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International Journal of Environmental AnalyticalChemistry

ISSN: 0306-7319 (Print) 1029-0397 (Online) Journal homepage: http://www.tandfonline.com/loi/geac20

A novel microfluidic device for fast extraction ofpolycyclic aromatic hydrocarbons (PAHs) fromenvironmental waters – comparison with stir-barsorptive extraction (SBSE)

Louise Foan, Florence Ricoul & Séverine Vignoud

To cite this article: Louise Foan, Florence Ricoul & Séverine Vignoud (2015) A novel microfluidicdevice for fast extraction of polycyclic aromatic hydrocarbons (PAHs) from environmentalwaters – comparison with stir-bar sorptive extraction (SBSE), International Journal ofEnvironmental Analytical Chemistry, 95:13, 1171-1185, DOI: 10.1080/03067319.2014.994617

To link to this article: http://dx.doi.org/10.1080/03067319.2014.994617

Published online: 04 Feb 2015.

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A novel microfluidic device for fast extraction of polycyclic aromatichydrocarbons (PAHs) from environmental waters – comparison with

stir-bar sorptive extraction (SBSE)

Louise Foan*, Florence Ricoul and Séverine Vignoud

CEA, LETI, Département des micro-Technologies pour la Biologie et la Santé, 17 rue des Martyrs, 38054,Grenoble Cedex 9, France

(Received 18 July 2014; final version accepted 13 November 2014)

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants renowned for theirubiquity in the environment and potential carcinogenicity. To verify compliance to theEuropean Drinking Water Directive (98/83/EC) and Water Framework Directive (2000/60/EC), these compounds have to be monitored in environmental waters. Conventional labora-tory analysis implies important costs and labour, and may lead to low sample representative-ness due to sampling, transport and storage prior to analysis. To date, no portable equipmentenables in situ PAH determination with adequate selectivity and sensitivity. To build a cost-effective and high-performance portable system, a microfluidic device for fast extraction ofPAHs with low volume samples has been developed and tested by solvent desorption. Pre-concentration recoveries were evaluated by high-performance liquid chromatography asso-ciated with multi-wavelength fluorescence detection (HPLC-FLD). The operational para-meters were optimised with 5 µg L−1 spiked solutions. The pre-concentration of 10 mLsolutions with the microfluidic device led to equivalent extraction recoveries to stir-barsorptive extraction (SBSE) for the high molecular weight PAHs (≥4 aromatic rings) inapproximately 50 times less time. The performance of the microchip has to be improvedfor the lightest PAHs (2–3 aromatic rings) by testing larger microchips and by using a morepolar material than polydimethylsiloxane. Influence of matrix effects was also investigated bytesting the device with artificial surface waters (mineral water supplemented with humicacids) and real samples (filtered lake water).

Keywords: PAH; environmental water; microfluidic device; extraction; solvent desorption;method development; matrix effects; SBSE

1. Introduction

Polycyclic aromatic hydrocarbons (PAHs) are products of thermal decomposition, formedduring incomplete combustion of organic materials and geochemical formation of fossil fuels.The main anthropogenic sources are power plants, domestic heating, waste incineration, indus-trial processes and, most importantly, motor vehicle exhaust emissions [1–3]. PAHs are con-sidered to be persistent organic pollutants (POPs) due to their slow rates of degradation, toxicityand potential for both long-range transport and bioaccumulation in living organisms [4].Carcinogenic, mutagenic and immunotoxic effects of PAHs, detrimental to human health,have frequently been reported [1–5]. These compounds are particularly monitored in environ-mental waters to check their compliance with the environmental quality standards (EQS),

*Corresponding author. Email: louise.foan@cea.frThis paper was presented at the ISEAC-38 Symposium held in Lausanne, Switzerland (June 17-20, 2014).

Intern. J. Environ. Anal. Chem., 2015Vol. 95, No. 13, 1171–1185, http://dx.doi.org/10.1080/03067319.2014.994617

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defined for eight PAHs in surface waters (continental, transitional and coastal) by the EuropeanWater Framework Directive (2000/60/EC, 2008/105/EC) [6,7]. Moreover, water intended forhuman consumption has to meet the minimum requirements laid down for five PAHs in theEuropean Drinking Water Directive (1998/83/EC) [8].

