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Characterisation of a micro-total analytical system for the determination of nitrite with spectrophotometric detection
Analytica Chimica Acta 387 (1999) 1±10
Characterisation of a micro-total analytical system for the determination of nitrite with spectrophotometric detection G.M. Greenway*, S.J. Haswell, P.H. Petsul Department of Chemistry, University of Hull, Hull, HU6 7RX, UK Received 17 August 1998; received in revised form 30 November 1998; accepted 8 December 1998
Abstract A micro-total analytical system (mTAS) is described for the determination of nitrite based on its reaction with sulphanilamide to form the diazonium salt which when coupled with N-(1-naphthyl) ethylene diamine yields an azo dye whose absorbance was measured at 526 nm. The reaction took place in an etched glass manifold containing channels 302 mm wide and 115 mm deep in which electro-osmotic ¯ow was used to move the reagents. The absorbance of the product was measured in situ using a micro-spectrophotometric-®bre optic detection system. The electro-osmotic ¯ow mixing characteristics of the reagents together with the production of the colorimetric complex have been investigated. In addition methods for obtaining sensitive detection in the micro-reactor are reported. Using the conditions established a linear calibration was obtained between 0 and 100 mM with a correlation coef®cient of 0.999. The RSD at 50 mM NOÿ 2 was 2.6% (n6) and the limit of detection obtained (3) was 0.20 mM NOÿ . # 1999 Elsevier Science B.V. All rights reserved. 2 Keywords: Micro ¯ow injection analysis; Nitrite; Miniaturisation; mTAS
1. Introduction The presence of low level chemical species in the environment, an increasing need for good industrial process control, developments in clinical diagnostics and numerous areas have lead to the search for improved, less expensive and more robust methods for measuring low levels of chemical analytes. Micrototal analytical systems (mTAS) have been suggested as a possible solution for such analysis as they are primarily concerned with the chemical reactions in small volumes of liquids where the measurement is *Corresponding author. Tel.: +44-01482-465475; fax: +4401482-466416; e-mail: [email protected]
based on the reaction between more than one reagent, with subsequent determination being achieved in situ and an appropriate detector. These micro ¯ow systems have the inherent advantages of being portable, requiring low reagent consumption, and are able to operate remotely [1]. The development of such systems has progressed more recently with the introduction of electro-osmotic ¯ow that provides a ¯exible and robust method of moving ¯uids through microchannels of typically less than 200 mm i.d. [2±4]. Fast controllable reactions have been shown to occur between picolitres of samples in such reactors [5] which to date have been mostly produced by photolithography techniques using substrates such as glass and silicon [5±7].
0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0003-2670(99)00047-1
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A range of different detection techniques have been investigated for mTAS including spectrophotometry and electrochemistry [8,9]. Daykin and Haswell [8] for example reported the development of a micro ¯ow injection system (mFIA) for the determination of phosphate using spectrophotometric detection [8], whilst Fiehn et al. [9] have demonstrated the use of ¯uidic ISFET microsystem for the determination of pH, nitrate, ammonia, sodium and potassium based on a sensor technology using micro¯uidic injectors and diodes. In this work a mFIA has been investigated and developed for the determination of nitrite. Electroosmotic ¯ow has been used to mobilise reagents in an etched glass manifold to produce a coloured spectrophotometrically active analytical product. The absorbance of the product was measured in the manifold using a micro-spectrophotometric-®bre optic detection system. Nitrite was determined by its reaction with sulphanilamide to form the diazonium salt which was coupled with N-(1-naphthyl) ethylene diamine to yield an azo dye whose absorbance was measure at 526 nm. 2. Experimental 2.1. Reagents and materials All the chemical standards and reagents used were analytical grade unless stated otherwise and the water used was high purity de-ionised (18 M cm resistivity) (Elgastat UHQ PS, Elga, High Wycombe, UK). The sodium nitrite was supplied by Fisher Scienti®c (Loughborough, UK), ammonium chloride, hydro¯uoric acid and ammonium ¯uoride being obtained from Merck (Poole Dorset, UK). The hydrochloric acid was supplied from Philip Harris (Shenstone, Lich®eld, UK), and the sulphanilamide and N-(1naphthyl) ethylene diamine (NED) was from Aldrich (Gillingham, Dorset, UK). Sodium formate and sodium acetate were from Avocado Research Chemical (Heysham, UK) and the nitric acid was R.P.NorTM mapur , (Fontenay, S/Bois, France). The Microposit chrome etch 18 and photoresist remover 1112A were from Shipley (Coventry, UK). The glass for the microreactor chips and cover plates were both Superwhite Crown B70 borosilicate glass (Instrument Glasses,
En®eld, UK). The silica ®bre optics were obtained from Opti¯ex (Doncaster, UK). Electronic components were from RS Components (Northhants, UK). The nitrite (10 mM NOÿ 2 ) stock solution was prepared from sodium nitrite. The pH of the ammonium chloride buffer (190 mM) was adjusted with ammonia to give a pH of 5. The sulphanilamide (1.16 mM) and N-(1-naphthyl) ethylene diamine (1.95 mM) were prepared as a mixed reagent in ammonium chloride buffer, with ®ve drops of concentrated hydrochloric acid being used to dissolve the sulphanilamide. 2.2. Device fabrication The photolithographic plates containing 25 devices of ®ve different geometries were obtained from photomask producers (Alignrite, Wales, UK). The method of wet etching was a modi®ed version of that described by Daykin and Haswell [8]. The glass plates were placed in 1% HF and 5% NH4F at 708C and agitated every 10 min for 1 min, followed by 1 min lateral stirring with a plastic rod during the etching process. The etching time for the manifold used in this work was 2 h after which the plate was removed from the bath and washed thoroughly with water. Before the etching solution was discarded the channels on the plates were measured using a DekTek3ST stylus surface pro®ler to ensure the required channel dimensions had been achieved. Three pro®les were taken at three different locations along the channel and an average of the channel width, depth and area were recorded. With the required channel sizes achieved the etching solution was carefully discarded. The plates were then exposed to strong UV-light for 1±2 h before being dipped into a photoresist remover for 2±3 h to remove the photoresist. The plates were washed with tap water and any excess photoresist and the chrome layer were then stripped off with the Microposit chrome etch 18 with a ®nal washed being carried out in de-ionised water. The etched plates were then cut into individual manifolds i.e. 25 (152 mm2) being obtained from the original plate. In the work by Daykin and Haswell [8] the manifolds had thin glass cover plates with 2 mm i.d. holes thermally bonded onto the manifolds. The solution reservoirs were then stuck on to the cover plates. Many problems associated with mechanical and chemical stability were encountered with this approach and
G.M. Greenway et al. / Analytica Chimica Acta 387 (1999) 1±10
therefore thick glass cover plates (17 mm) incorporating 2 mm i.d., predrilled holes as reservoirs were thermally bonded to the substrate so negating the need to glue reservoirs into the manifold. To achieve this the thick cover plate was carefully positioned such that the 2 mm holes (reservoir) were placed directly on the end of the etched channels in the substrate and were held ®rmly by Blu-tack (Bostik, Leicester, UK). The chip was placed on the silica support in the centre of a microwave ashing system (CEM microwave ashing system 300, NC, USA). During the bonding process the thicker cover plate was placed at the bottom to prevent the channel from being completely fused due to the weight of the cover plate pressing on the etched base plate. The thermal due to the weight of the cover plate pressing on the etched base plate. The thermal programme that was used was 758C for 30 min, 1508C for 30 min, 6808C for 30 min, 6508C for 30 min, 5708C for 30 min, 5058C for 30 min after which the oven was allowed to return to room temperature. The bonded chip was tested for successful fusion by ®lling reservoirs with water and determining a linear current voltage relationship over the range 0±1 kV. If the chips became blocked in the development work they were cleaned with mineral acid. 2.3. Procedure 2.3.1. Determination of the reagent flow characteristics To determine the ¯ow characteristics of the reagents and reaction products a second manifold prepared as above with a channel size of 325 mm wide and 30 mm deep was used. In this work the applied voltage, concentration and pH of the reagents were the selected variables. A traditional FIA system using the method described by Daniel et al. [10] was ®rst used to identify preliminary reaction conditions. These conditions are shown in Fig. 3. Using these conditions the optimum applied voltage and corresponding ¯ow rates were determined for the individual reagents using the manifold shown in Fig. 1. With 560 mM NH4C1 and a constant pH of 7.8 the NH4C1 was placed in reservoir A whilst water was placed in reservoirs B and E. Reservoir E also acted as ground for the ¯oating current. Voltage was applied across A() and B(ÿ) which pumped the reagent from A to B for a period of 30 min. The volume of NH4C1 in reservoir A was
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Fig. 1. Schematic diagram of the micro flow injection manifold for the determination of reagents flow characteristics. Channel size was 325 mm wide and 30 mm deep. The volume of the channel section from the junction BC to BE was 76 nl and BD to BE was 44 nl. Asulphanilamide, Bammonium chloride, CN-(1naphthyl) ethylene diamine, Dnitrite/nitrate/sample, Ewaste.
