Flow-injection chemiluminescence determination of ultra low concentrations of nitrite in water

ANALYTICA

CHIMICA ACTA ELSEVTER

Analytica Chimica Acta 316 (1995) 261-268

Flow-injection chemiluminescence determination of ultra low concentrations of nitrite in water Pave1 Mikugka

*,

ZbyGk VeEeFa, ZbynEk ZdrShal

Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic. Veceii 97, CZ-61142 Hmo, Czech Republic

Received 3 February 1995; revised 9 June 1995; accepted 22 June 1995

Abstract A new flow chemiluminescence method for the determination of ultra low concentrations of nitrite in water is presented. Nitrite reacts with H,02 in acid medium to form peroxynitrous acid that is subsequently detected as peroxynitrite by the chemiluminescence reaction with alkaline solution of luminol. The detection limit of nitrite is 1 X LO-” mol 1’ (for 50 ~1 samples) and the calibration graph is linear up to 1 X 10e5 M NO;. The relative standard deviations for 1 X lKh M and 3 x lO-R M NO; are 1.8% and 5.4%, respectively. The interferences of cations are eliminated by passing the sample through a cation-exchange column. Common anions do not interfere. Analysis time is 3 minutes. The results are in good agreement with a standard spectrophotometric method. Keywords: Chemiluminescence; Flow injection; Nitrite; Waters

1. Introduction Nitrite formed during the biodegradation of nitrate and ammoniacal nitrogen or nitrogenous organic matter is an important indicator of faecal pollution of natural waters. The determination of nitrite is of great importance because of its harmful impact on human health. The toxicity of nitrite is primarily due to its interaction with blood pigment to produce methemoglobinemia. The reaction between nitrite and secondary or tertiary amine results in the formation of N-nitroso compounds, some of which are known to be carcinogenic, teratogenic and mutagenic [l-3]. A lot of methods have been proposed for the

* Corresponding author. 0003.2670/95/$09.50

determination of nitrite in water. The predominant part of them is based on the spectrophotometric detection of azo dye resulting from the reaction of nitrite with the Griess type reagent [4-81. Spectrophotometric determinations are usually time consuming and sensitivity is mostly lower than sensitivity of subsequent methods. Various electrochemical [9-121, chromatographic [13-171 or fluorometric [16,18-201 methods have also been described. A direct chemiluminescence (CL) determination of nitrite has not yet been reported. Nitrite can be determined for example on the base of the quenching of peroxyoxalate chemiluminescence after HPLC separation [17]. Another method involves the oxidation of iodide by nitrite; formed iodine is extracted by a gas and detected by the CL reaction with an alkaline solution of luminol [21]. A further method

0 1995 Elsevier Science B.V. All rights reserved

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utilizes the acid decomposition of nitrite to NO and NO, that are transferred into an alkaline luminol solution by a stream of purified air and CL light is measured [22]. The majority of the methods used nowadays for the CL determination of nitrite is in general based on the conversion of nitrite to nitric oxide which is transferred into the gas phase and detected by the CL reaction with ozone. Ascorbic acid [23], iodide [24,25] or V’” [26] are recommended for the reduction of nitrite to NO. Altematively, NO is under vacuum released from an acidified solution of nitrite [27]. This paper describes a new indirect chemiluminescence determination of nitrite in water. Nitrite is oxidized by hydrogen peroxide in sulfuric acid medium into peroxynitrous acid that is detected as a peroxynitrite anion by the CL reaction with an alkaline solution of luminol.

2. Experimental

2. I. Apparatus A schematic diagram of our FIA system is represented in Fig. 1. Three syringe pumps (Laboratory Equipments, Prague, CR) are used for delivery of reagents and a carrier. The optimum flow-rates of the carrier, the reagent solution and the chemiluminescence solution are 100, 30 and 130 ,ul min-‘, respectively. The sample is introduced into the car-

Chimica Acta 316 (1995) 261-268

Fig. 2. A cross-sectional view of the chemiluminescence detector. CL, chemiluminescence solution inlet; RM, reagent mixture inlet; GC, glass capillary; FS, fused silica tube; AF, alurninium foil; PMT, photomultiplier tube; ST, stainless steel tee; BE, brass enclosure; ER, epoxy resin.

