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Determination of polycyclic aromatic hydrocarbons using lab on valve dispersive liquidliquid microextraction coupled to High performance chromatography M. Fernandez, S. Clavijo, R. Forteza, V. Cerdà

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Received date: 27 October 2014 Revised date: 28 January 2015 Accepted date: 3 February 2015 Cite this article as: M. Fernandez, S. Clavijo, R. Forteza, V. Cerdà, Determination of polycyclic aromatic hydrocarbons using lab on valve dispersive liquid-liquid microextraction coupled to High performance chromatography, Talanta, http: //dx.doi.org/10.1016/j.talanta.2015.02.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Determination of polycyclic aromatic hydrocarbons using lab on valve dispersive liquid-liquid microextraction coupled to high performance chromatography M.Fernandez, S.Clavijo, R. Forteza and V.Cerdà*1 Department of Chemistry. University of the Balearic Islands. 07122 Palma de Mallorca. Spain ABSTRACT In this work, dispersive liquid-liquid microextraction (DLLME) method was applied for high performance liquid chromatography (HPLC) determination of 15 PAHs in aqueous matrices.The extraction procedure was automated using a system of multisyringe flow injection analysis coupled to HPLC instrument with fluorescence detector. Factors affecting the extraction process, such as type and volume of extractionand dispersive solvent, extraction time and centrifugation step were investigated thoroughly and optimized utilizing factorial design. The best recovery was achieved using 100 µL of trichloroethylene as the extraction solvent and 900 µL of acetonitrile as the dispersive solvent.The results showed that extraction time has no effect on the recovery of PAHs. The enrichment factors of PAHs were in the range of 86-95 with limits of detection of 0.02–0.6µg L-1. The linearity was 0.2–600 µg L-1

for different PAHs. The

relative standard deviation (RSD) for intra- and inter-day of extraction of PAHs were

in

the

range

of

1.6–4.7

and

2.1–5.3,

respectively,

for

five

measurements.The developed method was used to assess the occurrence of 15 PAHs in tap water, rain waters and river surface waters samples.

KEYWORDS: dispersive liquid–liquid microextraction, experimental design, HPLC, Polycyclic aromatic hydrocarbons

1

Author for correspondence. E-mail: [email protected]

1. INTRODUCTION During the last decade the water quality has become one of the principal international concerns for the ecological fate of ecosystems and human health. Numerous pollutants need continuous monitoring, including polycyclic aromatic hydrocarbons (PAHs). These are a wide group of ubiquitous contaminants included to the European Union (EU) and United States Environmental Protection Agency (USEPA) priority pollutant list due to their toxicity and carcinogenic activity [1,2]. Their primary sources of contamination come from natural incomplete combustion processes and anthropogenic emissions, but these latter are generally considered to be the major source of these compounds input into the environment. They can reach surface waters in different ways, including atmospheric deposition, urban run-off, municipal and industrial effluents, oil spillage or leakage. PAH sare hydrophobic compounds (logKow=3–8) with very low water solubility [3]. As a result, it is necessary to incorporate a concentration step in the analytical

procedure,

prior

to

chromatographic

determination

in

the

environmental samples to improve the sensitivity of method. To resolve these problems, different techniques have been proposed. Therefore PAHs are generally extracted from water samples either by liquid–liquid extraction (LLE) [4,5] and solid-phase extraction (SPE) [6,7]. LLE is an effective technique for separation and preconcentration of analytes but requires large amounts of expensive and toxic solvents and uses multistep methods which lead to loss of analytes. SPE uses less solvent than LLE but the sample processing rates are slow and channeling reduces the capacity to retain analytes [8]. Another drawback in SPE, as in LLE, is the considerable amount of time needed and the manual operations involved [9]. Today, the attention is pointed towards the simplification of sample preparation with techniques that are environmentally friendly by reduction of the amount of organic solvents. In this sense microextraction methods have attracted much attention

in

the

recent

years.