measured by ®lling the reservoir to a premarked point bonding was with a micro syringe thereby replacing the amount that had been pumped out of the reservoir A. From the volume change over the 30 min the ¯ow rate was calculated. The procedure was repeated again for the next chosen voltage until the optimum applied voltage was determined. Keeping the optimum applied voltage and the pH constant the optimum concentration of the NH4C1 was determined and ®nally keeping the optimum applied voltage and concentration constant the optimum pH was determined. In a same manner, the ¯ow characteristics of the sulphanilamide, NED, nitrite standards and mixed reagent were determined. The mFIA manifold and con®guration for the ®nal analytical measurements is shown in Fig. 2. 2.4. mFIA system The mFIA system used is described schematically in Fig. 2 and was contained for experimental purposes in a custom built insulation box with two power supplies offering power outputs of up to 50 W with a maximum voltage of 1 kV (Advance Hivolt, West Sussex, UK). The current and voltage were monitored by a computing multimeter (model 1906, Thurlby Thandar Instrument, Huntingdon, Cambridgeshire, UK). A custom built light source with a green light emitting diode (LED) was attached using an SMA ®tting to a 110 mm i.d., 125 mm o.d. silica ®bre optic (Opti¯ex, Doncaster, UK) which transferred the light to and from the reaction channel in the micro-reactor. The receiving ®bre optics carried the absorbed light into a computer controlled diode array micro-spectrometer with SpecView windows software (microParts, Dortmund, Ger-
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Fig. 2. A schematic of the micro flow injection manifold for the determination of nitrite using diode array microspectrometer. Microreactor chip enclosed in insulation box. Microchannel size of 302 mm wide and 115 mm deep with fibre optics (110 mm) indicated by the thick heavy line, HVPS 1 and 2 are high voltage power supplies. Reservoir Amix reagent, Bnitrite/sample, Cwaste 1, Dwaste 2.
many). The ®bres were prepared by removing the plastic coated covering at the ends of the ®bre optics with NITRO1MORS all purpose paint-vanish remover (Winsford, UK). The bare ends of the ®bre optics were then cleaned and cut with a quartz crystal. The ®bre optics were carefully inserted into a water ®lled side channel of the chip and aligned using the diode array micro-spectrometer. The ®bre optics were ®xed in position by gluing with a general-purpose adhesive (Bostik, Leicester, UK) and silicon rubber glue (RS components, Northants, UK) and allowed to harden over night before being used. 3. Results and discussion 3.1. Characterisation of electro-osmotic flow properties of the reagents The electro-osmotic ¯ow characteristics of the reagents used in the colorimetric determination of nitrite were identi®ed using the manifold shown in Fig. 1 according to the procedure described in Section 2. The buffer used in the original method was ammonium chloride. Despite this giving good electroosmotic properties bubbles did occur around the electrodes due to formation of ammonia at voltage greater than 600 V. To overcome these dif®culties several other buffering systems were investigated, however, the pH of the borate was found to be too high toward the end of the scale (9±10) whereby EOF is greatly reduced. The sodium acetate and sodium formate
buffers gave a low EOF due to their low pH (<3). In characterising the buffer ¯ow a balance had to be established between the best conditions for EOF and the best conditions for the colorimetric reaction to occur. 3.1.1. Ammonium chloride The ¯ow characteristics of the NH4C1 are shown in Fig. 3(a). An optimum voltage of 350 V was found to deliver a ¯ow of 0.80 ml/min NH4C1, however, any voltage between 250±400 V was clearly capable of delivering a suitable ¯ow of NH4C1 (Fig. 3(a1)). As the applied voltage was increased beyond 400 V the ¯ow rate decreased and as the power increased above 600 V bubbles were formed around the electrodes and were being entrained into the channel forming air gaps and causing the current to ¯uctuate. The most suitable concentration for NH4C1 EOF was determined to be 190 mM (1% w/v), thereafter, as the concentration increased the ¯ow decreased due to the ions migrating to the electrodes and leaving the bulk solution behind (Fig. 3(a2)). The ¯ow rate for the reagent is also known to be dependent on the pH [11,12] with pH values between 4 and 10 being known to be suitable for EOF. In this study the highest ¯ow rate was obtained at pH 6.8 (Fig. 3(a3)). The decrease in the ¯ow observed at pH>7 was again due to bubble formation. 3.1.2. Sulphanilamide The ¯ow characteristics of sulphanilamide are shown in Fig. 3(b). The optimum applied voltage
G.M. Greenway et al. / Analytica Chimica Acta 387 (1999) 1±10
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Fig. 3. Characterisation of electro-osmotic flow of reagents: (a) EOF of ammonium chloride, (a1) NH4Cl: 560 mM, pH 7.8, (a2) 350 V, pH 7.8, (a3) 350 V, 190 mM; (b) EOF of sulphanilamide, (b1) sulphanilamide: 2.9 mM, pH 2.0, (b2) 400 V, pH 2.0, (b3) 400 V, 0.39 mM; (c) EOF of NED, (c1) NED: 5.4 mM, pH 4.0, (c2) 800 V, pH 4.0, (c3) 800 V, 0.05 mM; (d) EOF of nitrite, (d1) nitrite: 0.1 mM, pH 5.0, (d2) 400 V, pH 5.0, (d3) 400 V, 0.05 mM and (e) EOF of mixed reagent, (e1) mixed reagent: 1.16/1.95 mM, pH 4.0.
