Analytical potential of mesofluidic lab-on-a-valve as a ...a versatile front end to...

Trends in Analytical Chemistry, Vol. 30, No. 1, 2011 Trends

Analytical potential of mesofluidiclab-on-a-valve as a front end tocolumn-separation systemsManuel Miro, Hugo M. Oliveira, Marcela A. Segundo

The lab-on-a-valve (LOV) integrated microdevice has recently attracted much attention as a functional mesofluidic platform for

programmable, pressure-driven flow as compared to lab-on-a-chip counterparts. We review the current state of the art of LOV as

a versatile front end to column-separation techniques, namely, liquid chromatography (LC), gas chromatography (GC) and

capillary electrophoresis (CE) for automatic mesofluidic handling at the low-microliter level, in-line sample processing and

introducing the appropriate form of the analyte into the instrument for separation or detection.

The open architecture of the LOV monolith unit has been to date exploited to accommodate micro solid-phase extraction in a

renewable fashion, the so-called bead-injection analysis, encompassing reversed-phase materials and molecularly imprinted

polymers, and in-valve microscale affinity chromatography. A plethora of interfaces have been recently devised for reliable

injection of minute, well-defined volumes of analyte-containing solutions into LC,GC or CE

We illustrate these applications with representative examples in environmental and bioanalytical arenas.

ª 2010 Elsevier Ltd. All rights reserved.

Keywords: Lab-on-a-valve; Flow analysis; Bead-injection analysis; Capillary electrophoresis; Gas chromatography; Liquid chromatography;

Mesofluidic; Micro affinity chromatography; Micro solid-phase extraction

Manuel Miro*

Department of Chemistry,

Faculty of Sciences, University

of the Balearic Islands,

Carretera de Valldemossa km

7.5, E-07122 Palma de

Mallorca, Illes Balears, Spain

Hugo M. Oliveira,

Marcela A. Segundo,

REQUIMTE, Departamento de

Quımica, Faculdade de

Farmacia, Universidade do

Porto, Rua Anıbal Cunha, 164,

4099-030 Porto, Portugal

*Corresponding author.

Tel.: +34 971172746;

Fax: +34 971173426;

E-mail: [email protected]

0165-9936/$ - see front matter ª 2010

1. Introduction

Notwithstanding the fact that chroma-tography has been consolidated as a rou-tine and research tool for assays in a vastnumber of analytical fields, sample prep-aration is still the bottleneck for reliableand unbiased analysis. Multi-step con-ventional sample processing is the mosterror-prone part of the analytical processand accounts for some two-thirds of theoverall time of liquid chromatographic(LC), gas chromatographic (GC) or capil-lary electrophoretic (EC) assays [1].

The continual quest for novel sample-preparation techniques has led to thedevelopment of faster, more cost-effective,miniaturized versions of sample extractionwith green chemical credentials to replaceconventional methods [1,2]. Liquid-phasemicroextraction (LPME) techniques, andvariants thereof [i.e. single-drop LPME,dispersive LPME, solidified floating organicdrop, and hollow-fiber assisted LPME] aregaining ground prior to column-separa-tion systems [3–5] over time-consuming,labor-intensive traditional liquid-liquid

Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2010.08.007

extraction (LLE) with the added advantageof reduced exposure of the analyst toharmful organic solvents. However, thepredominant sample-processing methodsthat have been rapidly growing in recentyears as a consequence of the improvedenrichment factors, and, thus, sensitivityfor chromatographic assays as comparedto LLE and LPME, are solid-phase extrac-tion (SPE) and miniaturized versionsthereof including fibre/needle-based solid-phase microextraction (SPME), in-tubecapillary SPME, stir-bar sorptive extrac-tion (SBSE), and micro-solid phaseextraction (lSPE) in the format of packedmicrocolumns, syringes or pipette tips[1,2,6,7].

Much effort has gone into the develop-ment of not merely miniaturized but alsoautomated sample pre-treatment ap-proaches to simplify the overall analyticalprocess, capitalizing on robotic systems(e.g., Prospekt, Prospekt-2 and Symbiosisfrom Spark Holland or OS-2 from Merckfor lSPE prior to LC) [8] or flow-basedapproaches [9–11]. Automation of sampleprocessing exploiting the first and second

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Trends Trends in Analytical Chemistry, Vol. 30, No. 1, 2011

generation of flow analysis [i.e. flow injection (FI) andsequential injection (SI)], and related techniques (e.g.,multi-commuted or hybrid systems) coupled to chro-matographic techniques [viz., LC, ion chromatography,low- to moderate-pressure monolithic chromatography(so-called SI chromatography), GC or CE)] has attractedmuch attention from researchers over the past few years[12–14]. Coupling FI- or SI-based sample pre-treatmentto chromatographic systems introduced new ways ofimproving the overall efficiency of chromatographicmethods with minimal operator intervention and offer-ing potential incorporation of unit operations at will forchemical derivatization, sample clean up and/or analyteenrichment as integral or hyphenated modules of theflow network prior to separation and detection.