Thus,

methods

such

as

solid-phase

microextraction (SPME) [10,11], stir bar sorptive extraction (SBSE) [12,13],

liquid-phase microextraction (LPME) [14,15], and more recently dispersive liquid–liquid microextraction (DLLME) [16,17] as developed as alternatives techniques for classic extraction procedures in the determination of PAHs. Among these, the most simple, rapid and environmental friendly approach seems to be the DLLME procedure [18], introduced by Rezaee and co-workers [16]. In essence, DLLME is based to the rapid injection of an appropriate combination of solvents to an aqueous sample containing the analytes of interest in a conical test tube. The binary mixture of the solvents consists in a few microliters of a high density extraction solvent with very low water solubility and another, named disperser, with high miscibility in both extractant and water phases; in order to form a cloudy solution consisting of small droplets of extraction solvent which are dispersed throughout the aqueous phase. DLLME is low cost technique, with minimal organic solvents consumption, high recoveries and enrichment factor and very short extraction time (a few seconds).Their main limitation is its lack of selectivity due to the presence of interferences from matrix , especially in analysis of trace analytes in a complex sample [18]. So far, DLLME coupled with HPLC [11,19,20], supercritical fluid chromatography [21] and gas chromatography (GC) [10, 16, 22-24], in combination with various detectors are the most usefulness and popular methods for precise quantitative analysis of PAHs. The main objective of this study was to develop and validate a DLLME-HPLC method for simultaneous determination of 15 PAHs in waters samples. Special attention was given on the optimization of the DLLME procedure by careful evaluation of the type and volume of extraction and dispersive solvents, as well as the effect of extraction time using experimental design. In addition, the optimal conditions of the solubility of PAHs in water were evaluated.

2. EXPERIMENTAL 2.1. Chemicals and Reagents.

HPLC grade methanol, acetonitrile, and acetone were obtained from Scharlau, (Spain), together with reagent grade methylene chloride and chloroform. GC-

MS grade trichloroethylene was purchased from Sigma Aldrich Quimica SA, (Madrid, Spain). Millipore water obtained from a MilliQ Direct-8 purification system Millipore Iberica S.A.U., (Madrid, Spain) was employed to prepare the aqueous solutions after filtration through a 0.45-µm pore size cellulose filter. A PAH calibration mix standard of naphthalene (Nap), acenaphthylene (Acpy),acenaphthene (Acp),fluorene (Flu),phenanthrene (PA),anthracene (Ant), fluoranthene(FL),

pyrene(Pyr),

benz[a]anthracene(BaA),

chrysene(Chr),

benzo[b]fluoranthene (BbFl), benzo[k]fluoranthene (BkFl),benzo[a]pyrene (BaP), dibenzo[a,h]anthracene (DBA),benzo[g,h,i]perylene (BghiP)and indeno[1,2,3-cd]pyrene (IP), at 10 mg L-1in acetonitrile were bought from Supelco (Bellefonte, PA, USA), as a reagent kit containing all 16 priority PAHs listed by the United States Environmental Protection Agency.

2.2. Sample preparation A stock solution, containing 10 µg mL-1 of each analyte, was prepared by suitable dilution of standard PAH kit with acetonitrile and was stored at 4ºC. Water samples were prepared daily by spiking millipore water with analytes at known concentrations to study extraction procedure under different conditions in order to optimize the method. To validate the propose method for the separation and preconcentration of PAHs, different types of waters were analyzed. Tap water (TW) was sampled in the laboratory, rain water (RW) was collected in our university campus (University of Balearic Islands, Spain) and streamwater (SR) (Santa Margalida Mallorca), was collected in October 2012 in glass bottles and stored refrigerated. Before analysis, aqueous samples were diluted with 5% (v/v) acetonitrile as organic modifier,and stirred during 24 hours to prevent adsorption of PAHs on the PTFE tubing of the flow manifold in order toobtain quantitative recoveries andprevent the cross-contamination between samples. All solutions were filtered through a 0.45 µm cellulose filter.

