Membrane-Based Separation Techniques: Dialysis, Gas Diffusion and Pervaporation

CHAPT ER

8 Membrane-Based Separation Techniques: Dialysis, Gas Diffusion and Pervaporation Maria Dolores Luque de Castro

Contents

1. 2. 3. 4. 5. 6.

Introduction The General Membrane-Based Separation Module The Continuous Manifold Detectors Chemical Reactions Involved Dialysis 6.1 Generalities 6.2 Membranes 6.3 The module, the FIA manifold and other units 6.4 Applications of on-line dialysis 7. Microdialysis 7.1 Types of microdialysis probes 7.2 Detectors and microdialysed samples 7.3 Salient improvements in microdialysis 7.4 Calibration in microdialysis 7.5 Coupling MD to high-resolution separation techniques through dynamic approaches 8. Gas Diffusion 8.1 Applications of continuous gas diffusion 9. Analytical Pervaporation 9.1 The pervaporator 9.2 The auxiliary manifold 9.3 Detectors coupled to FIA–pervaporation 9.4 Coupling a pervaporator to a gas chromatograph: an alternative to headspace sampling 9.5 Coupling a pervaporator to capillary electrophoresis equipment Abbreviations References

Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00608-9

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r 2008 Elsevier B.V. All rights reserved.

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1. INTRODUCTION Sample preparation (SP), a crucial step and the bottleneck of most analytical processes, is gaining due recognition as a specialized area in analytical chemistry [1]. An increasingly important consideration when developing SP methods is the ability to automate the entire analytical process. In this respect, flow injection analysis (FIA) is a key tool for SP as it facilitates the implementation of the steps required to obtain the ‘‘analytical sample’’ [2]; that is, the solution to be led to the detector or injected into a high-resolution equipment. Although the latest generations of flow-based approaches possess attractive features for use in this field, their short lives have allowed for only modest development to date. The variety of techniques used to prepare liquid samples (or solids that are previously dissolved in part or in full) are primarily employed for cleanup and/ or preconcentration. Those involving the use of a membrane are the subject matter of this chapter and the next. All membrane-based separation techniques have the advantage that they can be incorporated in dynamic (mainly FIA) manifolds for continuous operation. Table 1 lists such techniques — which differ mainly in the physical state of the membrane (i.e., solid or liquid) — and states the material transferred through the membrane and the driving force of mass transfer through it in each case. Based on the different materials that are transferred and also the different driving forces, existing dialysis modes (viz. conventional or passive, Donnan, electrodialysis, microdialysis) are listed separately in the table. Liquid membranes, which are widely used at present to implement a variety of liquid–liquid extraction techniques or modes, are the subject matter of the next chapter, which follows the discussion of conventional continuous liquid–liquid extraction. Continuous filtration, which can be implemented by using a membrane or filter, but also filterlessly — by exploiting special geometric characteristics of the dynamic system or with the assistance of ultrasound — is discussed also in the next chapter. In most membrane-based separation processes the membrane separates two miscible or immiscible fluid phases that can be static or mobile. Most frequently, the two fluids are liquids, one constituting the liquid sample (often referred to as the ‘‘feed’’ or ‘‘donor solution’’) and the other the ‘‘receiver’’, ‘‘strip’’ or ‘‘acceptor solution’’. The membrane prevents mixing and, very often, direct contact between the two solutions, the latter function being particularly important when the donor and acceptor solutions are miscible. One major feature of solid, porous membranes — those used in the techniques discussed in this chapter — is the so-called molecular weight cut-off (MWCO), which is governed by pore size. The boundaries between membrane techniques, which have been established from particle size, as proposed by Porter [3], provide an indication of the relationship between MWCO and pore size.

2. THE GENERAL MEMBRANE-BASED SEPARATION MODULE Figure 1 shows the two main types of membrane separation modules: sandwich and tubular or hollow-fibre. The former consists of two blocks made of Perspex,

Membrane-Based Separation Techniques: Dialysis, Gas Diffusion and Pervaporation

Table 1

205

Features of continuous membrane-based separation techniques

Technique

Material transferred

Driving force

Material retained

Ions and lowmolecular-weight compounds

Concentration difference

Charged compounds Idem

Ionic strength gradient Electrical field

Microdialysis

Ions and lowmolecular-weight compounds

Concentration difference

Gas diffusion

Gases and vapours

Pressure difference

Analytical pervaporation

Gases and vapours

Pressure difference

Dissolved and suspended material with high molecular mass No-charged compounds No-charged compounds Dissolved and suspended material with high molecular mass Membrane impermeable gases and vapours No gas or vapour compounds

Soluble compounds in the immiscible phase Idem Compounds with reversible change of charge Soluble compounds in the immiscible phase Idem

Partition coefficient

Dialysis Conventional, passive dialysis

Donnan dialysis Electrodialysis

Continuous liquid–liquid extraction Conventional

Without typical units Supported liquid membrane extraction (SLME) Microporous membrane liquid–liquid extraction (MMLLE) Polymeric membrane extraction (PME) or membrane extraction with sorbent interface (MESI) Continuous filtration With filter

Idem Change of partition coefficient Partition coefficient Idem

Solvent and dissolved species

Gravity

With knotted-tube

Idem

Centrifugal

Ultrasound-assisted

Idem

Standing ultrasonic waves

Insoluble compounds in the immiscible phase Idem Compounds with permanent charge Insoluble compounds in the immiscible phase Idem

Suspended material variable particle size cut-offs Suspended, precipitate material Idem

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(1)

Acceptor stream

(A)

To detector Membrane

Waste Donor stream (sample)

(2) (a)

(b)

(c)

Figure 1 Different types of membrane-based units. (A) Sandwich-type (1), with different chamber designs (2): (a) parallelepipedal, (b) winding and (c) spiral. (B) Tubular or hollow-fibre type.

Teflon, aluminium or some other material having identical internally engraved conduits (usually semicircular, triangular or rectangular grooves 0.1–0.5 mm deep and 0.5–2 mm wide) that make up the inner chamber, the geometry of which varies from model to model. The membrane is placed between the two blocks, which must be joined tightly in order to avoid leakage. Each engraved microconduit has two holes on its ends that connect it with the manifold tubing. The tubular module consists of two concentric tubes, the inner one being a porous tube of an appropriate polymer through which the donor stream (the sample) is circulated internally while the acceptor stream is circulated externally or vice versa. The main variables influencing the performance of a membrane-based separation module are as follows: (i) membrane surface, which is a function of the particular membrane material; (ii) membrane path length, which should be as long as possible; (iii) membrane porosity, which is crucial in some techniques; (iv) membrane thickness, which should be a compromise between mass-transfer efficiency and membrane life, and (v) membrane geometry, which should result in a large contact area.

Membrane-Based Separation Techniques: Dialysis, Gas Diffusion and Pervaporation

(B)

207

Waste

Acceptor (or donor) stream

Membrane

To detector

Donor (or acceptor) stream

Figure 1 (Continued).

