dispersive liquid-liquid microextraction for determination of organic analytes

Trends Trends in Analytical Chemistry, Vol. 29, No. 7, 2010

Dispersive liquid-liquidmicroextraction for determinationof organic analytesAntonio V. Herrera-Herrera, Marıa Asensio-Ramos, Javier Hernandez-Borges,

Miguel Angel Rodrıguez-Delgado

One of the most important objectives of modern analytical chemistry is miniaturization, simplification and automation of the

whole analytical procedure, especially to speed up sample treatment, which is currently the bottleneck of analysis. Introduction

of dispersive liquid-liquid microextraction (DLLME) has greatly contributed to meeting this objective, due to its simplicity,

rapidity of operation and low consumption of solvents and reagents. DLLME has attracted much interest from scientists working

in separation science. Since its introduction in 2006 for preconcentration of organic analytes from water samples, a good number

of works have reported efficient, quick extraction of organic or inorganic analytes.

However, before using DLLME, there is a need to optimize carefully influential factors (e.g., types and volumes of extraction

and disperser solvents, extraction time, sample amount, pH, and salt addition).

The present review focuses on applications of DLLME for extracting organic analytes (e.g., pesticides, pharmaceuticals,

polychlorinated biphenyls, and polybrominated diphenyl ethers) from the time that DLLME was introduced to the end of

December 2009. We pay special attention to those works that represent an improvement in the technique and the most

challenging applications.

ª 2010 Elsevier Ltd. All rights reserved.

Keywords: Dispersive liquid-liquid microextraction; Endocrine disruptor; Organic analyte; Pesticide; Pharmaceutical; Polychlorinated biphenyl;

Polybrominated diphenyl ether; Preconcentration; Sample treatment; Solvent

Antonio V. Herrera-Herrera,

Marıa Asensio-Ramos,

Javier Hernandez-Borges,

Miguel Angel Rodrıguez-

Delgado*

Departamento de Quımica

Analıtica, Nutricion y

Bromatologıa, Facultad de

Quımica, Universidad de La

Laguna (ULL), Avenida

Astrofısico Francisco Sanchez,

s/n�, 38206 La Laguna

(Tenerife), Spain

*Corresponding author.

Tel.: +34 922 31 80 46;

Fax: +34 922 31 80 03;

E-mail: [email protected]

728

1. Introduction

Current trends in analytical chemistryare highly focused on improvement in thequality of analytical results, introductionof new technological developments withanalytical use and, especially, miniaturi-zation, simplification and automation ofthe whole analytical procedure, bearingin mind as the final objective the con-struction of l-total analysis systems(l-TAS) or lab-on-a-chip designs [1]. Onthe way to achieving that final objective,important advances have been made inminiaturization and simplification ofsample-pretreatment procedures. Theseadvances have focused on minimizingsample and reagent consumption (andthus the cost of the analysis), maintain-ing high selectivity and recoveries, andspeeding up the sample-treatment pro-cess, which is currently considered thebottleneck of analysis.

0165-9936/$ - see front matter ª 2010 Elsev

One of the techniques attracting specialattention is dispersive liquid-liquid mic-roextraction (DLLME), which was intro-duced in 2006 by Rezaee et al. [2] for thepreconcentration of organic analytes fromaqueous matrices. DLLME is generallybased on a ternary component solventsystem, in which extraction and dispersersolvents are rapidly introduced into theaqueous sample to form a cloudy solution.Extraction equilibrium is quickly achieved,due to the extensive surface contact be-tween the droplets of the extraction sol-vent and the sample. After centrifugation,extraction solvent is normally sedimentedat the bottom of the tube (if the density isabove that of water) and taken with amicrosyringe for its later chromatographicanalysis. Fig. 1 shows each of these steps.

In this first work [2], a combination oftetrachloroethene as extraction solventand acetone as disperser solvent wasused to extract polycyclic aromatic

ier Ltd. All rights reserved. doi:10.1016/j.trac.2010.03.016

mailto:[email protected]

http://dx.doi.org/10.1016/j.trac.2010.03.016

Figure 1. Steps involved in a dispersive liquid-liquid microextraction (DLLME) procedure.

Trends in Analytical Chemistry, Vol. 29, No. 7, 2010 Trends

hydrocarbons (PAHs) from water samples. Preliminarystudies were also carried out to examine the ability of thetechnique to extract organochlorine and organophos-phorus pesticides as well as substituted benzene com-pounds (benzene, toluene, ethyl benzene, and xylenes).The procedure was found to be extremely simple, quick,efficient, and with a very low consumption of solvents.Later applications showed that good results were alsoobtained for inorganic analytes [3].

The extraction efficiency of DLLME is influenced byseveral factors (e.g., types and volumes of extraction anddisperser solvents, extraction time, sample amount, pH,and salt addition). The extraction solvent should havedensity greater than that of water (although someapplications of lower-density solvents have also beenproposed) and also a low solubility in water. However,the disperser solvent should be soluble in the extractionsolvent and miscible in water, thus enabling the forma-tion of fine droplets of the extraction solvent in theaqueous phase. Chlorobenzene, carbon tetrachloride,tetrachlorethylene and carbon disulphide are frequentlyextraction solvents, while acetone, methanol, acetoni-trile and ethanol are normally used as disperser solvents.Because most extraction solvents are immiscible withwater, the technique most commonly preferred for thelater analysis of organic analytes is gas chromatography

(GC), while, if high-performance liquid chromatography(HPLC) is chosen, this should be taken into account inorder to exchange the solvent before injection. Theanalysis of inorganic analytes does not normally requiresuch a precaution.

Since the initial introduction of DLLME, severalmodifications of the technique have also providedgood results. In some cases, low-density solvents wereused as extractants and thus, after centrifugation, thefinal extraction drop collected was a ‘‘floating drop’’[4–7], while, if the solution was sufficiently cooledand the solvent had a low melting point, it was easierto collect a ‘‘solid floating organic drop’’ [8–12]. Thiscan be achieved using n-hexane, cyclohexane andcertain long-chain alcohols (e.g., 1-decanol, 1-undec-anol, 1-dodecanol, 1-octanol, and 2-dodecanol). Inother cases, disperser solvents are not introduced,since good, efficient dispersion may also take place notby injection but by dissolving depending on tempera-ture. This last case, which has been called termedtemperature-controlled dispersive liquid-phase mic-roextraction (LPME), is only applicable when the sol-ubility of the extraction solvent in the aqueous samplehas a high dependence on the temperature (smallvariations in temperature greatly change the solubil-ity). In an initial step, the extraction solvent, which is

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normally immiscible at ambient temperature, is addedto the sample. When the temperature is increased,complete dissolution normally takes place, and, after aslowly cooling, a cloudy solution is formed. Finally,after centrifugation, the extraction-solvent droplet iscollected at the bottom of the tube. This variation ofthe technique has been applied using only ionicliquids (ILs) as extractants [13–16], as we show later.In other cases, dispersion is by ultrasound [17] orrepeatedly aspiring and injecting the sample with thedisperser solvent [18,19].

The growing interest in this sample-pretreatmenttechnique can be observed in the increasing number ofarticles published since its introduction. Very recently, areview article by Sarafraz-Yazdi and Amiri [20] provideda general discussion of the different experimental set-upsof LPME procedures (including extraction principles,historical development and performance) without specialfocus on the different application areas of each of them(including DLLME).

Rezaee et al. [21] recently published another reviewarticle dealing with the evolution of the technique bystudying its combination with different analytical tech-niques. Also, Aristidis and Ioannou [3] recently reviewedthe potential use of the technique in inorganic analysis.However, so far, no specific review article has dealt withthe different applications of DLLME for organic analysis.That is why the aim of our work is to discuss the differentapplications of the technique for the extraction of or-ganic analytes {e.g., pesticides, pharmaceuticals, poly-chlorinated biphenyls (PCBs), polybrominated diphenylethers (PBDEs)} from matrices of different composition,focusing on extraction performance and experimentalapproaches. We took special care to include all thearticles published in Journal Citation Research (JCR)journals since DLLME�s introduction (up to December2009) and to detail in tables all the relevant experi-mental conditions for the types of organic analyte. Wealso discuss current trends and future applications.