PAHs in environmental waters are monitored by laboratory analyses which imply importantcosts and labour, and require sampling, transport and storage steps which can induce biases onthe final results due to analyte loss or sample contamination [9]. Yet no portable solution assuresselective measures of all PAHs at sub-µg L−1 levels. The most compact commercial systems usefluorescence detection to monitor accidental occurrence of hydrocarbons in waters (commercia-lised by AQUAMS©, CONTROS© and HACH©). The drawback of theses probes is that theymeasure a global fluorescence level of all hydrocarbons (non-selective techniques). On the otherside, a portable GC-MS equipped with a solid-phase micro-extraction (SPME) system devel-oped by INFICON© assures good selectivity, but does not enable sensitive measurements ofPAHs of heavy molecular masses (limitation of the SPME pre-concentration). During the lastdecade, several research teams have developed microfluidic devices for pre-concentration oforganic pollutants contained in water, using both solid-phase extraction [10–12] and liquid–liquid extraction [13]. However, the solid-phase extraction devices presented insufficient sensi-tivity for PAHs [10–12]. For example, Lafleur et al. [11] developed a centrifugal microfluidicplatform with a 143 µg L−1 detection limit for anthracene. As for the liquid–liquid extractiontechnique, it was only tested with compounds of low polarities (KOW < 5) and with concentra-tions non-representative of environmental levels [13].

In this context, the following study’s objective is to test an off-line miniaturised pre-concentration device that could be integrated in the future in a portable device for monitoringPAHs directly in environmental waters. Our work was carried out with a lab-on-a-chip, con-sisting of a silicon/glass microfluidic device functionalised with an adequate polymer for PAHextraction and concentration.

Polydimethylsiloxane (PDMS) was chosen for functionalising the device, as it is acommonly used polymer for PAH extraction [14,15]. Indeed, PDMS has a high affinityfor apolar compounds, with octanol/water partition coefficients higher than 10,000 (logKow > 4), which is the case of all PAHs except naphthalene and acenaphthene, the lightestcompounds [16]. PDMS has been used in many sorptive micro-extraction techniques such asfiber-packed needles [17], silicone membrane equilibrators [18] and thin film micro-extrac-tion devices [19]. In particular, stir-bar sorptive extraction (SBSE), commercially knownwith the trademark Twister® and based on extraction by sorption of dissolved compoundsfrom the aqueous phase by a magnetic bar covered with PDMS, is a performing extractiontechnique for PAH analysis at trace levels in environmental waters [16,20–36]. Analytedesorption can be performed with solvents [20,25–27,30,34,36]. Liquid desorption (LD) isgenerally followed by high-performance liquid chromatography associated with fluorescencedetection (HPLC-FLD) [20,25,30,36].

The following article presents the performances, in terms of recoveries and duration,obtained with a PDMS functionalised microfluidic device during the pre-concentrationprocedure, consisting of a sample extraction step followed by a solvent desorption stepwith a syringe pump. Analyses were performed by HPLC-FLD. First, operational parameterswere optimised with ultra-pure water spiked with 5 µg L−1 of 15 PAHs. As generallyperformed in the literature (e.g., [30]), desorption step was optimised before the extractionstep. Then, the optimal pre-concentration procedure with the lab-on-a-chip was comparedwith SBSE-LD-HPLC-FLD. Finally, to evaluate matrix influences, tests were carried outwith filtered artificial water (mineral water spiked with humic acids) and filtered naturalwater (lake water sample).

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2. Experimental

2.1 Chemicals, standards and material

Analytical grade solvents were used for all experimental steps: acetone (CHROMASOLV® Plus, forHPLC, ≥99.9%), acetonitrile (CHROMASOLV® gradient grade, for HPLC, ≥99.9%) and methanol(puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99.8% (GC)) were provided by SupelcoAnalytical (Sigma-Aldrich, St. Louis, MO, USA); dichloromethane (RPE, ACS for analysis, reag.Ph. Eur., reag., ≥99.9%) and n-pentane (RPE for analysis, reag. Ph. Eur., Reag. USP., ≥99%) wereobtained from Carlo Erba (Rodano, Italy) and Milli-Q water by Millipore (Billerica, MA, USA).

For microchip functionalisation, nonpolar dimethyl polysiloxane and dicumyl peroxide werepurchased from Sigma-Aldrich (St. Louis, MO, USA).