The best relative position of the donor and acceptor chambers depends on the particular technique. Thus, dialysis is favoured by placing the acceptor chamber below the donor chamber, and the opposite holds for gas diffusion and pervaporation; in liquid–liquid extraction, however, the best position depends on the relative density of the two immiscible phases.

3. THE CONTINUOUS MANIFOLD Coupling a continuous FIA manifold to a membrane-based separation module has opened up a host of prospects for conditioning the donor and/or acceptor solution to optimize mass transfer or implement post-mass transfer reactions in order to prepare the analytical samples for the next step of the overall process. With these aims in mind, injection valves can be used to insert samples (Figure 2A) and switching valves (Figure 2B and C) to select the appropriate

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(A) PP S

IV DS MSM

R1

W1

AS D

R2

(B)

W2

PP

R1

S/DS SV

MSM W1

C IV AS

R2

D

W2

Figure 2 Continuous flow configurations coupled to a membrane-based separation module (MSM) and, optionally, auxiliary reagents (R). (A) Continuous operational mode with injection of the sample into the donor stream. (B) Intermittent operation for preconcentration (the MSM is located in the loop of an injection valve IV). (C) Stopped-flow configuration with intermittent operation of one of the programmable pumps PP for preconcentration. AS, acceptor stream; C, carrier; D, detector; DS, donor stream; S, sample; SV, switching valve; W, waste.

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(C)

PP1

R1

S/DS SV W1 MSM

C

PP2 AS R2

D

W2

Figure 2 (Continued).

stream for each operational stage. The membrane-based separation module is usually placed in the transport–reaction zone (Figure 2A and C) or within the loop of an injection valve (Figure 2B). The sample can be injected into the channel through which the carrier acting as donor solution is circulated (Figure 2A) or, alternatively, can be continuously inserted by aspiration (Figure 2B and C), in which case the inserted volume is controlled through the flow rate and aspiration time. A higher efficiency of mass transfer entails stopping the acceptor stream, while the donor solution is continuously passing through the separation module for an accurately preset time, or stopping both streams if the sample is scant. This approach can be implemented in two ways, the simpler one being the use of a six-way valve as depicted in Figure 2B. The valve loop can be isolated in order to hold a static portion of acceptor liquid while the acceptor stream is driven directly to the detector during the enrichment mass-transfer time. The other choice is based on intermittent pumping, which entails synchronizing sample injection or aspiration with the stop-and-go sequence of pump PP2 to establish the flow of acceptor stream, while pump PP1 is allowed to work uninterruptedly (Figure 2C). In this way, a static microvolume of acceptor stream collects the analyte from a large sample volume over a programmed stop period. For small sample volumes, the donor, or both streams, can be stopped for proper mass transfer. In SIA manifolds, the donor and acceptor solutions are connected to two different ports of the multiposition selection valve and the acceptor line is connected to the detector [4]. The sample is aspirated into the holding coil and delivered to the donor port, where mass transfer to the acceptor solution occurs. Then, the acceptor solution is propelled to the detector. In order to expand the scope of these approaches, one can use more sophisticated manifolds containing

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additional components. For example, the acceptor stream can be driven by a second pump in a hybrid FIA–SIA manifold [4]. More drastic sample pretreatments such as hydrolysis or reagent addition require the use of additional pumps [5], which obviously complicates the experimental setup. No membrane-based separation modules have so far been coupled to lab-on-valve (LOV) devices. The variables most markedly influencing performance in continuous manifolds are as follows: (a) the flow rate, which dictates the membrane-solution contact time; (b) the composition of the donor — buffered, to avoid different behaviour of samples and standards — and acceptor phases, the latter of which should be selected as a function of the nature of the former [6]; (c) the relative direction of the flows (concurrent or countercurrent); (d) the temperature, which has a strong influence on some membrane-based techniques (particularly on those involving vapour formation). Other specific variables affect some techniques only and are discussed in the pertinent sections. Monitoring mass-transfer kinetics is crucial with a view to optimizing membrane-based systems. The main goals here are to identify the influential factors and establish an appropriate transfer time in order to ensure a high throughput without appreciably detracting from sensitivity. The kinetics of mass transfer can be monitored in two ways, namely: (a) by integrating separation and detection in a single module [7], which can be done by placing either a probe-type sensor (whether electroanalytical or optical) inside the acceptor chamber, with its active surface facing the membrane, or interfacing the acceptor chamber with an external detector via fibre optics, with optical detection; (b) by integrating, following separation, a retention step with detection, which can be accomplished by using a flow-cell packed with a suitable material (an ion-exchanger or sorbent) inside a non-destructive detector to quantify the continuous retention of the separated analyte or its reaction product. When mass transfer through the membrane fails to provide adequate concentrations of the target analytes, the process can be forced by increasing the flow rate of the acceptor stream in order to continuously deliver a clean acceptor for increased mass transfer and inserting a preconcentration module (e.g., a sorption column) downstream of the acceptor chamber [8]. When the concentrations of the target analytes in the sample exceed the upper limit of the linear range of the calibration curve, then a dilution or pseudo-dilution step must be used in order to fit the unknown concentration to this portion of the calibration curve and increase the precision of the measurements as a result. A number of alternatives to the usual prior sample dilution are possible and can be based on: (a) using a smaller loop for the injection valve; (b) changing the chemical conditions to a less favourable situation if a derivatization reaction is required, in order to reduce the yield of the monitored species; (c) using a thicker membrane; (d) increasing the flow rate of the donor and/or acceptor; (e) using a lower temperature in the donor chamber (with gas diffusion or pervaporation); and (f) enlarging the air gap between the sample and membrane by inserting an appropriate number of spacers (in pervaporation) [9].

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4. DETECTORS Most types of detectors have been used in combination with membrane-based separation modules, albeit to a different extent depending on both the type of analyte and separation technique involved. Thus, photometers are by far the most frequently used detectors to monitor the intrinsic absorbance of either the separated compounds or the products of their derivatization (for which FIA manifolds can be of great help) [10,11]. However, fluorescence or chemiluminescence-based molecular detection techniques are scarcely used in this context [12]. Atomic spectroscopic absorption and emission detectors are mainly used after liquid–liquid extraction. However, the use of electroanalytical techniques is usually associated with dialysis (particularly potentiometry and, to a lesser extent, conductometry and amperometry) [9,10]. Mass spectrometers are mainly used after microdialysis — the complexity of the compounds calls for such a sophisticated type of detector [13–15] — and also, occasionally, following dialysis or gas diffusion [16,17].

