Flow-through sorptive preconcentration with direct optosensing at solid surfaces for trace-ion analysis

Trends in Analytical Chemistry, Vol. 23, No. 1, 2004

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Flow-through sorptive preconcentration with direct optosensing at solid surfaces for trace-ion analysis Manuel Miro´, Wolfgang Frenzel This article highlights the potential of the solid-phase optosensing scheme as a microanalytical technique for the monitoring of ionic species at the low ng/ml level. We describe the background of solid-phase optosensing, critically comparing the performance of its applications in batch and flowing stream systems. We discuss in detail the current state of the art of flow-through solid-phase extraction (SPE)-based sensors, which are regarded as being a mature technique, and we outline its intrinsic and unique analytical features. We give special emphasis to the criteria to be fulfilled by both bead and membrane sorbent materials if used in a reversible fashion. We also thoroughly address the potentialities and limitations of different optrode cell designs devoted to absorbance/reflectance detection. We present the concept of renewable-bead injection as a powerful scheme that can be exploited with a wide variety of reagent-based assays, including those relying on irreversible reactions. As a practical example to compare the analytical performance of the variety of approaches and cell configurations described in the bulk of the text, we selected on-column monitoring of ultratrace levels of nitrite, which involved the reversed-phase extraction of the derivative compound resulting from the Shinn reaction. # 2003 Published by Elsevier B.V. Keywords: Flowing stream methods; Ionic species; Optosensing; Sorptive preconcentration Manuel Miro´* Department of Chemistry, University of the Balearic Islands, Faculty of Sciences, Carretera de Valldemossa, Km. 7.5, E-07122 Palma de Mallorca, Illes Balears, Spain Wolfgang Frenzel Institut fu¨r Technischen Umweltschutz, Technische Universita¨t Berlin, Str. des 17 Juni 135, D-10623 Berlin, Germany *Corresponding author; E-mail: mmirollado@hotmail. com; [email protected]

1. Introduction Classical spectrophotometric approaches in the liquid phase frequently lack either sensitivity or selectivity for trace-ion analysis. Although selectivity is typically enhanced through the synthesis of novel chromogenic agents, the implementation of instrumental modi¢cations, such as long-path optical cuvettes, does not usually yield the desired sensitivity improvement. Preconcentration has been demonstrated to be a suitable strategy to ful¢l

this goal. Extraction methods depending on solid-liquid interaction have proved to be more practical than those depending on liquid-liquid interaction, being faster in phase separation and not requiring a critical phase ratio, thus allowing higher enrichment factors. Yet, the traditional SPE concept, involving the elution of the retained species to perform the optical detection in the eluate phase, su¡ers from the inherent drawback of partial loss of the preconcentration capabilities gained during the sorption step. There is, therefore, a need for optical sensing at the solid surfaces, involving direct measurement of the light attenuation of the sorbent phase following the sorptive preconcentration of the analyte properly derivatised. Historically, the fundamentals of this technique, termed solid-phase absorptiometry and originally devised for the determination of micro amounts of transition metals in natural waters, can be traced back to the pioneering work of Yoshimura et al. [1]. For the ¢rst time, they established a linear relationship between the absorbance of the sorbent material loaded with the analyte or derivatisation product and the initial concentration of the species in the liquid phase [2]. The noteworthy features asserted by these researchers are the remarkable 100^1000-fold sensitivity enhancement attained with regard to the conventional photometric methods in homogeneous phase with precisions better than 10%, and the feasibility of

0165-9936/$ - see front matter # 2003 Published by Elsevier B.V. doi:10.1016/S0165-9936(04)00107-4