A standard mix (polycyclic aromatic hydrocarbons 16 solution, Supelco Analytical, Sigma-Aldrich, St. Louis, MO, USA), containing naphthalene (NAP), acenaphtylene (ACL), ace-naphthene (ACE), fluorene (FLR), phenanthrene (PHE), anthracene (ANT), fluoranthene(FTN), pyrene (PYR), benz(a)anthracene (B(a)A), chrysene (CHR), benzo(b)fluoranthene(B(b)F), benzo(k)fluoranthene (B(k)F), benzo(a)pyrene (B(a)P), dibenz(a,h)anthracene (D(ah)A), benzo(g,h,i)perylene (B(ghi)P) and indeno(1,2,3-cd)perylene (IND) at 10 µg mL−1 inacetonitrile, was used for calibration and for spiking the test solutions.

For SBSE extractions, Twisters® covered with 24 µL of PDMS (length: 10 mm, PDMSthickness: 0.5 mm) were purchased from Gerstel (Mülheim an der Ruhr, Germany).

Humic acid sodium salt (Supelco Analytical, Sigma-Aldrich, St. Louis, MO, USA) was usedto prepare artificial water containing dissolved organic matter, in order to evaluate matrix effectson the performance of the microfluidic device.

Syringe filters with 0.2-µm polyethersulfone membranes (PALL, Port Washington, NY,USA) were used for filtering the artificial and natural environmental water samples.

2.2 Fabrication and PDMS coating of the microfluidic device

Microfluidic silicon/glass chips of 4.8 mm × 16.8 mm (Figure 1(a)) were fabricated usingstandard micro-technology techniques: photolithography, DRIE (Deep Reactive Ion Etching),

Figure 1. Picture (a) and scanning electron microscope (SEM) images (b) of the microfluidic device; (c)laboratory assembly used for testing PAH pre-concentration with the microfluidic device.

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top anodic bonding etc., as described elsewhere [37]. The internal chamber contains rows ofhexagonal micro-pillars (Figure 1(b)). Fused-silica capillaries (100 µm inner diameter) wereglued to the inlets for fluidic connection. Extraction of organic compounds from a liquid matrixwith this type of device has been recently patented by our laboratory [38].

For the application to PAH extraction, the microfluidic silicon/glass chips were individuallyfunctionalised with PDMS. About 0.160 g of nonpolar dimethyl polysiloxane (OV-1) wasdissolved in 5.0 mL of n-pentane (32 g L−1). The mixture was stirred for 1 h to ensure completedissolution. A thermally activated cross-linking agent (dicumyl peroxide) was added to theliquid polymer phase (1 wt%). The mixture was stirred for 10 min under sonication. Each chipwas filled with the coating solution using a syringe pump at a flow rate of 10 µL min−1.Afterwards, the chip was placed in a 40°C bath, with the fused-silica capillary sealed by a GCseptum. After 5 min, the chip was then connected for at least 4 h to vacuum to ensure completeevaporation of the solvent. After coating, the chip was heated up to 180°C at 5°C min−1 andheld for 4 h under a nitrogen flow (2 bar).

Several OV-1 concentrations in n-pentane were tested in a preliminary experiment, as thethickness of the PDMS layer in the microchip is directly related to this parameter.Concentrations from 2 to 16 g L−1 revealed lower extraction recoveries than with 32 g L−1.Higher concentrations could not be implemented, due to the high viscosity of the solutions.Considering PDMS mass conservation after coating, microchip channel geometry and a PDMSdensity of 1 g mL−1, a 0.4 µm average thickness was obtained with the 32 g L−1 concentration.Hence, the advantage of the microfluidic device is its very high specific area (9 cm2) with a lowvolume of PDMS (0.4 µL). In comparison with the SBSE bars recovered with 24 µL of PDMS,polymer volume is divided by 60 and thickness by 1250, while specific area is increased by afactor 18. Contact of the samples with a high surface area of polymer helps to reduce extractiontime.

2.3 Pre-concentration with the microfluidic device

Extraction of the aqueous samples and LD by solvents were carried out with a syringe pump(Figure 1(c)). Extractions were performed with 10 mL of solution. As the pre-concentrationmicrochip is intended for a portable device, it is preferable to avoid a solvent exchange step, andtherefore desorption was only tested with water miscible solvents: acetone, acetonitrile andmethanol.

