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Analytica Chimica Acta 674 (2010) 206–210

Contents lists available at ScienceDirect

Analytica Chimica Acta

journa l homepage: www.e lsev ier .com/ locate /aca

pplication of dispersive liquid–liquid microextraction and spectrophotometricetection to the rapid determination of rhodamine 6G in industrial effluents

ourya Biparva, Elias Ranjbari, Mohammad Reza Hadjmohammadi ∗

epartment of Chemistry, University of Mazandaran, Babolsar, Iran

r t i c l e i n f o

rticle history:eceived 20 January 2010eceived in revised form 18 June 2010ccepted 19 June 2010vailable online 1 July 2010

eywords:ispersive liquid–liquid microextraction

a b s t r a c t

A rapid and effective preconcentration method for extraction of rhodamine 6G was developed by usinga dispersive liquid–liquid microextraction (DLLME) prior to UV–vis spectrophotometry. In this extrac-tion method, a suitable mixture of acetone (disperser solvent) and chloroform (extractant solvent) wasinjected rapidly into a conical test tube containing aqueous solution of rhodamine 6G. Therefore, a cloudysolution was formed. After centrifugation of the cloudy solution, sedimented phase was evaporated,reconstituted with methanol and measured by UV–vis spectrophotometry. Different operating variablessuch as type and volume of extractant solvent, type and volume of disperser solvent, pH of the sample

hodamine 6Gaste water samples

pectrophotometry

solution, salt concentration and extraction time were investigated. The optimized conditions (extrac-tant solvent: 300 �L of chloroform, disperser solvent: 3 mL of acetone, pH: 8 and without salt addition)resulted in a linear calibration graph in the range of 5–900 ng mL−1 of rhodamine 6G in initial solutionwith R2 = 0.9988 (n = 5). The Limits of detection and quantification were 2.39 and 7.97 ng mL−1, respec-tively. The relative standard deviation for 50 and 250 ng mL−1 of rhodamine 6G in water were 2.88% and

. Finawate

1.47% (n = 5), respectivelydifferent industrial waste

. Introduction

The industrial growth with changes in manufacturing processesas resulted by an increase in the volume and complexity ofastewater discharges to the environment [1]. Synthetic dyes have

ound comprehensive usage in textile dyeing, paper printing, foren-ic technology, color photography and as an additive in petroleumroducts. Xanthene dyes are a member of the class of syntheticyes. Rhodamine 6G with the chemical structure shown in Fig. 1, isderivative of the xanthene dyes, which is highly water soluble. It

s among the oldest and most commonly used synthetic dyes thatre used as a colorant in textile and foodstuffs. Most of syntheticyes are carcinogenic and others after transformation or degra-ation yield compounds such as aromatic amines, which may bearcinogenic or otherwise toxic [2].

The dye rhodamine 6G is injurious if swallowed by humans andnimals, and causes irritation to the skin, eyes and respiratory tract.he carcinogenicity, reproductive and developmental toxicity, neu-

otoxicity and chronic toxicity towards humans and animals haveeen experimentally proven [3]. The dye rhodamine 6G used in theresent study is widely employed in textile industries. Thus due tohe perilous nature and harmful effects of rhodamine 6G, making

∗ Corresponding author. Tel.: +98 112 5342380; fax: +98 112 5342350.E-mail address: Hadjmr@umz.ac.ir (M.R. Hadjmohammadi).

003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2010.06.024

lly, the DLLME method was applied for determination of rhodamine 6G inrs.

© 2010 Elsevier B.V. All rights reserved.

efforts to develop a simple method for determination of rhodamine6G is inescapable.

Sample preparation is an important step in analytical methodsand follows two main steps, the first is sample clean-up and sec-ond is preconcentration. So a combination of advanced instrumentswith novel sample preparation methods has enabled analysis oftrace amounts of analytes with higher accuracy. In the last decadesdesign and development of miniaturized alternative methods tothe older sample preparation techniques has been one of the mostimportant challenges for analysts [4].