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exploiting optosensing systems under irreversible sorption conditions. Solid-phase photometry has traditionally been associated with the electrostatic immobilization of the target species onto cross-linked copolymeric resins functionalised with sulfonic acid moieties (originally Dowex 50W-X2) or quaternary amino groups (viz. Dowex 1-X2). The unique features of this material are related to the retention of the complexed analyte not merely by primary electrostatic interactions but strong secondary hydrophobic as well as p^p stacking interactions amongst the aromatic rings of the complex and those of the support. Nevertheless, the major limitation of this sorbent resides in its intrinsic aromatic structure, which makes ultraviolet (UV) detection impracticable. Hence, a sort of ion exchanger with aliphatic or heterocyclic matrices, such as Sephadex QAE, or SP dextran-type beads comprising cross-linked polysaccharides partially oxidised and functionalised with quaternary aminoethyl or sulfopropyl groups were proposed as worthwhile alternatives [2,3]. In the recent years, silica-gel sorbents modi¢ed with either aromatic rings or alkyl chains, generally associated with SPE/clean-up protocols prior to chromatographic separation, have been widely described [4,5]. Obviously, optical transparency is an imperative condition for the sorbent material when solid-phase measurements are being handled. Hence, certain reversed-phase polymers used for the preconcentration of non-charged metal chelates in a sorption/elution mode, such as Dowex XAD or Lichrolut EN resins, are not su⁄ciently transparent to be recommended as stationary phases for on-column detection. From the analytical point of view, an e¡ective way to enhance the sensitivity of the measurements is to increase the sample/sorbent volume ratio. Hence, the most exploited procedure involves analyte enrichment from a large sample volume (typically 1 L) onto the minimum resin amount required to ¢ll the optical path of the cuvette [2]. However, the in£uence of thickness of the ion-exchanger layers has been thoroughly investigated to attain the maximum light-path length through the concentrate layer without causing an unduly increase in background attenuance [6]. Besides, positioning the packed material as close as possible to the detector window, and placing a hollow cylinder with a mirrored inner surface between the sample and the detector have proved to be excellent ways to reduce the background level [7].

between the bulk of the sorbent and the analyte properly derivatised. After ful¢lment of the steady-state regime of the heterogeneous batch reaction, which typically lasts 30 min, the sorbent phase is commonly isolated from the solution by ¢ltration or centrifugation. The coloured beads are then transferred to a quartz cuvette as a suspension with a few microlitres of solution. The attenuance readings of the solid layer are usually performed within 15^20 min of ¢lling the cell with the modi¢ed support particles to attain a dense, uniform packing. In order to take into account the light-scattering caused by the sorbent itself and the walls of the cuvette, the preparation of a reference resin should be performed under identical experimental conditions. From the above considerations, it is evident that the original concept of ion-exchange absorptiometry, in spite of its unquestionable improvement of sensitivity with regard to colorimetric reactions in solution, is not really applicable to routine analysis. It is extremely time- and labour-consuming because of the sorbent activation, reaction development, ¢ltration, washing, loading of the cell with particles, detection and, ¢nally, unloading for each determination. Furthermore, it requires skill and expertise to form a homogeneous solid layer in the cell, and the use of large volumes of sample solution together with high amounts of solid material is unfavourable. The incorporation of the solid material as a reactive surface in a £ow cell coupled to photometric detection has opened new avenues in the ¢eld of optical sensing, as demonstrated by the trailblazing papers of Yoshimura’s group in Japan [8], Valca¤rcel’s in Co¤rdoba [9], Ruzicka’s group in Seattle [10] and Capita¤n-Vallvey’s [11] in Granada. In these articles, special attention was paid to the integration of separation, concentration and detection in £ow systems. The basic idea behind this concept is on-the-£y monitoring of processes occurring within the solid-phase interfaced with optical detection. In this respect, there is obvious similarity to chemical sensors, and such integrated analytical methods can be viewed as £ow-through sensor systems [12]. The unique feature of such kinds of £ow-through sensors, mainly implemented in £ow-injection systems but also adapted to sequential injection procedures, is the miniaturisation of solid-phase spectrophotometry with the subsequent reduction of sample and reagent consumption. The improvement of the analytical performance in terms of accuracy, reliability, precision and sample throughput is also worth mentioning.

2. Evolution of solid-phase spectrophotometry 3. Packing bead materials Classical ion-exchanger spectrophotometry can be de¢ned as a non-chromatographic static technique consisting of establishing merely one distribution equilibrium

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In order to select a suitable sorbent for £ow-through sorbent-based spectrophotometric approaches, the