To avoid cross-contamination, the microchips were rinsed with 1 mL of dichloromethaneand 1 mL of acetonitrile at a flow rate of 0.5 mL min−1 between pre-concentration tests.

For the optimisation procedure, solutions of ultra-pure water spiked with 5 µg L−1 of PAHswere prepared daily in 40 mL VOA (EPA) amber glass vials and stored at 4°C betweenexperiments. The solubilisation and stability between t0 and t = 10 h with five solutions ofindividual PAHs (ACE, ANT, FTN, B(a)P and IND) at 5 µg L−1 in ultra-pure water werechecked by spectrofluorometry. The extraction and desorption operational parameters wereoptimised in the following order: elution solvent, desorption volume, desorption rate andextraction rate (Table 1).

The optimal parameters were tested on real samples to evaluate matrix effects. Artificialsolutions were prepared with mineral water (Evian®). A solution containing 10 mg L−1 ofdissolved organic matter (DOM) was prepared by dissolving humic acid sodium salts in themineral water under 600 rpm stirring for 24 h. The DOM solution was then filtered, diluted andspiked with 5 µg L−1 of PAHs. The extraction of the DOM solution was compared with spiked(5 µg L−1 of PAHs) and non-spiked mineral water (n = 3). Environmental water was also testedby sampling continental surface water at Saint-Quentin-Fallavier lake (Isère, France), which was

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transported and stored at 4°C, then filtered at 0.2 µm (all steps were performed in less than24 h). Extraction was tested on spiked (5 µg L−1 of PAHs) and non-spiked samples.

For the optimisation of the pre-concentration parameters and the tests with real samples, atotal of five different microchips were used. All experiments were carried out in triplicates.

2.4 Comparison with stir-bar sorptive extraction (SBSE)

The pre-concentration performances of the microfluidic device, in terms of PAH recoveries andduration of the analytical step, were compared with extraction by SBSE followed by LD. Bothtechniques were evaluated with analysis by HPLC-FLD.

Most SBSE extraction and LD operational parameters were chosen from optimisation in theliterature [20,25,30,36]. Stir-bars were conditioned under stirring at 1000 rpm in 4 mL ofdichloromethane/methanol (60:40, v/v) for 15 min and under sonication in 1.5 mL of thesame solution for 10 min. Extractions were performed in triplicate at 1000 rpm in 40 mLVOA (EPA) amber glass vials with 10 mL of ultra-pure water/methanol (95:5, v/v) spiked with5 µg L−1 of PAHs. Methanol addition is recommended to avoid adsorption of PAHs on the wallsof the vial during the extraction step. Desorption step was carried out under sonication for20 min in HPLC vial inlets containing 200 µL of acetonitrile.

SBSE extraction durations chosen in the literature are considerably variable: from 30 min to24 h [27,32]. Equilibration time depends on many factors (stir-bar PDMS volume, samplevolume, extraction temperature, stirring speed, etc.) [30]. Moreover, optimisation was not

Table 1. Experimental parameters used for the optimisation of pre-concentration with the microfluidicdevice.

Extraction Desorption Pre-concentration

Volume(mL)

Flow rate(mL min−1)

Time(min) Solvent

Volume(mL)

Flow rate(mL min−1)

Time(min)

Total timea

(min)Concentration

factorb

10 0.50 20 Acetone 0.5 0.05 10 30 2010 0.50 20 Acetonitrile 0.5 0.05 10 30 2010 0.50 20 Methanol 0.5 0.05 10 30 2010 0.50 20 Acetonitrile 0.1 0.05 2 22 10010 0.50 20 Acetonitrile 0.2 0.05 4 24 5010 0.50 20 Acetonitrile 0.5 0.05 10 30 2010 0.50 20 Acetonitrile 1.0 0.05 20 40 1010 0.50 20 Acetonitrile 1.5 0.05 30 50 710 0.50 20 Acetonitrile 0.5 0.01 50 70 2010 0.50 20 Acetonitrile 0.5 0.02 25 45 2010 0.50 20 Acetonitrile 0.5 0.05 10 30 2010 0.50 20 Acetonitrile 0.5 0.10 5 25 2010 0.50 20 Acetonitrile 0.5 0.50 1 21 2010 0.10 100 Acetonitrile 0.5 0.05 10 110 2010 0.25 40 Acetonitrile 0.5 0.05 10 50 2010 0.50 20 Acetonitrile 0.5 0.05 10 30 2010 0.75 13 Acetonitrile 0.5 0.05 10 23 2010 1.00 10 Acetonitrile 0.5 0.05 10 20 20

