optimization of the in do phenol blue method for the automated determination of ammonia in estuarine...

8/2/2019 Optimization of the In Do Phenol Blue Method for the Automated Determination of Ammonia in Estuarine Waters

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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



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).



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.



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).



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



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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