2. DLLME applications

2.1. PesticidesPesticide analysis is probably the field in which DLLMEhas found its major applications (see Table 1), water ofdifferent types (mainly tap, river, well and lake waters)being the matrix most commonly selected. In few cases,other aqueous matrices {e.g., wines [35] or fruit juices[40,44]} have also been analyzed, as have solid matrices[5,23,26,29,36,38,45,50] to a much lesser extent,probably because of the complexity of the samples andalso because of the need to develop a previous pretreat-ment procedure based mainly on solvent or waterextraction. This is the case for the extraction of pesticidesfrom watermelon and cucumbers [23], soils [26,36,45],

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apples [29], bananas [38], table grapes and plums [50],and tea leaves [5,26].

In the vast majority of the works included in Table 1,the chlorinated solvents used have several problems, themost important being their toxicity. However, theamount of solvents normally used in DLLME is muchreduced, diminishing risks and toxicity, which is a clearadvantage of its use. Although the technique was ini-tially ‘‘designed’’ for solvents with greater density thanthat of water, lower-density solvents have also been usedwith good results. As an example, Farajzadeh et al. [4]used cyclohexane, a non-chlorinated extraction solvent,which allowed the extraction of three organophosphoruspesticides from tap and well waters with extractionrecoveries of 68–105%. A special tube-shaped devicewas developed for this purpose, so that a mixture ofdisperser and extraction solvents (acetone and cyclo-hexane, respectively) was injected into the aqueousphase, giving rise, after centrifugation, to an organicphase that remained upon the aqueous phase. Then, asmall volume of water was injected through a septumlocated in the base of the device, so that the organicphase could be easily collected with a syringe. Enrich-ment factors obtained were in the range 100–150.

Another application was the work of Leong et al. [8],who developed a technique based on solidification of afloating organic drop (DLLME-SFO) for the determinationof six organochlorine pesticides in waters. The experi-mental procedure consisted of adding disperser andextraction solvents (acetonitrile and hexane, respec-tively) to the aqueous solution, so small hexane dropletswere formed. Then, the tube was deposited in crushed iceand the solidified organic solvent drop was transferredinto another receptacle, where it melted. Enrichmentfactors were of the order of 37–872.

It is important to mention that, in some of the worksincluded in Table 1 in which matrices other than waterwere analyzed, the DLLME procedure acted more as a‘‘cleaning step’’ or as a solvent exchanger than as a fullpreconcentration procedure. This takes place when, forexample, the eluate of solid-phase extraction (SPE) con-taining the analytes [35,36,41,56,57] or an organicsolvent that has been used to extract the analytes from asolid matrix [5,23,36,58–63] is mixed with a suitableextraction solvent and injected into an aqueous matrix.The eluate normally acts as disperser solvent. The mainquestion that comes up when this is so is whether theextraction solvent can extract analytes from the dis-perser-solvent extract in the same way as it does fromwater. In this sense, Zhao et al. [23] demonstrated thatthere were no obvious differences when using acetoni-trile and chlorobenzene as disperser and extraction sol-vents, respectively, to extract six organophosphoruspesticides (ethoprophos, parathion methyl, fenitrothion,malathion, chlorpyrifos and profenofos) from water-melon and cucumber. In this case, sample pretreatment

Table 1. Dispersive liquid-liquid microextraction (DLLME) of pesticides

Analyte Matrix Extraction solvent Dispersersolvent

Recoveries Separationtechnique

Comments Ref.

13 Organophosphoruspesticides

River, well and farmwater

Chlorobenzene(12 lL)*

Acetone(1 mL)*

E.R.: 78.9-107%R.R.: 93-118%E.F.: 789-1070-fold

GC-FPD Aqueous phase: 5 mL. Comparison betweenDLLME, SPME and single-dropmicroextraction. LODs: 3-20 pg/mL

[22]


Watermelon andcucumber

Chlorobenzene(27 lL)

Acetonitrile(1 mL)

E.R.: 67-111%R.R.: -E.F.: 41-50-fold

GC-FPD Pretreatment: 10 g sample, add 10 mLacetonitrile, 4 g MgSO4 and 1 g NaCl, shakeand centrifuge. Aqueous phase: 5 mL.DLLME was compared with a conventionalmethod. LODs: 0.5-20 lg/kg

[23]

Methomyl River and lake water Tetrachloroethane(20 lL)*

Methanol(0.5 mL)*

E.R.: -R.R.: 93.1-97.0%E.F.: 70.7-fold

HPLC-UV Aqueous phase: 5 mL, 30% (w/v) NaCl.Compared with other extraction methods(SPE, SPME and LPME), DLLME has widerlinear range, is faster and has lowerprecision. LODs: 1 ng/mL

[24]

10 Chlorobenzenes Tap, well and riverwater

Chlorobenzene(9.5 lL)*

Acetone(0.5 mL)*

E.R.: 71.1-81.3%R.R.: 87-121%E.F.: 711-813-fold

GC-ECD Aqueous phase: 5 mL. LODs: 0.0005-0.05 lg/L

[25]

5 Pyrethroid pesticides Tap, river, reservoirand groundwater

[HMIm][PF6](45 lL)

- (Seecomments)

E.R.: -R.R.: 76.7-135.6%E.F.: -

HPLC-UV Use of IL as the only extraction solvent andtemperature as the driving force for theextraction and phase separation. Aqueousphase: 10 mL, pH 6. LODs: 0.28-0.6 lg/L

[13]


Rain, river, reservoirand groundwater

[HMIm][PF6](50 lL)

- (Seecomments)

E.R.: -R.R.: 88.2-103.6%E.F.: 50-fold

HPLC-UV Use of IL as the only extraction solvent andtemperature as the driving force for theextraction and phase separation. Aqueousphase: 10 mL, pH 5. LODs: 0.17(methylparathion) and 0.29 (phoxim) ng/mL

[14]

6 Organosulfurpesticides

Tap, lake, river, welland farm water, soil,green tea and tealeaves

Carbon tetrachloride(10 lL)*

Methanol(0.8 mL)*

E.R.: -R.R.: 78.5-117.2%E.F.: 176-946-fold

GC-FPD Pretreatment: tea beverage, 50-fold dilution;soil, 0.5 g added to 10 mL water,ultrasonicate, filter and 25-fold dilution; tealeaves, 3 g added to 100 mL water,ultrasonicate, filter and 25-fold dilution.Aqueous phase: 5 mL. Comparison withhollow fiber liquid phase microextraction(HF-LPME). LODs: 0.21-3.05 lg/L

[26]

4 Chlorophenoxyaceticacids

River water Tetrachloroethylene(80 lL)

Tetrahydrofuran(1.92 mL)

E.R.: -R.R.: -E.F.: 131-156-fold

HPLC-UV Aqueous phase: 5 mL, 0.2 M HCl, 3% (w/v)NaCl. LODs: 2.3-3.3 ng/mL

[27]

Metacrate Tap, farm and riverwater

Dichloromethane(116 lL)*

Methanol(0.565 mL)*

E.R.: -R.R.: 96.7-101.8%E.F.: 118-fold

HPLC-UV Use of experimental design. Aqueous phase:6 mL, 8% (w/v) NaCl. LOD: 1 ng/mL

[28]

Captan, folpet andcaptafol

Apples Chlorobenzene(9 lL)

Acetone(1 mL)

E.R.: 76.1-93.0%R.R.: 93.0-109.5%E.F.: 824-912-fold

GC-ECD Pretreatment: 20 g sample, homogenize,centrifuge, filter the supernatant and dilutewith water. Aqueous sample: 5 mL. LODs:3.0-8.0 lg/kg

[29]

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Table 1. (continued)



Comments Ref.