Note: aPre-concentration time is the addition of extraction and desorption time;bTheoretical concentration factors obtained with extraction and desorption recoveries of 100% (ratio between the sampleand solvent volume).For each optimisation step, the parameter optimised is in bold type and the optimal values from previous steps are initalics. The final optimal pre-concentration conditions are highlighted in grey.

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carried out in many studies, and in some cases (e.g., [25]) non-equilibrium conditions wereused. Therefore, to evaluate the optimal SBSE extraction time with the chosen operationalparameters, tests were carried out with extractions of 1, 2, 8 and 24 h (n = 3), followed bythermal desorption and analysis by gas chromatography associated with tandem mass spectro-metry (TD-GC-MS/MS). TD was performed for this preliminary experiment, as the latterinduces no carry-over between analyses (desorption recovery of ~100 % for all PAHs), hencethe pre-concentration recoveries measured depend only on the extraction recoveries. Stir-barswere then frozen at −20°C and sent to the CEDRE (Brest, France) for analysis.

2.5 HPLC-FLD analysis

The PAH analyses were performed with a HPLC system consisting of a Waters 2695Separations Module associated with a Waters 2475 Multi-wavelength Fluorescence Detector.The system was equipped with a 250 mm × 4.6 mm I.D. SupelcosilTM LC-PAH C18 column(particle size 5 µm) and a 20 mm × 4.6 mm I.D. precolumn (particle size 5 µm) (SupelcoAnalytical, Sigma-Aldrich, St. Louis, MO, USA). Their temperatures were controlled by theWaters 2695 Separations Module. Empower 2 Software was used for data acquisition (Waters,Milford, MA, USA).

Analyses were carried out by injecting 20 µL of sample extract (automatic injection with theWaters 2695 Separations Module). Elution was carried out with a binary solvent gradient ofwater and acetonitrile (ACN) at a flow rate of 1.5 mL min−1. The gradient elution programmewas as follows: initial conditions with 60% ACN maintained for 5 min, followed by a 25-minlinear ramp to 100% ACN and finally a 10-min plateau at 100% ACN. The column temperaturewas set at 30°C (±0.5°C). To obtain the best selectivity and sensitivity, detection was performedwith three acquisition channels and selected fluorescence wavelengths. The excitation/emissionwavelength pairs (nm) chosen for each channel are given in Table 2. The acquisition ofsimultaneous chromatograms with different wavelengths assures better quantification of com-pounds which have similar retention times but different fluorescence properties [39]. Indeed,switching wavelengths between compounds with close retention times can induce biases ontheir quantification. Acenaphthylene was not quantified as this compound is not observed withfluorescence detection. Acenaphthene and fluorene were co-eluted and present the same specificfluorescence wavelengths, so they were quantified together.

Table 2. Analytical parameters for PAH determination by HPLC-FLD.

Channel Time (min) λexcitation (nm) λemission (nm) PAH Retention time (min)

A 0.0 233 320 NAPACE + FLR

6.810.1

12.5 365 462 FTN 13.5B 0.0 250 375 PHE

ANT11.111.8

14.0 302 431 B(b)FB(k)FB(a)PD(ah)AB(ghi)P

19.820.220.822.523.9

C 0.0 275 380 PYRB(a)ACHR

14.416.817.0

19.0 300 500 IND 23.7

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3. Results and discussion

3.1 HPLC-FLD calibration and validation

The three chromatograms obtained during the multi-wavelength analysis of a 10 ng mL−1

standard solution of PAHs in acetonitrile are presented in Figure 2.Detection and quantification limits were determined by studying the chromatogram obtained with

a 2 ng L−1 standard solution. The limit of detection (LOD) was calculated as equal to three times thebackground (S/N = 3) and the limit of quantification (LOQ) as 10 times the background (S/N = 10)[40]. LOQs are given in Table 3.