rier utilizing a manually operated six-way injection valve (Laboratory Equipments, Prague, CR). The sample-loop volume is 50 ~1. The carrier, distilleddeionized water, is mixed in a PTFE tee with the reagent solution which consists of H,O, in H,SO, medium. The reagent mixture then flows through a coiled PTFE reactor (0.6 mm I.D. X 30 cm length) and reacts with the CL solution (luminol in KOH medium) in a CL cell. Chemiluminescence spectra are recorded with a fluorescence spectrophotometer MPF-3 (Perkin Elmer) with its light source turned off. 2.2. Detector

(130) CL

HPP Waste r

RE

HPP

-

CA

HPP

-

00)

uw

Fig. 1. A schematic diagram of the FLA system. CL, chemiluminescence solution; RE, reagent solution; CA, carrier; HPP, highpressure pump; S, sample injection; D, cherniluminescence detector; RC, reaction coil.

A cross-sectional view of the chemiluminescence detector is shown in Fig. 2. A home-made flow CL cell is formed by two concentric capillaries. A glass capillary (1.0 mm I.D. X 1.4 mm O.D. X 20 mm length) held in a stainless steel tee is used as the body of the CL cell. The reagent mixture is entering through a reagent capillary (fused silica tube 0.5 mm I.D. X 0.6 mm O.D.) which is inserted concentrically into the glass capillary. The CL solution enters through the central arm of the tee, then flows around the reagent capillary and at its outlet is mixed with the reagent mixture to generate chemiluminescence light. The length of the glass capillary is limited by the diameter of the used photomultiplier tube (PMT)

MikuSka et al. /Analytica

window. The CL cell volume is 15.7 ~1. Both the glass and the reagent capillaries are glued with epoxy resin to the tee. The CL cell is adjusted in a vertical position (liquid flows from bellow upward) at a distance of 1 cm in front of the PMT (model 65 PK 518, Tesla, Prague, CR) that detects the light emitted during the CL reaction. The PMT is operated at 1000 V at the laboratory temperature with no wavelength discrimination. The PMT and the CL cell are held in a light-tight brass enclosure. The current from the PMT is fed to an electrometer and then recorded on a chart recorder. The peak height is measured as a CL signal. Millivolts as the CL signal are measured by a volt meter that is connected parallel to the chart recorder. Collection efficiency of the emitted light by the PMT is enhanced by gluing a small strip of aluminium foil on the glass capillary opposite the PMT. 2.3. Chemicals All solutions were prepared with distilled-deionized water. Luminol (Serva, Heidelberg, Germany) is pure and is used without further purification. NaNO, (Fluka, Buchs, Switzerland), concentrated HgS04, 30% H,O,, KOH, Na,-EDTA and other chemicals (all from Lachema, Brno, CR) are of analytical grade. The concentration of H,O, is determined by titration of the stock soIution using the thiosulfate-iodide method, The cation-exchange column (4 mm PTFE tube, 1.5 mm I.D.) is packed with H’ form DowexR SOW-XZ (200-400 mesh, wet exchange capacity 0.7 mequiv/ml) (Bio-Rad, Richmond, USA). 1 mm catex bed is plugged by a glass wool at each end of the column.

3. Results and discussion

levels [31,32]. Peroxynitrous acid (HOONO) was prepared by the action of ozone on an aqueous solution of alkali azide [33] or by the oxidation of aqueous nitrite with acidified hydrogen peroxide [28,31,34]. Peroxynitrous acid is produced almost instantly according to the equation: HONO + H,O, “_: HOONO + H ?O This reaction nitrite.

A few methods have been used for the preparation of peroxynitrous acid or peroxynitrite. The latter was synthesized by introducing NO into the alkaline solution of hydrogen peroxide [28-301 or by the ultraviolet photolysis of aqueous alkaline nitrate at high pH

was selected for the determination

3.2. Optimization

of

of reagent concentrations

The optimizations are made with respect to the maximum ratio of the CL signal-to-noise for the varying of the concentration of given reagent at constant concentrations of the rest compounds. Two concentrations of nitrite (8 X lo-’ M and 1 X lo-’ M) are used as standards during optimization measurements. The reaction of nitrite with hydrogen peroxide to form peroxynitrous acid needs the presence of an acid catalyst. We have chosen sulfuric acid because of its small volatility. With increasing concentration of H,SO, the CL signal increases, reaches a maximum for 0.3 M H,SO, and then falls down. Fig. 3 shows the effect of the H20, concentration on the CL intensity. We have chosen 4 mM H,Oz

t

l-

0000 3.1. Theory

263

Chimica Acta 316 (I 995) 261-268

0 01

0 O? [Hydrogen

0 03 peroxide]