Trends in Analytical Chemistry, Vol. 23, No. 1, 2004 extensive experience of SPE technology [4,5] can serve as a valuable source of information. Although SPE is generally associated with sample clean-up procedures prior to chromatographic separations, the fundamental mechanisms of the sorptive processes involved are identical. Accordingly, the choice of sorbent is mainly guided by the chemical structure of the target molecule to be sorbed, but su⁄ciently good chemical and mechanical stability under dynamic £ow conditions also need to be considered. In contrast with classical batch methodology, the application of permanent reactors in £ow systems involves repetitive cyclic operation, so that total reversibility of the sorption/elution process is an indispensable requirement. As a result, it is obviously inappropriate to have as packing materials sorbents that retain the target compound so strongly that it may only be recovered after destruction of the support matrix. Another relevant criteria to be taken into account in selecting sorbent materials is the extent of swelling and shrinking, which should be negligible when wetted with solutions of di¡erent polarity. Hence, displacement of the active microzone from the illumination area would be prevented. With this in mind, both copolymeric resins and polysaccharide-based organic sorbents with high degree of cross-linking are preferred, with the precaution that the di¡usion rate of the measurand into the bead pores should not to be unduly lower. Examples of applications reported to date involving the aforementioned packing resins can be classi¢ed according to the temporarily immobilized species on the solid-phase: 1. direct detection of the analyte without derivatisation, successfully applied to the determination of ultratrace levels of Cu(II) [13] as well as individual phenolic species [14] or active constituents [15] capitalized on their native UV absorbance; 2. on-line monitoring of the preconcentration behaviour of the product resulting from analyte derivatisation [16], also adapted to multielement, heavy-metal analysis exploiting ¢rstderivative spectrometry [17]; 3. retention of an analyte complex, which is afterwards derivatised on-column by the injection of a non-selective chelating agent [18]; and, 4. immobilization of a chromogenic reagent on the packed column used for on-column reaction and detection of the measurand [9,19]. Nowadays, silica-based sorbents available in widely varying polarity are regarded as practical alternatives to the above materials as a consequence of their high

Trends capacity, homogeneous particle and pore-size distribution and good mechanical stability [4,5,20]. Moreover, modi¢ed silica-gel beads do not exhibit the kinetic limitations of most polymeric resins derived from di¡usional transport of the target species into the pores of the matrix. However, users are advised not to operate them under concentrated acidic or alkaline conditions for a prolonged interval of time. Thus, octadecylchemically modi¢ed silica-gel matrices have been successfully used as a packing material for the optosensing monitoring of metal-PAR chelates [21] as well as heteropolyacid-forming species following on-column reduction to molybdenum blue [20].

4. Design criteria Selecting a suitable £ow-cell con¢guration for the implementation of sorbent-extraction optosensing is not a trivial task. In order to achieve high concentration factors, the target compound should be sorbed preferentially on a small portion of the support material. In contrast to batch applications [1,2,7] where a uniformly covered sorbent material is brought into the cuvette, in £ow systems, the immobilised analyte forms a longitudinally distributed concentration gradient, as shown schematically in Fig. 1. The pro¢le of the concentration gradient along the column is governed by several factors, including the distribution ratio of the compound between aqueous and solid phase, the capacity of the sorbent material, the packing density and homogeneity of the particles, and the geometrical dimensions of the sorbent bed. Under favourable conditions, the target species is retained in a narrow zone at the column head; hence, a sharp pro¢le is attained. With respect to the optical measurements, the ideal situation is when the area illuminated by the incident light beam matches the preconcentration section. As long as this is ensured, light attenuation for a given chemical assay and £ow-cell geometry depends only on the amount of compound retained on the column. A mismatch will cause either deviations from the Lambert-Beer law and reduced dynamic range, when the illuminated area is smaller than the retention zone, or an increase in the background attenuance, when the illuminated area is larger. Besides, the transmitted light beam should be geometrically as similar as possible to the sorption region to avoid sensitivity decreasing through optical dilution of the analyte (i.e., diminution of the e¡ective concentration on the solid-phase detected by the photosensor). It is evident from Fig. 1 that the highest sensitivity for a particular mass loading would theoretically be achieved when the light is focused on an in¢nitely thin sorbent layer on which the sample £ow impinges. http://www.elsevier.com/locate/trac

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Figure 1. Profile of the concentration gradient of the sorbed analyte along the column bed in flow-through configurations.