Atrazine Tap andreservoir waterand wastewater


Methanol(0.55 mL)

E.R.: -R.R.: 69.9-89.8%E.F.: -

HPLC-UV Aqueous phase: 10 mL, pH 5, 10% (w/v)NaCl. LOD: 0.601 ng/L

[30]

Triclosan andmethyltriclosan

Tap and riverwater andtreated and rawwastewater

1,1,1,-trichloroethane(40 lL)

Methanol(1 mL)

E.R.: 90-100%R.R.: 81.7-105.4%E.F.: 256 and 231-fold

GC-MS/MS Aqueous phase: 10 mL. Addition of N-methyl-N-(tert-buthyldimethylsilyl)trifluoroacetamide assilylation reagent to produce triclosanderivatization in the extraction step(advisable to improve the sensitivity ofthe method). LODs: 2-5 ng/L

[31]

4 Heterocyclicinsecticides

Tap, lake andfountain water

[HMIm][PF6](0.052 g)

Methanol(0.5 mL)

E.R.: 79-110%R.R.: -E.F.: 209-276-fold

HPLC-DAD Aqueous phase: 5 mL. LOQs: 0.53-1.28 lg/L

[32]

Triclosan,triclocarban andmethyl-triclosan

Reclaimed,irrigating, riverand domesticwater

1,2-dichlorobenzene(15 lL)

Tetrahydrofuran(1 mL)

E.R.: 84.5-95.3%R.R.: 64.3-121%E.F.: -

UHPLC-UV Aqueous phase: 5 mL, pH 7. LODs: 45.1-236 ng/L

[33]

5 Organochlorinepesticides

River, tap, seaand reservoirwater

Tetrachloroethylene(5.2 lL)

Tert-butyl methyl ether(7.8 lL)

E.R.: -R.R.: 54.0-119.4%E.F.:1885-2648-fold

GC-MS Aqueous phase: 10 mL, 3% (w/v) NaCl.The extraction and disperser solventsadded together (13 lL oftetrachloroethylene:tert-butyl methylether 4:6 v/v). LODs: 0.4-2.5 ng/L

[34]

7 Fungicides Red and whitewine

1,1,1,-trichloroethane(100 lL)

Acetone(1 mL)

E.R.: 61-99%R.R.: 78.3-107.3%E.F.: 156-254-fold

GC-ECD and GC-MS

Pretreatment: dilution (1:1 v/v), SPE(Oasis HLB), elution with 1 mL acetone(dispersive solvent). Aqueous phase:10 mL. LOQs: 30-120 ng/L (GC-ECD) and40-250 ng/L (GC-MS)

[35]

4 Sulfonylureaherbicides

Soil Chlorobenzene(60 lL)

Acetone(- see comments)

E.R: 30-55%R.R: 76.3-92.5%E.F.: 102-216-fold

HPLC-DAD Pretreatment: 10 g soil, add 20 mLacetone-0.15 M NaHCO3 (2:8 v/v),shake, filter and DSPE (0.15 g C18/10 mLfiltrate). Aqueous solution: 5 mL, pH 2.Acetone contained in the solution wasused as the disperser solvent. LODs: 0.5-1.2 ng/g

[36]


Melted snow,stream, riverand lake water


Acetonitrile(0.6 mL)*

E.R.: >70%R.R.: 85.58-119.6%E.F.: 100-fold

HPLC-UV Aqueous phase: 10 mL, pH 7. LODs:0.32-0.51 lg/L

[37]

8 Multi-classpesticides

Bananas [HMIm][PF6](88 mg)

Methanol(0.714 mL)

E.R.: 53-97%R.R.: 92-106%E.F.: -

HPLC-DAD Pretreatment: 1 g banana, add 5 mLacetonitrile, 2 g MgSO4, 0.5 g NaCl, 0.5 gsodium citrate tribasic dehydrate and0.25 g sodium hydrogen citratesesquihydrate, shake, evaporate andredissolve. Use of experimental design.Aqueous phase: 10 mL, pH 2.7, 28.9%(w/v) NaCl. LODs:0.320-4.66 lg/kg

[38]

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4 Chlorophenoxy acidherbicides

Tap and riverwater

1,1,2,2-tetrachloroethane(25 lL)

0.1 N NaOHsolution containing5% (v/v) methanol(0.050 mL)(see comments)

E.R.: -R.R.: 80.0-116.9%E.F.: -

UPLC-DAD Use of DLLLME. Aqueous solution:10 mL, 0.2 N HCl, 2.5% (w/v) NaCl.LODs: 0.10-0.95 lg/L

[39]


Tap and lakewater

N-hexane(10 lL)*


E.R.: -R.R.: 82.9-102.5%E.F.:37-872-fold

GC-ECD Method based on solidification of floatingorganic drop. Aqueous phase: 5 mL, 0.8 gNaCl. LODs: 0.011-0.11 lg/L

[8]

24 Multi-class pesticides Apple juice Carbon tetrachloride(100 lL)

Acetone(0.4 mL)

E.R.: 60-105%R.R.: -E.F.: 1-18-fold

GC-GC-MS Aqueous phase: 5 g (�5 mL). LODs: 0.06-2.20 lg/L

[40]

3 Amide herbicides Tap andreservoir water

Carbon tetrachloride(25 lL)

Acetone(1 mL, seecomments)

E.R.: -R.R.: 89-116%E.F.: 6593-7873-fold

GC-MS Pretreatment: 100 mL sample pH 2.0, SPE(MWCNTs), elution with 1 mL acetone(disperser solvent). Aqueous phase: 5 mL,pH 2. LODs: 0.002-0.006 lg/L

[41]

Benomyl Tap and wellwater


N,N-dimethylformamide(0.5 mL)*

E.R.: 87-89%R.R.: 90.0-92.0%E.F.: -

HPLC-FD Aqueous phase: 5 mL, pH 1.5. LOD:3.3 lg/L

[42]


Tap, river andwell water

Cyclohexane(100 lL)

Acetone(2 mL)*

E.R.: 68-105%R.R.: -E.F.: 100-150-fold

GC-FID andGC-MS

Use of extraction solvent lighter thanwater and non-chlorinated. Aqueousphase: 7.5 mL. LODs: 3-4 lg/L (GC-FID)and 0.003 lg/L (GC-MS)

[4]

8 Phenylurea herbicides River, tap andwell water

Carbon disulfide(103 lL)*

Acetone(2 mL)*

E.R.: -R.R.: 86-109%E.F.: 11-118-fold

HPLC-DAD Employment of toluene (45 lL) as co-solvent. Aqueous phase: 5 mL. LODs:0.01-0.5 lg/L

[43]

Chlorotoluron,diethofencarb andchlorbenzuron

Melted snowand tap and lakewater

[HMIm][PF6](65 lL)

- (See comments) E.R.: -R.R.: 86.3-106.5%E.F.: -

HPLC-UV Use of IL as the only extraction solventand temperature as the driving force forthe extraction and phase separation.Aqueous phase: 10 mL, pH 7, 15% (w/v)NaCl. LODs: 0.04-0.43 lg/L

[15]

Carbaryl and triazophos River, sea andtap water andapple, grape andpeach juice

Tetrachloroethane(15 lL)*

Acetonitrile(1 mL)*

E.R.: -R.R.: 80.4-117.9%E.F.: 87 and 276-foldrespectively

HPLC-FD Aqueous phase: 5 mL. LODs: 12.3-16.0 pg/mL

[44]

Carbendazim andthiabendazole

Lake, rain andwell water andsoil

Chloroform(80 lL)


E.R.: 51 and 71%,respectivelyR.R.: 84.0-94.0%(water) and 82.0-93.4% (soil)E.F.: 149-210-fold

HPLC-FD Pretreatment: 20 g soil, add 40 mL HCl0.1 M, shake and adjust pH to 7.0.Aqueous phase: 5 mL, pH 7, 10% (w/v)NaCl. LODs: 0.5-1.0 ng/mL (water) and1.0-1.6 ng/g (soil)

[45]


River, surfaceand tap waterand wastewater

Tetrachloroethylene(10 lL)

Acetone(1 mL)

E.R.:R.R.: 63-120%E.F.: 46-316-fold

GC-MS Aqueous phase: 10 mL. LODs: 1-25 ng/L [46]


Melted snowand everglade,river andreservoir water

[HMIm][PF6](50 lL)

- (See comments) E.R.:R.R.: 87.4-110.0%E.F.: 50-fold

HPLC-UV Use of IL as the only extraction solventand temperature as the driving force forthe extraction and phase separation.Aqueous phase: 10 mL, pH 6. LODs:0.04-0.43 lg/L

[16]


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Comments Ref.