0 04 IN)

Fig. 3. Effect of Hz02 concentration on the CL signal. (0) = 1 X lo-” M NO; (the range of the chart recorder is 2.0 V). (0) = 8 x lo-’ M NO; (the range of the chart recorder is 0.2 V); [H,SO,] = 0.3 M, [LMN] = 2 mM, [KOH] = 0.6 M, [Na,-EDTA] = 3 mM; FcA = 100 pl/min, F,, = 30 pl/min, FcL = 130 fil/min; L = 30 cm; tHOONO = 40 s.

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for other measurements. Higher H,O, concentrations cause decay of the synthesized HOONO [31]. Peroxynitrite anion as a strong oxidant reacts with luminol (LMN) to yield the CL light even without metal catalysts that are necessary for the CL reaction of LMN with H,O, 1351. The CL intensity first increases and after passing through a maximum (2 mM luminol) decreases with increasing luminol concentration. It could probably be due to partial absorption of the emitted light by unreacted luminol molecules. The CL reaction of peroxynitrite with LMN carries out most effectively in a strong alkaline medium. The amount of detected light increases with an increase in KOH concentration, passes through a maximum (0.6 M KOH solution) and then decreases again. An excess of H,O,, nonconsumed at the oxidation of nitrite, reacts with LMN in the CL cell. This undesirable CL reaction, arising from metal cations presented as trace impurities at used reagents, forms the high background level. Addition of 3 mM Na,EDTA into both the reagent and the CL solution suppresses background levels nearly to the dark current level of the PMT. 3.3. Effect of flow-rates The determination of trace nitrite concentrations requires highly stable flows of all three streams to

161

Reagent

solution

flow

rate

(pL/min)

Fig. 4. Effect of reagent solution flow-rate (F,) on the CL signal. FCA = 100 pl/min, FcL = FRE + FCA; tHOONO= 40 s; [NaNO,] = 1 X 10e5 M; concentrations of reagents, see Fig. 3.

Chimica Acta 316 (1995) 261-268

I

OO

400

200 Total

flow

600 rate

(yL/min)

Fig. 5. Effect of total flow-rate (FTOT) on the CL signal. FToT = FCA + FRE + FcL; FCA:FRE:FCL = 100:30:130; tHOONO= 40 s; [NaNO,] = 1 X lo-’ M; concentrations of reagents, see Fig. 3.

obtain stable and low-noise base lines. We used high-pressure syringe pumps because peristaltic and piston pumps are not suitable due to their pulse flow. The ratio of flow-rates of individual streams is first optimized. The flow-rate of the carrier (F&l is fixed at 100 pl/min and flow-rates of the reagent solution and the CL solution are tested. Diminishing of the reagent solution flow-rate (FRE) results in a lower dilution of nitrite concentration in the injected sample and the signal height increases simultaneously (Fig. 4). The best ratio of FCA:FRE is 100:30. The effect of the CL solution flow-rate (F,,) on the CL signal height is negligible in the range (FCL/(FCA + FRE)) = 0.5-1.0. We have chosen FcL e.g. 130 @/min, as the optimum. At =FcA +Fm this flow-rate of the CL solution the lowest noise of the base line was observed probably as a result of the best mixing of the reagent mixture and the CL solution within the given CL cell. The ratio of flow-rates of individual streams FC.: F,,: FRE = 100:30:130 is used at subsequent measurements. The total flow-rate (FTOT) across the CL cell is another factor that influences the CL signal. It is known that CL reactions of luminol are very fast. Therefore it is necessary to mix the CL solution with HOONO directly in the CL cell. FToT is then necessarily optimized so that most of the CL reaction proceeds inside the CL cell during the passage of analyte in front of the PMT. The dependence of the peak height on the total flow-rate is shown in Fig. 5.