The aim of this manuscript is to discuss the analyticalcapabilities of the third generation of flow analysis [i.e.the lab-on-a-valve (LOV) platform integrated into an SIfluidic system] as a front-end to column-separation sys-tems for solution handling and automated execution ofappropriate pre-treatments of troublesome samples,encompassing matrix isolation, analyte preconcentra-tion and derivatization reactions. The underlying objec-tive is to introduce the analytes optimally intochromatographic and detection systems in line with therequirements of the assays. To date, no overview hasbeen published on the current state of the art of LOV as asample-processing unit prior to liquid and gas column-separation techniques.

In this work, we briefly pinpoint the fundamentalprinciples of the mesofluidic LOV concept, whereuponthe various interfaces designed so far for coupling LOV

Beadsusp

Auto samplerD Vial

at-linecolumn

in-line

µAC

DCapillary

Buffer

Beam in

on-line

Figure 1. Sequential injection lab-on-a-valve (SI-LOV) assembly as couSP, Syringe pump; IV, Injection valve; HC, Holding coil; D, Detector; lAC

154 http://www.elsevier.com/locate/trac

with different degrees of automation to discrete sample-introduction chromatographic/electrophoretic instru-ments (i.e. LC, GC and CE) are described in detail. Thepotential of LOV as an emerging downscaled tool forsimplification of chromatographic assays and imple-mentation of expedient sample-processing procedures isillustrated with representative examples in the environ-mental and bioanalytical fields reported in the literatureover the past decade.

2. Fundamental principles of LOV-based assays

The inception of the third generation of FI originatedfrom the need to downscale reagent-based assays forminimal consumption of samples and consumables [15],but, contrary to lab-on-chip microdevices, with an openarchitecture to accommodate a plethora of unit opera-tions at will within the same mesofluidic platform on thebasis of programmable flow. Readers are referred tocomprehensive critical reviews for in-depth insight intothe fundamentals and distinct features of LOV for mini-aturization and simplification of analytical assays [16–20].

In short, the microfabricated LOV channel system(typically 1.6 mm i.d.) is a single monolithic structuremade of polymethylmethacrylate (Perspex), hard poly-vinylchloride (PVC), polyetheretherketone (PEEK) orpolyetherimide (Ultem), meeting the demands of assaysand mounted atop a multi-position selection valve of anSI network (Fig. 1). Designed to incorporate all necessarylaboratory facilities for a variety of homogeneous and

ension

IV

D

on-line

HC

SP

Carrier

Beam out

column

LOV

pled to column-separation systems using various interface modes., Microafinity chromatography.


heterogeneous-phase chemical assays, hence the namelab-on-a-valve, it was originally conceived [15] to facil-itate solution metering at the low-lL level, mixingsample with reagent(s), incubation of stacked zones bymeans of stopped-flow approaches and sample dilution.However, more recently, it has proved suitable toimplement microsolid-phase extraction (lSPE) proce-dures [21] and gas-liquid separation of evolved volatilespecies within the microfabricated channel system [22].Not least, the LOV platform houses a multipurpose flow-through cell [15,16] for real-time monitoring of thedevelopment of chemical reactions and separation pro-cesses. The integrated microconduit system has been sofar furnished with optical detection facilities, includingUSB charge-coupled devices, laser-induced spectrofluo-rimeters or luminometers and miniaturized atomicfluorescence spectrometers [19], where communicationsto the detector and/or the light source are made viaoptical fibers (see Fig. 1), and where the position of thefibers can tailor the path length of the flow-cell [15]. In-valve electrochemical detection has recently provedfeasible using the flow-through cell to house:� miniature ion-selective electrodes for potentiometric

assays [23];� three-electrode voltammetric units for scanning vol-

tammetry of multiple redox species [24]; or,� anodic/cathodic stripping analysis of trace elements

[25].The microconduit LOV unit is also amenable to oper-

ate with conventionally sized peripheral devices,including a wide variety of optical detection techniquesand modern analytical instruments {e.g., electrothermalatomic absorption spectrometry [16,19,21], inductivelycoupled plasma-atomic emission spectroscopy or massspectrometry [21], X-ray fluorescence spectrometry [26],and electrospray ionization mass spectrometry [27–29]}.However, over the past few years, coupling the meso-fluidic platform to chromatographic or capillary elec-trophoretic systems has increasingly attracted attentionin a variety of areas of research endeavor, and merits acomprehensive report on the role of LOV for acceleratingand miniaturizing bioseparations and sample handlingin multi-residue analysis.

3. Interfacing LOV with column-separationtechniques

A survey of recent literature in the field has revealed thatdiverse designs and configurations have been reportedwhere LOV is exploited as a front end to LC, GC or CE.The mesofluidic platform can be coupled to columnseparation systems in off-line, at-line, on-line or in-linefashions, terms that have been misused in several works.Fig. 1 shows the varied coupling strategies and Table 1sets out representative interface modes in real-worldapplications.

