Development of a simple device for dispersive liquid–liquid microextraction

Analytica Chimica Acta 655 (2009) 60–65Contents lists available at ScienceDirectAnalytica ChimicaActaj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /a caDevelopment of a simple device for dispersive liquid–liquid microextraction with lighter than water organic solvents:Isolation and enrichment of glycyrrhizic acid from licoricePayman Hashemi a ,∗,Somayeh Beyranvand a ,Reza Siah Mansur b ,Ali Reza Ghiasvand aa Department of Chemistry,Faculty of Science,Lorestan University,Falakol,aflak St,Khoramabad,Iran bAgriculture and Natural Resource Research Center,P.O.Box 348,Khoramabad,Irana r t i c l e i n f o Article history:Received 23June 2009Received in revised form 22September 2009Accepted 23September 2009Available online 27 September 2009Keywords:Dispersive liquid–liquid microextraction Lighter than water organic solvents Glycyrrhizic acidHigh performance liquid chromatography Multivariate optimizationa b s t r a c tA simple device was developed that makes the use of lighter than water organic solvents feasible in disper-sive liquid–liquid microextraction (DLLME)method.In the ordinary DLLME,the fact that a heavier than water organic solvent must be used,to be sedimented at the conical bottom of a centrifuge tube,limits the applications of this method in some extent.In the developed method,a glass tube with a narrow neck is inserted inside the centrifuge tube.After phase separation,the organic solvent is accumulated in the narrow neck of the device and therefore,can be simply collected by a micro-syringe.The DLLME method with the proposed device was tested for the enrichment of glycyrrhizic acid from aqueous extracts of licorice before analysis by a HPLC method.n -Hexanol and acetone were used as the organic and disperser phases,respectively.Effects of pH,salt concentration and phase volumes on the extraction of the analyte were optimized using a central composite (response surface)design.Under the optimized conditions (i.e.pH 1.3,ionic strength 0.2mol L −1,n -hexanol 140␮L and acetone 0.8mL)an extraction recovery of 104.1(±5.1)%and an enrichment factor of 54were obtained.The proposed method was successfully applied for the study of glycyrrhizic acid’s level of licorice roots grown in three regions of Lorestan province,Iran,with different climate conditions.© 2009 Elsevier B.V. All rights reserved.1.IntroductionLicorice (Glycyrrhiza globra L.)is a traditional herbal medicine with several health benefits such as antiinflammatory,antiul-cerous,antidotal,antiallergic,immunomodulating and antiviral properties [1–3].Glycyrrhizic acid (GA),is the main pharmacolog-ically active ingredient in the root of licorice which is about 170times sweeter than sucrose [4].GA is a weak acid containing three carboxyl and five hydroxyl groups.It is a glycosylated saponin with a pentacyclic triterpenic structure bound to two glucuronic acid units as shown in Fig.1[4].The GA content of the licorice depends upon several parameters such as the species,plant age,season of harvest and climate conditions.Its level ranges probably from 0.2to 83.7mg g −1[5–7].A number of methods such as reflux [6,7],solvent extraction [8,9],surfactant assisted pressurized liquid extraction [10],pressurized hot water extraction [11]and microwave assisted extraction [12]have been used for the withdrawal of GA from licorice roots before its analysis by HPLC.Methanol,ethanol and∗Corresponding author.Tel.:+986612202782;fax:+986612200185.E-mail addresses:payman hashemi@ ,hashemi.p@lu.ac.ir (P.Hashemi).water,or a mixture of them,have been among the most commonly used solvents for this purpose [1].However,other solvents such as tributyl phosphate,trialkylphosphine oxide,n -hexanol and 2-ethyl-hexanol have been also practiced [8].Microextraction methods have attracted much attention in recent years as alternatives for classic liquid–liquid and solvent extraction procedures.Solid phase microextraction (SPME)[13]and liquid-phase microextraction (LPME)[14],both with direct and headspace operations,have been extensively used in recent years.These techniques are simple and fast,miniaturize sample pretreat-ment processes and minimize the use of organic solvents.However,these techniques suffer from some problems such as sample carry-over,relatively high cost,fiber fragility and relatively low pre-cisions.Recently,dispersive liquid–liquid microextraction (DLLME)was introduced as an alternative to the classic liquid–liquid extrac-tion method [15].DLLME is simple,rapid and cost effective and provides high enrichment factors.DLLME has been applied for the analysis of a variety of trace organic pollutants and metal ions in the environmental samples [15–21].DLLME is based on a ternary com-ponent solvent system.In this method,generally,a small volume of a heavier than water organic solvent (such as carbon tetrachlo-ride or chlorophenol)is dispersed into fine droplets