5. CHEMICAL REACTIONS INVOLVED Some membrane-based separation processes require a (bio)chemical reaction to be effective; such a reaction can take place outside the continuous system, in the donor or acceptor stream (or donor or acceptor chamber) or in both. Using a derivatizing reaction prior to separation in dialysis is uncommon, but subjecting the dialysate to some reaction is a frequent step. Derivatizing reactions in the acceptor chamber have a two-fold purpose. First, in taking place at the interface, they facilitate transfer of the analyte by preserving a favourable concentration gradient, thus increasing the rate of the separation process. Second, the derivatizing reaction can also help adapt the transferred analyte to the particular detector used, thereby frequently increasing the selectivity and sensitivity of the continuous method. Gas diffusion and pervaporation frequently require derivatizing reactions prior to separation, usually to generate volatile products from the analyte. Acid– base reactions are by far the most frequently used in this context: an acid carrier is incorporated into the carrier stream to cause the release of gases such as CO2, SO2, HCN, H2S, Cl2 or NO2 from their respective analytes; alternatively, a basic carrier stream induces the formation of ammonia gas from samples containing the ammonium ion, a very common product of enzymatic reactions. Concerning derivatizing reactions in the acceptor chamber, in addition to the two-fold purpose of dialysis, absorption of the transferred gas analyte by the acceptor solution is substantially increased if the analyte has acid–base properties. Thus, gases such as CO2, SO2, HCN and NO2 can be readily dissolved in a basic acceptor, and so can NH3 in an acid solution; then, a variety of analytical indicators can be used to monitor the separation process. However, one can also monitor the pH change caused by the analytes potentiometrically or use more complex derivatizing reactions depending on the analyte–detector combination.

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Another alternative is direct transport of the analyte by a carrier gas to the detector (e.g., with hydride generation or cold mercury vapour generation). In liquid–liquid extraction, reactions prior to or in the donor chamber intended to favour mass transfer, allow the analyte to be converted into a nonionic or ionic product depending on whether it is extracted to an organic or aqueous phase, respectively. When the analyte is in the acceptor, the goals of derivatizing reactions are similar to those in dialysis. Filtration can require a prior reaction either to form the solid species or facilitate its filtering; a subsequent dissolution reaction is usually necessary in direct methods if the analyte is contained in the solid phase.

6. DIALYSIS 6.1 Generalities In conventional, passive dialysis, separation between solutes is based on a concentration gradient between two liquid miscible phases, of the species liable to cross the membrane. Miscibility between the donor and acceptor phases clearly distinguishes dialysis from liquid–liquid extraction, and also from other membrane-based separation techniques such as osmosis or ultrafiltration. In osmosis, it is the solvent rather than the solute which crosses the membrane — it is unclear, though, whether this phenomenon occurs concomitantly with dialysis. In ultrafiltration, occasionally referred to as ‘‘reverse osmosis’’, a solution is forced under pressure across a membrane with concomitant separation of its components. The driving force in this case comes from the pressure difference and it is the solvent, rather than the solute, which crosses the membrane against the concentration gradient. The theoretical principles of dialysis are beyond the scope of this chapter and are described in detail elsewhere [10]. Dialysis can be classified according to the dynamic state in which one or both phases are involved. The process may or may not be allowed to develop until mass-transfer equilibrium is reached: if the two phases are quiescent, the process must develop to completion; however, if one or both phases are in motion — or even if either is stopped over a given interval to increase the efficiency of the process — equilibrium is never reached and strict timing must be employed. In the latter instance, the process can be carried out with agitation of a constant volume of the two phases or by using streams of the two phases which may flow concurrently. This latter alternative is the more commonly used in analytical chemistry (mainly with FIA systems) as it affords separation with precision of 1–2% RSD (i.e., much better than that achieved with air-segmented or non-segmented continuous flow systems) [11]. Dialysis has been implemented in FIA manifolds in its four modes, namely: passive or conventional dialysis, active dialysis in both the Donnan and electrodialysis modes and microdialysis — which is also passive — the first and last being the most widely used. Depending on the way the sample reaches

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the donor compartment (viz. continuously or after splitting into several aliquots), operation can be continuous or pulsed — alternatively, the flow can be halted at the donor compartment. The driving force in dialysis is the analyte concentration gradient across the membrane (i.e., passive dialysis); if an external electric field is applied, electrodialysis occurs. When the analytes are charged species, they can be separated from the sample matrix and preconcentrated in an acceptor solution by Donnan dialysis. In this case, an appropriate ion-exchange membrane (e.g., Nafion) is used to separate the sample from the acceptor solution, of smaller volume and higher ionic strength. Ions of a given charge as a function of the membrane type are transported from the acceptor solution into the sample solution as a result of the existing ionic strength gradient, while co-ions from the sample solution, including analyte ions, diffuse in the opposite direction in order to maintain electroneutrality. Unlike passive dialysis, electrodialysis and Donnan dialysis afford preconcentration of the analyte as well.

6.2 Membranes Dialysis membranes are hydrophilic and mainly of the microporous, homogeneous and ion-exchange types. Microporous membranes are structurally similar to a conventional filter and operate basically on the same principle. Pore sizes are typically 1–10 nm and hence much smaller than those of conventional filters. Typically made of cellophane, cellulose acetate, polycarbonate, polysulfone, polyvinylidene fluoride, copolymers of acrylonitrile and vinyl chloride, polyacetal, polyacrylate, polyelectrolyte complexes, cross-linked polyvinyl alcohols and acrylic copolymers such as Nafion, porous membranes, which are the most widely used, are filled with both the donor and acceptor solutions. Homogeneous membranes are made of homogeneous films with interfaces distributed uniformly throughout. Mass transfer across them occurs via molecular diffusion, dialysis efficiency depending on the solubility and diffusivity of species across the membrane interface. However, homogeneous membranes have scarcely been used in FIA–dialysis approaches. Ion-exchange membranes have a submicroporous structure with no conventional macroscopic pores, but consist of film-forming polymers with positively or negatively charged ions attached to pore walls. An important, yet often ignored parameter in dialysis is the MWCO, which usually ranges from 14,000 to 3,000 (i.e., 0.003–0.01 mm). Ensuring optimal separation between macromolecular matrix components and the target analytes entails choosing a pore size providing the best possible compromise between a high membrane flux of analytes and adequate removal of interfering compounds. The membrane thickness and porosity (i.e., the number of pores per unit membrane area) are often not reported in the scientific literature even though they can have a significant influence on the dialysis efficiency, since obtaining high analyte fluxes obviously requires using thin, highly porous membranes [18].

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6.3 The module, the FIA manifold and other units Usually, dialysers are of the type shown in Figure 1, which were formerly marketed by Technicon and Tecator, and later by Gilson; however, FIA practitioners used to design their own modules for proper fitting to their needs, which led to the development of a number of units with only slight differences between them. Conventional FIA manifolds for coupling to dialysers are as depicted in Figure 2. Some special designs are commented on in Section 6.4. In addition to the indispensable units of an FIA–dialysis system, some approaches require other units to facilitate application of the particular analytical methodology. Such units are also occasionally of separative nature (e.g., a dialysis–liquid chromatograph assembly in which the dialyser is used as a precolumn system [19], an ion-exchanger column intended to increase the sensitivity [20] or a gas diffusion module for the simultaneous or sequential determination of two analytes in the same sample [21] or the insertion of gaseous reagents in addition to liquids into the main flowing stream). The detectors most frequently used in this context are optical in general and photometric in particular. Detection is usually preceded by a derivatizing reaction between the dialysated analyte and a reagent dissolved in the acceptor stream. This increases the efficiency of the separation process by effect of the dialysis equilibrium being gradually shifted as the free analyte is removed from the acceptor stream. Optical techniques such as fluorimetry and atomic absorption spectrometry have also occasionally been used in this context — the scant use of atomic techniques can be a result of organic colloidal or suspended materials in the sample usually not interfering with most determinations. In regards to electrochemical detectors, on-line dialysis is very useful with a view to increasing the selectivity, stability and lifetime of ion-selective and voltammetric electrodes. No FIA–dialysis–GC-coupled systems appear to have been reported; also, FIA–dialysis has been only occasionally used with high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) [20,21].