Tap, well, rainand river water

[C8MIm][PF6](35 lL)*

Methanol(1 mL)*

E.R.: >70%R.R.: 87.3-117.6%E.F.: 200-fold

HPLC-UV Aqueous phase: 5 mL. LODs: 0.1-5.0 lg/L [47]

8 Phenylurea herbicides River water Dichloromethane(60 lL)


E.R.: -R.R.: 91.2-104.1%E.F.: 68-126-fold

HPLC-DAD Use of PDLLME (partitioned DLLME) thatimproves the extraction efficiency bychoosing a dispersive solvent that can bepartitioned in the extractant droplets.Aqueous phase: 5 mL, 0.5% (w/v) NaCl.LODs: 0.10-0.28 ng/mL

[48]

4 Carbamate pesticides River, tap, rainand well water

Chloroform(70 lL)*

Acetone(1 mL)*

E.R.: 30-45%R.R.: 84.0-94.0%E.F.: 101-145-fold

HPLC-DAD Aqueous phase: 5 mL. LODs: 0.4-1.0 ng/mL

[49]

8 Multi-class pesticides Table grapesand plums

[HMIm][PF6](88 mg)

Methanol(0.714 mL)

E.R.: 64-100% (tablegrapes) and 58-105% (plums)R.R.: 88-106% (tablegrapes) and 89-105% (plums)E.F.: -

HPLC-DAD Pretreatment: 1 g sample, add 5 mLacetonitrile, 2 g MgSO4, 0.5 g NaCl, 0.5 gsodium citrate tribasic dehydrate and0.25 g sodium hydrogencitratesesquihydrate, shake, evaporate andredissolve. Use of experimental design.Aqueous phase: 10 mL, add 28.9% (w/v)NaCl. LODs:0.651-5.44 lg/kg (tablegrapes) and 0.902-6.33 lg/kg (plums)

[50]

5 Carbamates Rain, surfaceand groundwater

Trichloromethane(40 lL)

Acetonitrile(1 mL)

E.R.: 86.0-97.2%R.R.: -E.F.: 80-117-fold

HPLC-DAD Aqueous phase: 5 mL. LODs: 0.1-0.5 ng/mL

[51]

2 Phenoxyacetic acidherbicides

Tap and wellwater


Acetone(1 mL)*

E.R.: 90.21 and115.23%,respectivelyR.R.: 94.0-102.9%E.F.: -

HPLC-DAD Aqueous phase: 5 mL, pH 1.5, 10% (w/v)NaCl. LODs: 0.16 lg/L

[52]

Atrazine and simazine Tap, reservoirand groundwater


Methanol(0.55 mL)*

E.R.: -R.R.: 60.7-91.4%E.F.: -

HPLC-UV Aqueous phase: 10 mL, pH 5, 10% (w/v)NaCl. LODs: 0.1-0.004 lg/L

[53]


Tea N-hexane(24 lL to the 2 mL initialextraction mixture, seecomments)

Acetonitrile(0.5 mL)

E.R.: 83.3-117.4%R.R.: -E.F.: -

GC-FPD Pretreatment: 1 g powdered tea, add 2 mLof a mixture of acetonitrile and n-hexane(250:3 v/v). Then, 0.5 mL supernatant isadded to the aqueous phase. Aqueousphase: 5 mL. LODs: 0.030-1 lg/kg

[5]

Pentachlorophenol Tap and wellwater

Tetrachloroethylene(15 lL)*

Acetone(1 mL)*

E.R.: -R.R.: 86%E.F.: 90% (wellwater) and 94% (tapwater)

HPLC-DAD Aqueous phase: 5 mL, pH 3.0, 1% (w/v)NaCl. LODs: 0.03 lg/L

[54]

3 Carbamate pesticides Tap, river andrain water


Acetonitrile(1 mL)*

E.R.: 74.2-94.4%R.R.: 84.5-104.4%E.F.: 148-189-fold

HPLC-UV Aqueous phase: 5 mL. LODs:0.1-0.9 lg/L

[55]

E.R.: Extraction recovery; R.R.: Relative recovery; E.F.: Enrichment factor.*In this work disperser solvent is expressed as x mL containing y mL of extraction solvent.

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consisted of adding 10 mL of acetonitrile to 10 g ofsample together with 4 g of anhydrous MgSO4 and 1 g ofNaCl. The mixture was then shaken in a vortex mixerand centrifuged. Regarding the DLLME procedure, 27 lLof chlorobenzene (extraction solvent) were added to1 mL of acetonitrile taken from the previous extractionstep and the mixture was introduced into 5 mL of puri-fied water. After shaking and centrifuging, the sedi-mented chlorobenzene phase was collected and injectedinto the GC-flame photometric detection (GC-FPD) sys-tem. In addition, the method developed was comparedwith a conventional extraction method with acetonitrile,and, despite both methods exhibiting similar extractionrecoveries and precision, DLLME provided 10-foldgreater enrichment factors and better limits of detection(LODs) (0.10–0.19 lg/kg vs. 0.8–2.0 lg/kg).

Another example is the work of Moinfar et al. [5], whodeveloped a similar methodology, also for the extractionof organophosphorus pesticides (phorate, diazinon, dis-olfotane, methyl parathion, sumithion, malathion, fen-thion, profenphose, ethion and phosalone) from tea. Inthis work, 2 mL of acetonitrile:n-hexane (250:3 v/v)were added to 1 g of powdered sample and the mixturewas magnetically stirred for 45 min. Because the DLLMEdisperser-acting solvent (acetonitrile) and the extraction-acting solvent (n-hexane) had already been used to ex-tract the pesticides from the matrix, there was no need tofilter, to evaporate or to change the solvent ratio. Then,an aliquot of the supernatant was quickly introducedinto 5 mL of water, and, after collecting the n-hexanephase, it was injected into the GC-FPD instrument. LODswere 0.03–1 lg/kg and recoveries 83.3–117.4%.

Another strategy developed changed selectivity of theextracting system by employing a co-solvent. Saraji et al.[43] extracted eight phenyl-urea herbicides (tebuthiuron,diuron, propanil, fluometuron, siduron, linuron, thidi-azuron and diflubenzuron) from river, tap and well water.Acetone, carbon disulfide and toluene were used as dis-perser, extraction and co-solvent, respectively. Use of theco-solvent was initially justified as reducing the volume ofthe toxic extraction solvent for DLLME (carbon disulfide inthis case) and being needed to investigate its effects on theextraction efficiency. Despite toluene also having signifi-cant toxicity, the mixture provided a clear improvement ofthe extraction efficiency, leading to an increase of the peakarea of all analytes (more than 200 units for some of thepesticides), with LODs in the range 0.01–0.5 lg/L andenrichment factors 11–118. When developing this type ofprocedure, the density of the extraction/co-solvent mix-ture should also be adequate for collection of the finaldroplet. Despite these good results, no further use of co-solvents for extraction of organic analytes has been re-ported, probably due to the complexity of the system andto the good results achieved by other solvents.