2.3. Extraction Procedure For the DLLME procedure, an aliquot (4.00 mL) of an aqueous solution containing the analytes was analyzed. A mixture of 900 µL of acetonitrile as dispersive solvent and 100 µL oftrichloroethylene as extracting solvent was used. A cloudy solution was formed. Then the fine droplets of the extraction phase were settled at the bottom of tube. Then an aliquot of 20 µL of the separated phase were aspirated into aninjection loop for further analysis. 2.4. Liquid chromatographic analysis The analysis of the sample were performed by HPLC using a Jasco system constituted by an autosampler (AS-2055 Plus), two pumps (PU-2080 Plus), and a multi-wavelength fluorescence detector (FP 2020 Plus). Separations were carried out on a Vydac RP-18e (250 mm × 4.6 mm) column. The analytical signal was monitored and integrated using ChromNav software. Gradient elution, (see table 1), was programmed for total separation of 15 PAHs in 20 minutes (Fig 1). The injection volume was 20 µL and the mobile phase was composed of solvent A: acetonitrile–water (60:40) and solvent B: 100% acetonitrile at a flow rate of 1.5 mL min− 1. Table 1 Detection was performed at different selected fluorescence wavelengths programmed to obtain the better sensitivity and minimal interference for each compound. The excitation/emission wavelengths pairs (nm) were monitored as shown in table 2. Table 2

Figure 1

2.5. Instrumentation A scheme of the MSFIA system used for the extraction and injection ofPAHs in HPLC column is shown in Fig. 2. The manifold was constructed with PTFE

tubing of 0.8 mm i.d., except for a two long holding coil 500 cm HC1 and 300 cm HC2,made of 1.5 and 0.5 mm i.d., respectively.Supply tubes for syringe loading and waste dischargewere made of PTFE tubing of 1.5 mm i.d.

Figure 2

A multisyringe burette module, a valve modulewith one rotary eight-port multiposition valve and a rotary six-port micro injection valve (loop volume 20 microliters) from Crison SL (Alella, Barcelona) were used to distribute the liquid through the system. The multisyringe module was equipped with one glass syringe (Hamilton, Bonaduz, GR, Switzerland) of 5 mL, which had a three-way solenoid valve (N-Research, Caldway, NJ) at its head (V). A Lab on valve (LOV) microconduit (Sciware Systems, Spain) mounted atopthe eight-port

multiposition

selection

valve,

fabricated

with

KelF®

(polychlorotrifluoroethylene) and encompassing eight integrated microchannels (0.5mm i.d./14.0mm length) avoided the dispersion of solvent and promoted propelling to the extraction chamber (EC).The extraction process occurred at position 6 of LOV in a 5 mL commercial pipette tip adapted through a connector. The HPLC pump and the chromatography column were connected directly to the inject valve to perform the separation analysis. The operations sequence followed for the determination of PAHS is detailed in Table 3. It is very important the order in which the sample and solvent are introduced in the EC, obtaining the best results when the extraction solvent is dispensed followed by the dispersive solvent and sample at the end. Table3 2.6. Data processing Statistical computer package “Statgraphics Centurion XVI” was used to construct experimental design matrices and evaluate the results.

3. RESULTS 3.1. Study of solubility efficiency of PAHs in water Because the low solubility of PAHs in water, an organic solvent, such as methanol or acetonitrile or a surfactant is usually added to the sample to ensure good solubility of PAHs in the aqueous media and prevent their adsorption on the PTFE tubing of the flow manifold and glassware; otherwise, the PAHs could not be recovered quantitatively, and cross-contamination may occur. In this sense, the concentration of the organic solvent is a critical parameter because if it is low it may not be must be enough to solubilize the high molecular weight PAHs , whereas if it is high could cause the early elution for the low molecular weight PAHs.Thus, proper concentration of organic modifier in aqueous sample needs to be investigated by series experiments. In this work, acetonitrile was employed as the organic modifier to increase the solubility of PAHs in water. A full factorial design 23 was chosen as a screening method to estimate the relative influences of acetonitrile percentage, agitation mode and time and their possible interactions on the analytical response (peak area). The experimental run order was randomized to reduce the effect of extraneous variables. The values corresponding to the high and low levels were chosen according to preliminaries studies and literature data. Then the percentage of acetonitrile was evaluated at 0.5 and 5 %. Ultrasound and magnetic stir bar was the agitation techniques used. The agitation time was 15 minutes or 1 hour for the low level and 1 or 24 hours for the high level for ultrasound and magnetic stir respectively. An analysis of variance (ANOVA) was performed on the design to assess the significance of the model. The estimated effects of three main factor sand three two-way interactionswere visualized by means of Pareto charts (Fig 3). Figure 3 According to previous report, different results were observed for low and high molecular weight PAHs. For the less water soluble PAHs (e.g. BghiP) all factors were significant at the 95% confidence level while for the low molecular weight PAHs (e.g. Acp) only interaction of mode and time of extraction is significant, in addition the percentage of acetonitrile has a negative effect for these last. The optimization of the response for the individual analytes is not a successful way of optimization for a whole set of analytes with different physicochemical