6.4 Applications of on-line dialysis The earliest applications of FIA–dialysis (passive dialysis) were developed by Ruzicka and Hansen for the determination of inorganic phosphate and chloride in blood serum [22]. Despite their early implementation, applications in this field have been rather scant compared to other separation techniques used in combination with FIA. This may have resulted from dialysis being slow relative to most FIA operations, and also from dialysis efficiencies usually being quite low. Under typical experimental conditions, solute transfer in FIA–dialysis is at best less than 15% — occasionally less than 1%; this can be ascribed to the short time available for solute transfer in FIA. Only in the scantier used Donnan dialysis and electrodialysis can preconcentration or enrichment factors be

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considered; some authors, however, use these terms as synonym for efficiency in passive dialysis. The concept of permselectivity, which refers to the preferential permeation of one molecule through a membrane with respect to diffusing molecules in a mixture, is frequently misunderstood. Permselectivity is defined as the ratio of the final concentration of molecules to their initial concentration. Therefore, the adjective ‘‘semi-permselective’’ is absurd. In addition to the methods for highly concentrated analytes in biological fluids such as blood, urine or milk [10,11], where dialysis avoids the need for dilution and complex deproteination steps, some more recent methods warrant a brief description here. L-(–)-malic and L-(+)-lactic acids in wine have been simultaneously determined as depicted in Figure 3, by coupling dialysis–enzymic derivatization with photometric or fluorimetric detection in order to monitor the reduced form of the coenzyme nicotinamide adenine dinucleotide (NADH). Discrimination between the two analytes was accomplished by splitting the dialysate into two aliquots, a portion of the solution being trapped in the loop of an auxiliary injection valve and each aliquot being led to one of the bioreactors by means of a switching valve. The ensuing method is fast and simple; also it provides results consistent with those of official methods and can be easily used to monitor malolactic fermentation in wines. A study of dialysis selectivity and efficiency as a function of membrane features such as pore size and material was previously carried out by using an FIA–dialysis system on-line connected to a liquid chromatograph [18]. The FIA–dialysis combination was used as early as 1984 for non-determinative purposes such as examining drug–protein (sulphonamide–bovine serum albumin, BSA) binding interactions [23] in a manifold, which included a sandwich-type dialyser. After more than 20 years, similar studies involving carbamazepine–BSA have been carried out by dialysis sampling using a hollowfibre unit (Figure 4A) placed in the loop of the injection valve (which operates as shown in Figure 4B) and coupled on-line with a solid-phase extractor prior to fluorescence detection [24]. The evolution of the FIA–dialysis coupling over the PP

IV2

(1)

Buffer

D IV3 IV1

H2O Sample

R1

SV (2)

R2

W2

M W1 DU

Figure 3 Flow-injection manifolds for the simultaneous determination of malic and lactic acids in wine. D, detector; DU, dialysis unit; IV, injection valve; M, membrane; PP, peristaltic pump; R, enzymatic reactor; SV, selection valve; W, waste. Reprinted from Ref. [18]. Copyright (2001), with permission of Elsevier B.V.

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S (A) PTFE tube

Epoxy resin

C

Microdialysis tubes

(B) W

W

3

3 2

4

3’

C

1

2

4’

2’

5’

1’

4

2’ 3’

1’ 5

D

C

1

8’

DU

4’

5

D

DU

8

7’

6 7

S Sampling

5’

7’

6’

8’

8

6’

6 7

S Injection

Figure 4 (A) Hollow-fibre dialyser and (B) its location in the loop of the sampling valve and operation. C, carrier; D, fluorescence detector; DU, dialysis unit; S, mixed sample solution; W, waste. Reprinted from Ref. [24]. Copyright (2005), with permission of Elsevier B.V.

past 20 years can be envisaged by comparing the two methodologies. In biochemical analysis, it is the preferred choice for determining iron bioavailability by simulating gastrointestinal digestion in a continuous flow–dialysis assembly coupled on-line to an electrothermal atomic absorption spectrometer (ETAAS) and pH-meter as in Figure 5 [25]. A comparison of the tubular dialysers in Figures 4 and 5 reveals that the two operate in the opposite manner as regards to the donor and acceptor streams. Donnan dialysis has scarcely been used in combination with FIA even though when used in 1989 it provided a 100-fold enrichment factor for cations [26]. This was later on improved to values over 200 for an 8-min dialysis time [27]. A more recent application of this approach to the determination of lead in spiked

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Figure 5 Scheme of a continuous manifold with the hollow-fibre dialyser operating in an opposite manner to that in Figure 4A. The pH measurement cell and ETAAS autosampler are also shown. Reprinted from Ref. [25]. Copyright (2005), with permission of Springer-Verlag.

sweeteners provided recoveries greater than 90% [28]. The main reason for such limited application, which contrasts with the high efficiency achieved, is the steep ionic strength gradient required to facilitate the process, which accounts for the fact that Donnan dialysis has only been used prior to optical atomic detection and never with biological fluids [29]. Electrodialysis has also scarcely been coupled to FIA manifolds despite the apparent simplicity of the approach and the efficiency of the process, e.g., more than 37% for chloride [30] and 94% for copper(II) [31]. Possibly, the presence of high concentrations of other species with charge of the same type causes interferences in most cases. Less frequent, but also worth mentioning here, is the use of dialysis for reagent insertion into streams without dilution as proposed by Hwang and Dasgupta [32]. Finally, one completely ignored application of dialysis in continuous approaches is in membrane-protected flow-through electrodes to avoid deterioration and poor performance by eliminating direct contact between macromolecules and the active surface of the sensor [33]. Sequential injection analysis (SIA) has also been coupled to dialysis for purposes similar to those in FIA (i.e., analyte dilution and removal of highmolecular-weight sample components), albeit with substantially reduced sample and reagent consumption. Thus, chloride in milk has been dialysed, then conductometrically determined with an excellent precision (RSDo1%), but using sample and reagent volumes per analysis similar to those required by FIA

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computer detectors S DV

660 nm

1 RE

HC W pump

C

H- SE

Cl- SE

W

D

W

Figure 6 SIA–dialysis approach for the simultaneous determination of pH, chloride and nickel. C, coil; Cl–SE, chloride selective electrode; DU, dialysis unit; DV, stream directional valve; HC, holding coil; H+SE, pH selective electrode; Ref., AgCl/Ag reference electrode. According to Ref. [4]. Copyright (2004), with permission of Elsevier B.V.