In the past decade, a new type of solvent, IL, has beenintroduced into analytical chemistry as extractant [64].

ILs are low-melting salts that form liquids composedentirely of ions, which have generally been found to beless toxic, less volatile and less contaminating thanconventional solvents. These salts have also been used asextraction solvents in DLLME, although the number ofworks published is still very small [13–19,32,38,47,50,65–67]. Regarding IL-DLLME of pesticides, a rel-atively small number of works has come out, especiallyin recent years [13–16,32,38,47,50]. In most cases,water samples have also been analyzed using HPLC asthe separation technique [13–16,32,47]. We shouldmention that, in four of these works [13–16], no dis-perser solvent was used (only the IL [HMIm][PF6]), sincethe dispersion is temperature-assisted. Temperaturechanges make an IL completely disperse into the aque-ous solution forming extremely small drops that increasethe mass transfer into the IL phase and induce phaseseparation. The first application of this variation of theIL-DLLME technique was the work of Zhou et al. [13],who used temperature-controlled IL-DLLME to extractfive pyrethroid pesticides (cyhalothrin, deltamethrin,fenvalerate, taufluvalinate and biphenthrin) from differ-ent types of water samples (tap, river and reservoir wa-ter, and groundwater). The experimental procedureconsisted of adding 45 lL of the IL [HMIm][PF6] to theaqueous phase (10 mL, pH 6) that contained the ana-lytes and heating up to 70�C in a water bath. At thispoint, the IL was totally dissolved and analytes weresupposed to migrate into the IL phase. Then, the mixturewas cooled in a water-ice bath and it became turbid.After centrifugation, the IL phase could be collected,dissolved in mobile phase and injected into the HPLC-ultraviolet (HPLC-UV) system. This study obtained goodspiking recoveries (76.7–135.6%) and LODs in the range0.28–0.6 lg/L.

The use of an IL in DLLME prior to HPLC requiressuitable dilution of the droplet in the mobile phase –which is not always easily achieved because large vol-umes of organic solvents are normally required. In thesecases, it is important to study the composition of thesample solvent to be injected and its influence on peakefficiency.

The first application of IL-DLLME for the extractionof pesticides from matrices other than water was re-cently developed by Ravelo-Perez et al. [38,50] for theextraction of fruit extracts (bananas [38], and grapesand plums [50]) using HPLC-DAD for the chromato-graphic analysis. In these cases, disperser solventswere used together with the IL. In the first of theseworks [38], parameters affecting the IL-DLLME of eightpesticides (i.e. thiophanate-methyl, carbofuran, carba-ryl, tebuconazole, iprodione, oxyfluorfen, hexythiazoxand fenazaquin) were optimized by means of anexperimental design (central composite design). Theselected parameters were sample pH, NaCl percentage,IL amount ([HMIm][PF6]) and methanol volume (dis-


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Figure 2. High-performance liquid chromatography with diode-array detection (HPLC-DAD chromatograms) of (A) spiked and (B) non-spikedbanana sample after optimum ionic liquid-dispersive liquid-liquid microextraction (IL-DLLME) conditions. Peak identification:(1) thiophanate-methyl (100 lg/kg), (2) carbofuran (20 lg/kg), (3) carbaryl (50 lg/kg), (4) tebuconazol (50 lg/kg), (5) iprodione (20 lg/kg),(6) oxyfluorfen (50 lg/kg), (7) hexythiazox (500 lg/kg), and (8) fenazaquin (200 lg/kg) (Reprinted from [38] with permission from Elsevier).


perser solvent). In general, it is very frequent to findthat DLLME parameters are optimized by means of astep-by-step approach. However, in most cases, a step-by-step optimization is laborious and tedious because itnormally requires a large number of experiments.Furthermore, and more importantly, it does not con-sider possible interactions between factors. The finalprocedure of this work consisted of the ultrasound-


assisted extraction of 1 g of homogenized bananas withacetonitrile using different salts (MgSO4, NaCl, sodiumhydrogen citrate sesquihydrate and sodium citratetribasic dihydrate) to improve the recoveries. Aftercentrifugation and evaporation of the supernatant,reconstitution of the extract in water at pH 2.7 pro-vided the best media to develop the IL-DLLME proce-dure, which used 88 mg of [HMIm][PF6], 714 lL of

Table 2. DLLME of pharmaceuticals

Analyte Matrix Extraction solvent Disperser solvent Recoveries Separationtechnique

Comments Reference

Chloramphenicol Honey 1,1,2,2-tetrachloroethane(30 lL)*

Acetonitrile(1 mL)*

E.R.: -R.R.: 89.5-91.7%E.F.: 68.2-fold

HPLC-UV Pretreatment: 1 g of honey was mixedwith 5 mL of water and vortexed untilhomogeneous sample was obtained.Aqueous phase: 5 mL. LOD: 0.6 lg/kg

[68]

2 Tricyclicantidepressant drugs

Water andhuman plasma


Methanol(1 mL)

E.R.: -R.R.: 54.76-74.02%E.F.: 740-1000-fold

GC-FID Pretreatment: 0.5 mL plasma wasmixed with 1 mL methanol. Aftercentrifugation 0.05 mL were dilutedwith water to 5 mL. Aqueous phase:5 mL, pH 12. LODs: 0.005-0.01 lg/mL

[69]

Clenbuterol River, lake andstream water


Acetone(0.5 mL)*

E.R.: -R.R.: 97%E.F.: 175-fold

HPLC-UV Combined with later semi-automatedin-syringe back extraction. Aqueousphase: 5 mL, NaOH 1 M, 25% (w/v)NaCl. LOD: 4.9 ng/mL

[70]

Chloramphenicol andthiamphenicol

Honey 1,1,2,2-tetrachloroethane(30 lL)*

Acetonitrile(1 mL)*

E.R.: -R.R.: 89.5-93.6%E.F.: 68-88-fold

HPLC-UV Pretreatment: 1 g of honey was mixedwith 5 mL of water and vortexed untilhomogeneous sample was obtained.Aqueous phase: 5 mL. LODs: 0.1-0.6 lg/kg

[71]

7 Quinolones Swine muscle Dichloromethane(300 lL)*


E.R.: -R.R.: 93.0-102.6%E.F.: -

HPLC-DAD Pretreatment: 5 g sample, add 5 mLacetonitrile with 50 lL of 70-72%perchloric acid, 2 g anhydrousMgSO4 and 1 g NaCl. DLLME wascompared with dispersive-micro-SPE.Aqueous phase: 7.5 mL. LODs: 5.6-23.8 lg/kg

[58]

7-aminoflunitrazepam Urine Dichloromethane(250 lL)*

Isopropyl alcohol(0.5 mL)*

E.R: 92.3-103.7%R.R.: -E.F.: 20-fold

HPLC-MS/MS Pretreatment: centrifugation.Aqueous phase: 5 mL, 0.2 Mammonia, 5% (w/v) NaCl. LOD:0.025 ng/mL

[72]

3 Psychotropic drugs Urine Carbon tetrachloride(20 lL)*


E.R.: 96-104%R.R.: -E.F.: -

HPLC-UV Pretreatment: centrifugation.Aqueous phase: 5 mL, pH 10. LODs:3-8 ng/mL

[73]

4 Non-steroidal anti-inflammatory drug

Urine [BMIm][PF6](280 lL)

Methanol(0.720 mL)

E.R.: -R.R.: 99.6-107.0%E.F.: 74-85-fold

HPLC-UV One-step in-syringe DLLME.Aqueous phase: 10 mL, pH 3. LODs:8.3-32.0 ng/mL

