analytical stream, (as often encountered when joining a slow stream of concentrated reagent, as the case here, with a faster sample or main stream) may be the cause. A modification of the manil'old was then made (Fig. II where the reagent volumes were doubled keeping the final concentration the same as the original manual method, This manifold was found to give maximum colour development when heating to 35-40"C. low blank and high degree of reproducibility. The effect of ambient light on the colour development, if any, would be eliminated since the reaction is completed in the light-tight oil heating bath. Salt solution (3~o NaCI) was used for blank/wash and calibration. The amount of ammonia in this (less than 0.1/~g-at.NH,t-Nl -*) was determined and taken into account in the computation. For the analysis of samples containing less than 0.l.ug-at. NH.rNI -t, the ammonia in this salt solution should be reduced to a much lower level. Ultra-violet oxidation of the stock solution in the presence of 1 ml of H:O2 was found suitable to reduce the ammonia level to less than 0.02 ug-at. I - t. The reproducibility of the method was assessed by repeated (hourly) analysis of a known standard solution over a period of 12 h. The percentage standard deviation was found to be 1.4°; (standard deviation = 0.07). Calibration in 3~ salt solution was found linear in the region of 0.2-20ugat.NH,,-NI-*. Replicate analysis of standard made up in different salinities (35, 30, 25, 20, 15 and 107£,,) showed little or no salt effect (the ~%i;standard deviation for all replicates combined was was less than 2°0).
Benesch R. & Mangelsdorf P. 11972) Eine Methode zur colorimetrischen Bestimmung yon Ammoniak in Meerwasser. Helgoliindcr wiss. Meeresunters. 23. 365-375. Emmet R. T. 11968) Direct spectrophotometric analysis of ammonia in natural water by the phenol-hypochlorite reaction..Vac. Ship. Res. Decelop. CeJtt. Rep. 2570. Grasshoff K. & Johannsen H. (1972) A new sensitive and direct method for the automatic determination o1" ammonia in sea water. J. Copes. int. Explor. Mer. 34. 5t6-52l. Harwood J. E. & Huyser D. J. (19701 Automated analysis of ammonia in water. Water Res. 4. 695-704. Head P. C. 11971) An automated phenolhypochlorite method for the determination of ammonia in sea water. Deep-Sea Res. 18, 531-532. Johnston R. (1966) Determination of ammonia in sea water as rubazoic acid. hm Corn Explor. Sea. C.M. 1966/N: 11. Koroleff F. (1970) Direct determination of ammonia in natural waters as indophenot blue. lnj'ormation on techniques aml methods/or sea water amdysis, lnterlah. Rep. (3) 19-22. Liddicoat M. I.. Tibbits S. & Butler E. I. [1975) The determination of ammonia in sea water. Li,moL Oct'altogr. 20. 131-132. Newell B. S. (1967) The determination of ammonia in sea water. J. mar. biol. Ass. U.K. 47. 271-2g0. Prochfizkovfi L. (19641 Spectrophotometric determination of ammonia as rubrazoic acid with bispyrazolone reagent. Analyr. Chem. 36. 865-871. Slawyk G. and Maclsaac J. J. [1972) Comparison of two automated methods in a region of coastal upwelling. Deep-Sea Res. 19. 521-524. Soldrzano L. (1969) Determination of ammonia in natural waters by the phenolhypochlorite method. Limnol. Oceanogr. 14. 799-801.