Off-line coupling includes manual injection of thesample pre-processed in SI-LOV into the chromato-graphic/electrophoretic equipment [30]. The beauty ofthis approach is the ease of reconstituting the analyte-containing media after pre-treatment in either the mo-bile phase for reversed-phase LC or an organic volatilesolvent for GC. This is aimed to minimize potential LCband-broadening effects, overlapping peaks, co-elutionwith void volume and peak asymmetry, although off-linecoupling lacks automation.

At-line procedures are performed with a program-mable robotic station or dedicated/commercial auto-sampler [31], and no manual transfer of samples isrequired. The pretreated sample from LOV is delivered toa vial in a discontinuous mode with regard to the sep-aration process [i.e. there is a need to interrupt theseparation procedure during the sampling step (seeFig. 1)]. Previously developed flow-through methods forsample processing are easily automated in at-line LOVformat, as it does not require a dedicated flow interface.

On-line coupling refers to a further degree ofautomation, whereby the LOV and chromatographic/electrophoretic components are connected in a continuous-flow format via a flow interface {e.g., rotary injectionvalves for LC or GC or the internal flow-through cell ofLOV itself for CE (see Fig. 1) [32–34]}. The sample in-jected or processed in LOV is transported to the interface,exploiting programmable flow inherent to SI, fromwhich it is introduced into the separation system bypressure-driven flow in CE [34], mobile phase in LC [32]or gas stream in GC [33]. In contrast to at-line coupling,on-line methods foster repeated injections in CE, whileseparation is still in progress (i.e. there is no need to waitto detect the analytes with longer retention times beforeinjecting the next sample) [13]. In on-line chromato-graphic assays, LOV sample processing might be syn-chronized with separation by LC or GC, whereby twosamples could be concurrently handled in a fully auto-mated mode.

In-line coupling refers to the specific scenario wherethe separation process is accommodated within themicrominiaturized LOV module by incorporating a smallsorptive column {e.g. in-valve microaffinity chromatog-raphy (lAC) for selective uptake, preconcentration and/or separation of biomolecules [27–29,35–37] (see belowfor further details)}. The most significant drawback of in-line coupling, compared to at-line or on-line methods, isits inability to handle samples in parallel because theoverall analytical process encompassing sampling,derivatization, separation and occasionally detection[27,35] is confined in the integrated LOV unit.

4. Automated sample processing

In this section, we provide insight into the unique fea-tures of LOV as a mesofluidic platform for downscaling

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Table 1. Analytical approaches for coupling lab-on-a-valve (LOV) and related platforms to column-separation systems

Coupling mode, chromatographictechnique and detection system

Target analyte Sample matrix Manual sample treatment Automated LOV sampletreatment

Ref.

Off-line LC-ESI-MS-MS Female steroid hormones Urine Enzymatic hydrolysis with b-glucuronidase

Off-valve SPE [30]

At-line CE-UV Linear alkylbenzene sulfonates Treated effluent, estuarine water,wastewater and sea water

pH adjustment andcentrifugation

Off-valve SPE [31]

On-line CE-UV (indirect) Anions (sulfate, chloride, thiosulfate,nitrite, nitrate, citrate, fluoride,phosphate, bicarbonate, acetate)

n.a.a – – [34]

On-line CE-UV-FL and in-valve UV Pancreatic islet proteins Cellular perfusion liquids Dilution in HEPES buffer atpH 7.5

In-line derivatization withfluorogenic reagent(fluorescamine)

[38]

On-line LC-UV NSAIDs Surface water, raw wastewater,treated wastewater and urine

Filtration and pH adjustment In-valve SPE (BI) [32]

On-line LC-UV UV filters Swimming pool and seawater Acidification and filtration In-valve SPE (BI) [40]

On-line LC-UV Chlorotriazine herbicides anddegradation products

Crude soil extract, surface water andgroundwater

Ultrasonic extraction of soil Off-valve SPE (BI) [41]

On-line LC-UV Riboflavin (vitamin B2) Milk, energetic drink, pig liver Acidic/enzymatic digestionof pig liver

In-valve SPE (BI) [42]

In-line lAC-UV and lAC-ESI-MS Biotin-containing conjugates andbeta-galactosidase activity

Lysates from cultured human skinfibroblasts

Incubation with biotin-containing conjugates

Off-valve removal of freebiotin by ion-exchange

[27]

In-line lAC-ESI-MS Binding constants (Kd) between tPex5proteins and synthetic peptides

Five-component mixture of peptideswith variable affinity constants

– In-valve BI [28]

In-line lAC-ESI-MS Phosphomannomutase andphosphomannose isomeraseactivities

Lysates from cultured human skinfibroblasts

In-vitro enzymatic reactionof primary product withyeast transketolase

In-valve BI [29]