(viz. 142 and and 750 mL, respectively) [34]. Simultaneous monitoring of pH, nickel and chloride has also been accomplished with the SIA–dialysis manifold in Figure 6, where, following monitoring of the sample pH, the sample is mixed with a derivatizing reagent to monitor nickel photometrically and finally dialysed, the dialysate being led to a chloride selective electrode for potentiometric monitoring [4]. Like FIA–dialysis [10,11], the SIA–dialysis couple constitutes an excellent tool for monitoring evolving systems. Thus, Lapa et al. developed a method for the simultaneous biosensing of glucose and ethanol in beer fermentation by which portions of the dialysate were driven to bioreactors containing immobilized glucose oxidase and alcohol oxidase, and the resulting H2O2 was quantified amperometrically at 50 samples per hour [35]. In an SIA determination of reducing sugars in wine by the neocuproine/ Cu(II) method, the dialyser served the two-fold purpose of diluting and separating the sugars from high-molecular-weight coloured compounds [36]. No LOV–dialysis combination has so far been reported.

7. MICRODIALYSIS Microdialysis (MD) warrants separate discussion from dialysis on account of its special characteristics and the devices used for implementation. In MD, the dialysate constitutes the sample itself, so the place where the sample has been taken should be the sampled system, which can be a living system or even a large sample portion where the MD probe is inserted for sampling. The fibre is perfused slowly with a sampling solution (the perfusate), the ionic composition and pH of which should be close to those of the system under study.

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The dialysis membrane, which can be microporous (e.g., polytetrafluoroethylene, PTFE, or polypropylene of 0.1–1 mm pore size), but also homogeneous non-porous (polydimethylsiloxane, latex), is permeable to small molecules, but not to macromolecules such as proteins [37]. Compounds which can diffuse through the membrane are swept to either a collection vial or an on-line connected manifold for appropriate treatment or detection. The key features of MD, which are the basis of its wide field of applications, include: (1) the ability to collect samples with minimum disturbance of the sampling site; (2) the improved selectivity by effect of high-molecular-weight species and particulate matter being removed from the sample medium; (3) no need for a purification step prior to analysis — as no cellular matter is diffused, and hence absence of enzymatic breakdown of dialysated substances; (4) the ability to continuously monitor reactions in near-real time; and (5) the ability to calibrate probes and estimate absolute concentrations in extracellular fluids in some cases. This last is a major concern as both in vitro and in vivo calibration methods are frequently unable to mimic the dialysis of species from the sampling site. Although MD is widely believed to be a relatively recent sampling technique, the earliest dialysis probe was developed back in 1972 [38]. Originally, MD was designed for sampling brain tissue; subsequently, however, it has been used to sample other organs and fluids including blood, adipose tissue, muscle tissue, liver, glands or even plant tissues such as those of bananas. Also, MD has been employed in physiological, pharmacological, toxicological and behavioural studies to remove endogenous substances such as neurotransmitters and their metabolites or exogenous substances such as drugs and toxicants with both in vivo [39] and in vitro sampling [40]. MD-based sampling is a powerful tool for studying in vivo pharmacokinetics and drug metabolism, and also for monitoring bioprocesses as it allows the concentrations of target analytes in blood or other tissues to be determined with minor changes in their composition. The main advantages of MD sampling are as follows: (1) sample collection is expeditious — long-time sampling (over several days) from freely moving animals is possible; (2) multiple MD sampling in the same animal allows the distribution of a drug in different pharmacokinetic compartments to be established [38]. A new and promising trend in this field is the use of MD as an in-situ sample-processing technique for environmental research [41]. A very different application of MD sampling is as passive dosimeter for on-site, real-time monitoring of chemical contaminants in pore soil solutions [42]. Below are briefly described the typical MD experimental setup, ancillary equipment; the types of probes, continuous manifolds, and detectors used; major achievements; calibration and the main combinations of MD with other techniques (namely: MD–HPLC and MD–CE).

7.1 Types of microdialysis probes There are four types of MD probes, namely: the stainless steel concentric cannula design, flexible side-by-side cannula design, linear design and flow-through

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(A)

(B)

FT

(C) Extension of FS

DM

DM FS

(D)

PET DM

Figure 7 Microdialysis probes: (A) concentric, (B) flexible, (C) linear and (D) flow-through cannulas. DM, dialysis membrane; FS, fibre skeleton; FT, flexible tubing; PET, polyethylene tubing.

design [43]. The first one is a concentric cannula, also known as ‘‘pin-style, rigid cannula’’ (Figure 7A) consisting of an inner and outer length of stainless steel tubing. The inner cannula extends beyond the outer cannula and is covered with the dialysis membrane. This probe is mechanically stable and permits precise placement in the brain, but is not amenable to implantation in peripheral tissues [44]. The second design, which is used for intravenous implantation and is called the ‘‘pin-style flexible cannula’’ (Figure 7B) is based on a modification of the concentric cannula involving the use of side-by-side pieces of fused silica. In this design, one piece of fused silica extends beyond the other and is covered with the dialysis membrane [44,45]. The third design is the most useful probe for sampling peripheral tissues (Figure 7C). In it, a hollow-fibre dialysis membrane is connected to a small-bore tubing to form an inlet on one end and an outlet on the other. This probe can be implanted simply by threading through the target tissue. The fourth probe (Figure 7D) is

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constructed by inserting an MD fibre into a length of polyethylene tubing [46]. MD probes and membranes are commercially available under different trade names (e.g., Bioanalytical Systems Inc., CMA/MD, Enka Glantzoff, ESA). The continuous manifolds used to interface probes to detectors or highresolution equipment are invariably very simple.

7.2 Detectors and microdialysed samples MD probes have frequently been coupled to electrochemical detectors [47], highly sensitive radioimmunoassay detectors [48] and, especially, to mass spectrometers (MS) and tandem mass spectrometers (MS/MS) [49,50]. Biosensors have enabled direct monitoring of glucose [51–53] or glucose and lactate by using an integrated array consisting of glucose and lactate enzyme biosensors [53]. Glucose and lactate have also been determined with an amperometric enzyme biosensor consisting of an enzyme bilayer oxidase/osmium poly(vinylpiridine) redox polymer, a horseradish peroxidase ring and a split-disk plastic film carbon electrode [54].