characteristics. Taking into account this situation, it was necessary to identify a compromise zone where all the experimental responses satisfied the specifications imposed by the researcher to achieve the proposed aims. Therefore, a desirability function was used to predict the best experimental conditions considering that the response variables for all 15 PAHs were equally significant. The method consists in transforming the measured property of each response to a dimensionless desirability scale, defined as a partial desirability function; its value varies from zero, undesirable response, to one, optimal response. This enables the combination of results obtained for properties measured on different scales. Once the function is defined for each experimental response, an overall objective function, representing the global desirability function is defined as the weighted geometric average of the individual desirability functions [25]. In this case,the desirability function was defined by assigning a desirability value of 0.0 to minimum peak area, 1.0 for the maximum value. In order to achieve the highest desirability score (desirability 1), the software optimized the peak area of the target analytes with calculating the optimized model variables of agitation mode, agitation time and percent of acetonitrile in the aqueous sample.

Figure 4 shows the desirability chart using a magnetic stir bar as agitation mode. The optimized factors settings (see Table 4) provide a composite desirability of 0.87. Figure 4 Table 4

3.2. Optimization DLLME conditions In order to maximize extraction recovery (ER%) of PAHs, the conditions of the DLLME procedure should be optimized. The extraction recovery was determined from the following equation:

where Vf,Vaq and EF are the volume of the floating phase,the volume of aqueous phase and enrichment factor,respectively. The enrichment factor was calculated as following:

Cf and C0 are the concentration of analytes in the floating phase and aqueous phase, respectively. DLLME employs a mixture of a high-density solvent (extractant) and a water miscible polar solvent (disperser). Acetone,methanol and acetonitrile can be used as dispersers, whereas chlorinated solvents (e.g. chlorobenzene, carbon tetrachloride (CCl4),tetrachloroethylene (C2Cl4)) are usually employed as extractants. Extraction solvent should have properties such as lower density than water, low solubility in water, good extraction capability of analytes and chromatographic compatibility. According to these properties, chloroform, methylene chloride and trichloroethylene were studied. Dispersive solvent should be miscible with both water and extraction solvent and produce very fine droplets of extraction solvent, when the mixture of extraction and dispersive solvent was rapidly injected into the aqueous sample. Acetone, acetonitrile and methanol were tested as dispersive solvents. In order to evaluate volume of the extraction solvents, a fixed volume of dispersive solvents (900 µL) and two volumes of extraction solvents (50 and 100 µL) were examined Lower volumes were avoided due to the very small volume of the sedimented phase formed because of subsequent harmful effects on reproducibility. For the DLLME process, the extraction time is defined as the interval between injection of the mixture of disperser and extraction solvents, and starting of centrifugation. The effect of extraction time was tested for 30 and 120 seconds. Then, a combination of different extraction and dispersive solvents, volume of the extraction solvents, and extraction time were used by a combined factorial design (32 x 22), two category variables at three levels and two numerical variables to two levels as shown in Table 5. A total of 32 experiments were performed to explore important factors and their interactions, and these were run in random manner to minimize the effect of uncontrolled variables. The sum of the extraction recovery of PAHs was chosen as response variable.