In-line lAC-UV Mouse immunoglobulins (IgG) Mixture of BSA and IgG proteins – In-valve BI [35]

In-line lAC-UV and in-line lBIS-UV Human IgG, rabbit IgG and horse IgG n.a.a – In-valve BI [36]

In-line lBIS-FL Biotinylated single-stranded DNA n.a.a – In-valve, on-columnderivatization with fluorogenicreagent (OliGreen)

[37]

n.a., not applicable.aApplied to standard solutions; NSAIDs, Non-steroidal anti-inflammatory drugs; BI, Bead injection; PTV, Programmable temperature vaporizer; ECD, Electron-capture detector; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; lBIS, Micro-bead injection spectroscopy; lAC, Micro-affinity chromatography; FL, Fluorimetry; ESI-MS, Electrospray ionization-mass spectrometry.

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Table 2. Working operational conditions of lab-on-a-valve (LOV)-based solid-phase extraction (SPE) protocols coupled to column separation systems

Target analyte Solid phase (type, size,quantity, packing mode)

Sorbent conditioning Sample loading(volume/flow rate)

Sample matrixremoval

Elution procedure(solvent, volume, flow

rate)

Introduction intochromatographic system

Ref.

Female steroidhormones

C18 Hydra, 30–40 lm,50 mg, non-renewablepacked column

2 mL of MeOH + 1 mLof carriera

10 mL/1.2 mL/min

2 mL ofcarrier + 1 mL of air

150 lL of MeOH at0.3 mL/min

Off-line, with eluatereconstitution

[30]

Linear alkylbenzenesulfonates

C18 Hydra, 30–40 lm,50 mg, non-renewablepacked column

1 mL of MeOH + 1 mLof water

50 mL/3 mL/min

1 mL of air 100 lL of MeOH at3 mL/min

At-line, with partialintroduction of eluatethrough a vial placed inCE carrousel

[31]

NSAIDs Oasis HLB, 30 lm, 4.5 mg,BI renewable column

150 lL of 95% (v/v)MeOH + 150 lL of10 mmol/L HCl

18b mL or 1c mL/3 mL/min

1 mL of10 mmol/L HCl

150d lL of 95% (v/v)MeOH at 0.5 mL/min

On-line, by filling loopof LC injection valve(entire eluate)

[32]

UV filters Oasis HLB, 30 lm, ca. 6 mg,BI renewable column

650 lL of 95% (v/v)MeOH + 1350 lL of10 mmol/L HCl

9 mL/8 mL/min

750 lL of 10 mmol/LHCl

600 lL of 95% (v/v)MeOH at 6 mL/min

On-line, by filling loopof LC injection valve(heart-cut approach)

[40]

Chlorotriazineherbicides andmetabolites

Oasis HLB, 30 lm,2.7 mg + SupelMIPtriazinese, 59 lm, 7.0 mg, BIrenewable column

500 lL of MeOH + 500lL of water

10f mL or 1e mL/1 mL/min

800 lL ofwatere,f + 800 lL oftoluenee

170 lL of MeOH at0.3 mL/min

On-line, by filling loopof LC injection valve(entire eluate)

[41]

Riboflavin (vitamin B2) SupelMIP riboflavin, 58 lm,11.2 mg, BI renewablecolumn

975 lL of 50% (v/v)MeOH + 565 lL ofwater

1 mL/1 mL/min

2 mL of water 312.5 lL of 50% (v/v)MeOH + 1%CH3COOH at 0.5 mL/min

On-line, by filling loopof LC injection valve(heart-cut approach)

[42]

Polychlorinatedbiphenyls

Bond Elut Plexa, 40–55 lm,3 mg, BI renewable column

300 lL of EtAc + 300 lLof MeOH + 300 lL of50% (v/v) MeOH

12g mL/0.5 mL/min

500 lL of air 80h lL of EtAc at 0.3 mL/min

On-line, by transfer ofthe entire air-segmentedeluate to PTV injector

[33]

aWater acidified with phosphoric acid (pH 4.5).bEnvironmental samples.cUrine.dStopped-flow of 10 s per aliquot of 50 lL.eSoil extracts.fWaters.gCorresponding to 6 mL of sample diluted 1:1 in MeOH.hStopped-flow of 15 s per aliquot of 40 lL;BI, Bead injection; PTV, Programmable-temperature vaporizer; EtAc, Ethyl acetate.

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and automating sample processing in homogeneous orheterogeneous phase prior to chromatographic or elec-trophoretic separations. Tables 1–3 survey the mainreal-world applications in the environmental and bio-analytical arenas, including practical operational detailsof LOV-based sample pretreatment, coupling mode to LC,CE and GC and relevant analytical figures of merit.

