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Estuarine, Coastal and Shelf Science (1983) 17, 219-224
Optimization of the indophenol blue method
for the automated determination of ammoniain estuarine waters
R. F. C. Mantoura and E. M. S. Woodward
Institutefor Marine Environmental Research, FVospect Place, The Hoe, Plymouth,Devon PLI 3DH, U.K.
Received 16 July 1982 and in revised form 9 November 1982
Keywords: ammonia; analysis; estuarine
Existing automated methods for the determination of ammonia in natural waters
suf fer from serious ‘salt error’ in estuaries because of changes in pH, ionic strengthand optical properties with salinity. A modified automated indophenol bluemethod is described which minimizes the ‘salt error’ to less than 8% over the entire
salinity of estuaries.
Introduction
Ammonia is an important constituent of the nitrogen cycle in natural waters and its
involvement in the biogeochemical processesof estuaries s receiving increased attention
(Wollast, 1981; Knox et al., 1981). Most of the methods developed for the analysis of
ammoniaare basedon the spectrophotometric determination of the indophenol blue (IPB)
complex formed by the reaction of ammoniawith phenol and hypochlorite, in alkaline pH
(Berthelot reaction; Solorzano, 1969; Riley, 1975; Krom, 1980). There are several auto-
mated IPB procedures for the analysis of ammonia n seawater (Head, 1971; Grasshoff
& Johannsen, 1972; Benesch & Mangelsdorf, 1972; Le Corre & Triguer, 1978; Loder &
Glibert, 1976; Reusch-Berg & Abdullah, 1977; Folkard, 1978), but these are not entirelysuitable for estuarine usebecause hey suffer interferences from changes n salinity (Sasaki
& Sawada, 1980), pH (Harwood & Huyser, 1970; Krom, 1980) and alkalinity commonly
encountered in estuarine waters. The salinity dependenciesare also nconsistent: Liddicoat
et al. (1975) and Loder & Glibert (1976) have reported higher sensitivity in freshwater
relative to seawater, whereas the opposite was noted by Head (1971) and Benesch &
Mangelsdorf (1972). In other modifications, nonlinear salinity dependencewas observed,
with maximum sensitivity varying between 8%0 (Le Corre & Treguer, 1976), and 15%0
(Grasshoff & Johannsen, 1972). In this paper we describe an automated method for ammo-
nia which overcomes most of the salt errors by use of a highly pH-buffered formulation.
Reagents
Reagent No. 1: dissolve separately 10 g of phenol (AnalaR, BDH) in 40 ml ethanol (95%)and 0.16 g sodium nitroprusside catalyst (AnalaR, BDH) in 100ml deionized water and
combine. Store in amber bottle and prepare fresh daily.
219
0272-7714/83/080219+06$03.00/0 0 1983 Academic Press Inc . (London) Limited
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220 R F. C. Mantouta 6 E. M. S. Woodward
Reagent No. 2: dissolve 30.0 g tri-sodium citrate dihydrate (Na,C,H,0,.2H,O; AnalaR,
BDH), 0.20 g DTT (dichloro-s-triazine-2,4,6-( IH, 3H, 5H)-trione sodium salt dihyd-rate; Koch-Light) and 20 *O ml of 4.3 M NaOH and make up to 100 ml with ammonia-free
water. Prepare fresh daily.
Ammonia standards: dissolve 0.099 g of ammonium sulphate (AnalaR, BDH) in 1.0 1
deionized water; add 5 ml chloroform preservative. Store up to a month in refrigerator.
This ammonia stock standard is 1500 pg-at. NH,-N 1-l. Working standards may be
prepared by volumetric dilution into ammonia-free water or by standard addition into
GFX-filtered estuary water.
Ammonia-free water is prepared by passing deionized water through a column of
Amberlite IR-120 (hydrogen form) and used immediately as the blank, reagent diluent or
wash in the automatic analyser.
Manifold
The reaction manifold describing the automated determination of ammonia is shown in
Figure 1. Two alternative modes of sampling are shown, discrete and continuous. Discrete
5 ml samples contained in ashed (450 “C) glass vials are sampled from an autosampler
(Hook & Tucker model A40-II; 1.5 min sample/wash). For high resolution work in the
estuary, the continuous sampling mode is preferred. We use a custom-built filtration block
(Morris et al., 1978) fabricated from stainless steel and supporting a 47 mm Whatman
GF/C filter. Sample and reagent streams are pumped through Technicon Tygon flow-rated
tubes fitted onto an Ismatek pump (model MP-13, Switzerland) with the exception of the
reagent 1 which requires solvent-resistant ‘Solvaflex’ tubing. Glass transmission tubes are
used throughout. The sample stream is segmented with acid-scrubbed air. After mixing
of sample and reagents, the IPB complex is developed in a delay coil (4 *6 min; coil diam.