Table 5 Figure 5 shows the pareto chart of this design and indicates the important factors affected on recoveries. The obtained results indicate that the type and volume of extraction solvent, the type of dispersive solvent, also the interaction between both solvents and the interaction between dispersive solvent and the volume of solvent of extraction were the most significant variables with a positive effect on the recovery of PAHs. This is due to volume of the dispersive solvent increased the solubility of the PAHs in water, rising the extraction efficiency and the mass transferring between the organic and the aqueous phases. The selected solvents were: acetonitrile (disperser) and trichloroethylene (extractant) with a volume of 900 and 100 µL respectively. Figure 5 Because the mixture of acetonitrile and trichlorethylene has a density significantly higher than water, we have observed that fine droplets containing the extracted analytes settle in a few seconds at the bottom of the conical tube used for extraction, without requiring a centrifugation step. 3.3. Performance of the analytical procedure The analytical performance of the proposed MSFIA-DLLME-HPLC method was evaluated in terms of repeatability, linearity, correlation coefficient, detection and quantification limits and enrichment factors under optimized experimental conditions, as summarized in Table 6.The linear calibration of targeted PAHs was examined in the range of 0.2–600µg L-1, and good linear behavior was seen.The correlation coefficient (R2) and relative standard deviation (RSD %)for intra- and inter-day of extraction of PAHs were determinedin a solution containing 0,2 µg L-1 of each PAHs.The coefficient of determination (R2) ranged from 0.9989 to 0.9999 for PAHs compounds. The relative standard deviation(RSD %) for intra- and inter-day of extraction of PAHs were in the range of 1.6–4.7 and 2.1–5.3, respectively, for five measurements.The limits of detection (LODs) values of the analytes for the preconcentration of 4 mL sample volume, which were calculated as three times signal to noise ratio (S/N = 3), ranged from 0.02 to 0.61 µg L-1 for spiking waters. As it can be seen, the method has sufficient sensitivity for the determination of PAHs in environmental water samples. Nevertheless, it is possible to enhance the sensitivity further by injecting larger volumes, increasing the size of the syringe.

Table 6

3.4. Application to Real Samples The MSFIA-DLLME-HPLCproposemethod was applied for thedetermination of 15 priority PAHs in watersamples and immediately analyzed as described above, todemonstrate the applicability and reliability of the method. Threedifferent kinds ofreal samples, tap water, rain water and stream surface water were analyzed. To estimate the matrix effect of water samples,all the samples were spiked with 0.3 µg L-1 of PAH individual standardsto calculate the recovery of the targeted compounds. The sample results and therecovery study were performed in triplicate (see Table 7). Table 7 No PAHs were found in the analysis of tap water samples. However some PAHs were found in rain or stream water samples. Only PAHs of 2 or 3 rings were detected. This is due to their solubility in water, which decreases with the increase of molecular weight and then five or six ring PAHs in aqueous medium only found in highly contaminated sites. The relative recoveriesof the analytes in tap, rain water, and stream surface were 92–102, 89–106 and 92–103%, respectively. The recovery results obtained for the three real samples were similar. This demonstrated that DLLME-HPLC method was notsignificantly affected by the sample matrices.

4. CONCLUSIONS An analytical methodology for the determination of PAHsin aqueous sample was developed. The results showed goodperformance of the analytical protocol. Quantification limitsachieved by the method allowthe application of the procedure below the levels imposed by existing regulations. The automated MSFIA-DLLME proposed procedure offers significant saving ofreagents and time compared to other techniques used, being capable of providing a rapid isolation of the target analytesas was demonstrated by processing real samples.

For the environmental sample determination, the results of DLLME showed the successful application for separation and preconcentration of low concentration PAHs compounds in water samples.

Aknowledgments. The authors acknowledge financial support from Spanish Ministry of Economy and Competitiveness (MINECO) through Project CTQ2013-47461-R cofinanced by FEDER funds. The Conselleria d’Economia, Hisenda, I Innovació of the Government of the Balearic Islands is acknowledged for the allowance to competitive groups (43/2011). M.Fernández thanks to the Conselleria d'Educació, Cultura I Universitats from the Government of the Balearic Islands for a PhD stipend co-financed by Fondo Social Europeo.