4.1. Homogeneous phaseSI and LOV should be regarded as advanced platforms forreproducible, expedient sample presentation to column-separation systems and for pressure-assisted CE usingsyringe pumps, as nicely demonstrated by the seminalwork in 2002 [34]. Wu et al. interfaced a conventionalLOV micromachined unit with a dedicated CE systemusing the flow-though cell component of the LOV intowhich the separation silica capillary and cathode wereinserted [34]. The outflow channel of the LOV was fur-nished with an open/close solenoid valve for controlledpulsed-flow hydrodynamic injection, as facilitated bypressurizing the flow chamber with the aid of the syringepump. Conductivity bias in electrokinetic injection forsamples of varied ionic strength is thus entirely cir-

Table 3. Analytical figures of merit of lab-on-a-valve (LOV) mesofluidic m

Target analyte Working range

Female steroid hormones n.g. (0.33–5.4 lg/L)a

Linear alkylbenzene sulfonates 4 ng/L–10 mg/LAnions (sulfate, chloride, thiosulfate,nitrite, nitrate, citrate, fluoride,phosphate, bicarbonate, acetate)

0.5–3.0 mmol/Lb

0.034–3.5 mmol/Lc

0.014–1.4 mmol/Ld

Pancreatic islet proteins 3.43–6.87 lmol/Le

0.39–1.96 pmol/Le

260–781 nmol/Le

NSAIDs 0.4–40 lg/LUV filters 5–160 lg/LChlorotriazine herbicides anddegradation products

0.1–10 lg/L

Riboflavin (vitamin B2) 0.45–5.0 mg/LPolychlorinated biphenyls 2–100 ng/LBeta-galactosidase activity 46.1 (nmol of product)/h/(mg

Binding constants (Kd) between tPex5proteins and synthetic peptides

10�7–10�5 mol/Lg

Phosphomannomutase andphosphomannose isomeraseactivities

2.0 and 2.8 (lmol of productmin/(mg of protein)f

Mouse immunoglobulins (IgG) n.g.Human IgG, rabbit IgG and horse IgG 0.10–1.00 g/LBiotinylated single-stranded DNA 0–0.993 mg/L

n.g., not given.aValues found in samples.bSample introduction by electrokinetic injection for chloride and sulfate acSample introduction by head column field amplification for chloride aniodSample introduction by head column field amplification for sulfate anioneValues for insulin, proinsulin and c-peptide, respectively.fMean value found for real samples.gPeptide in the sample medium.


cumvented. The hydrodynamic injection of a samplezone of low conductivity prior to the application ofvoltage would lead to electrokinetic sample preconcen-tration by head-column field amplification/samplestacking with no need for manual manipulation of thecapillary injection site [34]. In-line activation, flushingand rejuvenation of the capillary and the exchange orrefreshment of the background electrolyte buffer usingthe same instrumentation can be deployed as desired byresorting to computer-controlled operating protocols.Pressure bursts of well-defined volumes of electrolyte todecrease residence times or washing out of the capillaryas soon as the target peaks were recorded fostered high-throughput separations [34,38]. A field-deployable SI-CEinstrument based on similar principles was recently re-ported for unattended monitoring of anions and cationsin creek streams [39].

Dual optical detection attaching an optical fiber CCD-spectrophotometer to the LOV flow-through cell, besidesthe in-line capillary detector for recording of isolatedspecies, has been exploited for real-time observation ofautomatic CE hydrodynamic injection [38]. Demon-strating an assay of anionic surfactants, Ruiz-Jimenez

ethods involving chromatographic/electrophoretic separations

LOD Precision (RSD%) Ref.

0.18–1.8 ng/L <11 [30]1–15 ng/L <5.4 [31]n.g. <14 [34]

n.g. <6.8 [38]

0.02–0.67 lg/L <11 [32]0.45–3.2 lg/L <13 [40]0.02–0.04 lg/L <5.5 [41]

0.05 mg/L <5.5 [42]0.5–6.1 ng/L <9 [33]

of protein) f n.g. n.g. [27]

n.g. n.g. [28]

)/ n.g. <10 [29]

6 mg/L n.g. [35]5 mg/L n.g. [36]11.1 lg/L n.g. [37]

nions.n..

Figure 2. Coupling of lab-on-a-valve (LOV) with capillary electrophoresis (CE) for automated sample derivatization, expedient hydrodynamicinjection into CE and dual optical detection for separation and determination of islet proteins (Reproduced from [38] by permission of The RoyalSociety of Chemistry).


and Luque de Castro [31] were able to quantify twoparameters concurrently, namely, total content andindividual species of linear benzene sulfonates. Not least,preliminary derivatization of analytes for proper sepa-ration and/or detection of optically-inactive species usingclassical SI methodology would be readily surveyed byin-valve optical detection. This was nicely illustrated byWu et al. [38] for in-line fluorogenic labeling of isletproteins and optimization of the derivatization protocolin terms of sample-to-reagent volume ratio and degree ofzone overlap via programmable fluidic control. Fig. 2illustrates the configuration of LOV with dual opticaldetection and in-line sample derivatization in homoge-neous phase as a front end to CE for monitoring of smallproteins.

