Analytrca Chrmrca Acta, 232 (1990) 345-349
Elsevler Science Pubhshers B.V., Amsterdam - Pnnted m The Netherlands
345
Formation kinetics of the pink azo dye
in the determination of nitrite in natural waters
SU-CHENG PA1 and CHUNG-CHENG YANG
Instrtute of Oceanography, Natronal Tarwan Unrversrty, Tarper (Tarwan)
JOHN P RILEY *
Oceanography Laboratones, Department of Earth Suences, Unrversliy of Lwerpool, Lwerpool (UK) (Received 8th November 1989)
ABSTRACT
The kinetics of the reactlon for the formatlon of the pmk azo dye m the determmatlon of mtnte m both fresh water and sea water was stud& at different acldltles, temperatures, and concentrations of N-l-naphthylethylenedlamme (NED). It was found that the reactlon 1s conslderably faster m sea water than m fresh water, and that mcrease m the acldlty shghtly mcreases the molar absorptlvlty A concentration of NED (4.29 X 10m6 M) between the extremes described m the hterature and 4 2 x 10m3 M sulphamlanude are recommended for both manual determination and flow-mqectlon analysis with respect to rapld reactlon and a low reagent blank
Nitrite is usually determined in natural waters, including sea water, by modifications of the classi- cal Greiss-Ilosvay technique. Nitrite is reacted with an aromatic primary amine and the resulting diazonium ion is coupled with another aromatic amine to give a pink azo dye, the absorbance of which is measured. Most workers now use meth- ods based on a reagent combination of sulphanila- mide and N-1-naphthylethylenediamine (NED), first suggested by Shinn [l]. They have the ad- vantages of simplicity, sensitivity and speed, com- plete reaction being achieved in a few minutes at room temperature. There are considerable dif- ferences between the ratios of the concentrations of the reagents which have been used in the vari- ous published procedures. However, very little at- tention has been paid to the kinetics of colour formation and to those factors which affect them, 0003-2670/90/$03 50 0 1990 - Elsevler Science Pubhshers B.V.
probably owing to the excellent performance of the method.
Reaction kinetics become very important if the method is adapted to flow-injection analysis, as in most instances the time allowed for the sample to react with reagents in the manifold is much shorter than in manual procedures. As a consequence, colour development is frequently not complete by the time the sample reaches the detection device. In the authors’ experience, many factors affect the final response in flow-injection analysis for nitrite, including reagent concentrations, acidity, temper- ature and the salt matrix. The effects of variations of these factors have been studied in a series of batch experiments in which the formation of the azo dye was monitored using a dipping probe calorimeter following addition of the NED re- agent (kinetics of the iuitial diazotization reaction
were not examined as the reaction is very rapid). The examination of the results enabled the condi- tions for the coupling reaction to be optimized so that the procedure could be adapted for flow-in- jection analysis.
EXPERIMENTAL
Reagents
A 1% (w/v) solution of sulphanilamide reagent (SUL) was prepared by dissolvmg 1 g of sulphanilamide in 10 ml of 2.5 M hydrochloric acid and diluting to 100 ml with water. A 0.1% (w/v) solution of N-1-naphthylethylenediamine reagent (NED) was prepared by dissolving 0.1 g of NED in 100 ml of water. A stock standard nitrite solution (2.5 mmol 1-l) was prepared by dissolv- ing 0.1725 g of sodium nitrite in 1 1 of distilled water. It was used to prepare a working standard solution in which 1 ml = 0.25 pmol NO;.
A freshly collected and filtered sample of coastal sea water was stored in a polyethylene bottle until required. Its nitrite content was found to be ca. 0.2 PM.
Instruments
Colour formation was monitored with a Brink- man PC-800 dipping probe calorimeter with a light path equivalent to 1 cm and fitted with a filter having its maximum transmission at 540 nm. The probe was mounted in a magnetically stirred reaction vessel which consisted of a modified Pyrex beaker which was mounted in a thermostatically controlled bath (*2“(Z). The readings from the calorimeter were checked periodically by measur- ing the absorbance of the solution with a Shimadzu 2100 double-beam spectrophotometer at the same wavelength, after colour development was com- plete.
Procedure
To the dry Pyrex beaker were added consecu- tively 1 ml of working standard solution (0.25 pmol of NO;), (47 - x - y) ml of distilled water [or 40 ml of sea water and (7 - x - y) ml of distilled water] and x ml of 0.25 M hydrochloric acid. When the solution had reached the desired
temperature, 1 ml of SUL reagent was added. The mixture was stirred for 3 min and y ml of NED reagent was then added rapidly. The recorder was immediately switched on and the change in ab- sorbance with time was recorded.