REFERENCES

Figure 1. On-line DLLME–HPLC HPLC chromatograms of water spiked with 0.02 µg/L of each PAH. PAHs were preconcentrated from 4 ml of sample. The elution order and retention time are shown in Table 4.

Figure 2. Schematic illustration of the DLLME-MSFIA-HPLC DLLME HPLC for the analysis of PAHs in water. MIV: microinjection valve, LOV: lab on valve, DS: dispersive

solvent, Es: extraction solvent, S: sample V: valve, S: syringe pump and EC: extraction chamber.

Diagrama de Pareto Estandarizada para BghiP

Diagrama de Pareto Estandarizada para Acp

+ -

A:% AcN AB

+ -

BC B:agitation mode

AC

AB

B:agitation mode

C:agitation time

C:agitation time

A:% AcN

BC

AC 0

10

20 30 Efecto estandarizado

40

0

50

0,5

1

1,5 2 Efecto estandarizado

2,5

Figure 3. Pareto carts (for Acp and BdhiP) of the main effects obtained from full factorial design. Grafico de deseabilidad agitation mode=2

Deseabilidad

1 0,8 0,6 0,4 0,2 0 -1

-0,6

-0,2 % AcN

0,2

0,6

1

1 0,6 0,2 -0,2 -0,6 -1 agitation time

3

Figure 4. Desirability charts for optimized variables.

Diagrama de Pareto para CRF

B:Sdispersive

Sig. en 5% No sig.

AB A:Sextraction C:Vextraction BC AC BD AD D:textraction CD 0

10 20 30 Contribución a la variación (%)

40

Figure 5. Pareto chart of the main effects obtained from combined factorial design for DLLME optimization.

Highlights A dispersive liquid-liquid microextraction method has been applied to 15 PAHs Allows its application below the levels imposed by existing regulations The automated MSFIA-DLLME method offers significant saving of reagents and time It has been successfully applied to determine PAHs in water samples

1

Directive 2008/105/EC, 2008, Official Journal of the European Communities L- 348/84 (24th December, 2008), Council Directive (16th December, 2008) on environmental quality standards in the

eld of water policy, amending

and subsequently repealing Council Directives 82/176/EEC, 83/513/EEC, 84/156/EEC, 84/491/EEC, 86/280/EEC and amending Directive 2000/60/EC of the European Parliament and of the Council. 2

Directive 2006/0129 of the European Parliament and of the 649 Council on environmental quality standards in the field of water policy and amending Directive 2000/60/EC, European Commission, Brussels, 2006.

3

M. Zander, Angewandte Chemie International Edition in English, 25 (1986) 379-380.

4

D.M. Brum, R.J. Cassella, A.D. Pereira Netto, Talanta, 74 (2008) 13921399.

5

L. Tavakoli, Y. Yamini, H. Ebrahimzadeh, S. Shariati, Journal of Chromatography A, 1196–1197 (2008) 133-138.

6

I. Urbe, J. Ruana, ibid.778 (1997) 337-345.

7

H.-D. Liang, D.-M. Han, X.-P. Yan, ibid.1103 (2006) 9-14.

8

Z. Sosa Ferrera, C. Padrón Sanz, C. Mahugo Santana, J.J. Santana Rodrıg ́ uez, TrAC Trends in Analytical Chemistry, 23 (2004) 469-479.

9

I. Rodrıg ́ uez, M.P. Llompart, R. Cela, Journal of Chromatography A, 885 (2000) 291-304.

10 A.J. King, J.W. Readman, J.L. Zhou, Analytica Chimica Acta, 523 (2004) 259-267. 11 M.-M. Zheng, B. Lin, Y.-Q. Feng, Journal of Chromatography A, 1164 (2007) 48-55. 12 Z. Qin, L. Bragg, G. Ouyang, J. Pawliszyn, ibid.1196–1197 (2008) 89-95. 13 E. Pérez-Carrera, V.M.L. León, A.G. Parra, E. González-Mazo, ibid.1170 (2007) 82-90. 14 Y.-N. Hsieh, P.-C. Huang, I.W. Sun, T.-J. Whang, C.-Y. Hsu, H.-H. Huang, C.-H. Kuei, Analytica Chimica Acta, 557 (2006) 321-328. 15 N. Ratola, A. Alves, N. Kalogerakis, E. Psillakis, ibid.618 (2008) 70-78.