From theoretical considerations of batch procedures [2], it follows that the sensitivity is also a¡ected by both the volume ratio between sample and sorbent bed and the path-length of the packed £ow-through cell. However, in contrast to conventional spectrophotometry or ion-exchanger absorptiometry, direct proportionality between light absorbance and path length does not occur in £ow applications, because a larger amount of solid material is required for a thicker layer, so the concentration factor is reduced. In addition, increasing the path length to 1^2 cm (without considering multi-re£ection e¡ects) increases light scattering, which, in turn, adversely a¡ects the signal-to-noise ratio and the precision of the transmittance measurements. Furthermore, the performance of the £ow-through sensor deteriorates through the build up of back-pressure. However, path lengths below 1 mm create a large longitudinal distribution of the analyte and cause low breakthrough volumes, so they too are not recommended. Di¡erent types of £ow-through cells, originally designed for liquid-phase measurements, have been employed for solid-phase spectrophotometric measurements. In most papers, standard £ow cells with an optical path of 1 cm, such as the Hellma 178.010-OS/QS (¢rst used by Yoshimura’s group [8]) and the Hellma 138-OS/QS with 1^2 mm path-length (exploited by Valca¤rcel’s group [9,17] and Molina-Diaz’s group [14,15]), were packed with solid materials. In order to avoid excessive back-pressure, the packing material

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does not usually take up the entire volume of the cell cavity, in spite of the possible contribution of the solution within this volume to the absorbance measurements. Very similar con¢gurations have been applied to photoluminescence detection, but they are outside the scope of this review. Readers are referred to the other papers/monographs [22^24] and references therein for both fundamentals and practical applications. The suitability of the Hellma 138-OS £ow-cell for implementation in a sorbent-extraction optical ¢bre con¢guration was investigated [25]. The troublesome packing of the cell to obtain a uniform bed layer was the main drawback. Instead of achieving a plain surface at the sorbent head, a dome-shaped surface was frequently detected because of the cell geometry. As a result, preferential £ow channels appeared at the edges, with the subsequent retention of the coloured species out of the observation ¢eld. Yet, the widespread use of this cell in £ow-through optosensing schemes may be attributed to its ready adaptation to conventional photometers, as opposed to sorbent-¢lled tubular microcolumns. In the latter case, some modi¢cations of the cell holder are required to ensure the proper reactor positioning as well as the use of high-intense ¢bre optic light-sources to account for the high background attenuance [10]. Nevertheless, tubular microcolumns do not su¡er the above-mentioned problems of classical £ow-through cells and are better suited to optrode designs. When light is transmitted radially through the

Trends in Analytical Chemistry, Vol. 23, No. 1, 2004 column and perpendicularly to the £ow direction an additional advantage arises from the fact that Schlieren E¡ect interferences are less pronounced [26]. Taking these considerations into account, a robust, versatile, purpose-made transducer cell was devised to accommodate tubular microcolumns. The unrivalled features of the novel design are the feasibility of accepting cylindrical reactors with inner diameters in the range 0.5^7 mm and the adaptation of the illumination area to the particular column dimension. The assembled device was arranged in a £ow-injection fashion, with the aim of adapting several common spectrophotometric assays of ionic species to sorbent-extraction optosensing at octadecyl-chemically modi¢ed silica-gel sorbents [27]. To minimize the light loss by the wide-angle scattering at the cylindrical walls and at the sorbent bed, a novel £ow-through, prism-shaped, sorbentpacked optrode was designed and applied to nitrite monitoring at the low ng/ml level [25]. The noteworthy features of this con¢guration, schematically shown in Fig. 2, are the collection of the maximum amount of transmitted light together with

Trends the ability to attain a regular bead packing, leading to improvements in sensitivity and signal-to-noise ratio compared to classical geometries. A typical pro¢le obtained in £ow-through solid-phase absorptiometric systems involves four zones, detailed in Fig. 3. The evaluated signal is either the di¡erence between baseline and plateau levels or the slope of the sorption step. The outstanding feature of the latter approach is prevention of transducer saturation by choosing the proper integration time. As observed in this picture, the signal deviation caused by the Schlieren E¡ect at the elution step does not distort the analytical on-column measurements, as opposed to sorption/elution methods relying on post-column, liquid-phase detection [28].

5. Sorption membranes In recent years, there has been increasing interest in the development of extrinsic optical-¢bre membranebased chemical sensors, since they combine the

Figure 2. Flow-injection sorbent-extraction optosensing manifold for the ultratrace determination of nitrite. Front (a) and side (b) views of the prismaticsquare flow-through cell used for spectrophotometric on-column detection are shown in the figure. IV, injection valve; SV, Switching valve; RC, Reaction coil; LED, Light-emitting diode; W, waste.