Liquid-phase microextraction (LPME) was developed as a sol-vent minimized pretreatment technique which is fast, simple,and inexpensive. LPME combines extraction, preconcentration,and sample introduction in one step [5,6]. It is based on dis-tribution of the analytes between a few microliters of organicsolvent and the aqueous sample solution. Recently, a novel liquid-phase microextraction modality termed as dispersive liquid–liquidmicroextraction (DLLME) has been developed by Rezaee et al. [7].It is based on a ternary component solvent system like homoge-neous liquid–liquid extraction and cloud point extraction. In thismethod, the appropriate mixture of extraction solvent and disper-

sive solvent is injected into aqueous sample rapidly by syringe,and a cloudy solution is formed. The analyte in the sample isextracted into fine droplets of extraction solvent. After extraction,phase separation is performed by centrifugation and the enrichedanalyte in the sedimented phase is determined by chromatog-

P. Biparva et al. / Analytica Chimic

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Fig. 1. Molecular structure of rhodamine 6G.

aphy or spectrometry methods. From commercial, economicalnd environmental point of view, DLLME offers several impor-ant advantages over conventional solvent extraction methods:aster operation, easier manipulation, no need of large amountsf organic extraction solvents, low time and cost, high extractionecovery and enrichment factor and less stringent requirementsor separation. Up to now, DLLME has been widely used for thenalysis of organic compounds in environmental water samples,ncluding polycyclic aromatic hydrocarbons [7], organophospho-us pesticides [8], organosulfur pesticides [9], chlorophenols [10],hthalate esters [11] and polybrominated diphenyl ethers [12]. Inddition, its application has been extended in the field of tracelemental analysis in environmental water samples [13,14]. Thus,LLME could be considered as an alternative technique to fit theseurposes because of its simplicity and applicability in almost allnalytical laboratories.

The present paper describes a new, simple and reliablextractive-spectrophotometric method for determination of rho-amine 6G using DLLME. To the best of our knowledge, thistudy may be the first report describing the application of DLLME-pectrophotometric method for determination of a dye in wateramples. The effects of various experimental parameters, such ashe kind and volume of extraction and disperser solvent, pH of theample solution, extraction time and salt effect have been studied.he applicability of presented method for the analysis of industrialaste water samples has also been investigated.

. Experimental

.1. Instrumentation

Recording the absorption spectra and absorbance measure-ents were carried out with a Cecil CE5501double beam UV–visible

pectrophotometer (Cecil Instruments Ltd., Cambridge, UK) using.5 cm quartz cells. A Jenway 3030 pH-meter, (Bibby Scientific Ltd.,taffordshire, UK) equipped with a combine Ag/AgCl glass electrodeas used to check the pH of the solutions. A Denley bench cen-

rifuge model BS400 (Denley Instruments Ltd., Billingshurst, UK)as used to accelerate the phase separation.

.2. Reagents

All chemicals used in this work, were of analytical reagentrade and were used without further purification. Double dis-illed deionized water was used throughout which was produced

y a Milli-Q system (Millipore, Bedford, MA, USA). Carbon tetra-hloride, chloroform and dichloromethane as extraction solvent,ethanol (for spectroscopy), acetone (for spectroscopy) and ace-

onitrile (HPLC grade) as dispersive solvent, were obtained fromluka (Buches, Switzerland). The rhodamine 6G, Sodium chloride,

a Acta 674 (2010) 206–210 207

potassium chloride and calcium dichloride were purchased fromMerck (Darmstadt, Germany). A stock standard solution of rho-damine 6G (500 mg L−1) was prepared in double distilled/deionizedwater. The working standard solutions were prepared in doubledistilled/deionized water too.

2.3. Dispersive liquid–liquid microextraction procedure

For DLLME, 10.0 mL aliquot of water sample was placed in a15 mL screw cap glass tube with conic bottom and spiked at thelevel of 500 ng mL−1 of rhodamine 6G. A mixture of 3 mL of acetone(as disperser solvent) and 300 �L CHCl3 (as extraction solvent) wasinjected into a sample solution by using 10.0 mL syringe rapidly,so that a cloudy mixture was formed. The cloudy solution wascentrifuged for 5 min at 3000 rpm. Accordingly, fine droplets ofextraction phase were sedimented. The sedimented phase at thebottom of conical test tube (about 250 �L) was entirely transferredinto a vial using 100 �L HPLC syringe for evaporation of solvent. Theextract was evaporated to dryness at 75 ◦C in an oven. The residuewas dissolved in 1.00 mL methanol and was conveyed to a UV–visspectrophotometer to measure its absorbance at �max (530 nm).