P. MikuZa

et al./Analytica

Chimica Acta 316 (19951 261-268

of HOONO synthesis kept constant the gradual lengthening of reaction coil with increasing flow-rates of carrier and the reagent solution. It is obvious that the not complete the moment when the the CL cell even at smallest examined have chosen FTOT = 260 Fl/min for time saving. Analysis time is about 3 minutes and the residence time of analyte in the CL cell is about 3.6 s at this

5

FI-OT

I

--t

&------

3.4. Effect of length of reaction coil

100

The length of the reaction coil at constant flowrates of the carrier and the reagent solution determines the time of HOONO synthesis, e.g. the resultant concentration of HOONO that is mixed with the CL solution in the CL cell. The relationship between the CL signal for 8 X 10e7 M NO, and the length of reaction coil is shown in Fig. 6. The exit HOONO concentration at the reagent capillary outlet decreases with increasing length of the coil as a result of the short life time of the produced HOONO [30,34]. For further measurements we have selected 30-cm coil because of the increasing time drift of base line at shorter coil. 3.5. Interferences The effect of various potential interferents concurrently presented in water on the determination of nitrite by the proposed method is investigated (Table 1). According to their character the tested concomiTable 1 Interferences

265

Length

zoo of

reaction

tants are divided into three groups. The first group is formed by oxidants (permanganate, hypochlorite, dichromate, peroxoborate). A reaction of MnO; or Cr,O;with luminol is accompanied by an emission of chemiluminescence light. They interfere positively both in the presence and in the absence of nitrite in the injected samples. Hypochlorite without nitrite shows only weak emission of CL light from its reaction with LMN. In the presence of nitrite hypochlorite preferentially reacts with NO; and the amount of emitted CL light is lower. The second group contains metal cations (Co’+, Cu2+, Cr” ‘, Fe’+, Fe3+) which catalyze the CL

of ions Ratio ’

Peak ’

Ion *

Ratio b

NO;

1.oo 1.38 0.88 1.00 1.10 1.20 1.12 0.99 1.Ol 1.15

14.3 1.1 0.4 0.0 3.2 2.7 1.4 0.0 0.1 0.5

HSOj BrINO; ClHCO, HPO$ Ca’ + Mg’+ SCN-

1.00

0.0

1.05 0.61 1.00 1.00 0.99 1.00 0.92 0.99 0.96

0.0

CU-” Cr”

* I X 1W5 M solution of ion is injected. h Ratio of the peak heights of (ion + nitrite)/nitrite. ’ Peak height (cm) of ion alone (the range of the chart recorder

(cm)

Fig. 6. Effect of length of reaction coil on the CL signal. [NaNO,] = 8 X lo-’ M; concentrations of reagents and flow-rates, see Fig. 3.

Ion “

MnO; Cl0 B,O;0-0: G? + Fe’ 4. Fe’ .

co11

is 2.0 V).

Peak ‘

0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

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Fe*+ is the limited concentration of iron in drinking water according to the Czech hygienic rules). Breakthrough of Fe2+ ions is observed after the passage of 119 ml of tested solution (equivalent to 2380 injections).

B

3.6. Spectrum

of chemiluminescence

light

A

~ 400

500

600

Fig. 7. Chemiluminescence spectra. Curve A, emission spectrum of the CL reaction of luminol with peroxynitrite ([NaNO,] = 0.1 M; reagent solution: 0.01 M H,O,, 0.3 M H,SO,, 3 mM Na,-EDTA; CL solution: 2 mM luminol, 0.6 M KOH, 3 mM Na,-EDTA). Curve B, emission spectrum of the CL reaction of luminol with H,O, /Co’+ ([Co” ] = 1 X 1O-5 M; reagent solution: 0.01 M H,O,, 0.3 M H,SO,; CL solution: 2 mM luminol, 0.6 M KOH).