in the sample solution by the aid of a water miscible solvent as disperser.After0003-2670/$–see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2009.09.034P.Hashemi et al./Analytica Chimica Acta655 (2009) 60–6561Fig.1.The chemical structure of glycyrrhizic acid.phase separation followed by centrifugation,the organic solvent is sedimented at the bottom of the centrifuge tube as a micro-drop that can be withdrawn by a micro-syringe.However,the fact that in this method a heavier than water,normally chlorinated,highly toxic,solvent has to be used limits,in some extent,the applications of this method.In this paper,a simple device composed of a glass tube with a narrow neck,is presented that makes the use of lighter than water organic solvents feasible.The method is then applied for the enrich-ment and HPLC determination of GA in licorice roots and study of the effect of climate conditions on its level.A recent study in our laboratory indicated that the climate conditions can significantly influence the essential oil composition of cumin seeds[22].How-ever,to the best of our knowledge,there is no report on the effect of climate conditions on the GA content of licorice roots.A central composite(response surface)design is used for efficient optimization of the microextraction conditions.Central composite design is a multivariate optimization technique that pro-duces a detailed quantitative model for the response(s).This model can predict how a response relates to the values of various factors and it usually requires less number of experiments compared to a univariate procedure[23].2.Experimental2.1.Reagents and materialsGlycyrrhizic acid(GA)monoammonium salt was prepared from ACROS Organics(Newjercy,USA).HPLC grade acetonitrile,n-hexanol,organic solvents and other chemicals were purchased from Merck(Darmstadt,Germany)and used as received.Double-distilled water was used throughout.A0.01mol L−1solution of GA in ethanol was prepared and used as stock solution for the preparation of working standards.2.2.ApparatusHPLC analysis of the samples was conducted on a Shimadzu (model L-10AD)instrument consisting of two reciprocating pumps, a DGU-14A in-line degasser,a model CT10-10AC oven,a high pres-sure manual injection valve(20␮L injection loop)and a UV/VIS (model SPD-10A)detector.The software used for the data acquire-ment and processing was Class-vp v.R6.1.The analytical column was a25cm×4.6mm i.d.RP-8column(Shim-Pack CLC-C8)packed with5␮m particles and equipped with a1cm guard column(C8-B197)packed with10␮m particles of the sametype.Fig.2.The proposed NNGT device for lighter than water organic solvents.A25␮L HPLC micro-syringe(F-LC,SGE,Australia)was used for the sample withdrawal and injection.A totally glass Fisons(UK) double distiller was used for preparation of doubly distilled water.For ultrasonic irradiation of the samples,an ultrasonic water bath(22kHz,model5RS,Sonica,Italy)was used.An Eppendorf AG centrifuge(model5810,Hamburg)was used for centrifugation of the extracts.For DLLME with lighter than water organic solvents a home-made narrow neck glass tube(NNGT)was used(Fig.2).The glass tube was about10cm in length and12and4mm in diameters of the body and neck,respectively.The NNGT was placed inside a15mL polyethylene centrifuge tube for the centrifugation and phase separation.2.3.Field samplesLicorice(G.globra L.)root samples were collected from three regions of Lorestan province in the west part of Iran.This province has a variation from warm to cold climates.The regions selected for this study were Alashtar with cold,Khoramabad with temperate and Poldokhtar with tropical climates.Some characterizations of the experimental sites and collection time of the samples have been summarized in Table1.From each region,at least10root samples were collected.The collected samples were dried in room temper-ature,ground into about1mm pieces,homogenized and stored in capped bottles before the extraction.2.4.ProceduresFor direct ultrasound assisted solvent extraction of GA from licorice samples,1.0g of the ground sample was transferred into a glass tube and25mL water was added.The test tube was capped,thermostated at55◦C,and sonicated for15min in the ultrasonic water bath.The supernant solution was decantated and the extraction procedure was repeated on the residue once more. The supernant solutions were added together,filtered through a 0.45␮m membranefilter and stored at4◦C before analysis.62P.Hashemi et al./Analytica Chimica Acta655 (2009) 60–65Table1Some characterizations of the experimental sites and collection time of the licorice samples.Region Climate Altitude(m)Mean annual temperature Mean annual rainfall Harvest timeAleshtar(Honam)Cold160012.9◦C576mm June2007 Khoramabad(Sarab Changaei)Temperate112517.9◦C438mm June2007 Poldokhtar(Ghashghaei)Tropical71322.9◦C433mm June2007The same procedure was used for preliminary extraction of the samples before the isolation and preconcentration by the