35 mm, length 4.4 m, 40 turns) immersed in an oil bath at 50 “C. Following cooling to room
Colorimeter630 nm 50 mm f/c
Icy”-” I o-10 I IR2 ML- /
,I.. .042 1 1 $ ./
Waste 4 I I 3’40 ’
I 1---
Figure 1. Manifold for the automa tic determination of ammo nia (# on pump tube Rl indi-
cates ‘Solvaflex’ tubing).
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Automated determination of ammonia 221
TAB LE 1. Ana lytical performance of the automated NH, analyser
Linear detection rangeReprod ucibility (% SD of
10 replicate s at 3 pg-at N L-l)
Detection limi t (S/N = 2)
Delay time
Response time (95%)
Sample/wash times
Sample throughput
0.2-18 B-at N 1-1
kl,O%
0.02 B-at N L-1
11.7 minutes
2.5 minutes
1 ‘5 minutes
20 h-’
temperature, the IPB complex is measuredat 630 nm with a Chemlab Colorimeter (model
Mk III, Hornchurch, U.K.) and the absorbance ecorded on a chart recorder. On occasion,
a Technicon II Auto Analyser@ calorimeter (model SCIC-AA II) was also deployed. Theentire system is palletized for easeof transport and operation in the field. The analytical
performance figures basedon the Chemlab Colorimeter are summarized n Table 1.
Results and discussion
Magnesiumprecipitation
Since estuarine waters bear a greater chemical resemblance o seawater han to river water,
we evolved our method by careful consideration of the automated IPB methods developed
for the analysis of ammonia in seawater (Head, 1971; Grasshoff & Johannsen, 1972;
Benesch & Mangelsdorf, 1972; Grasshoff, 1976; Le Corre & Treguer, 1978; Loder &
Glibert, 1976; Reusch-Berg & Abdullah, 1977; Folkard, 1978). These show an inconsist-
ency in the formulation of reagents which may in part explain the variations in salt error.
Although all the methods employ tri-sodium citrate chelator to avoid precipitation of
magnesiumhfdroxide at alkaline pH, there is a stoichiometric deficiency of citrate with
respect to magnesium n two of these reports (20% in Grasshoff & Johannsen, 1972; and
46% in Le Corre & Treguer, 1978). We overcame the precipitation problem commonly
encountered in the automated methods (Head, 1971) by ensuring a stoichiometric excess
of citrate (~120%). Since surfactants are not used, the entire system requires occasional
wash (every N 10 h) with 1M HCl followed by 1M NaOH.
Salt errorExperiments were conducted with waters obtained from the Tamar Estuary (U.K.) and
in mixtures of River Tamar water with seawater. Most of the ‘salt error’ in estuarine
samplesoriginates from poor pH buffering (Sasaki & Sawada, 1980) rather than ionic
strength. Despite the river water being more acidic (pH = 7.5) than seawater (pH = 8. l),
the final pH of the reaction mixture was higher in the freshwater samples.This arises rom
the lower alkalinity (- O-8 meq 1-i) and hence buffering capacity of river water relative
to sea water (alkalinity = 2.3 meq 1-i). Since the IPB reaction is sensitive to the pH
of the medium (Harwood & Huyser, 1970; Riley, 1975; Krom, 1980), pH variations in
estuarine waters must be minimized by the use of buffered reagents operating about the
optimum pH of cv 10.6. At this pH, citrate (pK, = 3.13, 4.76, 6.40) is ineffective asbuffer. Grasshoff & Johannsen (1972) utilized unspecified amounts of borate (pK, =
9.23), but we experienced solubility problems which may explain why these authors
omitted it in a subsequent report (Grasshoff, 1976). Amino sulphonate buffers such as
CAPS (cyclohexl-amino propane sulphonic acid; pK, = 10.4) were also unsuitablebecauseof limited solubility and contamination with ammonia.
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222 R. F. C. Mantoura & E. M. S. Woodward
140 ’40 ’ I I I I I I
(a)a)120 -20 -
,... --** . . . . . ,*/... --** . . . . . ,*/ c--c------___-------__
+..*..* 4cccc -* . ...** . ...*-I-I-
---** 4cc* 4cc
.+ .+ .*......
ioo(~++*oo(~++*
‘;‘.....*;‘.....* -----__----__.a.-. -*-,-*L1*-T l*.-. -*-,-*L1*-T.