[65]

Lovastatin andsimvastatin

Tap, lake andriver water

[HMIm][PF6](500 lL)

- (See comments) E.R.: -R.R.: 80.5-112.0%E.F.: -

HPLC-UV Aqueous phase: 50 mL. Dispersionwas made by an ultrasonic bath andcooling into an ice bath (absence ofdisperser solvent). LODs: 0.17-0.29 ng/mL

[17]


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methanol and 28.9% (w/v) of NaCl (in this case, largeamounts of NaCl were necessary to induce a largesalting-out effect). The combination of these two pro-cedures (acetonitrile extraction of the fruit andreconstitution of the evaporated extract in water)provided a suitable arrangement that allowed appli-cation of DLLME as part of the sample-pretreatmentprocedure for extraction of pesticides from complexsamples (e.g., fruits). Mean recovery percentages were53–97% with RSD values below 8.7%, which repre-sent LODs of 0.320–4.66 lg/kg, well below maxi-mum residue limits of the European Union (EU MRLs).Fig. 2 shows the HPLC-DAD chromatogram of spiked

Figure 3. Comparison of extraction efficiencies from water (A) and muscl(DMSPE) and dispersive liquid-liquid microextraction (DLLME) approaches


and non-spiked banana samples at the maximumabsorption wavelengths of the pesticides after thewhole extraction procedure (including DLLME). As canbe seen, at the beginning of the chromatogram, thereappeared a wide peak, corresponding to the IL – oneof the main drawbacks of using IL-DLLME, asalready reported [14–19,32,38,47,50,65–67] –although, in this case, it did not interfere at all in theseparation.

The second of these works [50] extended the applica-tion of the method for the extraction of the same groupof pesticides from grapes and plums. Mean recoveryvalues were 64–100% for table grapes and 58–105% for

e matrix (B) processed using dispersive-micro-solid-phase extraction(Reprinted from [58] with permission from Elsevier).


plums, with LODs very close to the previous ones, alsobelow EU MRLs.

In both works, the method was also applied to theanalysis of commercial fruit samples, in which residuesof pesticides could be found in some, but always belowtheir MRLs.

2.2. PharmaceuticalsAnother important group of analytes that have been theobjective of DLLME procedures are pharmaceuticals (seeTable 2) {e.g., antibiotics [58,68,71], tricyclic antide-pressant drugs [69], clenbuterol [70], hypnotic drugs[72], psychotropic drugs [73], anti-inflammatory drugs[65] and cholesterol lowering agents [17]}. As can beseen, not only water samples have been analyzed[17,69,70] but also more complex ones {e.g., honey[68,71], urine [65,72,73] and pig muscle [58]}.

Chen et al. [68] applied DLLME to determine chlor-amphenicol, a broad-spectrum antibiotic banned byinternational legislation, in honey. For suitable methoddevelopment, it was necessary to dilute the honey withwater and to vortex the solution until it became homo-geneous. The high viscosity of the initial sample pre-cluded the formation of the droplets, so dilution was thebest approach. A mixture of acetonitrile and 1,1,2,2-tetrachloroethane (as extraction solvent) was injectedinto an aliquot of 5 mL of the homogeneous dilutedhoney sample. After centrifugation, the final extractiondroplet was analyzed by HPLC-UV. Mean relativerecoveries were in the range 89.5–91.7% with RSD lessthan 5.1%.

The proposed DLLME method was compared with LLE[74] and SPE [75] procedures, which were previouslydescribed in the literature to extract chloramphenicolfrom foods (meat, seafood, egg, honey, milk, plasma andurine) and a biological sample (honey), respectively. Interms of enrichment factors, DLLME was found to bebetter than LLE, but slightly worse than SPE. Concerningextraction time and solvent saving, DLLME was found tobe superior in both cases. In a later work of the samegroup, the methodology was extended to the simulta-neous extraction of chloramphenicol and its analoguethiamphenicol [71], for which relative recoveries were89.5–93.6% and RSD less than 6.3%.

Regarding the extraction of pharmaceuticals fromsolid matrices, Tsai et al. [58] used a modified DLLMEmethod combined with HPLC-DAD to determine quino-lones in pig muscle. About 5 g of tissue were extractedwith acetonitrile (containing 70 lL of 70–72% per-chloric acid), which was used as disperser solvent. In thiscase, 300 lL of dichloromethane were added and themixture was quickly introduced into 7.5 mL of deionizedwater. In this case, DLLME was used as more a cleaningstep than an extraction procedure. The effect of bothextraction solvent volume and pH of water was investi-gated. Increase of dichloromethane volume resulted in

higher extraction recoveries, but the cloudy suspensionof droplets may not be well formed, and the ternarycomponent system should be vortex mixed. However,larger volumes of dichloromethane resulted in largervolumes of the settled phase but also in decreases in theenrichment factor. By contrast, water pH was found tohave little influence on the partition behavior of quino-lones. The method developed achieved relative recoveriesgreater than 93%. From Fig. 3, it is clear that absoluterecovery values for the water samples were higher thanthose for muscle tissue. Apart from that, dispersivemicro-SPE (DMSPE) was also applied to the acetonitrileextract and both methods were compared. Relativerecoveries obtained with DMSPE were also in the range96.4–105.4%. Results showed that both procedureswere comparable and could be used to determine quin-olones in muscle with confidence (Fig. 3).

Regarding the use of IL-DLLME, Mao et al. [17]developed the only work in which dispersion was by anultrasonic bath to determine cholesterol-lowering agents(lovastatin and simvastatin) in water. HPLC-UV was usedfor analyte quantification. Of water, 50 mL (approxi-mately 10 times the volume most commonly used inDLLME procedures) were mixed with 0.50 mL of[HMIm][PF6] and sonicated for 400 s. Then, the samplewas cooled into an ice bath for 20 min and gentlyshaken. Ultrasonic initial temperature, volume of IL,water pH, cooling time and salt effect were investigated.Under the optimum extraction conditions, the methodprovided LODs of 0.17 ng/mL for lovastatin and 0.29 ng/mL for simvastatin with relative recoveries of 80.5–112.0%. The use of IL in DLLME resulted in the presenceof a wide peak at the beginning of the chromatogram, asalso happened on other occasions for different types ofanalytes. However, it did not interfere with correctidentification and quantification of the analytes.

2.3. PCBs and PBDEsDLLME has also been applied to the extraction of PCBsand PBDEs, which are among the most widespread,persistent environmental pollutants. Table 3 showsdetails of the articles published regarding these analytes.Both types of compounds are lipophilic, so they have avery low solubility in water. So far, the technique hasbeen used for the determination of PCBs by HPLC-UV[76–78] and PBDEs by GC-ECD [56,59,60,72] and GC-MS [61]. From Table 3, it is clear that only on a fewoccasions were there analyses of complex samples {e.g.,soils [60] or aquatic organisms in which bioaccumula-tion of these analytes may occur [59,61]}. Chloroben-zene [59–61,77] and tetrachloroethane [56,76,78] werepreferred in all cases as extractant solvents (volumes lessthan 35 lL), while acetonitrile [56,76] and acetone [59–61,77] were used as disperser solvent, except in one casein which tetrahydrofurane was used [78]. Concerningthe application of IL-DLLME, PBDEs and PCBs have yet to


Table 3. DLLME of PCBs and PBDEs



Comments Reference

4 Polybrominated diphenylethers

Tap, lake water andlandfill leachate

Tetrachloroethane(20 lL)

Acetonitrile(1 mL)

E.R.: -R.R.: 87.0-119.1%E.F.: 268-305-fold

HPLC-UV Aqueous phase: 5 mL. LODs: 12.4-55.6 pg/mL

[76]