Coupling LOV with hybrid-flow approaches [e.g.,multi-syringe FI analysis (MSFIA)] opened new avenuesin on-line hyphenation of SPE (see also below for furtherdetails) to reversed-phase LC or GC for appropriate pro-cessing of the eluate in homogeneous phase [32,40–42],or delivery of minute, well-defined volumes of eluate viaair segmentation into large-volume [programmable-temperature vaporizer (PTV)] GC injector [33], respec-tively. Fig. 3 shows the MSFIA-LOV-PTV-GC set-up fordetermination of trace-level concentrations of polychlo-rinated biphenyls in solid-waste leachates. It should benoted that conventional on-line SPE-reversed-phase LCcoupling relies upon column-switching methods [43],

wherein the sorbed species are eluted with the mobilephase itself, rather than with a methanolic/ethanoliceluent of improved elution strength, because of undueband broadening of the most polar compounds wheneverthe latter eluent is used. However, the marriage of MSFIAwith LOV has proved to ensure both optimal conditionsfor retrieval of concentrated species and efficient LC bandfocusing. This has been accomplished by automatic post-SPE dilution of up to 175 lL of the polar organic eluentwith a water stream provided concurrently by one of theliquid drivers of the multi-syringe device (see Fig. 4 for thesystem configuration), thereby rendering a final eluateplug that closely matches the initial chemical composi-tion of LC mobile phase [32,40–42].

4.2. Heterogeneous phaseBesides the successful miniaturization achieved onapplications concerning homogenous chemistry, LOV isparticularly useful for handling heterogeneous, solid-liquid systems as a front end to separation techniques.This has been aimed at implementing lSPE protocols(Table 2 and references therein) or monitoring interac-tions between biomolecules, including on-columndetection [36,37,44].

The coupling of LOV-SPE to LC, GC or CE has beendevoted to assays in complicated matrices, namely thosebelonging to environmental, food or biological samples.As illustrated in Tables 2 and 3, over the past few years,


Figure 3. Lab-on-a-valve (LOV) platform coupled to programmable temperature vaporizer-gas chromatography (PTV-GC) for preconcentration and determination of trace-level concentrations ofpolychlorinated biphenyls in solid-waste leachates. LOV, Lab-on-a-valve; MPV, Multi-position selection valve; IV, Injection valve; MSP, Multi-syringe pump; HC; Holding coil; PTV, Programmabletemperature vaporizer; SV, Solenoid valve; GC, Gas chromatograph; ECD, Electron-capture detector (Reproduced from [33] with permission of the American Chemical Society).

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the marriage of LOV and SPE has gained widespreadacceptance in uptake and preconcentration of a broadspectrum of analytes, including emerging pollutants inenvironmental samples [31–33,40,41] or biologicallyrelevant molecules in urine [30,32] or food [42], prior totheir separation and quantification with CE-UV [31], LC-ESI-MS2 [30], LC-UV [32,40–42] or GC-ECD [33]. Insome instances, a conventional, packed-bed column fil-led with octadecyl chemically-modified silica gel (seeTable 2) has been attached at a peripheral port of themonolithic LOV device. By taking advantage of the easeof handling micro amounts of bead suspension throughthe precisely machined channels of the LOV device, aplethora of tailor-made protocols capitalizing on theso-called bead-injection (BI) approach [45] have beendesigned, comprising column packing, sorbent condition-ing, sample loading and matrix removal, analyte elutionand column removal with sorbent disposal (see Table 2for experimental details). The BI approach offers twomain advantages, not matched by any other automatic,flow-based SPE scheme:� the automatic renewal of sorbent, without any inter-

vention of operator or replacement of devices or phys-ical parts of the system, so as to circumvent theprogressive deactivation and tighter packing of per-manent in-line SPE cartridges; and,

� the accurate metering of sorbent and eluate quantitiesby resorting to bi-directional programmable flow, asprecisely controlled by the syringe pump, along withthe possibility of introducing all eluate or merely afraction of it via heart-cut injection protocols [42] inthe separation technique for further processing.The transfer of the entire volume of eluate into the

separation system is essential to reach low limits ofdetection (LODs), especially when using low-sensitivitydetectors (e.g., UV spectrophotometers coupled to LC).This approach, along with the handling of a well-definedvolume of sample P 10 mL and in-line dilution as de-tailed above, fostered the determination of non-steroidalanti-inflammatory drugs [32] and chlorotriazine herbi-cides and metabolites thereof [41] in environmentalsamples at the low-lg/L level (see Table 3). Low LODswere also attained through the automatic, on-linetransfer of all eluate to the PTV-GC-ECD system, asproposed by Quintana et al. [33] for determination ofpolychlorinated biphenyls in raw solid-waste leachatesat the 2–100 ng/L range.