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for moving sulphanilamide was found to be 400 V, however, the ¯ow rate at this voltage was only 0.38 ml/ min (Fig. 3(b1)). The lower ¯ow rates obtained at these applied voltages are due to a low EOF, a result mainly due to the acidic conditions of the reagent. The best concentration of sulphanilamide in terms of obtaining the highest ¯ow rate was 0.39 mM (Fig. 3(b2)), however, the concentration range between 0.39 and 0.98 mM was not optimum for the reaction and the coloured azo dye was not formed. Higher concentrations causes resistance in the channel causing ¯ow to reduce, however, a much higher concentration of 1.18 mM sulphanilamide was used because it immediately formed the coloured azo dye when added to the nitrite even at low ¯ow rates. The optimum pH of 1.18 mM sulphanilamide was 3.9 and gave a ¯ow rate of 0.52 ml/min (Fig. 3(b3)). The diazotisation and coupling reaction is known to be pH sensitive and is better carried out at pH<4. Thus, the conditions of 1.18 mM sulphanilamide at pH 3.9 offers good compromise conditions. 3.1.3. N-(1-naphthyl) ethylene diamine (NED) The hydrophobicity of NED meant that a constant ¯ow rate of 0.4 ml/min was found over a large voltage range and only began to increase at voltages exceeding 800 V (Fig. 3(c1)). Good ¯ow control was achieved for the given concentration range (Fig. 3(c2)) at high voltages with the maximum ¯ow rate obtained at 0.05 mM, however, a reaction between the 1.18 mM sulphanilamide and the 0.05 mM NED did not produced an immediate coloured azo dye. It was only at higher concentration (>1 mM) that an obvious coloured azo dye was formed. The optimum pH was 4.5 with a pH>5 showing a decrease in ¯ow rate (Fig. 3(c3)). This was important because it was found that when the pH of NED was >5 the coupling reaction did not occur. 3.1.4. Nitrite standard The voltage required to deliver a suitable ¯ow rate for the nitrite standard was found to be between 300 and 600 V with 400 V being the optimum (Fig. 3(d1)). A linear ¯ow was found between 0.005 and 0.05 mM NOÿ 2 , thereafter, the ¯ow ¯uctuated (Fig. 3(d2)). The optimum concentration which gave the highest ¯ow rate was 0.05 mM and the optimum pH was
4 (Fig. 3(d3)). In the analytical method ammonium chloride buffer at pH 4 controlled the EOF overriding these effects and ensuring reproducible injections. 3.1.5. Mixed reagents To overcome the dif®culties observed in moving the NED and sulphanilamide by EOF it was decided to premix the two reagents. A mixture of 1.18 mM sulphanilamide and 1.95 mM NED was prepared in 190 mM NH4C1 at pH 4 to facilitate the movement yet still allow the required reaction to occur. The mixed reagent was colourless and stable for at least 12 h. The best applied voltage found to deliver a good ¯ow rate (0.8 ml/min) for the mixed reagent was 600 V, although any voltages between 300 and 600 V would be indeed suitable (Fig. 3(e)). The increase in ¯ow rate above 600 V lead once again to Joule heating and an increase in bubble formation with subsequent evaporation. 3.1.6. Reaction product (azo dye) The product formed in the channel had a different ¯ow characteristics to the reagent and analyte and therefore needed a different applied voltage to move it around in the channel. This was established by carrying out the reaction outside the reactor and introducing the product to the reservoir for the ¯ow rate to be determined. The pH of the azo dye dropped to <2 making it dif®cult to move by EOF. Although, applied voltages between 100 and 1000 V were used there was no evidence of the dye moving. It was observed that the coloured azo dye formed in the channel was not able to move to the detection point and that the coloured azo dye, when left for more than 2 days decolourised at applied voltages>400 V indicating that any azo dye formed in the channel had to be moved by using lower applied voltages. To achieve appropriate analytical conditions a buffer system (NH4C1) was used to aid mobility of the coloured product. 3.2. Characterisation of detection method 3.2.1. Detector configuration The detection system was designed to detect the absorbance of the azo dye product but this depended also on the arrangement of the ®bre optics, the sensi-
G.M. Greenway et al. / Analytica Chimica Acta 387 (1999) 1±10
tivity of the LED and the spectrophotometer detection system. It was found that the best results were obtained by actually ensuring that the ends of the ®bres were carefully cut to give a de®ned ¯at surface so that the light coming out of this surface is not diffracted but intensely focus into the channel onto the receiving ®bre optics. The light intensity is greatly improved when the ends of the ®bre optics are glued into the channel and in direct contact with the solution providing a good alignment of the ®bres as this was the key in obtaining acceptable sensitivity. The intensity of the green LED used was 0.25 candela with a viewing angle of 258 which meant that if the ®bre optics surface was carefully cut there was no need for a focusing lenses. 3.2.2. Reagent mixing As the absorbance ef®ciency is measured along the microchannel in which the reaction occurs (as shown in Fig. 2) signal intensity is dependent on the length of sample plug in the channel. Experiments were performed where the reaction was carried out outside the reactor and the product was introduced directly into the channel, thus ensuring complete mixing had occurred. In these experiments the absorbance was shown to be proportional to sample plug length in the region of 1±9 mm (r20.996) with values up to 1.5 absorbance units for a 9 mm plug of 100 mM nitrite solution. However, when the reaction took place in the microreactor the absorbances obtained were found to be much lower. This was due to the incomplete mixing of the reagents as they converged in the mFIA manifold. To understand the processes that were occurring it is important to realise that in this reaction the coloured product is formed almost immediately, but the colour then continues to intensify. The nitrite solution was introduced at a T-junction into the detection channel (which already contains the mixed reagent) as illustrated in Fig. 2. From the results obtained it appeared that once the coloured dye (product) has been formed by diffuse mixing at the interface between the solutions it effectively blocked further contact of the mixed reagent with the nitrite due to the lack of mixing in such systems (Reynold numbers<1). The product slug produced was less than 2 mm and was then diluted on the resumption of the reagent mixture giving a much lower absorbance than would be
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expected. Further experiments with coloured dyes and different shaped intersections (T and Y) seemed to con®rm mixing beyond that expected by natural diffusion was not occurring. For the `Y' type (458) junction mixing there was some evidence of movement as far as the intersection and a close look at the intersection with a 9x eye piece magnifying glass con®rmed the lack of mixing. At the intersection the two dyes formed distinctively two layers and did not mixed when water was pumped into the channel to help the mixing. This con®rmed the suggestion that mixing was not occurring and therefore the reaction was as expected to be, a diffusion limited interfacial reaction. Thus it became apparent that the subsequent analytical procedure must accommodate the non-mixing or interfacial reaction characteristics of mFIA systems. 3.2.3. Optimisation of the injection time The effect of the injection time of the 100 mM nitrite into the mixed reagent can be seen in Fig. 4. As can be seen from the graph an injection time<10 s was not long enough to inject a suf®cient amount into the measurement channel and secondly an injection time>50 s was too long so that by the time the measurement was taken the coloured azo dye has moved out of the measurement channel leaving a new column of nitrite occupying the length of the main channel. The maximum absorbance was obtained at 30 s, although 20 and 40 s also gave good response. For this work 20 s injection was chosen for fast sample throughput. 3.2.4. Detection mode The microspectrometer (Fig. 2) was supplied by microParts, Dortmund, Germany. This consist of a Hamamatsu S5463-256 photodiode array contained in a grey aluminium housing with a spectral range of 380±780 nm (dispersion of 0.12 nm/mm). Light is introduced to the diode array via a ®bre of 50 mm. The electronic components consisted of microcontroller (Siemens SAB 80C166), A/D converter (Burr Brown ADS 7807U), and had a resolution of 16 bits with integration times of 40±2560 ms possible. The system was used in the Scan mode for this work and Window software SpecView was used to collect the data, which was transferred into ASCII-File to be
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Fig. 4. Investigation of the sample injection time (scan mode), 100 mM nitrite was used with 3 absorbance reading taken (n3) for each injection. Chosen max526 nm.