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Figure 3. Recording of a typical transient readout for sorbent absorptiometric optosensing schemes. The basic operations of the analysis cycle protocol are depicted under the peak signal.

advantages of optical ¢bres with the speci¢city and selectivity of the sensing element [29,30]. The implementation of these sensors in £ow systems has opened new perspectives in the ¢eld of optical sensing through automation of both sample conditioning and sensor regeneration steps as well as ready miniaturisation and ease of handling [12]. Relevant advances in solid-phase di¡use re£ectance monitoring of ionic species were made by Ruzicka and Hansen [31], who exploited the optosensing concept with covalently modi¢ed cellulose pads for pH detection in miniaturised microchannels. However, the most serious drawback of these optrodes is poor performance in trace-ion analysis because sorbentextraction enrichment schemes are not explored. Hence, higher sensitivity could be achieved by applying membrane-based preconcentration techniques, as demonstrated with active phosphorilated cellulose discs applied to the ultratrace determination of transition metals [32]. New immobilization procedures, involving the entrapment of sorbent beads properly modi¢ed within micro-PTFE ¢brils, have been extensively investigated and o¡er unique preconcentration capabilities. Commercially available disc sorbents (e.g., Empore discs) comprising chelating moieties, ion-exchanger resins or reversed-phase materials, were demonstrated to be well suited to in-¢eld screening di¡use re£ectance-based methods [33].

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Since the chemically modi¢ed silica gel or polymeric matrix particles are tightly bound within the inert PTFE support, the extent of swelling and shrinking upon changing the solvent or solution polarity is negligible, while good mechanical stability, uniform packing and chemical inertness are also guaranteed. Hence, the reagent-phase leaching, described as one of the most severe shortcomings of physical entrapped methodologies in optrode membranes [34], is readily solved. Unlike bead-based £ow-through sensors, which require particle sizes larger than 30^40 mm to avoid excessive back-pressure and light attenuation [20], particle diameters in the range 8^12 mm that exhibit a higher capacity for the analyte and allow for more e⁄cient mass transfer kinetics are feasible in £ow-disk-based optosensing systems, without causing an undue increase in the £ow impedance. Moreover, since the analyte is retained on an in¢nitely thin layer, extremely high enrichment factors are to be expected. Nevertheless, two basic requirements should be ful¢lled by the sensing microzone when adapted to di¡use re£ectance measurements: 1. it should be white, opaque and matt, thus, serving as an ideal re£ecting layer wherein mirrorlike re£ectance is minimized; and, 2. its thickness should be larger than the penetration depth of the light beam into the bulk for it

Trends in Analytical Chemistry, Vol. 23, No. 1, 2004 to be used as a di¡use re£ector, although this condition can be circumvented by accommodating a metallised re£ector on the back of the membrane. The performance of di¡erent sandwich-shaped, £owthrough cells, able to integrate separation procedures with on-line detection via perpendicular bifurcated optical ¢bres [35] or angled (45 ) single-strand ¢bres [36], was assessed in terms of versatility, robustness, detection limits, in£uence of refractive index changes and ease of construction/operation. In order to attain the desired sensitivity by analyte enrichment, it is imperative to focus accurately the incident light to the retention zone, collect the maximum percentage of multiple internal re£ected light, and ensure disk compatibility with £ow systems. The recent design of a novel cell well suited for membrane-based preconcentration procedures [37] is of particular interest (see Fig. 4 for further details). The outstanding asset of this con¢guration is the ability to hinder excessive membrane £attening with a resultant slowing of pore clogging. Moreover, fast mass-transfer kinetics is assured as solutions not only cross the membrane but £ow through the bulk of it. The strong dependence of both light-beam intensity and illumina-

Figure 4. Schematic view of the flow-through sandwich-shaped cell for disc-phase solid-phase preconcentration with optical sensing at the active membrane exploiting diffuse reflectance spectroscopy.

Trends tion geometry on the sensor performance has been established by exploiting reversed-phase extraction schemes for long-term nitrite monitoring at the low ng/ml level. Thus, the use of high-intensity re£ection probes together with the membrane arrangement as close as possible to the optical ¢bre and limitation of the sorption to the observation area were proposed as practical measures aiming to both enlarge the dynamic range and improve the signal-to-noise ratio of the determination. Although this type of measuring arrangement has also been called optrode [37], we stress here that, because of its inherent preconcentration capability, its fundamentals di¡er signi¢cantly from typical optosensing systems, which use as a sensing zone a purpose-made ionophore and/or organic indicator-containing porous membrane [38].