In other situations, where the enrichment factor isnot an issue, a heart-cut approach was adopted. Forexample, in the determination of riboflavin (vitamin B2)in troublesome foodstuffs [42], commercially-availablemolecularly imprinted polymers (MIPs), tailored forsupramolecular recognition of riboflavin, were utilized ina BI fashion for clean up of crude protein-rich samplesand selective uptake of the target vitamin with no needfor any ancillary sample treatment, followed by elution

with 312.5 lL of 50% (v/v) methanol/water + 1% (v/v)acetic acid. Nonetheless, merely 67 lL at the eluateheading were injected into the liquid chromatograph,providing results in agreement with certified values ofstandard reference materials {i.e. infant milk (NIST1846)} but also pig liver (BCR 487) extract, for whichthe MIP was not originally devised. In this particularwork, the automatic sorbent renewal fostered by BIproved essential for attaining unbiased results, as severecarry-over effects were observed when reusing MIPbeads in successive analysis of raw-milk-based samples.This application, along with the pioneering work ofBoonjob et al. [41], demonstrated that manipulation ofnon-spherical, heterogeneously-sized materials is feasibleinside LOV conduits in a repeatable fashion, despite thestate of the art so far, where the need for suspensionscontaining spherically-shaped particles with a narrowsize distribution has been frequently emphasized [16,21].To this end, a long-lasting stable suspension of triazine-selective MIP chunks in a bead container attached toLOV was achieved by tailoring the composition (densityand viscosity) of the suspending media via modulation ofthe methanol or acetonitrile/water ratio (in this case,60% (v/v) methanol/water was selected) so as to preventbeads floating or settling in the LOV microchannels [41].Also, for riboflavin-selective MIPs, the chunks weredispersed prior to their aspiration into LOV by providinga burst of elution solvent through the particles depositedat the bottom of the container [42]. This promotedformation of an adequate, reproducible dispersionduring the time interval required for bead withdrawal,providing repeatable column packing (RSD = 1.8%,n = 10).

A large number of works exploiting AC in a minia-turized BI-LOV fashion were reported recently [27–29,35–37]. Frontal AC (FAC) is commonly employed toassess the interactions between proteins and analytes bycontinuous infusion of the sample through a columncontaining a covalently-attached protein, and by moni-toring the elution of analytes. The breakthrough timeper analyte is correlated inversely with its bindingstrength. Estimation of binding constants is feasible oncethe column capacity is known.

FAC was successfully miniaturized for determinationof binding constants between synthetic peptides and trun-cated Pex5 proteins from human or from Trypanossomabrucei, demonstrating it to be a useful tool in drugdevelopment against parasitic diseases [28]. The LOVapparatus used in this study featured full automationalong with repetitive reloading of beads with immobi-lized protein at a well-defined column capacity, infusionof a precise sample volume, and continuous delivery ofthe eluted solution into an MS detector.

A similar arrangement was devised to monitor theefficiency of capturing biotin-containing conjugates onstreptavidin-agarose beads and ascertaining the efficiency


Figure 4. Multi-syringe flow-injection lab-on-a-valve (MSFI-LOV) manifold for determination of riboflavin in foodstuff. LOV, Lab-on-a-valve;MSP, Multi-syringe pump; LC, Liquid chromatograph; S, Syringe; Vi Three-way commutation valve; A, Air; CS, Conditioning solvent (50%(v/v) MeOH/H2O); BS, Bead suspension in conditioning solvent; C, Carrier solution (H2O); D, Diluent (H2O); W, Waste; CC, Central channel;EL, Eluent (50% (v/v) MeOH/H2O + 1% (v/v) CH3COOH); B, Channel for discarding beads; Sa, Sample/standard solution; HC, Holding coil;P, Chromatographic pump; IV, Injection valve; MC, Monolithic chromatographic column; D, Diode-array detector (Reproduced from [42] withkind permission from Springer Science+Business Media).


of their release upon perfusion of free biotin [27]. In thiscase, initial measurements were performed off-line, asfractions of eluate were collected and later processed atan ESI-MS detector.

Further studies were devoted to the assessment ofbiotin-containing conjugates elution rates, profiting fromdirect coupling of the ESI-MS detector to the affinitycolumn assembled inside the LOV conduits. Affinitycapture-elution protocols were also automated using thisconfiguration for evaluation of enzyme activities in celllysates [27,29]. A synthetic molecule, combining threeparts, designated ‘‘handle’’, ‘‘linker’’ and ‘‘substrate’’,was converted to a ‘‘handle-linker product’’ by the targetenzyme and both entities were selectively retained onstreptavidin-coated beads if the ‘‘handle’’ was biotin.


After elution, the substrate and product molecules weredetermined by ESI-MS, thereby facilitating concurrentestimation of the activities of several enzymes.