We did not study the mechanism of the described CL reaction in detail. We only measured the emission spectrum of this CL reaction (line A, Fig. 7) and compared it with the emission spectrum obtained from the reaction of LMN with H,O, using Co’+ as a catalyst (Iine B). Similar shapes of both spectra suggests that the light emitting species, e.g. excited 3-aminophthalate ion [39], is the same in both CL reactions. The emission maximum of both spectra is at 454 nm. A displacement of the emission maximum to higher wavelength values in comparison with the early reported spectrum [39] is probably due to higher pH values of the CL oxidation of luminol in our case. 3.7. Calibration

reaction of LMN with H,O,. Cu2+ and Fe3+ are almost quantitatively masked with EDTA. Co’+, Fe*+ and Cr3+ are shaded only partially because the stability constant of Co*+, and Fe2+ complex with EDTA is about 2 and 4 orders lower in comparison with the Cu*+ complex 1361 and Cr3’-EDTA complex is kinetically slow to form [37]. The third group includes current water ions such as bromide, hydrogensulfite, iodide, nitrate, chloride, hydrogencarbonate, dihydrogenphosphate, Ca2+ and Mg *+ . The effect of thiocy anate is investigated because of its presence in vegetables 1381. With the exception of iodide these ions do not interfere in larger extent. Negative interference of iodide results from a direct reaction of iodide with nitrite in acid solution. It is obvious that the determination of NO, by the proposed method is influenced mainly by the presence of cations in the sample. The interferences of cations are eliminated by passing the sample through the cation-exchange column. The column is situated at the entrance of the sample to the sample loop. A performance of the column is investigated by passing a 5 X lo-’ M FeSO, solution (5 X 10m6 M

Under the optimum concentrations of reagents and at FToT = 260 $/min the detection limit of nitrite for a signal-to-noise ratio of 3 is 1 X 10e9 mol l-‘, e.g. 50 fmol NO, in 50 ~1 of injected sample. A calibration graph (Fig. 8) is linear in the

Fig. 8. Calibration graph. Solid line (a), calibration curve in the range of nitrite concentrations 1 X10-‘-5X lo-’ M (bottom x-axis and right y-axis); dash line (O), calibration curve for nitrite concentrations up to 1 X 10e4 M (top x-axis and left y-axis); concentrations of reagents and flow-rates, see Fig. 3.

P. Mikus’kn et al./Ana&ica 1.10'M

3.10' M

l.lWM

Chimica Acta 316 (19951 261-268

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show that the concentrations of nitrites in various kinds of samples are in good agreement for both methods (all data with except of one result are lying inside the 95% confidence interval).

4. Conclusion

Fig. 0. Background CL emission and CL signal. The range of the chart recorder is 0.5 V for 1 X 10eh M and 3 X lo-’ M nitrite and 0.05 V for 1 X10-s M nitrite: concentrations of reagents and flow-rates, set Fig. 3.

concentration range 1 x 10-*-l X 10m5 M NO; (line A: y = 1.3864 X 10 *x + 22; r = 0.9995). The curvature of the graph at higher nitrite concentrations (> 1 X 10 -’ M, line B) is due to the lack of the HZ02 amount for sufficient oxidation of NO,. The relative standard deviations for 15 repeated measurements of 1 X 10e6 M and 3 X 10-a M NO; are 1.8% and 5.4%, respectively. Fig. 9 shows the records of the background CL emission and typical CL signals for three different nitrite concentrations.

The proposed method was applied for the determination of nitrites in real water samples. The concentrations of nitrites determined by the present method are compared with the standard spectrophotometric method [8]. The results, listed in Table 2,

of nitrites in real water samples

Sample

Tap water (No. 1) Tap water (No. 2) Tap water (No. 3) Well water (No. 1) Well water (No. 2) River water (No. 1) River water (No. 2) ” The present method. ” The spectrophotometric

[NOT ] (lo-’

mol I-‘)

a

b

2.1 25.8 0.5 1.7 4.0 3.1 52.6

1.7 17.4 1.4 1.5 3.1 3.6 60.4

method [8]

Acknowledgements The authors thank Prof. Vlastimil Kubaii and Dr. Milan Vrchlabsky of the Department of Analytical Chemistry, Faculty of Sciences, Masaryk University in Brno for lending the fluorescence spectrophotometer.

References

3.8. Analysis of real samples

Table 2 Determination

A new flow-injection chemiluminescence determination of nitrite is proposed. Nitrite is oxidized to peroxynitrous acid that is subsequently detected by the CL reaction with an alkaline solution of luminol. The method provides sensitive and fast determination of sub pg 1-l levels of nitrite in various kinds of water samples.

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