7.3 Salient improvements in microdialysis The performance of this membrane-based microtechnique has been improved by the use of cyclodextrins (CDs), an addition to perfusate which has salting-out effect for biological samples and allows low-molecular-mass components to be separated from biopolymers. CDs are also involved in the selective transport of the target analytes in chelated form [55]. Traditional MD shortcomings such as the need for time-consuming calculations in order to compensate for partial recovery of some analytes and depletion near the sampling site have been circumvented by developing a new sampling mode known as ‘‘ultraslow MD’’ [53], in which the flow rate is reduced from 2 mL/min to 100–300 nL/min. One effective alternative to conventional MD for complex systems is dual MD, which requires a more complex dynamic manifold than single MD. A complex biological sample is brought into contact with the first stage of MD in order to remove high-molecular-weight contaminants. The dialysate, which contains only medium-molecular-weight target compounds and low-molecular-weight contaminants, is then connected on-line to the second stage of MD to remove low-molecular-weight contaminants. A clean fraction of the sample, containing medium-molecular-weight target species, is thus obtained that can be directly analysed with an appropriate technique [50]. One other dual MD approach involves two MD membranes sandwiched between three polymer layers. In this case the sample passes sequentially through the first and second MD stage [49]. Continuous subcutaneous glucose monitoring is also possible by coupling an MD probe to a miniaturized thermal flow-through biosensor. To this end,

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the perfusor outlet is directly connected to the sample loop of the FIA miniaturized thermal biosensor [52].

7.4 Calibration in microdialysis Calibration is one of the most important issues in MD. At the perfusion rates typically used in MD, no equilibrium across the dialysis membrane is reached. The concentration of analyte collected in the dialysate is a fraction of the concentration in the sampled system. The efficiency of the microseparation step (viz. relationship between the concentration in the dialysate and that in the sampled system) is dependent on the type of membrane used and its length, the geometry of the probe, the perfusion flow rate, the sample matrix and the physical properties of the analyte [43]. Various approaches based on retrodialysis [56], extrapolation to zero flow rates [57], point of no net flux [58] and slow perfusion rates [45] have been developed to overcome these drawbacks.

7.5 Coupling MD to high-resolution separation techniques through dynamic approaches Reversed-phase or ion-exchange HPLC is the preferred choice for analysing MD samples [59]. Usually, short (r5 cm) microbore (1 mm i.d.) columns have been advocated for the analysis of microdialysates on the grounds of their combined high sensitivity and throughput. Conventional HPLC columns are also convenient to use here as they require no special devices such as low flow-rate pumps, low dead-volume injectors or reduced-volume detection cells. Microdialysates are protein-free and hence amenable to direct injection into a chromatograph or CE system. This enables direct coupling of MD to either techniques via a very simple dynamic manifold [60]. On-line MD–HPLC or MD–CE systems are useful for pharmacological studies. Near-real time information on the concentration of several analytes can be obtained simultaneously, and the separation and detection systems can be optimized for the target analyte. However, MD is a continuous sampling method and HPLC and CE require discrete samples; therefore, the dialysate must be collected over a fixed time interval in order to deliver the required sample volume, and each sample represents an average concentration obtained over the preset interval. As a result, temporal resolution is dependent on the sample requirements of the chromatographic system. Further improving temporal resolution and dialysis efficiency entails analysing submicrolitre volumes on-line. For this reason, CE is suitable for the analysis of MD samples [43]. Three types of on-line interfaces have so far been used to couple MD to HPLC or CE, namely: flow gap [61], flow gated [62] and attachable electrode interfaces [63]. Most of the ensuing applications use FIA configurations to develop the steps required in between MD and HPLC or CE separation; however, no applications involving SIA or LOV have been reported to date, even though these approaches are best suited to the small volumes provided by MD.

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8. GAS DIFFUSION The gas diffusion technique relies on the permeability of microporous membranes to gases. The process is typically kinetically controlled, each gas having its own diffusion constant, which is related to the so-called coefficient of permeability through the solubility constant in Henry’s law [64]. The mechanism of transport across a gas diffusion membrane comprises three consecutive steps, namely: (1) sorption of volatile components at the membrane surface; (2) diffusion of sorbed components through the polymer matrix and (3) evaporation from the polymer into the vapour phase on the permeate side of the membrane. The efficiency of the gas diffusion process is governed mainly by the intrinsic properties of the polymers used for membrane preparation. Glassy polymers (e.g., cellulose) have been shown to be less permeable than rubbery polymers (e.g., polydimethylsiloxane). Also, because the contribution of solubility to permeability prevails in non-glassy polymers, permeability increases with increased molecular mass of permeants. Selectivity depends on the molecular dimensions of the permeating species. Thus, hydrophobic gas diffusion membranes are usually made of PTFE and similar to those used in processes such as ultrafiltration and pervaporation. The gas diffusion module can be of the two types shown in Figure 1; i.e., sandwich or tubular. Similarly to dialysis — they only differ in the type of membrane used — modules have traditionally been supplied by Technicon, Tecator and, more recently, Gilson, and incorporated into both FIA and SIA manifolds. However, some users prefer to design or even make their own modules. Such is the case with the module recently reported by Iida et al., of the tubular type and very small dimensions, in which the ultrathin hollow-fibre — gas-permeable tubing of poly-4-methyl-1-pentene — is 190 mm in i.d. and 250 mm in o.d. [65]. The typical FIA manifolds coupled to gas diffusers are similar to those in Figure 2, and those typically used in SIA systems very much like that in Figure 6. No LOV–gas diffusion approaches have so far been reported. When a gas phase of analyte is formed in LOV, it is removed from the sample matrix by means of a gas–liquid separator [66].

8.1 Applications of continuous gas diffusion The earliest applications of FIA–gas diffusion were in the clinical field and involved the determination of carbon dioxide in plasma by using a straightforward manifold where the biological fluid was injected into a sulfuric acid stream and the diffused analyte accepted by an HCO3 /CO23 buffer containing an acid–base indicator [67]; that of ammonia in whole blood and plasma with direct potentiometric sensing [68] or photometric detection with the aid of an acid– base indicator [69] and the determination of ammonia in undiluted urine [70].