10 Polychlorinatedbiphenyls

Well, river andseawater samples


Acetone(0.5 mL)*

E.R.: -R.R.: 92-114%E.F.: 378-540-fold

GC-ECD Aqueous phase: 5 mL. LODs: 1.0-2.0lg/L

[77]

Decapolybromodiphenylether

Tap, lake and riverwater

Tetrachloroethane(22 lL)

Tetrahydrofuran(1 mL)

E.R: 89.9-95.8%R.R.: -E.F.: 153-fold

HPLC-UV Aqueous phase: 5 mL. LOD: 0.2 ng/mL [78]


Well, river, sea,leachate, and cloversamples

1,1,2,2-tetrachloroethane(22 lL)*

Acetonitrile(1 mL)*

E.R.: -R.R.: 62.1-110.4%E.F.: 6838-9405-fold

GC-ECD Pretreatment: 1.0 g of dried plant wasmixed with 4 g anhydrous MgSO4,extracted with 10 mL hexane/acetone(1:1 v/v), evaporated, redissolved withacetonitrile and diluted to 100 mL withMilli-Q water. Later SPE and elution with1 mL acetonitrile (disperser solvent).Factors influencing SPE (i.e. flow rate ofthe sample solution, breakthroughvolume, ionic strength, elution solventvolume and type) were optimized.Aqueous phase: 5 mL. LODs: 0.03-0.15 ng/L

[56]

4 Polychlorinated biphenyls Fish: perch, pomfretand yellow-fin tuna


Acetone(1 mL)*

E.R.: -R.R.: 81.2-108.4%E.F.: 87-123-fold

GC-ECD Pretreatment: 1 g of sample was mixedwith 3 g anhydrous sodium sulfate andextracted with 10 mL acetone. 1 mL ofacetone extract was mixed withchlorobenzene and then injected into5 mL of water. Aqueous phase: 5 mL.LODs: 0.12-0.35 lg/kg

[59]

5 Polychlorinated biphenyls Soil Chlorobenzene(30 lL)

Acetone(1 mL)

E.R.: -R.R.: 82.30-113.6%E.R.: -

GC-ECD Pretreatment: 1.0 g of soil was extractedwith 10 mL acetone. 1.0 mL of acetoneextract was mixed with chlorobenzeneand then 1.0 mL of mixture was injectedinto 5 mL of water. Aqueous phase: 5 mL.LODs: 0.20-0.50 lg/kg

[60]


Aquatic animaltissue: frog, snailand fish


Acetone(0.75 mL)*

(See comments)

E.R.: 95.0-123.6%R.R.: 75.3-127.8%E.F.: -

GC-MS Pretreatment: the muscle tissue wasfrozen (cleanup by freezing), mechanicalextracted (acetone and anhydrous sodiumsulfate) and frozen again (-80 �C), then0.75 mL supernatant (acetone) wassubjected to DLLME. Aqueous phase:5 mL. LODs: 2.4-4.9 lg/kg

[61]


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be studied, even though conventional solvents havedemonstrated good recovery values.

Hu et al. [59] determined a group of PCBs in perch,pomfret and yellow-fin tuna. For this purpose, 1 g offish was weighed, mixed with 3 g of anhydrous Na2SO4

and extracted with 10 mL of acetone by shaking for30 min. The solution was stored overnight at �80�C todeposit lipids. An aliquot of 1 mL of this acetone extract(containing 30 lL of chlorobenzene) was injected into5 mL of water. After centrifugation, the sedimentedphase was transferred to a vial, evaporated by nitrogenand redissolved in n-hexane for injection into the GCsystem. The method allowed the analysis of PCBs in fishat trace levels with LODs in the range 0.12–0.35 lg/kg.Factors affecting microextraction efficiency (e.g., thekinds and the volumes of extraction and disperser sol-vents and salt effect) were optimized by a step-by-stepapproach, as in the rest of the works shown in Table 3that extracted PCBs and PBDEs by DLLME [56,59–61,76–78]. Later, Liu et al. [61] applied the samemethod to determine PBDEs in frog, snail and fish using4 g of anhydrous Na2SO4, 33 lL of chlorobenzene and0.75 mL of acetone extract. The LODs were slightlyhigher (2.4–4.9 lg/kg).

Regarding the extraction of PBDEs by DLLME, it isworth pointing out the work of Liu et al. [56] for theiranalysis of well water, river water, seawater, leachate,and clover samples. In this case, an SPE-DLLME-GC-ECDprocedure obtained LODs at the ng/L level. In the caseof solid samples, about 1 g of dried plant was mixedwith 4 g of anhydrous MgSO4 and extracted with10 mL hexane/acetone (1:1 v/v). Then, the extract wasevaporated and redissolved in acetonitrile and diluted to100 mL with Milli-Q water. After concentration andpurification by SPE, 1 mL of elution solvent (acetoni-trile) was mixed with the extractant solvent (22 lLtetrachloroethane) and injected into 5 mL of water.Factors influencing SPE (i.e. flow rate of the samplesolution, breakthrough volume, ionic strength, elution-solvent volume and type) were optimized. Relativerecoveries, enrichment factors and LODs were in theranges 62.1–110.4%, 6838–9405 and 0.03–0.15 ng/L,respectively.

2.4. Other applicationsDLLME has also been applied to determine other organiccompounds, mainly contaminants, in diverse matrices.Table 4 shows recent applications. So far, these com-pounds, together with those indicated in previous tables,are the only organic analytes that have been extracted byDLLME. Endocrine disrupters [10,18,57,59,80–83],antioxidants [62,84], PAHs [9,11,66,67,85,86] andother organic compounds of different natures [7,8,63,87–104] have been analyzed by DLLME, IL-DLLME andDLLME-SFO. In most of the works in Table 4, extractionparameters were again optimized by a step-by-step

approach [7–9,11,18,19,57,66,67,79–83,85–89,91–100,102,104]; however, efficient experimental designmethodologies have also been carried out, but to a lesserextent [10,62,63,84,90,97].

Endocrine disrupters are a widespread group of sub-stances of different natures that can interfere with thefunction of the endocrine system and produce neuro-logical and reproductive failures. For their extraction,DLLME is an alternative to traditional methods. Sincesewage-treatment and effluent-treatment plants consti-tute sources of endocrine-disrupter pollution, waters,mainly tap, river and well water, have been analyzed.Chlorinated solvents have been used as extractant sol-vents (chlorobenzene, carbon tetrachloride and chloro-form), except in two works where IL [BMIm][PF6] [18]and 1-decanol [10] were used.

It is worth mentioning the work of Fattahi et al. [57],in which 19 chlorophenols were analyzed by GC-ECD inwater samples, combining SPE (with Bond Elute PPLsorbent) and DLLME. Concerning the extraction proce-dure, after passing 100 mL of water through the SPEcartridge, elution was with 1 mL of acetone (that wouldlater act as disperser solvent). Afterwards, 13 lL ofchlorobenzene as extraction solvent and 50 lL of aceticanhydride (derivatization reagent) were added and then5 mL of the aqueous phase containing 0.5% (w/v)K2CO3 were also poured. Extraction recoveries were 25–83%. Fig. 4 shows the chromatograms corresponding tonon-spiked (A) and spiked (B) river-water samples ob-tained after the SPE-DLLME-GC-ECD methodology. Thiscombination of SPE and DLLME with GC-ECD providedenrichment factors of 4390–17870 and better LODsthan other methods proposed in the literature (usingSPE-GC-ECD, SPME-GC-MS and LPME-GC-MS), includinganother similar work developed by the same authors inwhich only DLLME was applied to extract the samegroup of chlorophenols from waters [79]. The authorsalso claimed that the method was valid for separationand preconcentration of chlorophenols from salinesolution up to 10% (w/v) due to the insignificant effectthat ionic strength had on the extraction recoveries. Oneof the most interesting features of this last work [79] isthat simultaneous extraction and derivatization byacetylation of the chlorophenols took place, which wasalso an important advantage of the technique. Acetyla-tion is one of the procedures widely employed to convertchlorophenols into less polar compounds and it increasesthe extraction efficiency and improves GC peak shapes.Apart from this work, several others also simultaneouslycombined a derivatization reaction with DLLME[35,79,83,88,89,99,100].