Calibration curves were prepared for six levels (2, 10, 50, 100, 500, 1000 ng mL−1) and eachcalibration level was injected in triplicate. The linearity range for PAH analysis extended from40 pg to 20 ng of injected compound, except in the case of B(k)F for which the signal saturatedover 10 ng of injected compound. Multi-wavelength synchronous acquisition is very satisfyingas all regression coefficients were higher than 0.9998 (Table 3).

Repeatability of the instrumental method was also very satisfying, even for very lowconcentrations. Indeed, the 2 ng mL−1 standard solution showed relative standard deviations(n = 3) lower than 2.8% (Table 3).

3.2 Optimisation of the pre-concentration with the microfluidic device

The three eluting solvents tested showed similar recoveries for all PAHs (Figure 3). Acetonitrilewas therefore chosen for the microchip desorption step, as this eluent is the HPLC solvent and isless toxic than methanol, which is a carcinogen.

Figure 2. Chromatograms obtained with multi-wavelength acquisition ((a): channel A, (b): channel B,(c): channel C) after injection of a 10 ng mL−1 standard solution of 16 PAHs in acetonitrile.Note: Retention times are indicated on each peak. Corresponding wavelengths used are given in Table 2. In(a): ACE and FLR are co-eluted (only ACE is indicated on the chromatogram). In (c): the second and thirdpeaks identified are B(a)A and CHR respectively.

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Concerning the influence of the eluent volume, two trends appeared (Figure 4): the lightestPAHs (NAP → PYR) showed maximum recoveries with 0.5 mL of acetonitrile, whereas theheavier PAHs (B(a)A → B(ghi)P) recoveries increased with the volume of solvent. However,there is barely any difference between 0.5 mL and 1.5 mL for the heavy PAHs, and highereluent volume induces lower concentration factors. Hence, 0.5 mL of eluent appears as theoptimal volume for desorbing PAHs from the PDMS microfluidic device.

Eluent flow rate, tested between 0.01 and 0.50 mL min−1, showed maximum recoveries forall PAHs with 0.05 mL min−1 of acetonitrile. Microscope observations showed that with lowflow rates (≤0.02 mL min−1), the microchip is not fully wetted by the solvent, in accordancewith Mora et al. who showed the difficulty to fill micro-channels with polar solvents due to thehydrophobicity of the PDMS surface [41]. At high flow rates (≥0.10 mL min−1), it seems that

Table 3. Instrumental LOQs, calibration data and repeatability obtained during HPLC-FLD analysis of 15PAHs.

LOQa (ng mL−1) Linearity range (ng) Calibrationb R2 RSDc (%)

NAP 1.0 0.045–21.1 y = 652 615x + 47 680 0.99991 2.1ACE+FLR 0.19 0.084–39.9 y = 5 063 805x + 619 395 0.99995 0.3PHE 0.13 0.044–20.7 y = 6 590 017x + 391 597 0.99996 0.5ANT 0.04 0.044–20.7 y = 19 066 633x + 1 787 805 0.99990 0.4FTN 0.48 0.042–20.1 y = 2 789 540x + 93 481 0.99993 2.6PYR 0.27 0.042–19.7 y = 10 438 728x + 251 183 0.99997 1.7B(a)A 0.21 0.044–20.9 y = 13 185 126x + 575 242 0.99996 2.8CHR 0.35 0.043–20.3 y = 15 237 936x + 572 414 0.99985 0.8B(b)F 0.07 0.043–20.3 y = 11 453 322x + 150 256 0.99988 0.8B(k)F 0.01 0.042–10.0 y = 99 704 632x + 4 112 482 0.99992 0.6B(a)P 0.03 0.042–20.1 y = 26 118 334x + 1 245 433 0.99997 1.9D(ah)A 0.04 0.043–20.5 y = 10 157 149x + 441 612 0.99997 1.0IND 0.46 0.042–19.9 y = 4 637 940x + 95 251 0.99997 0.4B(ghi)P 0.16 0.042–20.1 y = 12 618 555x + 117 283 0.99997 0.8

Note: aLOQ evaluated as 10 times the background with the 2 ng mL−1 standard (expressed in ng of PAH per mL ofacetonitrile); by = peak surface (µV s), x = PAH mass injected (ng); cRelative standard deviation obtained with threeinjections of the 2 ng mL−1 standard solution.

Figure 3. Optimisation of the solvent desorption step: recoveries obtained with different types of water-miscible eluting solvents.