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Enzymatic systems with integrated separation and detection have been employed to determine analytes such as creatinine in undiluted blood serum [71]. The most frequent field of application of FIA–gas diffusion is environmental analysis, where it has enabled the determination of chlorine dioxide in water with chemiluminescence detection [72]; inorganic arsenic species in surface seawater by hydride-generation–ICP [73]; sulfide in water with an S2 -selective electrode [74] and ammonia in water by photometry with an acid–base indicator [75,76], UV-molecular absorption [77] and chemiluminescence [78], to name a few. Recent applications of the FIA–gas diffusion combination include the determinations of the previous analytes in addition to novel methods for determining chlorine dioxide based on the fluorescence quenching of chromotropic acid [79]; creatinine using creatinine deiminase immobilized on chitosan [80] and urea in alcoholic beverages (rice wine) using the above-described microgas diffuser to separate CO2 by urease catalysis and photometric detection using an indicator [65]. The multisyringe FIA mode [81] has been used in combination with gas diffusion to separate sulfide from urban wastewaters containing suspended solids without the need for batch sample treatment. The stagnant acceptor solution was a mixture of N,N-dimethyl-p-phenylene diamine and Fe(III) which, in the presence of the diffused analyte, forms Methylene Blue, the dye being transferred to a miniaturized flow-through light-emitting diode-based fibre optic plug-in spectrophotometer for quantitation [82]. SIA systems have also been coupled to gas diffusers to isolate volatile analytes. One of the earliest methods of this type was that developed for the photometric determination of ammonium in environmental samples using an indicator [83]. More recent is the method for free chlorine involving the formation of a coloured compound with o-dianisine, which allows, with minor changes in the operating conditions, the obtainment of two dynamic ranges, namely: 0.6–4.8 mg ClO /L, which is suitable for determining the analyte in water; and 0.047–0.188 g ClO /L, which is suitable for bleaches. The throughput was 15 and 30 samples per hour, respectively, [84]. The SIA–gas diffusion system of Figure 8A has been proposed for the determination of free and total sulfur dioxide in wines based on the formation of the well-known coloured product of the analyte with formaldehyde and p-rosaniline. An acid solution was added to the sample prior to its passage through the donor chamber of the gas diffuser in order to facilitate SO2 formation. For the free SO2 determination, the sample was directly aspirated into the holding coil; for the total SO2, the sample was processed following in-line hydrolysis of bound SO2 with an alkali solution [85]. A similar method based on FIA–gas diffusion based on the same reaction and in-line hydrolysis of the bound analyte in the manifold of Figure 8B was previously developed [86], that facilitated comparing FIA and SIA coupled to gas diffusion. Both methods were found to provide similar linear ranges for the determination of the two analyte forms, namely: 5–300 and 2–35 mg/mL with FIA and 25–250 and 2–40 mg/mL with SIA for total and free SO2, respectively; the sampling rate was 50 and 20 samples per hour (total and free analyte) for the former method and 16 samples

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(A)

PP1 R3

W2

GDU

HC

R1

D

W3 W4

SV

1

2 W1

PP2

7

3

PP3

MC

8

4

6

X

R2

5 S Y

DC

S

Z

N

W5

(B) R3 RC2 R2

GDU

RC3

W2

D

W3

W1 MC

BR S

IV

C

RC1 R1

PP

Figure 8 Determination of free and bound SO2 in wines. (A) SIA–gas diffusion manifold. D, photometric detector; DC, dilution coil; GDU, gas diffusion unit; HC, holding coil; PP, peristaltic pump; R1, 0.8 M hydrochloric acid; R2, 4 M hydrochloric acid; R3, colour-forming reagent (p-rosaniline+formaldehyde); S, sample; SV, switching valve; W, waste; X, Y, Z, merging points. (B) FIA–gas diffusion manifold. BR, basic reagent; C, carrier; IV, injection valve; MC, mixing chamber; N, basic solution; R1, acid solution; R2, p-aminoazobenzene (chemically similar to p-rosaniline, but having single amino group); R3, formaldehyde; RC, reaction coil (other abbreviations as in Figure 8A). According to Refs. [85,86]. Copyrights (2001) and (1991), with permission of Elsevier B.V. and the American Chemical Society, respectively.

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per hour (both analyte forms) for the SIA method. The reproducibility, expressed as RSD, was 0.4% and 1.2% with FIA and SIA, respectively. Finally, the higher sophistication and automation in SIA affords lower reagent consumption.

9. ANALYTICAL PERVAPORATION Analytical pervaporation can be defined as a combination of continuous evaporation and gas diffusion through a gas permeable membrane. Both processes take place in a single step and in the same unit. The volatile analyte (or its volatile reaction product) present in a heated donor phase evaporates through a porous membrane and is collected in an acceptor stream for appropriate detection. An air gap is used between the sample in the donor chamber and the membrane to avoid clogging of the membrane pores when processing dirty samples and also to permit the presence in the donor chamber of species such as high-molecular-weight components, acids, bases, organic solvents, etc., which could damage the membrane on contact with it [87–92]. This is the most salient advantage of analytical pervaporation over its industrial counterpart and other membrane-based non-chromatographic techniques such as dialysis and gas diffusion.

9.1 The pervaporator An analytical pervaporation module differs from the general devices in Figure 1A in that the donor chamber is larger in order to contain both the sample and headspace in contact with the membrane. Basically, it consists of the following parts (Figure 9A): (a) an upper acceptor chamber fitted with inlet and outlet orifices through which the acceptor stream (liquid or gas) is circulated and in which the pervaporated analyte (or its volatile reaction product) is collected; (b) a thin membrane support; (c) spacers of varying thickness, if necessary, to increase the volume of the corresponding chambers; and (d) a donor chamber (lower part of the unit) for circulation of the feed stream [87–92]. The spiral rather than round shape of the acceptor chamber allows its volume to be reduced from 1 mL to 150 mL (Figure 9B). The conventional pervaporation unit is usually made of methacrylate [92], which is a transparent material facilitating continuous checking of the performance of the device. The different parts of the pervaporator are aligned by inserting metal rods in the drilled orifices — see (f), (g) and (i) in Figure 9A— and closer contact is achieved by screwing them together with four screws between two metallic supports — (e) in Figure 9A. Typical connectors, (h), are used to integrate a pervaporator in an FIA manifold. Integrating pervaporation with detection requires altering the acceptor chamber in order to place a probe sensor.

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(A) (e)

(a) (i)

(g)

(f)

(h)) (b)

(c) (i) (d)

(e)

(B)

Figure 9 (A) Components of a pervaporator (for details, see text). (B) Schematic of the spiral acceptor chamber.

The efficiency with which several components in a liquid mixture can be separated is a function not only of differences in vapour pressure, but in their permeation rate through the membrane.

9.2 The auxiliary manifold Analytical pervaporation can be carried out in a continuous way if the sample reaches the pervaporator via a continuous manifold or in a discrete mode if the sample is introduced by injection (with the aid of a valve or a syringe).

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The former choice option allows simplification and miniaturization in the preliminary operations and results in markedly improved analytical quality and productivity. After separation, the pervaporate is always either subjected to other steps preceding detection or driving to the detector via a continuous FIA manifold in both cases [93]. The simplest way of making pervaporation to work as a continuous separation technique — and the only way used so far — is by inserting a pervaporator into an FIA system. With liquid samples, coupling via a dynamic approach is essential to drive the sample either by injection or aspiration to the donor chamber. In addition, (bio)chemical and/or physical steps (namely, reactions converting the analyte into the most suitable form for evaporation, physical dispersion, etc.) can also be performed in the manifold, before the sample reaches the donor chamber; a detector can be placed behind the pervaporator in order to monitor non-volatile species [94,95]. With solid samples, the donor chamber operates as a multifunctional device in which the sample is weighed and, following adjustment to other parts of the pervaporator, is subjected to leaching, masking and (bio)chemical derivatization [96–104]. The acceptor chamber also requires in-line coupling to a dynamic manifold for driving the pervaporated species to the detector, a preconcentration unit or a high-resolution system. Even when the detector is integrated in this chamber [105], coupling with a continuous manifold is desirable in order to flush the chamber between samples. Derivatizing reactions, preconcentration steps, etc., also require the continuous manifold–upper chamber arrangement [106–108]. Discrimination between species can thus be readily achieved [89,94,109–113]. Placing the upper chamber in the loop of a conventional low-pressure injection valve as in Figure 2B allows the acceptor fluid to remain still during pervaporation if the valve is kept in its loading position. After a preset time long enough to ensure adequate enrichment of the acceptor fluid with pervaporated analyte, the valve is switched to its inject position and the stream drives the loop content to the detector. When the detector is integrated with the pervaporator through a probe [105] placed at the top of the acceptor chamber, facing the membrane, the valve allows monitoring mass transfer through the membrane (i.e., the kinetics of the pervaporation process). In addition to typical variables of continuous membrane-based separation techniques, pervaporation is influenced by the following factors: (a) Sample agitation. Magnetic stirring facilitates the removal of gases from the donor stream, and so does ultrasound, even more strongly — however, it is not recommended because it can cause leakage and losses of the gas phase as a result. (b) The presence of chemically inert beads of appropriate size in the donor chamber boosts the transfer of volatile species into the air gap with both liquid and solid samples because the residence time, in the former instance is increased by effect of the winding path the liquid has to go through and the beads separate the particles of solids, thus providing an increased surface area to remove the gas from the sample to the air gap. (c) Volume of the air gap. The smaller the gap is, the smaller the amount of analyte required to go through the membrane will be.