Fig. 5. Effect of changing the stop flow mode time on absorbance for injection of 100 mM NOÿ 2 (measurement recorded in scan mode).
analysed in an Excel spreadsheet. To optimise the absorbance signal the electro-osmotic ¯ow was stopped when the product was in the channel (stop ¯ow mode) and the dye allowed to increase in intensity, this allowed the signal to be adequately detected. The absorbance was found to increase the longer the product was in the channel until a steady state was reached but a hold time of 20 s was chosen as this gave acceptable limits of detection (Fig. 5). Subsequent work was carried out using a 30 s loading of the mixed reagents then 20 s injection of nitrite followed by 20 s in the stopped ¯ow mode, the measurement was then made and the cycle repeated.
3.3. Analytical characteristics Using the optimised conditions a calibration curve was obtained. The initial results using the manifold shown in Fig. 2 (Mode 1) with the two negative electrodes placed in reservoir C gave poor reproducibility (r20.973) as can be seen in the r2 value in Table 1. The reproducibility was increased by changing the set up of the electrodes in the manifold so that there were two negative waste reservoirs (Mode 2) to avoid the previous situation where the two negative electrodes were placed in the same reservoirs. In the new con®guration (Fig. 2) the reservoir A() to
G.M. Greenway et al. / Analytica Chimica Acta 387 (1999) 1±10
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Table 1 Calibration details Analytical parameters
mFIA
Conventional FIA a
b
Mode 1
Mode 2
Equation R2 LOD (mM)
y0.0014x0.0118 0.973 1.8
y0.0072xÿ0.0007 0.999 0.20
y0.0048xÿ0.0008 0.999 0.20
Standard (mM) 1.0 10.0 50.0
(RSD) 7.9 14.6 11.5
8.7 5.3 2.6
4.0 1.4 0.6
c
a
Mode 1: two negative electrodes in waste reservoir (C). Mode 2: two negative electrodes in separate waste reservoir (CD). c Where y is absorbance and x is concentration in mM. b
reservoir D(ÿ) was used to load the reagents and reservoirs B() to reservoir C(ÿ) was used to inject the sample. In each calibration run, the absorbance measurement of the mixed reagent was zero reference (or blank corrected) to compensate any dye formation arising from trace nitrite before the nitrite standard was injected and measured. A linear calibration was obtained between 0 and 100 mM with an equation of the line being y0.0072xÿ0.0007 where y is the absorbance and x is the concentration in mM with a correlation coef®cient of 0.999. The limit of detection (3) was approximately 0.20 mM NOÿ 2 . These results were comparable with those achieved by the conventional FIA in which the calibration was linear (r20.999) between 0 and 100 mM and the LOD (3) was 0.20 mM (Table 1). The reproducibility was not as good as for the conventional FIA system where the %RSD was below 5% for all standards. In the micro ¯ow system although the RSD was 2.6% for the 50 mM NOÿ 2 standard and 5.3% for the 10 mM NOÿ 2 standard it decreased to 8.7% for the 1 mM NOÿ 2 standard. This lack of reproducibility is thought to be due to the possibility of hydrodynamic ¯ow competing with the EOF. To overcome this we are now investigating the reproducibility when the channel size in the micro reactor is reduced to 200 mm. 4. Conclusion This paper has shown that it is possible to detect the nitrite by a spectrophotometric method in a micro ¯ow
injection analysis system with acceptable sensitivity and reproducibility. In developing such a system a compromise has to be made between the conditions required for good EOF (for example a pH range between 2 and 9) and conditions required for the diazotisation reaction. The optimisation could be carried out more ef®ciently by measuring both the EOF and absorbance. The method enables a sampling rate of 50 samples/h with a signi®cant reduction in reagent volumes and waste products. Further work needs to be carried out in the more fundamental area of reagent mixing and reaction characteristics to improve the system. Work is also currently being carried out to extend the method to the determination of nitrate by using in situ cadmium reduction of the nitrate to nitrite. Samples of natural waters will also be analysed after ®ltration through a 0.25 mm membrane ®lters. Acknowledgements The authors would like acknowledge the contributions made to this work by other members of the micro-reactor group at Hull, especially, Lorna Nelstrop and George Doku. They would also like to thank the Government of Papua New Guinea for the ®nancial support to Peter Petsul. References [1] S.J. Haswell, Analyst 122 (1997). [2] K. Seiler, Z.H. Fan, K. Fluri, D.J. Harrison, Anal. Chem. 66 (1994) 3485.
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[3] A. Meulemans, F. Delsenne, J. Chromatogr. B 660 (1994) 401. [4] N. Chiem, D.J. Harrison, Anal. Chem. 69 (1997) 373. [5] D.J. Harrison, A. Manz, Z. Fan, H. Ludi, H.M. Widmer, Anal. Chem. 64 (1992) 1926. [6] D.J. Harrison, Z. Fan, K. Seiler, Anal. Chim. Acta 283 (1993) 361. [7] D.J. Harrison, P.G. Glavina, Sensors and Actuators B 10 (1993) 10724. [8] R.N.C. Daykin, S.J. Haswell, Anal. Chim. Acta 313 (1995) 155.
[9] H. Fiehn, S. Howitz, M.T. Pham, T. Vopel, M. Burger, T. Wegner, in: A. van den Berg, P. Bergveld (Eds.), Micro Total Analysis Systems, (1995) 289. [10] A. Daniel, D. Birot, M. Lehaitre, J. Poincin, Anal. Chim. Acta 308 (1995) 413. [11] M.F.M. Tavares, V.L. McGuffin, Anal. Chem. 67 (1995) 3687. [12] Q.H. Wan, J. Phys. Chem. B 101 (1997) 4860.