16 M. Rezaee, Y. Assadi, M.-R. Milani Hosseini, E. Aghaee, F. Ahmadi, S. Berijani, Journal of Chromatography A, 1116 (2006) 1-9. 17 X. Zhao, L. Fu, J. Hu, J. Li, H. Wang, C. Huang, X. Wang, Chromatographia, 69 (2009) 1-5. 18 C. Ojeda, F. Rojas, ibid.74 (2011) 651-679. 19 M.T. Pena, M.C. Casais, M.C. Mejuto, R. Cela, Analytica Chimica Acta, 626 (2008) 155-165. 20 L. Oliferova, M. Statkus, G. Tsysin, O. Shpigun, Y. Zolotov, ibid.538 (2005) 35-40. 21 J. Rein, C.M. Cork, K.G. Furton, Journal of Chromatography A, 545 (1991) 149-160. 22 K. Li, H. Li, L. Liu, Y. Hashi, T. Maeda, J.-M. Lin, ibid.1154 (2007) 74-80. 23 A. Mehdinia, E. Khojasteh, T. Baradaran, A. Jabbari, Journal of Chromatography A, 1364 (2014) 20-27. 24 W. Tseng, P. Chen, S. Huang, Talanta, 120 (2014) 425-432. 25 G.A. Lewis, D. Mathieu, R. Phan-Tan-Luu, Pharmaceutical Experimental Design Marcel Dekkler, New York (1999)

Table 1. Optimized HPLC solvent gradient program of PAH analytes Time (min) 0 6 14 20 26

Solvent composition % solvent A 100 100 0 0 100

% solvent B 0 0 100 100 0

Table 2. Optimized fluorescence conditions for PAHs determination. Time (min) 0 4.5 6.5 7.5 9.5 12.0 14.0

Ex wavelength (nm) 267 280 247 250 238 270 294

Em wavelength (nm) 330 330 357 420 418 390 425

17.8

245

500

PAH component Nap Acp, Flu PA Ant FL, Pyr BaA, Chr BbFl, BkFl, BaP, DBA, BghiP IP

Table3. Time schedule and sequence of operations for on-line DLLME–HPLC determination of PAHs in aqueous solutions time (min) 0-0.5

description

0.5-2.5 2.5-6.5 6.5-8.3

empty the syringe

MSIA flow rate 10mL min-1

siryng e valve OFF

positio n LOV -

positio n MVI -

conditioning sample tubing

5mL min-1

ON

3

-

-1

pickup sample pickup DS

1mL min

ON

3

-

-1

ON

4

-

-1

0.5mL min

8.3-8.5

pickup ES

0.5mL min

ON

5

-

8.5-8.8

dispense into EC

15mL min-1

ON

6

-

8.8-8.9

filling loop injection

0.5mL min-1

ON

2

LOAD

8.9-9.0

injection of analytes to C18

-

-

-

9.0-30

HPLC run

-

-

-

INJEC T -

Table 4. Optimized factor settings and the individual and composite desirability for PAHs solubility. Factor Optimum % acetonitrile extraction time h extraction mode Compound Nap Acp Flu PA Ant FL Pyr BaA Chr BbFl BkFl BaP IP DBA BghiP Composite

Optimum 5 24 stir bar Desirability 0.5755 0.5346 0.4460 0.8369 0.7122 0.9999 1.0000 0.9995 0.9997 0.9995 0.9998 1.0000 1.0000 1.0000 1.0000 0.8736

Table 5.Factors and value level used in the experimental design factor

type

Role

low

high

levels

A:S extraction

Categoric

Controlable

-

-

1,2,3

B:S dispersive

Categoric

Controlable

-

-

1,2,3

C:V extraction

Continuous

Controlable

-1

1

-

D:t extraction

Continuous

Controlable

-1

1

-

Table 6.Linear range, limits of detection, enrichment factor, reproducibility of the method and PAHs retention times. Compound

tret(min)