proposed DLLME method.However,in this case only0.1g of the ground sam-ple was applied.For performing the DLLME method,the sample acidity was adjusted on0.05mol L−1(pH of about1.3)by addition of some hydrochloric acid solution.The ionic strength of the sample was also adjusted on0.4mol L−1using a sodium nitrate solution.In a microvial,0.8mL acetone(as the disperser)and140␮L n-hexanol (as the acceptor organic solvent)were mixed and the mixture was withdrawn by a2mL glass syringe,equipped with a10cm needle, and rapidly injected into4.0mL of the sample in the proposed NNGT device.A uniformly distributed cloud was achieved by further suc-tion and reinjection of about2mL of the cloudy sample using the same syringe.The mixture was then centrifuged at3000rpm for 8min for the phase separation.Since n-hexanol is a lighter than water solvent,the extraction phase was collected at the solvent top.By careful addition of some water drops,the organic phase was conducted to the narrow neck of the NNGT.This phase was then carefully withdrawn by a micro-syringe and injected into the HPLC column,after three times dilution by ethanol in a microvial.For the HPLC separation of GA and other co-extracted com-pounds,a gradient elution with a mixture of solvents A(1% solution of acetic acid in water)and B(acetonitrile)with aflow rate of1.0mL min−1was used.The elution program was as fol-lows:0–6min,20–30%B;6–8min,30–50%B;8–15min,50–60% B;15–17min,60–80%B;17–23min,80%B;23–25min,80–20%B. The chromatograms were acquired at254nm.For optimization of the extraction conditions by the proposed DLLME method a half fraction central composite(response surface) design was performed using Minitab statistical software[24].Four factors of n-hexanol(organic solvent)volume,acetone(disperser) volume,pH of the aqueous phase and salt(sodium nitrite)concen-tration were included in the design.The extraction recovery(E%) was considered as the dependent variable to be optimized using the model.Table2shows the abbreviations and levels of each factor included in the design.For calculation of the extraction recovery(E%)of the proposed method,100times of the amount extracted of GA(mmol)into the organic phase was divided by its initial amount in the sample.For calculation of the enrichment factor(EF),the GA concentration in the receiving phase was divided by its initial concentration in the sample.The detection limit of the method was calculated from 3 ,using the calibration curve data.The standard deviation of the blank(s b)was estimated from that of the residuals(s y/x)for this calculation.Table2The factors included in the central composite design and the low and high levels for each factor.Parameter Abbreviation Factor’s levels−+Extractant(n-hexanol)volume(␮L)EV12575 Disperser(acetone)volume(␮L)DV700500 Sample pH pH 3.5 2.0 Ionic strength(mol L−1)IS0.150.053.Results and discussion3.1.Ultrasound assisted solvent extraction of GAIt is known that ultrasound power can substantially improve the extraction efficiency of a solvent extraction method[25].There-fore,the extraction of GA from licorice root samples was conducted by the assistance of this power and in an ultrasonic bath.In some preliminary studies,different solvents such as ethanol,methanol, water and different mixtures of them were tested.It was found that water and mixtures of water and an alcohol are more effi-cient solvents than the pure alcohols.Water wasfinally selected as the solvent,because it was more appropriate for performing the subsequent DLLME procedure.Maximum extraction efficiency with water was obtained at a temperature of55◦C,after15min sonica-tion in the ultrasonic bath.For ensuring the complete extraction of GA from the samples,four more successive extractions were performed on the residue of thefirst extract.It was found that, respectively,84.4%and15.1%of GA is extracted in thefirst and second steps and extraction in the following steps is negligible. Therefore,extraction in two successive steps was considered as quantitative and used in subsequent experiments.For an appropriate separation of the licorice roots components, different gradient elution programs were used for the HPLC col-umn.In the literature,usually long programs,up to140min[9], have been reported for an appropriate separation.In this work theTable3The conditions used and extraction recoveries(E%)obtained for the central compos-ite design experiments.No.Parameters a E% DV EV IS pH17001250.05 2.045.83 26001500.1 2.7553.25 36001000.1 1.2544.91 4700750.05 3.522.20 56001000.1 2.7541.58 65001250.15 2.049.32 78001000.1 2.7553.62 84001000.1 2.7526.96 97001250.15 3.527.61 106001000.1 4.2516.36 11500750.15 2.034.64 127001250.15 2.053.90 135001250.05 3.532.01 145001250.15 3.534.45 156001000 2.7541.30 16600500.1 2.750.00 176001000.1 2.7544.47 18700750.05 2.018.65 197001250.05 3.544.91 20500750.05 2.025.44 21700750.15 3.518.27 22500750.15 3.525.38 23700750.15 2.037.49 246001000.1 2.7537.91 25500750.05 