Characterisation of a micro-total analytical system for the determination of nitrite with spectrophotometric detection G.M. Greenway*, S.J. Haswell, P.H. Petsul Department of Chemistry, University of Hull, Hull, HU6 7RX, UK Received 17 August 1998; received in revised form 30 November 1998; accepted 8 December 1998
Abstract A micro-total analytical system (mTAS) is described for the determination of nitrite based on its reaction with sulphanilamide to form the diazonium salt which when coupled with N-(1-naphthyl) ethylene diamine yields an azo dye whose absorbance was measured at 526 nm. The reaction took place in an etched glass manifold containing channels 302 mm wide and 115 mm deep in which electro-osmotic ¯ow was used to move the reagents. The absorbance of the product was measured in situ using a micro-spectrophotometric-®bre optic detection system. The electro-osmotic ¯ow mixing characteristics of the reagents together with the production of the colorimetric complex have been investigated. In addition methods for obtaining sensitive detection in the micro-reactor are reported. Using the conditions established a linear calibration was obtained between 0 and 100 mM with a correlation coef®cient of 0.999. The RSD at 50 mM NOÿ 2 was 2.6% (n6) and the limit of detection obtained (3) was 0.20 mM NOÿ . # 1999 Elsevier Science B.V. All rights reserved. 2 Keywords: Micro ¯ow injection analysis; Nitrite; Miniaturisation; mTAS
1. Introduction The presence of low level chemical species in the environment, an increasing need for good industrial process control, developments in clinical diagnostics and numerous areas have lead to the search for improved, less expensive and more robust methods for measuring low levels of chemical analytes. Micrototal analytical systems (mTAS) have been suggested as a possible solution for such analysis as they are primarily concerned with the chemical reactions in small volumes of liquids where the measurement is *Corresponding author. Tel.: +44-01482-465475; fax: +4401482-466416; e-mail: [email protected]
based on the reaction between more than one reagent, with subsequent determination being achieved in situ and an appropriate detector. These micro ¯ow systems have the inherent advantages of being portable, requiring low reagent consumption, and are able to operate remotely [1]. The development of such systems has progressed more recently with the introduction of electro-osmotic ¯ow that provides a ¯exible and robust method of moving ¯uids through microchannels of typically less than 200 mm i.d. [2±4]. Fast controllable reactions have been shown to occur between picolitres of samples in such reactors [5] which to date have been mostly produced by photolithography techniques using substrates such as glass and silicon [5±7].
0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0003-2670(99)00047-1
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A range of different detection techniques have been investigated for mTAS including spectrophotometry and electrochemistry [8,9]. Daykin and Haswell [8] for example reported the development of a micro ¯ow injection system (mFIA) for the determination of phosphate using spectrophotometric detection [8], whilst Fiehn et al. [9] have demonstrated the use of ¯uidic ISFET microsystem for the determination of pH, nitrate, ammonia, sodium and potassium based on a sensor technology using micro¯uidic injectors and diodes. In this work a mFIA has been investigated and developed for the determination of nitrite. Electroosmotic ¯ow has been used to mobilise reagents in an etched glass manifold to produce a coloured spectrophotometrically active analytical product. The absorbance of the product was measured in the manifold using a micro-spectrophotometric-®bre optic detection system. Nitrite was determined by its reaction with sulphanilamide to form the diazonium salt which was coupled with N-(1-naphthyl) ethylene diamine to yield an azo dye whose absorbance was measure at 526 nm. 2. Experimental 2.1. Reagents and materials All the chemical standards and reagents used were analytical grade unless stated otherwise and the water used was high purity de-ionised (18 M cm resistivity) (Elgastat UHQ PS, Elga, High Wycombe, UK). The sodium nitrite was supplied by Fisher Scienti®c (Loughborough, UK), ammonium chloride, hydro¯uoric acid and ammonium ¯uoride being obtained from Merck (Poole Dorset, UK). The hydrochloric acid was supplied from Philip Harris (Shenstone, Lich®eld, UK), and the sulphanilamide and N-(1naphthyl) ethylene diamine (NED) was from Aldrich (Gillingham, Dorset, UK). Sodium formate and sodium acetate were from Avocado Research Chemical (Heysham, UK) and the nitric acid was R.P.NorTM mapur , (Fontenay, S/Bois, France). The Microposit chrome etch 18 and photoresist remover 1112A were from Shipley (Coventry, UK). The glass for the microreactor chips and cover plates were both Superwhite Crown B70 borosilicate glass (Instrument Glasses,
En®eld, UK). The silica ®bre optics were obtained from Opti¯ex (Doncaster, UK). Electronic components were from RS Components (Northhants, UK). The nitrite (10 mM NOÿ 2 ) stock solution was prepared from sodium nitrite. The pH of the ammonium chloride buffer (190 mM) was adjusted with ammonia to give a pH of 5. The sulphanilamide (1.16 mM) and N-(1-naphthyl) ethylene diamine (1.95 mM) were prepared as a mixed reagent in ammonium chloride buffer, with ®ve drops of concentrated hydrochloric acid being used to dissolve the sulphanilamide. 2.2. Device fabrication The photolithographic plates containing 25 devices of ®ve different geometries were obtained from photomask producers (Alignrite, Wales, UK). The method of wet etching was a modi®ed version of that described by Daykin and Haswell [8]. The glass plates were placed in 1% HF and 5% NH4F at 708C and agitated every 10 min for 1 min, followed by 1 min lateral stirring with a plastic rod during the etching process. The etching time for the manifold used in this work was 2 h after which the plate was removed from the bath and washed thoroughly with water. Before the etching solution was discarded the channels on the plates were measured using a DekTek3ST stylus surface pro®ler to ensure the required channel dimensions had been achieved. Three pro®les were taken at three different locations along the channel and an average of the channel width, depth and area were recorded. With the required channel sizes achieved the etching solution was carefully discarded. The plates were then exposed to strong UV-light for 1±2 h before being dipped into a photoresist remover for 2±3 h to remove the photoresist. The plates were washed with tap water and any excess photoresist and the chrome layer were then stripped off with the Microposit chrome etch 18 with a ®nal washed being carried out in de-ionised water. The etched plates were then cut into individual manifolds i.e. 25 (152 mm2) being obtained from the original plate. In the work by Daykin and Haswell [8] the manifolds had thin glass cover plates with 2 mm i.d. holes thermally bonded onto the manifolds. The solution reservoirs were then stuck on to the cover plates. Many problems associated with mechanical and chemical stability were encountered with this approach and
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therefore thick glass cover plates (17 mm) incorporating 2 mm i.d., predrilled holes as reservoirs were thermally bonded to the substrate so negating the need to glue reservoirs into the manifold. To achieve this the thick cover plate was carefully positioned such that the 2 mm holes (reservoir) were placed directly on the end of the etched channels in the substrate and were held ®rmly by Blu-tack (Bostik, Leicester, UK). The chip was placed on the silica support in the centre of a microwave ashing system (CEM microwave ashing system 300, NC, USA). During the bonding process the thicker cover plate was placed at the bottom to prevent the channel from being completely fused due to the weight of the cover plate pressing on the etched base plate. The thermal due to the weight of the cover plate pressing on the etched base plate. The thermal programme that was used was 758C for 30 min, 1508C for 30 min, 6808C for 30 min, 6508C for 30 min, 5708C for 30 min, 5058C for 30 min after which the oven was allowed to return to room temperature. The bonded chip was tested for successful fusion by ®lling reservoirs with water and determining a linear current voltage relationship over the range 0±1 kV. If the chips became blocked in the development work they were cleaned with mineral acid. 2.3. Procedure 2.3.1. Determination of the reagent flow characteristics To determine the ¯ow characteristics of the reagents and reaction products a second manifold prepared as above with a channel size of 325 mm wide and 30 mm deep was used. In this work the applied voltage, concentration and pH of the reagents were the selected variables. A traditional FIA system using the method described by Daniel et al. [10] was ®rst used to identify preliminary reaction conditions. These conditions are shown in Fig. 3. Using these conditions the optimum applied voltage and corresponding ¯ow rates were determined for the individual reagents using the manifold shown in Fig. 1. With 560 mM NH4C1 and a constant pH of 7.8 the NH4C1 was placed in reservoir A whilst water was placed in reservoirs B and E. Reservoir E also acted as ground for the ¯oating current. Voltage was applied across A() and B(ÿ) which pumped the reagent from A to B for a period of 30 min. The volume of NH4C1 in reservoir A was
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Fig. 1. Schematic diagram of the micro flow injection manifold for the determination of reagents flow characteristics. Channel size was 325 mm wide and 30 mm deep. The volume of the channel section from the junction BC to BE was 76 nl and BD to BE was 44 nl. Asulphanilamide, Bammonium chloride, CN-(1naphthyl) ethylene diamine, Dnitrite/nitrate/sample, Ewaste.