-....-... A... A
a...... *a......
*......-....
*......
80 -0 -a..........
*--* . . . ...**--* . . . ...**-....,....,
601
-I
60
0.5 ..5 -
(b)b)
04 -4 -
0.3 -.3 -
Refractwe index blankefractw e index blank
5 IO 15 20 25 30 35Sallnlty %I
Figure 2. (a) The effect of salinity on the sensitivity of standard addition s of ammo nia in
laboratory mixed waters (0) and in waters from the Tamar estuary (A) expressed as 96 of
response in river water. For compa rison, the salt error curves reported by Grasshoff &
Johann sen (1972) and Loder & Glibert (1976) are also shown (. . . and ---, respectively).
(b) Contribution of refractive index and organic absorbance to the optica l blan ks in the
Chemlab Colorimeter. River water-seawater mixture (0) de-ionized water-seawater
mixture (0).
Since phenol has a pK, = 10 0, then the IPB reaction could be made self-buffering
provided the unreacted phenol is in excessof the alkalinity. The concentration of phenolin the final mix with seawater eported in the literature varies between 0.0014 M (Grasshoff
& Johannsen, 1972) and 0.050 M (Reusch-Berg & Abdullah, 1977). A fIna phenol concen-
tration of 0.06 M was sufficient, since even in the presence of 1008 ug- at NH,-N l-1, the
IPB reaction will consume only 3% of the phenol leaving most of the phenol to act as a
pH buffer. Ethanol was used to solubilixe the high concentration of phenol used in our
system. The salt error of our method, asdetermined by standard addition of ammonia ntowaters of different salinities, is shown n Figure 2(a). When compared with other methods,
our method displays minimal salt error (-8%) even though the final pH of the river water
mixture (pH 10.9) was greater than seawater (pH 9.9).
Although Liddicoat et al. (1975) reported that the IPB reaction is light sensitive, wefound that varying the ambient ight levels had no effect. The optimum (highest sensitivity)
temperature was 50 “C, which according to Benesch & Mangelsdorf (1972), should not
cause nterference from amino acids. DTT was used in preference to commercial hypo-
chlorite as the chlorinating agent becauseof its greater stability in solution (Grasshoff &Johannsen,1972; Krom, 1980).
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Automated determination of ammonia 223
1-
3-
2-
I-
I.
3
Distance (km )
Figure 3. Axial distribution of ammo nia and salinity in the Tamar Estuary, 25 Augu st 1981.
Corrections due to salt error are apparent in the ammonia peaks (- - - ) in the more saline
waters, whereas the optica l blank corrections (. .) are linearly related to salinity.
Optical blanks
In addition to the chemical effects of varying salinity, there are optical interferences in
calorimetric analysiswhich are peculiar to estuarine samples.Saline waters and river waters
have, in the absenceof calorimetric reagents, an apparent absorbancearising from:
(1) refractive bending of light beamsby sea salts- ‘refractive index blank’ (Atlas et al.,
1971; Loder & Glibert, 1976);
(2) Background absorbanceby dissolved organics of riverine origin; the former is a func-
tion of the optical geometry of the light beam and the flow cell, and the latter is related
to the organic loading of river water.
As shown by Figure 2(b), both are linearly related to salinity, which makesoptical blankcorrections easy to apply to estuarine samples.
Although the ‘Chemlab’ Colorimeter performed satisfactorily during continuous analysis
of estuarine waters, it suffered from serious optical interferences during discrete analysis.
The problem lies with the flow cell geometry which gives poor flushing between dense
saline samples,and deionized water wash. This gives rise to Schlieren effects and a noisy
absorbance race. For discrete analysis, the more costly Technicon Colorimeter with its
superior flow cell geometry (dead volume w 150 .rl) is recommended, since it does not
suffer from Schlieren effects, and has a lower refractive index blank.
The axial concentration of ammonia n the Tamar Estuary, shown in Figure 3, varies
markedly and this emphasizes he importance of continuous analysis n chemical studiesof estuaries. The contribution of the optical blank and effects of salt error are also shown.
Acknowledgements
We thank Mrs C. M. Goodchild for assistancen the early phaseof this work. This work
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224 R F. C. Mantoura & E. M. S. Woodward
forms part of the Estuarine Ecology Programme of the Institute for Marine Environmental
Research, a component of the Natural Environment Research Council, and was partlysupported by the Department of the Environment under Contract DGR 4801684.
References
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