2.4. Calculations

In order to calculate the extraction recovery (ER), as analyticalresponses, the following equation was used:

ER = Cset × Vset

C0 × Vaq× 100

where Cset and C0 are the concentrations of analyte in settled phase(1.00 mL methanol) and initial concentration of analyte in aque-ous sample, respectively. Cset was determined from a calibrationgraph which was obtained by direct determination of absorptionspectrum of standard solutions with concentrations in the rangeof 0.5–10 mg L−1. Vset and Vaq are the volume of settled phase andaqueous sample, respectively.

3. Results and discussions

For obtaining good sensitivity, precision and selectivity forextraction and determination of rhodamine 6G, the importantparameters which influence the efficiency of DLLME procedureshould be optimized, separately. All the reported results are theaverage of duplicate measurements.

3.1. Selection of extraction solvent and disperser solvent

The selection of a suitable extraction solvent is very impor-tant for the DLLME process. The extracting solvent has to meettwo properties: to extract the analytes well and to have den-sity higher than that of water [15]. Hence, carbon tetrachloride[16] (density, 1.59 g mL−1), chloroform (density, 1.48 g mL−1) anddichloromethane (density, 1.32 g mL−1) were considered for thispurpose.

As for the choice of dispersive solvent in DLLME, the miscibilityin organic phase (extraction solvent) and aqueous phase (samplesolution) is a key factor, which can disperse extraction solventinto very fine droplets in aqueous phase. Acetonitrile, acetone andmethanol were compared as disperser solvent in the extraction

of rhodamine 6G. For obtaining good efficiency, all combinationsusing CCl4, CHCl3, CH2Cl2 (200 �L) as extractant with acetone,acetonitrile, methanol (3.0 mL) as dispersive solvent were tried.According to the results shown in Table 1, acetone as the dis-perser solvent and chloroform as the extraction solvent provided

208 P. Biparva et al. / Analytica Chimica Acta 674 (2010) 206–210

Table 1Effect of extraction solvent and disperser solvent on extraction recovery obtainedfrom DLLME techniquea.

Extraction solvent Disperser solvent

Acetonitrile Methanol Acetone

CCl4 16.6b ± 2.6 3.1 ± 1.3 5.8 ± 0.7CHCl3 59.9 ± 2.3 61.8 ± 1.6 83.0 ± 3.6CH2Cl2 49.1 ± 3.9 38.1 ± 2.0 79.6 ± 3.2

a Extraction conditions: water sample volume,10.0 mL; disperser solvent vol-ume, 3 mL; extraction solvent volume, 200 �L; concentration of rhodamine 6G,500 ng mL−1.

b Extraction recovery (%).

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ig. 2. Effect of extraction solvent volume on extraction recovery. Extraction con-itions: water sample volume, 10.0 mL; disperser solvent (acetone) volume, 3 mL;oncentration of rhodamine 6G, 500 ng mL−1; extraction solvent: CHCl3.

aximum extraction recovery of 83.0%. Therefore, we selected ace-one/chloroform as a suitable set for subsequent experiments.

.2. Effect of extraction solvent volume

To consider the effect of the extraction solvent volume onxtraction recovery, different volumes of chloroform were tested.herefore, the volume of disperser solvent (acetone) was fixedt 3.0 mL and the volume of chloroform was ranged from 100 to00 �L. According to the Fig. 2, it was clear that extraction recov-ry showed better recovery by increasing the volume of CHCl3 up to00 �L, but after the volume of 300 �L, enhancement is not remark-ble. So, 300 �L chloroform was chosen as an optimum volume.

.3. Effect of disperser solvent volume

For obtaining optimized volume of disperser solvent, extrac-ions were carried out by changing the volume of acetone in theange of 1.0–4.0 mL. The represented results in Fig. 3, shows thathe extraction recovery firstly increased by increasing volume of

ig. 3. Effect of disperser solvent volume on extraction recovery. Extraction condi-ions: water sample volume, 10.0 mL; extraction solvent (CHCl3) volume, 300 �L;oncentration of rhodamine 6G, 500 ng mL−1; disperser solvent: acetone.

Fig. 4. Effect of pH of the sample solution on DLLME extraction of dye. Extrac-tion conditions: water sample volume, 10.0 mL; disperser solvent (acetone) volume,3 mL; extraction solvent (CHCl3) volume, 300 �L; concentration of rhodamine 6G,500 ng mL−1.

acetone and then reached a maximum plateau in the range of3.0–4.0 mL. It seems that at a lower volume of acetone consump-tion, cloudy state was not formed well and the extraction solvent(CHCl3) could not be well dispersed among aqueous solution in theform of very little droplet, which resulted in poor extraction recov-ery. Therefore, in the following experiments, 3 mL of acetone wasused as the optimal dispersive volume.