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With liquid samples, the best way to decrease the volume of the air gap is by lengthening the waste line in order to increase overpressure in the donor chamber, which raises the solution level.

9.3 Detectors coupled to FIA–pervaporation The best way of monitoring pervaporated species is by inserting a probe-type sensor in the acceptor chamber with its active side facing the membrane. The most salient advantages of integrating pervaporation and detection are as follows: (a)

(b)

(c)

The response time is shortened with respect to the typical use of the sensor behind the separation module as there is no need to transport the analytes to the detector. The kinetics of mass transfer across the membrane can be monitored to obtain a better understanding of the pervaporation process and facilitate optimization. The system can be dramatically miniaturized as leaching, derivatization, separation and detection can be done in a single module for solid samples.

Pervaporators are amenable to coupling to any type of detector via an appropriate interface such as a transport tube, a microcolumn packed with adsorptive or ion-exchange material, etc. The acceptor stream can be either liquid or gaseous depending on the characteristics of the particular detector. The detectors most frequently used here are of the spectroscopic (atomic or molecular), electroanalytical (potentiometric, voltammetric), electron capture and flame ionization types. The low selectivity of some of them is offset by the high selectivity of the pervaporation step, which makes the overall analytical process selective enough for analyses in complex matrices. However, the potential of pervaporation for sample insertion into water-unfriendly detectors such as mass spectrometers or devices such as those based on microwave-induced plasma remains unexplored.

9.4 Coupling a pervaporator to a gas chromatograph: an alternative to headspace sampling Coupling a pervaporator to a gas chromatograph is one of the most promising uses of pervaporation and it warrants a more detailed discussion based on the advantages of pervaporation over both static and dynamic headspace sampling. In the static approach, the sample is placed in a closed chamber and heated until the volatile compounds in the headspace reach equilibrium with the sample. Then, a portion of the vapour phase is injected into the chromatograph for analysis. The dynamic headspace or purge-and-trap mode requires continuous removal of the gas phase from the chamber. Because separating the volatile analytes from the sample is a slow process, an intermediate trap is needed in order to concentrate the analytes before introduction into the chromatograph.

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Pervaporation provides a number of advantages over headspace techniques that can be summarized as follows: (a)

(b) (c)

(d) (e)

The thin air gap above the sample requires very small amounts of the analytes to establish equilibrium with the sample and mass transfer across the membrane. Continuous removal of volatilized analytes displaces the equilibrium and increases the separation efficiency. Continuous removal of the pervaporated analytes to a preconcentration column, if used, allows fresh portions of acceptor gas to come into contact with the diffused species, thus displacing the mass transfer equilibrium. The separation step can be totally or partially automated (with liquid and solid samples, respectively) with minimal purchase and maintenance costs. Unlike the purge-and-trap mode, no water vapour condenser is required; nor is a hydrophobic sorbent as no water crosses through the hydrophobic membrane at the usual working temperatures.

9.5 Coupling a pervaporator to capillary electrophoresis equipment Recently, pervaporation was coupled on-line to CE for the individual separation of volatile analytes or products in the pervaporate. This combination has been used with liquid samples (wines) to determine volatile acidity and sulfur dioxide [114]; solid foods (fish, meat and sausage) to determine biogenic amines [115]; and slurry samples (yoghurt, juice and yoghurt–juice mixtures) [116] to determine four aldehydes plus acetone. The FIA manifolds used varied depending on the physical state of the sample. An FIA–CE interface designed by the authors was employed. Figure 10 shows the experimental setup required for each method and a detail of the FIA–CE interface, which consists of a methacrylate vial with an internal volume lower than that of commercial vials (0.45 mL vs. 3.5 mL) and two orifices to which the ends of PTFE tubes were glued. The upper orifice is the inlet and the orifice at the bottom of the vial – the outlet; the latter is used to both unload the liquid in the vial after injection into the capillary and to flush the vial between successive loads of pervaporate. As can be seen in Figure 10, the acceptor chamber is placed in the loop of an injection valve in order to facilitate halting the acceptor stream in order to enrich the stagnant solution as required — usually a compromise between sensitivity and sample throughput must be made in this respect. A switching valve preceding the injection valve allows a water stream to flush the upper dynamic manifold, including the interface, from which the stream circulating through it is aspirated for more reproducible performance. The FIA manifold used as an auxiliary of the donor chamber does not differ for liquid or slurry samples; however, it is not required for solids as these are weighted in the donor chamber and the reagents, if required, are injected through appropriate septa. Pervaporation invariably results in preconcentration and cleanup. The former is a function of the volatility and vapour pressure of the target analyte or reaction

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SR

PP

IV WS A OCV

SV

PU M

S R WB

MST

CE

W

Figure 10 Scheme of the pervaporator–CE coupling for solid samples (solid lines only), slurries (with additional dotted lines), liquid samples (solid, dotted and dashed lines). A, acceptor; CE, capillary electrophoresis equipment; IV, injection valve; M, pervaporation membrane; MST, magnetic stirrer; OCV, open–closed valve; PP, peristaltic pump; PU, pervaporation unit; R, reagent; S, sample; SR, syringe; SV, switching valve; W, waste; WB, water bath.

product, and the latter is a consequence of the removal of analytes from the sample matrix. No pervaporator appears to have been coupled to an SIA or LOV system to date.

ABBREVIATIONS BSA CD CE ETAAS FIA GC HPLC ICP LOV MD MS MWCO NADH SIA SP

Bovine serum albumin Cyclodextrin Capillary electrophoresis Electrothermal atomic absorption spectrophotometer Flow injection analysis Gas chromatography High-performance liquid chromatography Inductively coupled plasma Laboratory on valve, lab-on-valve Microdialysis Mass spectrometry Molecular weight cut-off Reduced form of nicotinamide adenine dinucleotide Sequential injection analysis Sample preparation

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