For experiments at high temperatures, the water was preheated to a temperature higher than the desired value using a microwave oven, and then allowed to cool to the desired temperature. This eliminated problems of bubble formation.
RESULTS AND DISCUSSION
Effect of acidity
The effects of variation of the acidity on the formation of the azo dye were monitored for 10 min in 40-ml samples of fresh water and sea water, both spiked with 0.25 pmol of nitrite, after adding the reagents and then diluting with water to a final volume of 50 ml. The final acidity ranged from 0.005 to 0.045 M at intervals of 0.005 M, whereas the fmal concentrations of NED and SUL reagents were fixed at 77 PM and 1.1 mM, respectively.
The absorbance vs. time curves were all ex- ponential, corresponding with the equation of A =A,,[1 - exp( -kt)], where A,,, and A are the absorbances at the end of the reaction and at time t respectively; contour plots from the data are shown in Fig. la and b. Allowance was made for the nitrite originally present in the sea water and blanks were deducted. It was found that the apparent molar absorptivity for both fresh water and sea water were almost identical at the same acidity, but tended to increase with increasing acidity (see Table 1). At an acidity of 0.005 M the value was ca. 44000 1 mol-’ cm- ’ at 540-543 nm, whereas it was 50400 1 mol-’ cm-’ at 0.025 M and 51600 1 mol-’ cm-’ at 0.045 M. The values found at the higher acidities in this experiment were similar but slightly higher than that sug- gested by Parsons et al. [2] and Strickland and Parsons [3], who gave an empirical factor equiv- alent to a molar absorptivity of ca. 48000 1 mall’ cm-’ at 540 nm at an acidity of 0.023 M. How- ever, the value for similar acidities was found to be 8% higher than that given by Grasshoff et al.
DETERMINATION OF NITRITE IN NATURAL WATERS 341 0 20 40 60 80 100 120 140 160 180 Reactlon time (s) lb) 50 , 0 20 40 60 80 100 120 140 160 180 200 Reaction time (s)
Ftg. 1. Contour plots to emphastze the effect of final actdrty on the formatron of the pmk azo dye Data pomts (absorbance X 103) were drgttrzed from the colour formatron curves. The arrow marks the actdity suggested by Strtckland and Parsons [3]. The final concentrattons of mtnte, SUL and NED were 5 PM, 1.1 mM and 77 PM, respecttvely. (a) Fresh water; (b) sea water. Temperature, 29°C.
[4], who gave a value of 46 000 1 mol- ’ cm- ’ at 543 nm at an acidity of 0.023 M.
It is evident from Fig. 1 that the rate of colour formation in the sea-water samples was consider- ably faster than in the fresh-water samples at all acidities. The average time for 90% colour devel- opment, t(90%), was 60 f 3 s in fresh water at acidities above 10 mM, compared with 40 f 2 s in sea water at all acidities (Table 1). Variation of the
TABLE 1
Molar absorptivitres (e) of the pink azo dye and ttmes requtred for 90% colour development at vanous acidities at 29 o C
Acidtty c (540 nm) a 00%) (s) (mM) (1 mol-‘cm-‘)
Fresh water Sea water
5 44000 70 40 10 48400 65 40 15 49600 63 39 20 50000 63 39 25 50400 60 40 30 50700 58 39 35 50900 58 39 40 51200 59 40 45 51400 58 40
’ Measurements were made wrth a Shunadzu 2100 double-beam spectrophotometer usmg a l-cm cell 10 mm after adding the NED reagent. The c values were calculated after deductton of the reagent blank.
final acidity produced no significant effect on the rate of colour development. However, the molar absorptivity increased by 12.7% as the acidity was increased from 5 to 15 mM (Table 1). Simulta- neously, the wavelength of maximum absorption increased slightly. Further increases in the acidity led to only minor increases in molar absorptivity.
Effect of NED concentration
The effect of the concentration of NED on the rate of colour development from 5 PM nitrite was
0, . I . I ’ 1 - -I
ii 0 100 200 300 400
I= Final concentration of NED (PM)
Ftg. 2. Ttme reqmred for 90% colour formatron wrth a sample contammg 0.25 pmol of mtnte at vartous concentrattons of NED at 29OC. The final acidity was 25 mM and the con- centratron of SUL was 1.1 mM. (a) Fresh water; (b) sea water.
g 80, g 70- 5 E 60 - ‘0 - 50- ; p 40- 8 30- a b ii o! . I, I . I * I I 20 30 40 50 60 70 F Temperature (“C)
Ftg. 3 Time requtred for 90% colour formation with a sample contamtng 0.25 pmol of mtnte at dtfferent temperatures The fmal actdtty, [SUL] and [NED] were kept constant at 25 mM, 1 1 mM and 77 PM, respecttvely. (a) Fresh water; (b) sea water
measured at a final acidity of 0.025 M and 1.1 mM SUL. It was found (Fig. 2) that the time required for 90% colour development bears a hy- perbolic relationship to the NED concentration, suggesting a first-order reaction. The values of t(90%) for sea water were only ca. 75% of those for fresh water.