6. Bead injection: the new generation The concept of bead injection (BI), classically associated with sequential injection (SI), was initially introduced as a powerful tool for the automation of immunoassays exploiting renewable surfaces in £owing stream systems [39]. The miniaturised, di¡use re£ectancebased chemical sensors capitalizing on this principle, also adaptable to classical spectrophotometric procedures, use minute amounts of beads, on which the reagent is adsorbed and the analyte becomes preconcentrated and monitored via optical ¢bres in jet-ring-con¢gured £ow-through cells [40]. In these specially designed cells, the liquid escapes radially through a circular gap narrower than the diameter of the beads, thus trapping the active sensing entities. After each analytical run, spent sorbent particles are disposed by £ow reversal and the sensor surface is regenerated by injecting a new plug of a fresh bead suspension. A particular bene¢t obtained with this scheme is the signi¢cant reduction of material consumption and at lest a 10-fold decrease of the volume of chemical waste as compared with conventional spectrophotometry. Furthermore, high sensitivity is warranted because of the compensation of both the short optical paths and reaction times with the immobilization of the measurand on a small surface area of the sensor wherein the reaction is driven by an excess of solid reagent. Further advantages in comparison to £ow-through packed-bed reactors that are presented in the sections above derive from its renewable nature, and they include: 1. suitability for implementing reagent-based assays without requiring full reversibility of the sorption/elution process; 2. long-term operation because sorbent compaction and clogging do not take place; and, http://www.elsevier.com/locate/trac

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3. preservation of sorption behaviour because surface contamination and deactivation do not occur. As a result, this concept is a propitious alternative to £ow-through ion-exchanger spectrophotometric procedures using eluent agents with high ionic strength to elute quantitatively strongly retained species, thus reducing drastically the lifetime of the sensing microzone [19]. The inability to desorb the iron(II)-1,10-phenanthroline complex from iminodiacetate-based chelating resins, probably because of the contribution of secondary interactions between the charged chelate and the matrix support, was overcome using the renewable microcolumn scheme [41]. However, two fundamental prerequisites to be ful¢lled for the bead material to be explored in a renewable fashion are the feasibility of forming a stable suspension and reproducible manipulation throughout the automated system. Consequently, we highly recommend ensuring bead-size homogeneity and spherical shape of the reagent-supporting entities in order to prevent compact settlement in the channels of the SI assembly [42]. Hence, conventional, reversedphase, chemically modi¢ed, silica-gel lumps are not really suitable for this purpose as a result of their irregular shape and size distribution. However, poly(styrene-divinylbenzene) beads containing pendant octadecyl groups on the copolymer surface completely satisfy the foregoing criteria, being perfectly spherical and of uniform 35-mm size. Their straightforward use as renewable reagent-carrying sensing particles in SI-BI-jet-ring-cell con¢guration was demonstrated in trace-level monitoring of Cr(VI) exploiting di¡use re£ection spectroscopic procedures [40]. The high transparency and perfectly spherical shape of Sephadex-type ion exchangers as well as particular chelating beads containing iminodiacetate moieties (viz. Chelex-100 from Bio-Rad Laboratories) assured both their applicability as sensing entities and their ease of handling in the £ow network, as described for the spectrophotometric £ow-injection-BI determination and speciation of iron species [43,44]. The implementation of the renewable-bead optosensing concept in £ow-injection manifolds furnished with commercially available £ow-through cells, such as those typically employed to accommodate packed-bed reactors (viz. Hellma 138-OS), has also been reported [43]. A further improvement in the automation of the micro£uidic handling of suspended beads as renewable carriers of reactive groups or immobilized reagents was gained through the introduction of the novel Lab-onValve (LOV) principle [45]. It is based on integrating a set of microchannels within a miniaturised system placed atop a conventional multi-position valve, thus allowing many unit operations to be implemented

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readily. This rigid, compact SI-LOV coupling behaves as a portable, versatile laboratory, which is specially adapted for real-time sorbent-extraction optosensing schemes via in-valve integrated microcolumn reactors equipped with optical ¢bre-detection facilities. The state of the art of this novel micro£ow system, which is able to handle bead particles with high precision, has been reviewed by Wang and Hansen [46]. Readers are referred to this publication, and to illustrations and references therein [46] for details regarding the potential of the SI-BI-LOV scheme as a microminiaturized analytical technique, well suited to on-line sample pre-treatment.