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the contact time between the solvent and the polymeric phase is not sufficient to reachequilibrium.

Optimal desorption parameters therefore appeared to be 0.5 mL of acetonitrile at a flow rateof 0.05 mL min−1, resulting in a desorption step of 10 min and a maximum pre-concentrationfactor of 20. These values were set for the optimisation of the flow rate of the extraction step(Figure 5). The lightest PAHs (NAP → PYR) did not present any clear trend between flow ratevalues. In this case, equilibrium seems reached whatever the flow rate. On the contrary, heavyPAHs (B(a)A → B(ghi)P) recoveries increased between 0.1 and 0.5 mL min−1, then decreasedfor 0.75 mL min−1. This result can be explained by the low wettability of the microchip at thelowest flow rate (0.1 mL min−1) and the short contact time at 0.75 mL min−1. The choice of anoptimal sample flow rate of 0.5 mL min−1 induced an extraction step of 20 min, and therefore atotal desorption time of 30 min.

3.3 Comparison with stir-bar sorptive extraction

Influence of SBSE extraction time on PAH extraction recoveries was evaluated by TD-GC-MS/MS for five compounds of variable molecular masses, constituted by 2 (ACE), 3 (ANT, FTN)and 5 (B(a)P, IND) aromatic rings (Figure 6). For the lightest PAHs (ACE, ANT, FTN),recoveries higher than 90% were reached after 2 h of extraction, whereas for the 5-ringcompounds, extraction times superior than 8 h were necessary. As equilibrium was reachedfor all compounds after 24 h, this extraction time was chosen for comparing SBSE with ourmicrofluidic device.

Pre-concentration with the microfluidic device using the optimal parameters was comparedwith SBSE (Figure 7). As shown in the literature [20,25,30,36], SBSE associated with LD

Figure 4. Optimisation of the solvent desorption step: recoveries obtained in function of acetonitrileelution volumes.

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Figure 5. Optimisation of the solution extraction step: recoveries obtained in function of sample flowrates.

Figure 6. SBSE extraction kinetics determined by TD-GC-MS/MS (n = 3).

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enables high PAH recoveries of 50–90%. Reproducibility is also very satisfying, with relativestandard deviations lower than 3.5% (n = 3).

The microfluidic device also shows high recoveries for the heavy PAHs (B(a)A → B(ghi)P),with values between 64% and 83%, whereas the lightest PAHs are recovered at less than 50%.Reproducibility is not as good as SBSE, but remains satisfying with relative standard deviationsunder 9.1% (n = 3). The microfluidic device is therefore operational for extraction of the highmolecular weight PAHs, with quantification limits in water of 0.4 to 29 ng L−1 (for B(k)F andIND respectively). Even if the recoveries are low, intermediate compounds (PHE, ANT, FTN,PYR) also present low quantification limits of 9 to 60 ng L−1 (for ANT and FTN respectively).Only the compounds with two aromatic rings present high LOQs: 180 ng L−1 for ACE+FLRand superior to 1 µg L−1 for NAP.

The low recoveries of light PAHs in the microfluidic device can be explained by their loweraffinity for PDMS and the reduced PDMS volume in comparison with SBSE. Indeed, with theapproximation that the partitioning coefficients between PDMS and water (KPDMS/W) areproportional to octanol–water partition coefficient (KO/W), it can be stated that [42]:

Rextraction %ð Þ ¼ 1

1þ β=KO=W(1)

with Rextraction the theoretical extraction recovery of a PAH characterised by its octanol–waterpartition coefficient (KO/W) and β the ratio between the sample volume and the PDMS volume.

The theoretical extraction recoveries with the SBSE technique and our innovative device arepresented in Table 4. For SBSE, all values are higher than 83%, showing good recoveries evenfor 2-ring compounds. This is due to the important volume of PDMS used (24 µL). With themicrofluidic device, theoretical recoveries of the heavy compounds (log KO/W > 5.5) are very

Figure 7. Recoveries obtained during the optimal pre-concentration procedure with the microfluidicdevice and comparison with SBSE.

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satisfying, with values higher than 94%, whereas the most polar compounds theoretical extrac-tion recoveries decrease very importantly with their octanol–water partition coefficient.