measured by ®lling the reservoir to a premarked point bonding was with a micro syringe thereby replacing the amount that had been pumped out of the reservoir A. From the volume change over the 30 min the ¯ow rate was calculated. The procedure was repeated again for the next chosen voltage until the optimum applied voltage was determined. Keeping the optimum applied voltage and the pH constant the optimum concentration of the NH4C1 was determined and ®nally keeping the optimum applied voltage and concentration constant the optimum pH was determined. In a same manner, the ¯ow characteristics of the sulphanilamide, NED, nitrite standards and mixed reagent were determined. The mFIA manifold and con®guration for the ®nal analytical measurements is shown in Fig. 2. 2.4. mFIA system The mFIA system used is described schematically in Fig. 2 and was contained for experimental purposes in a custom built insulation box with two power supplies offering power outputs of up to 50 W with a maximum voltage of 1 kV (Advance Hivolt, West Sussex, UK). The current and voltage were monitored by a computing multimeter (model 1906, Thurlby Thandar Instrument, Huntingdon, Cambridgeshire, UK). A custom built light source with a green light emitting diode (LED) was attached using an SMA ®tting to a 110 mm i.d., 125 mm o.d. silica ®bre optic (Opti¯ex, Doncaster, UK) which transferred the light to and from the reaction channel in the micro-reactor. The receiving ®bre optics carried the absorbed light into a computer controlled diode array micro-spectrometer with SpecView windows software (microParts, Dortmund, Ger-
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Fig. 2. A schematic of the micro flow injection manifold for the determination of nitrite using diode array microspectrometer. Microreactor chip enclosed in insulation box. Microchannel size of 302 mm wide and 115 mm deep with fibre optics (110 mm) indicated by the thick heavy line, HVPS 1 and 2 are high voltage power supplies. Reservoir Amix reagent, Bnitrite/sample, Cwaste 1, Dwaste 2.
many). The ®bres were prepared by removing the plastic coated covering at the ends of the ®bre optics with NITRO1MORS all purpose paint-vanish remover (Winsford, UK). The bare ends of the ®bre optics were then cleaned and cut with a quartz crystal. The ®bre optics were carefully inserted into a water ®lled side channel of the chip and aligned using the diode array micro-spectrometer. The ®bre optics were ®xed in position by gluing with a general-purpose adhesive (Bostik, Leicester, UK) and silicon rubber glue (RS components, Northants, UK) and allowed to harden over night before being used. 3. Results and discussion 3.1. Characterisation of electro-osmotic flow properties of the reagents The electro-osmotic ¯ow characteristics of the reagents used in the colorimetric determination of nitrite were identi®ed using the manifold shown in Fig. 1 according to the procedure described in Section 2. The buffer used in the original method was ammonium chloride. Despite this giving good electroosmotic properties bubbles did occur around the electrodes due to formation of ammonia at voltage greater than 600 V. To overcome these dif®culties several other buffering systems were investigated, however, the pH of the borate was found to be too high toward the end of the scale (9±10) whereby EOF is greatly reduced. The sodium acetate and sodium formate
buffers gave a low EOF due to their low pH (<3). In characterising the buffer ¯ow a balance had to be established between the best conditions for EOF and the best conditions for the colorimetric reaction to occur. 3.1.1. Ammonium chloride The ¯ow characteristics of the NH4C1 are shown in Fig. 3(a). An optimum voltage of 350 V was found to deliver a ¯ow of 0.80 ml/min NH4C1, however, any voltage between 250±400 V was clearly capable of delivering a suitable ¯ow of NH4C1 (Fig. 3(a1)). As the applied voltage was increased beyond 400 V the ¯ow rate decreased and as the power increased above 600 V bubbles were formed around the electrodes and were being entrained into the channel forming air gaps and causing the current to ¯uctuate. The most suitable concentration for NH4C1 EOF was determined to be 190 mM (1% w/v), thereafter, as the concentration increased the ¯ow decreased due to the ions migrating to the electrodes and leaving the bulk solution behind (Fig. 3(a2)). The ¯ow rate for the reagent is also known to be dependent on the pH [11,12] with pH values between 4 and 10 being known to be suitable for EOF. In this study the highest ¯ow rate was obtained at pH 6.8 (Fig. 3(a3)). The decrease in the ¯ow observed at pH>7 was again due to bubble formation. 3.1.2. Sulphanilamide The ¯ow characteristics of sulphanilamide are shown in Fig. 3(b). The optimum applied voltage
G.M. Greenway et al. / Analytica Chimica Acta 387 (1999) 1±10
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Fig. 3. Characterisation of electro-osmotic flow of reagents: (a) EOF of ammonium chloride, (a1) NH4Cl: 560 mM, pH 7.8, (a2) 350 V, pH 7.8, (a3) 350 V, 190 mM; (b) EOF of sulphanilamide, (b1) sulphanilamide: 2.9 mM, pH 2.0, (b2) 400 V, pH 2.0, (b3) 400 V, 0.39 mM; (c) EOF of NED, (c1) NED: 5.4 mM, pH 4.0, (c2) 800 V, pH 4.0, (c3) 800 V, 0.05 mM; (d) EOF of nitrite, (d1) nitrite: 0.1 mM, pH 5.0, (d2) 400 V, pH 5.0, (d3) 400 V, 0.05 mM and (e) EOF of mixed reagent, (e1) mixed reagent: 1.16/1.95 mM, pH 4.0.