7. Critical comparison In order to compare the variety of strategies described above, we selected the determination of trace concentrations of nitrite in environmental samples exploiting the reversed-phase preconcentration of the azo-dye resulting from Griess-Ilosvay reaction as a practical example. Table 1 includes the analytical ¢gures of merit of reported approaches. A sequential injection sorption/ elution system based upon analyte derivatisation following an iterative methodology of segmentation [47] is also considered, with the aim of comparing traditional in-line enrichment schemes with the integrated £ow-through sorbent-packed optrodes. Although the batch procedure with polyurethane foam [48] features high enrichment factors and lower attenuation levels than Dowex-type resins, the large sample volumes (viz. 100 ml) and equilibration times (viz. more than 30 min for a single measurement) that it requires restrict its applicability to environmental monitoring. The adaptation of sorbent-extraction methodologies to automated systems expedited the operational sequence and considerably reduced manipulation by the analyst. However, sorption/elution schemes su¡er from undesirable analyte post-column dispersion and, often, and the performance is impaired as a result of the pressure drop along the reactor, notably when organic eluents are employed. Hence, the pressure at the £ow-cell outlet has to be slightly increased to avoid bubble formation. Moreover, analytical sensitivity is frequently limited by the pre-elution e¡ect, which causes analyte breakthrough at moderate sample-loading times. To overcome these drawbacks, integrated £ow-through sensors are called for. Particular designs such as prismatic-square £ow-through cells are especially suited to minimising Schlieren E¡ects, decreasing the optical dilution of the analyte and severely reducing the background attenuation [25]. According to the data presented in Table 1 for the optosensing bead-based system and the classical on-line

Methodology [Reference]

Optical detection

Optical pathlength (mm)

Sorbent material

Sample volume (ml)

Linear dynamic range (ng/ml N-NO-2)

Enrichment factor

Detection limit (3s, ng/ml N-NO2)

Noteworthy features

Drawbacks

Static batch technique [48]

Solid-phase absorptiometry

10

Polyurethane foam parallelepipeds

100

1.5–43

140

1.5

High enrichment factors and low background attenuation. Suitable for irreversible reactions

Sorption-elution (SIA) [47]

Eluate absorbance

10

Silicagel-C18 beads (Waters, 55–105mm)

1–10

4–49 (1 ml)

17 (1 ml)

0.1 (10 ml)

Iterative segmentation (mass calibration)

0.25–6 (10 ml)

170 (10 ml)

Time/Labour-consuming. High amount of chemical waste generated Eluate detection (Analyte dispersion, bubble generation and Schlieren effects)

Suitable for environmental monitoring

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Prismatic-microcolumn optrode (FIA) [25]

Solid-phase absorptiometry

1

Silicagel-C18 BondElut beads (Varian, 30 mm)

2.5

0.3–30

93

0.14

Sandwich-cell membranebased optrode (FIA) [37]

Diffuse reflectance

10–20 mm (2penetration depth of light beam)

PTFE extraction discs (Empore 3M) with octadecyl chains or sulfonate moieties

2.5

0.9–12.2

140

0.03

Minimization of Schlieren effects, optical dilution and background level Straightforward configuration and solid-phase renewal. Large reactive surface. Absence of flowresistance

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Table 1. Analytical performance of solid-phase optosensing approaches devoted to the monitoring of ultratrace levels of nitrite exploiting reversed-phase extraction

Packing reproducibility

Extremely thin sensed layer

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enrichment procedure, a comparable limit of detection and similar sensitivity are attained in spite of the lower sampling volume (2.5 ml vs. 10 ml) and shorter path lengths (1 mm vs. 10 mm) used in the £ow-through sorbent-based optrode. The major pitfall of this con¢guration is the inherent hindrance to reproducible packing of the £ow-through cell, which in turn results in deterioration of the applicable dynamic working range. The transfer of the solid-phase absorptiometric assay into the format of di¡use re£ectance spectroscopy on a thin, sorbent embedded PTFE membrane improved the sensitivity of the measurements, despite the short optical path-length of the light beam, because of the high local concentration of sorbed analyte [37]. The design of a novel, open-sandwich, £ow-through cell that can accommodate membranes comprising adsorbent particles of small size ensured large reactive areas without £ow resistance or displacement of the retention zone from the observation ¢eld, e¡ects that are frequently detected in bead-extraction optosensing techniques.

Acknowledgements Manuel Miro¤ expresses his appreciation to the MECyD (Ministerio de Educacio¤n, Cultura y Deporte), Spain, for allocation of a post-doctoral stipend, making it possible for him to engage in research during a stay at the Technical University of Berlin, Germany.

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