3.525.32 265001250.05 2.040.31 276001000.2 2.7548.02 Opt b8001400.2 1.374a For the full names of the parameters and their units,see Table2.b The predicted optimized conditions by the model and the predicted E%.P.Hashemi et al./Analytica Chimica Acta 655 (2009) 60–6563separation time was reduced to about 20min by selecting appropri-ate solvents and a suitable gradient program with the appearance of GA peak at 14.7min in the chromatogram.3.2.DLLME of GAIn some preliminary experiments it was found that chlorinated solvents cannot efficiently extract GA from its aqueous solutions.Therefore,some lighter than water organic solvents,such as ethy-lacetate,cyclohexane,petroleum ether,tributyl phosphate and n -hexanol were examined for this purpose.Since withdrawal of the organic solvents collected as a thin layer on the sample surface was very difficult or even impossible,a simple NNGT device was fabri-cated and inserted inside the centrifuge tube (see Fig.2).By using this device,the organic phase was conducted to a narrow space (the neck of the device)and therefore could be easily suctioned by a micro-syringe.For protonation of the carboxylic acid group of GA and its better extraction to the organic phase,the test solutions were acidified to pH ing acetone as the disperser indicated a more efficient extraction of the analyte by using n -hexanol as the solvent.This solvent is known as an efficient solvent for solvent extraction of GA from aqueous licorice leaching solutions [8].Therefore,n -hexanol was used as the extraction solvent in subsequent experiments.Dif-ferent water miscible organic solvents such as methanol,ethanol and acetone were tested as disperser for the DLLME system.Since more stable and uniform cloudy solutions were shaped with ace-tone,this solvent was selected as the disperser.3.3.Optimization by the central composite designAfter performing some preliminary experiments,optimization of the extraction conditions by the proposed DLLME method was performed by a half fraction central composite design.The data given in Table 3show the extraction recoveries expressed as E %for 27experiments designed by using the MINITAB software [23].Table 4shows the regression coefficients for each term in the model and the t -Student’s statistics and probability values for the significance of the terms.It is obvious that n -hexanol volume (EV)and pH,with the smallest p -values,are the most signif-icant factors.The quadratic term of EV 2and the interaction between IS and pH were also significant in 95%confidence level.Fig.3depicts the response surface plots of extraction recovery modeling for some of the factors.The curvature in these kinds of plots can indicate possible interactions between factors.According to Fig.3a,in a high ionic strength the extraction efficiency increases by decreasing the pH.On the other hand,in a low ionic strength lower extraction efficiencies are obtained in low pH levels.ThisTable 4Estimated regression coefficients for E %obtained for the central composite design.Term Coef T P Constant 41.318510.1930.000DV 2.3055 1.6090.134EV 9.4766 6.6120.000IS 1.6589 1.1570.270pH −5.5212−3.8520.002DV 2−0.6020−0.3960.699EV 2−4.0184−2.6430.021IS 20.48990.3220.753pH 2−3.0160−1.9840.071DV ×EV 1.8951 1.0800.302DV ×IS −0.9382−0.5340.603DV ×pH −0.6453−0.3680.720EV ×IS −1.3723−0.7820.449EV ×pH −1.5833−0.9020.385IS ×pH−3.9907−2.2730.042Fig.3.The response surface plots for the extraction recovery (E %)as a function of IS and pH (a)and EV and pH (b).is a demonstration of the interaction between IS and pH that is in concurrence with the results of Table 4.No considerable interaction of such is observed between EV and pH in Fig.3b,as expected.The response surface model with the estimated regression coef-ficients (see Table 4)was used for the response optimization,including all the factors.The conditions for the predicted optimum point have been shown in the last row of Table 3.To evaluate the validity of the prediction by the model,three experiments were performed under the suggested optimized conditions and a mean extraction recovery of 70.6±2.6was obtained which shows a fair agreement between the predicted and experimental results.3.4.Effects of some individual factorsIn the optimized conditions suggested by the central compos-ite design,the extraction of GA was still not quantitative.Further studies showed that gentle shaking of the formed cloudy solution or suction and reinjection of a part of it by a syringe increases the extraction recovery.The results indicated that only one time of the suction and reinjection was sufficient for achievement of a uni-form cloudy solution and quantitative recovery of the analyte.This method was more efficient than gentle shaking of the NNGT and therefore,was used in all subsequent experiments.By using this method,about 100%recoveries could be obtained.After performing a multivariate optimization procedure,it is often desirable to study,in some details,the