AC has also been utilized as immuno-SPE material forselective uptake of one specific analyte or group ofmolecules by perfusion of sample through the affinitysorbent, where retention of analytes takes place, fol-lowed by a change of the perfusion media (pH, ionicstrength, competitive ligands) with concomitant elutionof retained analytes. This type of protocol has also beenimplemented in the LOV format using protein A-coatedsepharose beads for selective separation of mouseimmunoglobulin (IgG) [35]. In this case, miniaturizationand in-valve integration of optical detection at the outletof the affinity column provided a 60-fold decrease in the


LOD attained (6 mg/L) when compared to a large con-ventional column mounted atop the LOV device andequipped with the same detection system. Miniaturiza-tion also fostered shortening of the assay cycle from30 min to 2 min, while the volume of waste was reducedfrom 30 mL to 2 mL.

Most importantly, LOV has proved itself an excellenttool for implementing protocols that are not amenable toconventional AC equipment. Generally, interactions be-tween bioligands and the stationary phase in AC arefollowed by monitoring of eluted compounds. Ruzickaand co-workers proposed to survey biomimetic interac-tions in a different way by on-column probing after in-valve packing of affinity sorbent and during perfusion ofsample and eluent. This configuration was first describedin one of the works mentioned above [27], wherein theelution kinetics of biotin-containing conjugates was alsofollowed by measuring the decrease in absorbance at330 nm. The affinity column was assembled in-line bycreating a minute column (volume �6 lL), trapped in-side one of the channels of the LOV device, in such a waythat the bead surface was probed by two optical fibersfacing each other to provide light-absorbance measure-ments.

Dual-phase detection was also explored for separation,binding, and elution of IgGs on protein G-coated se-pharose beads [36]. Here, the optical fibers were placedat the outlet of the affinity column to probe elutedanalytes or facing the bead column for on-columninterrogation and monitoring of analyte capture. Thetwo approaches were complementary and essential forbioligand-interaction studies, as improved LODs (byabout 10 times) were attained in column-effluentmeasurements, while the remaining analytes trapped onthe beads could be easily detected by on-column probing.Diagnosing this problem correctly is essential to avoidcarry-over effects or the appearance of dummy peaksupon changes of eluent composition.

The preconcentration and fluorimetric detection ofbiotinylated, single-stranded DNA (b-ssDNA) on strep-tavidin-coated sepharose beads in LOV might be re-garded as an appealing approach to assay DNAelongation [37]. The optical fibers were configured atright angles, the bead column (volume �2.2 lL) wasassembled between them and perfused by b-ssDNAsolution followed by a DNA fluorescence probe (Oli-Green) solution. The signal obtained was proportional tothe mass of DNA retained in the column. In this case,on-column detection was selected over eluent probingbecause it allowed detection of strongly-bound or irre-versibly-bound molecules, as occurs with biotinylatedDNA and streptavidin-derivatized surfaces.

However, on-column LOV probing for absorbancemeasurements has some drawbacks, namely:

� high background signals, due to the intrinsic absor-bance of beads giving rise to reduced signal-to-noise(S/N) ratios;

� limited capacity of the column; and,� minute optical path length, which leads to lower sen-

sitivity, compared to direct measurements of eluate[36].Another source of error is the inhomogeneous distri-

bution of analytes through the sorptive column, with alarger concentration in a narrow zone at the head of thesorbent bed [46,47]. If there is variability in gradientdistribution between standards and real samples and theoptical fibers do not probe the overall effective retentionmicrozone, the quantification of target biomoleculeswould be biased.

5. Conclusions and future trends

The examples presented here have undeniably demon-strated that LOV is increasingly attracting interest as aprogrammable, flow-based, mesofluidic platform for a vastnumber of sample treatments prior to column-separationmethods. The coupling mode (i.e. on-line, in-line,at-line or off-line) reflects the degree of automationattained, which should be offset by the expected cost ofanalysis and the investment required upon purchasingand maintaining the equipment. Besides, we expectseveral unit operations, encompassing membrane-basedsampling and sample preparation [e.g., (micro)dialysis,gas diffusion or pervaporation], gas-liquid separation oreven advanced oxidation processes for analyte derivati-zation or degradation of organic compounds, to gainmomentum from the association between LOV and sep-aration techniques (LC, CE and GC), which leaves roomfor numerous analytical applications and a wide openroad for potential usage in a plethora of areas of researchendeavor. Another promising, yet unexploited, area isthe coupling of short reversed-phase or dynamically-coated, ion-exchange, monolithic columns or discs toLOV devices for either SPE or low-to-moderate-pressurechromatographic separation of a small number of ana-lytes in troublesome matrices following LOV-basedsample processing.

AcknowledgementsManuel Miro and Marcela A. Segundo are grateful tothe Spanish Ministry of Science and Innovation andFundacao para a Ciencia e Tecnologia for supporting theSpanish-Portuguese Integrated Action HP2008-0045and E-48/09, respectively. Manuel Miro extends hisappreciation to the Spanish Ministry of Science andInnovation for financial support through ProjectCTM2010-17214.



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