To improve the PAH recoveries, the β ratio will have to be reduced (less sample or morePDMS) or the affinity of the PAHs for the extraction phase (Kpolymer/W) will have to beincreased. Tests with the PDMS functionalised microchips could be performed with a samplevolume of 1 mL. However, trace concentrations are not quantifiable with such a low volumesample. To reach the ng L−1 LOQ levels required to comply with the European Directives onenvironmental waters, new microchip geometries, containing more PDMS, will therefore betested. Moreover, the microfluidic device will be functionalised with other polymers presentinga higher affinity for polar compounds, for example by combining polymers of differentpolarities, as recently developed in sorptive methods [19,43].

3.4 Evaluation of matrix effects

PAH concentrations in the mineral water and filtered lake water samples were under thequantification limits. Spiked artificial and natural waters showed lower recoveries for allPAHs than the spiked ultra-pure water (Figure 8). The ionic content and the presence ofdissolved organic matter therefore interferes with PAH sorption in the PDMS layer. In particular,heavy PAH recoveries are relatively low, with values under 50%. Mineral water spiked with10 mg L−1 of DOM showed significantly lower recoveries than mineral water for the 5- and 6-ring compounds (D(ah)A, IND, B(ghi)P). In the case of spiked lake water, all heavy PAHrecoveries (B(a)A → B(ghi)P) were significantly lower than in the spiked mineral water and inthe spiked 10 mg L−1 DOM solution. These results can be explained by the fact that highmolecular weight PAHs present a strong affinity with the dissolved organic matter whichnaturally occurs in surface waters, as previously shown by Xia et al. [44]. The same phenom-enon has been observed for SBSE [45–47]. Therefore, to take into account the matrix effects of

Table 4. Theoretical extraction recoveries at equilibrium determinedwith Equation (1) for the microfluidic device and SBSE.

log KO/Wa

Rextraction (%)

Microchip SBSE

NAP 3.30 6.7 82.7ACE 3.92 23.0 95.2FLR 4.18 35.3 97.3PHE 4.46 51.0 98.6ANT 4.45 50.4 98.5FTN 5.16 83.9 99.7PYR 4.88 73.2 99.4B(a)A 5.66 94.3 99.9CHR 5.81 95.9 99.9B(b)F 5.78 95.6 99.9B(k)F 6.11 97.9 100.0B(a)P 6.13 98.0 100.0D(ah)A 6.75 99.5 100.0B(ghi)P 6.63 99.3 100.0IND 6.70 99.4 100.0

Note: aOctanol/water partition coefficients are given in [16].

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natural samples, internal standards will have to be used while carrying out measurements in situwith the portable device.

4. Conclusion

Our project aimed to develop a portable device to analyse PAHs in environmental waters. Thisstudy focuses on the development of a microfluidic device for fast and low-solvent consumingpre-concentration of the aromatic hydrocarbons. Analysis by HPLC associated with multi-wavelength fluorescence detection was calibrated and validated in terms of sensitivity andrepeatability. Pre-concentration parameters (for the sample extraction and the solvent deso-rption) were optimised with spiked solutions of 15 PAHs. Performance of the microfluidicdevice was compared with a reference method, SBSE. Our lab-on-a-chip pre-concentration is 50times faster than SBSE. It is efficient for heavy PAHs (log KO/W > 5.5) but has to be improvedfor the more polar compounds, for example by testing larger microchips and by using a morepolar material than PDMS. Chip to chip reproducibility will also be improved by full-waferfunctionalisation. Besides, stability and aging tests will be performed to check the robustness ofthe technique. Matrix effects were shown with artificial and natural waters. The presence ofdissolved organic matter significantly reduces the recoveries of the 4- to 6-ring compounds.Internal standards will therefore have to be integrated in the future portable system. Moreover,further automation of the pre-concentration step is previewed with a fully integrated lab-on-valve system.

AcknowledgementsWe acknowledge Julien Guyomarch from the CEDRE (Centre de documentation, de recherche etd’expérimentations sur les pollutions accidentelles des eaux) for his expertise on SBSE analysis andRomain Richard from the University of Toulouse for collecting the natural samples at Saint-Quentin-Fallavier.

Figure 8. Recoveries obtained with the artificial and natural environmental waters.Note: NAP is not represented, as the concentrations measured were under the quantification limit.

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FundingThis work was supported by the French National Research Agency (ANR) through Carnot funding.

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