3.4. Effect of pH

pH of solution is an important factor during liquid–liquid extrac-tion (LLE) process involving analytes that possess an acidic or basicmoiety. The ionic form of a neutral molecule formed upon depro-tonation of a weak acid or protonation of a weak base normallydoes not extract through the organic solvent as strongly as does itsneutral form. Thus, pH should be adjusted to ensure that neutralmolecular forms of the analytes are present prior to performingthe microextraction step. In this step, the effect of pH of solutionon the amount of extracted rhodamine 6G was investigated in therange of 2–12. As it can be seen in Fig. 4, the best pH for extractionof rhodamine 6G was 8.0 in which the dye was completely in itsmolecular form.

3.5. Effect of salt addition

Addition of salt often improves extraction of analytes in con-ventional liquid–liquid extraction, as a result of the salting-outeffect. This has been used universally in solid-phase microextrac-tion (SPME) and LLE. Generally, addition of salt enhanced extractionof analytes, because the presence of the salt reduced the solubilityof the analytes in water and forced more of them on to the fibercoating for SPME or the organic phase for LLE. To evaluate the possi-bility of salting-out effect, extraction recovery was studied with theNaCl and CaCl2 concentrations in the range from 0% to 10% (w/v).The results showed that the addition of salts had no meaningfuleffect on the extraction recovery in the studied range. Hence, allthe extraction experiments were performed without the additionof salt.

3.6. Effect of extraction time

In miniaturized preconcentration methods such as SPME andLPME, extraction time is one of the most consequential parame-ters. The time of extraction is defined as an interval time between

injection of mixture of disperser solvent (acetone) and extractionsolvent (chloroform) before starting to centrifuge. In this workeffect of extraction time was examined from 0 to 20 min. Theobtained results showed that the variations of extraction recov-ery against the extraction time were not significant. In DLLME,

himica Acta 674 (2010) 206–210 209

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Table 2Tolerance limits of interfering species in the determination of 200 ng mL−1 rho-damine 6G.

Interference Tolerance ratio (w/w) Added as

Na+ 2000 NaClK+ 2000 KClCa2+ 2000 CaCl2Mg2+ 1000 Mg(NO3)2

Cl− 2000 NaClF− 1000 NaFNO3

− 2000 KNO3

SO42− 800 Na2SO4

HPO42− 800 Na2HPO4

CO32− 800 Na2CO3

Al3+ 400 Al(NO3)·9H2OFe3+ 400 FeCl3Mn2+ 500 MnSO4·H2OZn2+ 1000 ZnCl2Cd2+ 1000 Cd(NO3)2·4H2OCr3+ 500 Cr(NO3)3·9H2ONi2+ 1000 Ni(NO3)2·6H2OPb2+ 800 Pb(NO3)2

Co2+ 1000 Co(NO3)2·6H2OCu2+ 1000 Cu(CH3COO)2

TT

B

P. Biparva et al. / Analytica C

he surface area between the extraction solvent and the aqueoushase is significantly large, so that the transfer of the analyte fromhe aqueous phase into the extraction phase is carried out quickly.herefore, the time of extraction was very succinct because equi-ibrium state was obtained very fast. On the other hand, the mostime-consuming step in DLLME is the centrifuging of sample solu-ion in the extraction procedure, which was about 5 min.

.7. Quantitative analysis

After optimization of all parameters, quantitative characteristicsf the proposed method were studied. These included calibrationurve equation, determination coefficient, limit of detection (LOD)nd limit of quantification (LOQ). For the purpose of quantita-ive analysis, a calibration curve for rhodamine 6G was obtainedy spiking the standard directly into distilled water and extract-

ng under the optimal conditions. Linearity was observed over theange of 5–900 ng mL−1 of rhodamine 6G in the initial solution.he equation for the line was A = 0.80 (±0.03)C − 0.004 (±0.001)ith a determination coefficient (R2) of 0.9988 where A is the

bsorbance and C is the concentration of rhodamine 6G in �g mL−1.he limit of detection and quantification, defined as LOD = 3Sb/mnd LOQ = 10Sb/m (where Sb and m are, standard deviation of thelank and slope of the calibration graph after preconcentration,espectively), were 2.39 and 7.97 ng mL−1 (n = 10), respectively. Theelative standard deviations for 50 and 250 ng mL−1 of rhodamineG were 2.88 and 1.47 (n = 5), respectively. The preconcentrationactor, defined as the ratio of the concentrations of analyte in theettled phase and in the aqueous sample solution (concentrationsfter and before preconcentration), was 10.

