Water Research Vol. 12. pp. 399 to 402 0043-1354/78/0601-0399 $02.

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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 resulting in a very unsatisfactory sensitivity compared

The determination of ammonia based on a reaction
with phenol and hypochlorite, in which a blue-
colourcd 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).
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

Although the details of the mechanism are un- Water samples were fdtered through 0.45/~m membrane
known, the following overall reaction is generally filters, Interstitial water was obtained by centrifugation
accepted for the indophcnol formation: of sediment samples at 23,000g. Extinction values were

2 (~}'-O-+ NH3 + 3CtO" " - O ' - ~ N : X ~ O + 2H:,O+OH-+3Ct-

Catalysts of different nature are used for the reaction,
especially sodiumnitroprusside and potassiumferro-
cyanide. 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 spectro-
scopy 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),
399
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¢ esti-
mated 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 determina-
tions were carried out in triplicate. All reagents were analyti-
cal grade chemicals.
SELECTION OF REACTION CONDITIONS
Reagent concentrations
Optimal reagent concentrations were selected by varying
400 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 tempera-
ture. "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 pro-
cedure for salicylate method

Phenol method Salicylate method

Phenol 0.5% = 0.053 M --

Sodium salicylate -- 4 % = 0-24 M
NaOCI 0.15% = 0.021 M 0.077% = 0.01 M
NaOH 0.1% = 0.025 M 0.04% = 0.01 M
K4Fe(CN)6.3H20 0.02% = 4.7 x 10 -4 M 0.2% = 4.7 x 10 -3 M
pH 10.8 12.0
Reaction time 40 min 60 min
Measuring wavelength 635 nm 660 nm
Equivalent optical density 0.030/zg- 1 N H 3-N cm- 1 0.029#g- 1 NH 3-N cm -
Blank extinction 0.022 cm- t 0.030 cm- t
Colour stability At least 12 h At least 12 h
pK, ofindophenol 7.91 + 0.20 10.44 + 0.23

phenol or salicylate, NaOH, NaOCI and catalyst concentra-
tions over a wide range, covering most of the values men-
tioned in the literature.
Every concentration of phenol or salicylate was combined
with different NaOH concentrations; to each of these com-
binations 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 pre-
ferred 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 con-
taining 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 Dark
Time E Ebla,k E - Ebla,k E Ebj,nk E - Ebla.k

1h 0.607 0.030 0.577 0.182 0.012 0.170

2h 0.609 0.031 0.578 0.267 0.016 0.251
3h 0.608 0.031 0.578 0.392 0.020 0.372
4h 0.610 0.031 0.579 0.512 0.022 0.490
8h 0.610 0.032 0.578 0.517 0.022 0.495
12 h 0.612 0.036 0.576 0.532 0.024 0.508
24 h 0.609 0.041 0.568 0.540 0.030 0.510
48 h 0.458 0.044 0.414 0.546 0.036 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.
E66o
o5oo
I I I [ I I I 1 I i I I
0 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 41°C 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.
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
Sample Salicylate
Lake Vechten
(interstitial water) 8.80 (0.11)*
River Vecht 6.33 C0.09)
River Rhine 2.15 (0.09)
* Initial yellow colour due to iron present.
401
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 calibra-
tion 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 re-
producible. 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
The following ions have been tested separately for inter-
ference in the salicylate method (standard procedure,
20/~g of NH3-N present).
Ca 2+ up to 5 mg in final volume
Mg 2 + up to 1 mg in final volume
S 2- up to 150/zg in final volume
Fe 3+ up to 25/zg in final volume
Cu 2+ up to 100/~g in final volume
F- up to 20/zg in final volume
SiO~--Si up to 100/zg in final volume
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
I
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 absorp-
tion 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 experi-
ments with NH~-N added to several types of natural water.
Using samples from different sources, all NH3-N was re-
covered, indicating no significant interferences.
Next to this, comparison with other methods, especially
the highly specific enzymatic method, gives reliable informa-
tion 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.
Method
Enzymatic Salicylate Nessler
after distillation
8.91 (0.15) 10.21 (0.17) 10.75 (0.21)
6.12 (0.11) 6.78 (0.11) 6.79 (0.15)
1.99 (0.19) 2.43 (0.12) 2.51 (0.17)
402 H. VERDOUW,C. J. A. VANECHTELDand E. M. J. DEKKERS
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 sensi-
tivity was less: equivalent optical density was 0"020/ag-1
NH3-N cm- i. This effect appeared to be caused by a lower-
ing 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
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 con-
centration 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 determina-
tions 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 un-
necessary. 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
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 addi-
tion of extra NaOH.
Several phenomena occurring in indophenol forma-
tion 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.
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.
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