Effect of temperature on rate of colour develop- ment
The time for 90% colour development was mea- sured over the range 29-60° C under constant conditions (acidity 0.025 M; [SUL] 1.1 mM; [NED] 77 PM). The results (Fig. 3) show that the rate of colour development can be efficiently enhanced by an increase m temperature. In flow-injection analysis, the resolution of the peak could be im-
proved if the manifold were to be equipped with a heating bath. In practice, however, problems are likely to occur in the photometry stage as a result of bubble formation if temperatures in excess of 50 o C are used. For this reason it is preferable to speed up the reaction by increasing the concentra- tion of NED rather than by raising the tempera- ture.
DISCUSSION
The reagent conditions laid down in several standard manual nitrite methods are given in Ta- ble 2, from which it can be seen that the final NED concentrations used range from 74 to 148 PM. These correspond to t(90%) values for fresh water and sea water (m parentheses) of 63 (44) and 35 (28) s, respectively (Fig. 2). These dif- ferences in the rates of colour development are, of course, of no consequence m manual methods. In flow-injection analysis, however, an optimum pho- tometric response can only be achteved if the reactions can be made very rapid. Hence, the NED concentration will play a key role in decid- ing the peak response. In fact, very high NED concentrations (up to 1342 PM) have been recom- mended in several of the published flow-injection procedures (Table 3), even though they always lead to high reagent blanks. Consideration of the data in Figs. 1 and 2 led to the conclusion that the flow-mlection determination of nitrite could be carried out using a much lower final concentration
TABLE 2
Companson of reagent concentrattons m standard manual procedures for the determmatton of mtnte
Parameter Reference I51 131 161 a [71 b 141 181 Sample volume (ml) 40 50 50 50 50 40 Reagent volume (ml) 2 2 2 2 2 1 Ftnal volume (ml) 50 52 52 52 52 50 Wavelength (nm) 543 543 540 543 540 540 Acu&ty (mM) 28 23 97 23 23 45 WLI (mM) 1.4 1.1 2.2 11 11 4.6 [NJ=1 (PM) 92 74 148 74 74 150 a Method 354.1. b Method 419.
DETERMINATION OF NITRITE IN NATURAL WATERS 349
TABLE 3
Comparison of final SUL and NED concentratrons m automated procedures for the determmation of mtnte
Parameter Reference WI a 161 b 171 c 191 PO1 Pll Id II d Id II d Id II d Acrdity (mM) PC P P P 150 200 P WJLI MM) 6.2 11.6 3po.3 40.4 3!3 40.4 13 9 9.7 25.9 [NED1 (PM) 331 617 1007 1342 1007 1342 924 643 859 ’ Method 353.1. b Method 353.2. ’ Method 418F. d I and II = nitrate-mtnte mamfolds for Automated System I and II, respecttvely. ’ p: Phosphonc acid rather than HCl IS used.
(0)
( b) 4
Rg. 4. Peaks for determmatton of 0, 1, 2 and 4 pM mtrite m fresh and sea water by flow-mJectton analysts usmg the recom- mended reagent concentrations and flow-rates, compared wtth the stopped-flow signal for 4 pM mtnte (a) Fresh water; (b) sea water.
of NED. It was found (Fig. 4) using the stop-flow technique, that 90% colour development could be attained in lo-15 s with both fresh and sea water in a flow-injection manifold with final conditions of acidity 67 mM and SUL and NED concentra- tions of 4.2 mM and 4.29 PM, respectively. This was achieved by use of a 2% solution of SUL in 15% hydrochloric acid and a 0.3% solution of
NED and a flow-rate ratio of 2.5 : 0.1 : 0.1 (sample : SUL : NED).
The authors thank Mr. Liang-Saw Wen and Mr. Chun-Mao Tseng for their kind assistance with the experimental work. This project was sponsored by the National Science Council of the Republic of China, under contact number NSC79- 02009~002a-05. REFERENCES 1 2 3 4 5 6 7 8 9 10 11
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