.8. Interference studies

The effect of potential interferences, encountered in real sam-les, on the extraction recovery of 200 ng mL−1 rhodamine 6Gtandard solution in the presence of various amounts of individ-al interfering ions and some dyes, were examined. The tolerance

evel was defined as the maximum concentration of the foreignpecies causing a change in the analytical signal not higher than 10%hen it was compared with the signal of dye lonely. The obtained

esults are given in Table 2.The results showed that the recover-es of the analytes, in the presence of interfering ions at the ratioshat usually occur in real samples are almost quantified by usingLLME method. Two similar dyes, fluorescein and eosin Y, could be

olerated up to 15 and 10 ratio, respectively.

able 3he application of presented method for determination of rhodamine 6G in different sam

Samples Rhodamine 6G added ( ng mL−

Sample 1b –100.0200.0

Sample 2c –50.0

100.0

Sample 3d –50.0

100.0

Waste water of plastic production plant –50.0

100.0

DL: below the detection limit.a Mean value ± standard deviation.b The waste water effluent of textile industry (Iran, Ghaemshahr).c The waste water effluent of textile industry (Iran, Tehran).d Textile industry waste water treatment plant effluent.

Ag+ 500 Ag(NO3)Fluorescein 15Eosin Y 10

4. Recoveries of the method and samples analysis

To evaluate the accuracy and applicability of the proposedmethod, the extraction and determination of rhodamine 6G in dif-ferent water samples, i.e., the textile waste waters effluents andwaste water of plastic production plant were performed. All thesamples were spiked with dye standard at two levels, and wereextracted subsequently by using the DLLME technique and finallythe extracts were analyzed by UV–vis method. Three replicateexperiments were carried out for each concentration level. Theexperimental results are shown in Table 3. Recovery, R, was cal-culated as follows:

R(%) = Cspiked − Cunspiked

Cadded× 100

where C , C and C are the value of dye measured

spiked unspiked addedin spiked sample, value of dye in unspiked sample and amount ofadded dye, respectively. The results showed that recoveries fromthe samples were from 97.6% to 104.3% with RSD (n = 3) less than3.8%. This indicated that matrix did not influence the proposed

ples (sample volume: 10.0 mL, final volume: 1.00 mL, n = 3).

1) Rhodamine 6G found ( ng mL−1) Recovery (%)

BDL –97.6 ± 1.2a 97.6 ± 1.2a

196.0 ± 4.1 98.0 ± 2.3

112.9 ± 0.9 –162.5 ± 2.0 99.2 ± 3.2214.6 ± 2.4 101.7 ± 2.4

BDL –51.5 ± 1.2 103.0 ± 2.5

102.4 ± 2.8 102.4 ± 2.8

201.8 ± 1.7 –251.9 ± 1.8 100.2 ± 3.7306.1 ± 4.0 104.3 ± 4.0

210 P. Biparva et al. / Analytica Chimic

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[[15] E. Zeini Jahromi, A. Bidari, Y. Assadi, M.R. Milani, M.R. Jamali, Anal. Chim. Acta

585 (2007) 305–311.[16] NIOSH, Pocket Guide to Chemical Hazards, U.S. Department of Health and

ig. 5. The absorption spectra of (a) sample blank (b) and (c) the spiked concentra-ions of rhodamine 6G (A) textile waste water sample (B) waste water sample oflastic production plant.

LLME method for preconcentration of rhodamine from real sam-les. Fig. 5 shows the typical UV–vis spectra of the extracted dyerom textile and plastic industrial waste water samples before andfter spiking with two levels of dye standard.

. Conclusion

In this paper, a novel mode of DLLME extraction technique cou-led with spectrophotometric detection was developed for rapid

[

a Acta 674 (2010) 206–210

determination of rhodamine 6G. Up to our knowledge, only fewmethods are available for determination of rhodamine 6G [17] andthis method offers a simple way for the determination of rhodamine6G in different samples. The results demonstrated that the pro-posed method had good recoveries and reproducibilities. Therefore,this technique is feasible for quantitative analysis of rhodamine 6Gin real samples, and could be used in routine analysis.

Acknowledgement

The authors are grateful to associate Dr. M. J. Chaichi from Spec-troscopic Laboratory for technical assistance.

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