8/2/2019 Effect of Temperature on the Formation of In Do Phenol
Blue in a Spectrophotometric Method for the Determinatio
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Water Res. Vol. 19. No. 11, pp. 14-/.3-1445, [985 0043-1354 85
53.00 + 0.00printed in Great Britain. All rights reserved Copyright
C 1985 Pergamon Press Ltd
EFFECT OF TEMPERATURE ON THE FORMATION OFINDOPHENOL BLUE IN A
SPECTROPHOTOMETRIC
METHOD FOR THE DETERMINATION OF AMMONIAB. M. STEWART
Department of Agriculture. Freshwater Biological Investigation
Unit, Greenmount Road, Muckamore.Antrim BT41 4PX, Northern
Ireland(Received April 1985)
Abstract--Temperature was found to be an important factor in a
modified Berthelot reaction used forthe spectrophotometric
determination of ammonia in freshwaters. The effect of temperature
on thereaction was investigated over the range 4.5--55:C and it was
shown that the rate of colour formationincreased with increasing
temperature but associated with this was a significant decrease in
the sensitivityof the method.The initial temperature of the
reaction, on mixing of reagents, was shown to determine the
finalabsorbance of the solution and any further increase in
temperature only decreased the time to reachmaximum absorbance.Key
words--ammonia, indophenoI blue, sensitivity, spectrophotometric,
temperature
INTRODUCTIONOne of the standard methods used for the
deter-minati on of ammo niu m nitrogen in freshwaters is
themodified phenol-hypochlorite method of Berthelot(1859) (APHA,
1975). The method was optimized inorder that recourse to the
calibration curve for eachdetermination became unnecessary
(Scheiner, 1976).Phenol, sodium nitroprusside, citrate solution and
anoxidizing solution of sodium hypochlorite in basicmedium are
sequenti ally added to the sample andallowed to react for 45 min.
The resu lting blue colouris measured spectrophotometrically as
indo pheno l at635 nm against stan dard solutions.
Some authors of automated methods, based onthe Berthelot
reaction (O'Brien and Fiore, 1962;Technicon Control s Inc., 1959,
1960; Millar, 1967;Montu ra and Woodward, 1982) raised the
tem-perature of the reaction mixture to increase the rateof
formation of indophenol. In a modified, manualversion of the method
(Weatherburn, 1967) it wasshown that at temperatures of 20, 25, 37
and 75C,the sensitivity of the method was greatest at 75Cwhile time
to reach maximum absorbance was great-est at 37C. Heating times
were shown to be animpor tant factor when reaction times and
sensitivitiesof a modified Berthelot reaction were compared at
60and 100C (Rommers and Visser, 1969). Other obser-vations showed
that formation of products in theBerthelot reaction were dependant
on temperatureand proceeded faster at 15~C than at 25C (Gravit zand
Gleye, 1975). In the determin ation of methyl-amine, using a
modified Berthelot reaction (Carr andDass, 1981), the rate of
chromophor e formation wasgenerally enhanced by increasing the
reaction tem-
perature. However as the temperature was raised, thefinal
intensity of the blue chromophore decreased.Although a temperature
effect was not reported byScheiner (1976), we observed that, when
using thisparicular modified Berthelot method, an increase
inreaction temperature increased the rate of formationof indophenol
blue. However, associated with theincrease in temperature was a
significant decrease inthe sensi tivity of the method . Thi s
effect was firstnoticed while investigating the possibility of
ammo-nia loss from freshwater samples when amm oniu m
Nconcentrations of river waters analysed on site werecompared to
those analysed on returning to thelaboratory. Although
concentrations in the field didnot differ significantly from those
in the laboratory,it was noticed that the absorba nces of standards
andsamples prepared at the lower temperature on sitewere
consistently greater tha n those prepared at thehigher temperature
of the laboratory (Fig. 1). Thisapparent temperature effect was
investigated undercontrolled conditions in the laboratory.
E X P E R I M E N T A L A N D RESULTSInitially, the effect of
temperature on the rate of
colour formatio n of a 500/ag NH4N 1 I so lution wasinvestigated
by monitoring the formation of indo-phenol blue
spectrophotometrically in a 4-cm thermo-statted cell at 635 nm. Ab
sorbances were measuredagainst a distilled water blank using a Pye
Unican1800 spectrophotometer. This was repeated over arange of
temperatures with a Grant thermostaticallycontrolled, circulating
water-bath being used to con-trol the temperature of the cuvette
compartment ofthe spectrophotometer. Figure 2 shows that as the
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