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for moving sulphanilamide was found to be 400 V, however, the ¯ow rate at this voltage was only 0.38 ml/ min (Fig. 3(b1)). The lower ¯ow rates obtained at these applied voltages are due to a low EOF, a result mainly due to the acidic conditions of the reagent. The best concentration of sulphanilamide in terms of obtaining the highest ¯ow rate was 0.39 mM (Fig. 3(b2)), however, the concentration range between 0.39 and 0.98 mM was not optimum for the reaction and the coloured azo dye was not formed. Higher concentrations causes resistance in the channel causing ¯ow to reduce, however, a much higher concentration of 1.18 mM sulphanilamide was used because it immediately formed the coloured azo dye when added to the nitrite even at low ¯ow rates. The optimum pH of 1.18 mM sulphanilamide was 3.9 and gave a ¯ow rate of 0.52 ml/min (Fig. 3(b3)). The diazotisation and coupling reaction is known to be pH sensitive and is better carried out at pH<4. Thus, the conditions of 1.18 mM sulphanilamide at pH 3.9 offers good compromise conditions. 3.1.3. N-(1-naphthyl) ethylene diamine (NED) The hydrophobicity of NED meant that a constant ¯ow rate of 0.4 ml/min was found over a large voltage range and only began to increase at voltages exceeding 800 V (Fig. 3(c1)). Good ¯ow control was achieved for the given concentration range (Fig. 3(c2)) at high voltages with the maximum ¯ow rate obtained at 0.05 mM, however, a reaction between the 1.18 mM sulphanilamide and the 0.05 mM NED did not produced an immediate coloured azo dye. It was only at higher concentration (>1 mM) that an obvious coloured azo dye was formed. The optimum pH was 4.5 with a pH>5 showing a decrease in ¯ow rate (Fig. 3(c3)). This was important because it was found that when the pH of NED was >5 the coupling reaction did not occur. 3.1.4. Nitrite standard The voltage required to deliver a suitable ¯ow rate for the nitrite standard was found to be between 300 and 600 V with 400 V being the optimum (Fig. 3(d1)). A linear ¯ow was found between 0.005 and 0.05 mM NOÿ 2 , thereafter, the ¯ow ¯uctuated (Fig. 3(d2)). The optimum concentration which gave the highest ¯ow rate was 0.05 mM and the optimum pH was
4 (Fig. 3(d3)). In the analytical method ammonium chloride buffer at pH 4 controlled the EOF overriding these effects and ensuring reproducible injections. 3.1.5. Mixed reagents To overcome the dif®culties observed in moving the NED and sulphanilamide by EOF it was decided to premix the two reagents. A mixture of 1.18 mM sulphanilamide and 1.95 mM NED was prepared in 190 mM NH4C1 at pH 4 to facilitate the movement yet still allow the required reaction to occur. The mixed reagent was colourless and stable for at least 12 h. The best applied voltage found to deliver a good ¯ow rate (0.8 ml/min) for the mixed reagent was 600 V, although any voltages between 300 and 600 V would be indeed suitable (Fig. 3(e)). The increase in ¯ow rate above 600 V lead once again to Joule heating and an increase in bubble formation with subsequent evaporation. 3.1.6. Reaction product (azo dye) The product formed in the channel had a different ¯ow characteristics to the reagent and analyte and therefore needed a different applied voltage to move it around in the channel. This was established by carrying out the reaction outside the reactor and introducing the product to the reservoir for the ¯ow rate to be determined. The pH of the azo dye dropped to <2 making it dif®cult to move by EOF. Although, applied voltages between 100 and 1000 V were used there was no evidence of the dye moving. It was observed that the coloured azo dye formed in the channel was not able to move to the detection point and that the coloured azo dye, when left for more than 2 days decolourised at applied voltages>400 V indicating that any azo dye formed in the channel had to be moved by using lower applied voltages. To achieve appropriate analytical conditions a buffer system (NH4C1) was used to aid mobility of the coloured product. 3.2. Characterisation of detection method 3.2.1. Detector configuration The detection system was designed to detect the absorbance of the azo dye product but this depended also on the arrangement of the ®bre optics, the sensi-
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tivity of the LED and the spectrophotometer detection system. It was found that the best results were obtained by actually ensuring that the ends of the ®bres were carefully cut to give a de®ned ¯at surface so that the light coming out of this surface is not diffracted but intensely focus into the channel onto the receiving ®bre optics. The light intensity is greatly improved when the ends of the ®bre optics are glued into the channel and in direct contact with the solution providing a good alignment of the ®bres as this was the key in obtaining acceptable sensitivity. The intensity of the green LED used was 0.25 candela with a viewing angle of 258 which meant that if the ®bre optics surface was carefully cut there was no need for a focusing lenses. 3.2.2. Reagent mixing As the absorbance ef®ciency is measured along the microchannel in which the reaction occurs (as shown in Fig. 2) signal intensity is dependent on the length of sample plug in the channel. Experiments were performed where the reaction was carried out outside the reactor and the product was introduced directly into the channel, thus ensuring complete mixing had occurred. In these experiments the absorbance was shown to be proportional to sample plug length in the region of 1±9 mm (r20.996) with values up to 1.5 absorbance units for a 9 mm plug of 100 mM nitrite solution. However, when the reaction took place in the microreactor the absorbances obtained were found to be much lower. This was due to the incomplete mixing of the reagents as they converged in the mFIA manifold. To understand the processes that were occurring it is important to realise that in this reaction the coloured product is formed almost immediately, but the colour then continues to intensify. The nitrite solution was introduced at a T-junction into the detection channel (which already contains the mixed reagent) as illustrated in Fig. 2. From the results obtained it appeared that once the coloured dye (product) has been formed by diffuse mixing at the interface between the solutions it effectively blocked further contact of the mixed reagent with the nitrite due to the lack of mixing in such systems (Reynold numbers<1). The product slug produced was less than 2 mm and was then diluted on the resumption of the reagent mixture giving a much lower absorbance than would be
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expected. Further experiments with coloured dyes and different shaped intersections (T and Y) seemed to con®rm mixing beyond that expected by natural diffusion was not occurring. For the `Y' type (458) junction mixing there was some evidence of movement as far as the intersection and a close look at the intersection with a 9x eye piece magnifying glass con®rmed the lack of mixing. At the intersection the two dyes formed distinctively two layers and did not mixed when water was pumped into the channel to help the mixing. This con®rmed the suggestion that mixing was not occurring and therefore the reaction was as expected to be, a diffusion limited interfacial reaction. Thus it became apparent that the subsequent analytical procedure must accommodate the non-mixing or interfacial reaction characteristics of mFIA systems. 3.2.3. Optimisation of the injection time The effect of the injection time of the 100 mM nitrite into the mixed reagent can be seen in Fig. 4. As can be seen from the graph an injection time<10 s was not long enough to inject a suf®cient amount into the measurement channel and secondly an injection time>50 s was too long so that by the time the measurement was taken the coloured azo dye has moved out of the measurement channel leaving a new column of nitrite occupying the length of the main channel. The maximum absorbance was obtained at 30 s, although 20 and 40 s also gave good response. For this work 20 s injection was chosen for fast sample throughput. 3.2.4. Detection mode The microspectrometer (Fig. 2) was supplied by microParts, Dortmund, Germany. This consist of a Hamamatsu S5463-256 photodiode array contained in a grey aluminium housing with a spectral range of 380±780 nm (dispersion of 0.12 nm/mm). Light is introduced to the diode array via a ®bre of 50 mm. The electronic components consisted of microcontroller (Siemens SAB 80C166), A/D converter (Burr Brown ADS 7807U), and had a resolution of 16 bits with integration times of 40±2560 ms possible. The system was used in the Scan mode for this work and Window software SpecView was used to collect the data, which was transferred into ASCII-File to be
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Fig. 4. Investigation of the sample injection time (scan mode), 100 mM nitrite was used with 3 absorbance reading taken (n3) for each injection. Chosen max526 nm.