R2

EF

4.2 6.0 6.2 7.4 8.7 10.0 10.7 12.9 13.3 14.8 15.4 16.0 17.5 18.1 16.9

0.9989 0.9997 0.9999 0.9998 0.9997 0.9997 0.9999 0.9995 0.9998 0.9997 0.9999 0.9999 0.9999 0.9997 0.9998

87.56 86.93 92.06 92.36 95.61 86.08 87.62 90.51 88.05 89.78 95.45 90.87 88.35 91.83 91.24

Nap Acp Flu PA Ant FL Pyr BaA Chr BbFl BkFl BaP DBA BghiP IP

RSD % (n=5) inter intra day day 4.69 4.34 4.13 3.82 3.84 2.61 4.72 5.31 1.67 2.89 4.23 4.73 3.74 4.51 3.48 3.86 3.01 2.95 2.58 3.74 1.63 2.13 2.35 3.46 4.37 3.86 2.96 3.59 2.85 2.97

LOQ (µg L-1 ) 0.61 0.52 0.09 0.16 0.04 0.41 0.26 0.08 0.07 0.08 0.02 0.05 0.14 0.09 0.06

Table 7. Recoveries for three different kind of spiked waters tap water analytes Nap Acp Flu PA Ant FL Pyr BaA Chr BbFl BkFl BaP DBA BghiP IP

C nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

RR %±RSD 93± 4 96± 3 98± 2 93± 2 98± 2 96± 4 94± 2 97 ± 3 98 ± 2 96 ± 3 102 ± 2 97± 4 99± 1 97± 4 92± 3

rain water C 0.38 nd nd 0.43 0.46 0.51 0.42 nd nd nd nd nd nd nd nd

RR %±RSD 89± 4 97± 2 95± 3 98± 3 106± 3 96± 3 95± 3 98± 2 96± 2 97± 2 99± 4 95± 3 102± 3 94± 3 95± 2

stream surface waters C RR %±RSD 0.41 92± 3 0.13 96± 2 nd 97± 3 0.35 95 ± 2 0.52 99 ± 3 0.29 94 ± 2 0.29 97 ± 1 nd 96 ± 3 0.48 99 ± 2 nd 100 ± 4 nd 97 ± 3 nd 98 ± 2 nd 103 ± 2 nd 96± 3 0.31 96± 2

nd: not detected; RR: recoveries; C: (µg L-1)

Figure 1. On-line DLLME–HPLC chromatograms of water spiked with 0.02 µg L-1 of each PAH. PAHs were preconcentrated from 4 ml of sample.The elution order and retention time are shown in Table 4.

Figure 2. Schematic illustration of the DLLME-MSFIA-HPLC for the analysis of PAHs in water. MIV: microinjection valve, LOV: lab on valve, DS: dispersive solvent, Es: extraction solvent, S: sample V: valve, HC holding coil, S: syringe pump and EC: extraction chamber.

Figure 3. Pareto charts (for Acp and BdhiP)of the main effects obtained from full factorial design.

Figure 4. Desirability charts for optimized variables.

Figure 5.Pareto chart of the main effects obtained from combined factorial design for DLLME optimization.The extraction time has no significant effect on the results and 30 s was chosen in further studies as the minimum time needed for self-phase separation. In the literature is reported that a centrifugation step is required to sediment the extraction solvent at the bottom of the tube and taken with a microsyringe for its later chromatographic analysis [26,27].

Highlights A dispersive liquid-liquid microextraction method has been applied to 15 PAHs Allows its application below the levels imposed by existing regulations The automated MSFIA-DLLME method offers significant saving of reagents and time It has been successfully applied to determine PAHs in water samples

Graphical abstract

Determination of polycyclic aromatic hydrocarbons using lab on valve dispersive liquid-liquid microextraction coupled to high performance chromatography

Determination of polycyclic aromatic hydrocarbons using lab on valve dispersive liquid-liquid microextraction coupled to high performance chromatography.

In this work, dispersive liquid-liquid microextraction (DLLME) method was applied for high performance liquid chromatography (HPLC) determination of 1...
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