individual effect of some important factors.This will give a better view of the effects of the experimental conditions on the response which is useful in method development practices.Here,the individual effect of pH was studied because of its significance.The suction and reinjection method was used during the study for a better homogenization.64P.Hashemi et al./Analytica Chimica Acta655 (2009) 60–65Fig.4.Influence of pH on the extraction recovery (E %)and enrichment factor (EF)of GA in the DLLME procedure,under the optimized conditions.Fig.4indicates the large variation of E %and EF with pH.This indi-cates that protonation of the carboxylic group of GA occurs in quite low pH values.In such acidic conditions,GA gets uncharged and is extracted,more efficiently,into the fine droplets of the organic phase.The individual effect of ionic strength was also studied because of its importance in natural samples.The results showed a relatively small influence of ionic strength on the extraction efficiency of GA.The E %increased moderately with increasing the salt concentra-tion showing a flat maximum at about 0.4mol L −1.However,the E %variation in the region from 0.2to 0.5mol L −1was statistically insignificant.A more or less similar,but less significant,trend was observed for EF.3.5.Analytical performanceReplicated experiments in the optimal conditions (i.e.0.8mL acetone,140␮L n -hexanol,0.2mol L −1sodium nitrate,pH 1.3and using the suction and reinjection method)for the extraction of 4.0mL of 2×10−5mol L −1GA resulted in an extraction recovery of 104.1%with a relative standard deviation of 5.1%(for n =5)and an enrichment factor of 54.The calibration curve was linear over a range of 2×10−5to 1×10−3mol L −1GA,with a R 2of 0.999.The detection limit of the method for 3 was calculated to be 2×10−7mol L −1of GA.3.6.The DLLME of licorice samplesThe developed DLLME method was applied for extraction of GA from water extracts of licorice samples collected from three dif-ferent regions of Lorestan province,Iran,with different climate conditions (Table 1).The data,summarized in Table 5,indicate good agreement between the results of the DLLME and direct injections of the samples.It should be noted that for the DLLME of the licorice samples,some precipitate was observed between the two sepa-rated phases after centrifugation.This precipitate was somewhat dispersed in the supernant organic phase when a salt concentration of 0.2mol L −1was used.This problem,however,could be over-come by increasing the salt concentration to 0.4mol L −1.Fig.5compares the chromatograms of a licorice sample,before and after preconcentration by the proposed method,under the optimizedTable 5The results of the DLLME-HPLC analysis of the extracts of three licorice roots under the optimized experimental conditions.Licorice sample GA (mg g −1),DLLME GA (mg g −1),direct E %EF Alashtar11.18(±0.23)11.81(±0.02)94.749Khoramabad 11.28(±0.01)11.80(±0.04)95.651Poldokhtar20.51(±0.72)20.70(±0.15)99.152Fig.5.The chromatograms of the extracts of Khoramabad’s licorice sample,before (a)and after (b)preconcentration by the DLLME method,under the optimized con-ditions.S,stands for the solvent (n -hexanol)peak in chromatogram b.conditions.Evidently,the DLLME method has not only preconcen-trated but also purified the analyte in a considerable extent (note the scales of the vertical axes).It is due to the fact that hydrophilic (ionic)compounds are not extracted by the organic phase.Since in the preliminary extraction by water,lipophilic species has been already removed,only GA and a few other components are pre-concentrated by the DLLME method.Due to the high enrichment factors,the method can be used for the determination of trace levels of GA in licorice samples.Performing a one-way ANOVA test on the results indicated that climate conditions have signifi-cant effect on the GA level.Obtaining an F -ratio of 299indicates the high significance of this effect.The test showed that the GA level in the sample from tropical region was significantly higher than those of the temperate and cold regions.However,there was not a significant difference between the temperate and cold regions.4.ConclusionThe simple DLLME device proposed in this work could be successfully applied for the extractions with lighter than water solvents.The proposed device overcomes the necessity for using toxic chlorinated organic solvents in the ordinary DLLME method and extends,largely,the area of organic solvents applicable in this technique.Application of the proposed method for the extraction of GA from licorice samples was successful and could both pre-concentrate and partially purify the analyte.The proposed method requires much less amounts of the sample and is quite appropriate for trace analysis applications.The DLLME device in this work was used only as a case study,but the proposed device can be easilyP.Hashemi et 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