Fig. 5. Effect of changing the stop flow mode time on absorbance for injection of 100 mM NOÿ 2 (measurement recorded in scan mode).
analysed in an Excel spreadsheet. To optimise the absorbance signal the electro-osmotic ¯ow was stopped when the product was in the channel (stop ¯ow mode) and the dye allowed to increase in intensity, this allowed the signal to be adequately detected. The absorbance was found to increase the longer the product was in the channel until a steady state was reached but a hold time of 20 s was chosen as this gave acceptable limits of detection (Fig. 5). Subsequent work was carried out using a 30 s loading of the mixed reagents then 20 s injection of nitrite followed by 20 s in the stopped ¯ow mode, the measurement was then made and the cycle repeated.
3.3. Analytical characteristics Using the optimised conditions a calibration curve was obtained. The initial results using the manifold shown in Fig. 2 (Mode 1) with the two negative electrodes placed in reservoir C gave poor reproducibility (r20.973) as can be seen in the r2 value in Table 1. The reproducibility was increased by changing the set up of the electrodes in the manifold so that there were two negative waste reservoirs (Mode 2) to avoid the previous situation where the two negative electrodes were placed in the same reservoirs. In the new con®guration (Fig. 2) the reservoir A() to
G.M. Greenway et al. / Analytica Chimica Acta 387 (1999) 1±10
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Table 1 Calibration details Analytical parameters
mFIA
Conventional FIA a
b
Mode 1
Mode 2
Equation R2 LOD (mM)
y0.0014x0.0118 0.973 1.8
y0.0072xÿ0.0007 0.999 0.20
y0.0048xÿ0.0008 0.999 0.20
Standard (mM) 1.0 10.0 50.0
(RSD) 7.9 14.6 11.5
8.7 5.3 2.6
4.0 1.4 0.6
c
a
Mode 1: two negative electrodes in waste reservoir (C). Mode 2: two negative electrodes in separate waste reservoir (CD). c Where y is absorbance and x is concentration in mM. b
reservoir D(ÿ) was used to load the reagents and reservoirs B() to reservoir C(ÿ) was used to inject the sample. In each calibration run, the absorbance measurement of the mixed reagent was zero reference (or blank corrected) to compensate any dye formation arising from trace nitrite before the nitrite standard was injected and measured. A linear calibration was obtained between 0 and 100 mM with an equation of the line being y0.0072xÿ0.0007 where y is the absorbance and x is the concentration in mM with a correlation coef®cient of 0.999. The limit of detection (3) was approximately 0.20 mM NOÿ 2 . These results were comparable with those achieved by the conventional FIA in which the calibration was linear (r20.999) between 0 and 100 mM and the LOD (3) was 0.20 mM (Table 1). The reproducibility was not as good as for the conventional FIA system where the %RSD was below 5% for all standards. In the micro ¯ow system although the RSD was 2.6% for the 50 mM NOÿ 2 standard and 5.3% for the 10 mM NOÿ 2 standard it decreased to 8.7% for the 1 mM NOÿ 2 standard. This lack of reproducibility is thought to be due to the possibility of hydrodynamic ¯ow competing with the EOF. To overcome this we are now investigating the reproducibility when the channel size in the micro reactor is reduced to 200 mm. 4. Conclusion This paper has shown that it is possible to detect the nitrite by a spectrophotometric method in a micro ¯ow
injection analysis system with acceptable sensitivity and reproducibility. In developing such a system a compromise has to be made between the conditions required for good EOF (for example a pH range between 2 and 9) and conditions required for the diazotisation reaction. The optimisation could be carried out more ef®ciently by measuring both the EOF and absorbance. The method enables a sampling rate of 50 samples/h with a signi®cant reduction in reagent volumes and waste products. Further work needs to be carried out in the more fundamental area of reagent mixing and reaction characteristics to improve the system. Work is also currently being carried out to extend the method to the determination of nitrate by using in situ cadmium reduction of the nitrate to nitrite. Samples of natural waters will also be analysed after ®ltration through a 0.25 mm membrane ®lters. Acknowledgements The authors would like acknowledge the contributions made to this work by other members of the micro-reactor group at Hull, especially, Lorna Nelstrop and George Doku. They would also like to thank the Government of Papua New Guinea for the ®nancial support to Peter Petsul. References [1] S.J. Haswell, Analyst 122 (1997). [2] K. Seiler, Z.H. Fan, K. Fluri, D.J. Harrison, Anal. Chem. 66 (1994) 3485.
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[3] A. Meulemans, F. Delsenne, J. Chromatogr. B 660 (1994) 401. [4] N. Chiem, D.J. Harrison, Anal. Chem. 69 (1997) 373. [5] D.J. Harrison, A. Manz, Z. Fan, H. Ludi, H.M. Widmer, Anal. Chem. 64 (1992) 1926. [6] D.J. Harrison, Z. Fan, K. Seiler, Anal. Chim. Acta 283 (1993) 361. [7] D.J. Harrison, P.G. Glavina, Sensors and Actuators B 10 (1993) 10724. [8] R.N.C. Daykin, S.J. Haswell, Anal. Chim. Acta 313 (1995) 155.
[9] H. Fiehn, S. Howitz, M.T. Pham, T. Vopel, M. Burger, T. Wegner, in: A. van den Berg, P. Bergveld (Eds.), Micro Total Analysis Systems, (1995) 289. [10] A. Daniel, D. Birot, M. Lehaitre, J. Poincin, Anal. Chim. Acta 308 (1995) 413. [11] M.F.M. Tavares, V.L. McGuffin, Anal. Chem. 67 (1995) 3687. [12] Q.H. Wan, J. Phys. Chem. B 101 (1997) 4860.