Also regarding analysis of endocrine disrupters, Lopez-Darias et al. [10] extracted bisphenol A, 4-cumylphenol,4-tertbutylphenol, 4-octylphenol and 4-n-nonylphenolfrom seawater using 1-decanol as extractant solvent andacetonitrile as disperser solvent (150 lL of mixture


able 4. Other applications of DLLME

nalyte Matrix Extraction solvent Dispersersolvent


Comments Reference

ndocrine disrupters9 Chlorophenols Well, tap and

river waterChlorobenzene(10 lL)*

Acetone(0.5 mL)*

E.R.: 28.7-90.6%R.R.: 80.8-117.9%E.F.: 287-906-fold

GC-ECD Simultaneous derivatization (aceticanhydride). Aqueous phase: 10 mL, 0.5%w/v K2CO3. LODs: 0.010-2.0 lg/L

[79]

9 Chlorophenols Well, tap andriver water


Acetone(1 mL,seecomments)

E.R.: 25-83%R.R.: 71-121%E.F.: 4390-17870-fold

GC-ECD Pretreatment: SPE (Bond Elute PPL,elution with 1 mL acetone). Simultaneousderivatization (acetic anhydride).Aqueous phase: 5 mL, pH 2.5, 0.5% w/vK2CO3. LODs: 0.0005-0.1 lg/L

[57]

Phthalate esters Tap, mineraland river water

Chlorobenzene(9.5 lL)*

Acetone(0.5 mL)*

E.R.: 68.1-88.9%R.R.: 81-117%E.F.: 681-889-fold

GC-MS Aqueous phase: 5 mL. LODs: 2-8 mg/L [80]

Phthalate esters Lake, tap andmineral water


Acetone(0.75 mL)*

E.R.: 84-113%R.R.: -E.F.: 45-196-fold

HPLC-UV Aqueous phase: 5 mL. LODs: 0.64-1.8 ng/mL

[81]

isphenol A River and tapwater

Chloroform(142 lL)

Acetone(2 mL)

E.R.: -R.R.: 93.4-98.2%E.F.: -

HPLC-UV Aqueous phase: 10 mL. LOD: 0.07 lg/L [82]

Phenols Tap, river andwaste water

[BMIm][PF6](50 lL)

- (Seecomments)

E.R.: 94.9-108.2%R.R.: -E.F.: -

HPLC-UV The sample solution was mixed with theIL and dispersion was made by aspirationof 1 mL with syringe and injection intoremaining solution three times. Aqueousphase: 1.5 mL. LODs: 0.68-10 lg/L

[18]

isphenol A Tap andreservoir water


Acetone(0.5 mL)*

E.R.: 84.6-95.9%R.R.: -E.F.: -

GC-MS Simultaneous derivatization (aceticanhydride). Aqueous phase: 5 mL, 0.5%(w/v) K2CO3. LOD: 0.01 lg/L

[83]

Phenols Seawater 1-decanol(See comments)

Acetonitrile(See comments)

E.R.: -R.R.: 85.5-118%E.F.: 123-175-fold

HPLC-UV 150 lL of acetonitrile:decanol (15.7:1 v/v) were used. Comparison with single-drop microextraction and DLLME wasmore efficient for the determination in seawater. Use of experimental design.Aqueous phase: 5 mL. LODs: 0.2-1.6 ng/mL.

[10]

ntioxidantsrganox 1010,rganox 1076 andrgafos 168

Tap water Carbon tetrachloride(40 lL)*

Acetonitrile(2 mL)*

E.R.: -R.R.:78-110%E.F.: 64-220-fold

HPLC-DAD Comparison between step by stepapproach and experimental design tooptimize the microextraction and similarexperimental conditions were obtained.Aqueous phase: 5 mL. LODs: 3-7 ng/mL

[84]

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6

3

B

4

B

5

AIII

Irganox 1010and Irgafos 168

Polyolefinspolymers

Carbon tetrachlorid(200 lL)

Acetonitrile(2 mL)

E.R.: 74.8-102%R.R.: -E.F.: -

HPLC-UV Pretreatment: 10 mg of polymer wereixed with acetonitrile and carbon

etrachloride. The mixture was heated 3 ht 100 �C. After cooling and filtering,mL of water were injected in the

olution. Comparison between step bytep approach and experimental design toptimize the microextraction and similarxperimental conditions were obtained.ODs: 6-16 mg/kg

[62]

PAHs16 PAHs River, well and

surface waterTetrachloroethene(8.0 lL)*

Acetone(1 mL)*

E.F.: -R.R.: 82-111%E.F.:605-1060-fold

GC-FID he capability of DLLME for thextraction of some organic compoundsi.e. organochlorine pesticides,rganophosphorus pesticides andubstituted benzene compounds) waslso investigated. Aqueous phase: 5 mL.ODs: 0.007-0.030 lg/L

[85]

12 PAHs Tap, well, creekand river water

[BMIm][NTf2](See comments)

- (Seecomments)

E.R.: 84-115%R.R.: -E.F.: 184-935-fold

HPLC-UV he IL was formed in situ by a metathesiseaction. Water spiked with analytes wasixed with 38 lL BMIm-Cl. Later, IL wasispersed and dissolved and then anqueous solution of LiNTf2 (417 lL,.2 mg/L) was added. Comparison withraditional IL-DLLME and DI-SDME, theroposed method providing highernrichment factors. Aqueous phase:0 mL. LODs: 0.02-34.5 lg/L

[66]

4 PAHs Tap water andsnow

1-undecanol(20 lL)

Acetone(0.48 mL)

E.R.: -R.R.: 94.6-105.9%E.F.: 87-290-fold

GC-FID omparison with liquid phaseicroextraction based on solidification ofoating organic drop. DLLME was foundetter in the extraction time. Aqueoushase: 8 mL. LODs: 0.10-0.35 lg/L

[9]

8 PAHs Tap, river andwell water,grape and applejuice

1,1,2,2-tetrachloroethane(16 lL)

Acetone(1 mL)*

E.R.: -R.R.:74.9-123.0%E.F.: 296-462-fold

HPLC-FD queous phase: 5 mL. LODs: 0.001-.01 lg/L

[86]

18 PAHs Tap, bottled,fountain, well,river, rainwater,and treated andraw water

[OMIm][PF6](50 lL)*

Acetone(1 mL)*

E.R.: 90.3-103.8%R.R.: 30.4-103.9%E.F.: -

HPLC-FD ce-water bath for 2 min beforeentrifugation. Comparison with LLE andfficiency of LLE was affected in minorxtension by the nature of water samplesqueous phase: 10 mL, 10% (v/v)-propanol. LODs: 0.03-2.0 ng/L

[67]


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Te(osaLTrmda0tpe1CmflbpA0

IceeA2




Comments Reference

4 PAHs Waste, lake,undergroundand tap water

1-dodecanol(100 lL)

Methanol(0.2 mL)

E.R.: 88-108%R.R.: -E.F.: 88-118-fold

HPLC-UV Before centrifugation, the drop wassolidified by an ice-bath. Comparisonwith DLLME using 100 lLtetrachloromethane as extraction solventand with LPME. LPME provided the worstresults. Aqueous phase: 10 mL. LODs:0.045-1.1 ng/mL

[11]

Amines and nitrogenous compounds4 Anilines River and lake

waterTetrachloromethane(25 lL)*

Methanol(0.5 mL)*

E.R.: -R.R.: 85.4-111.7%E.F.: 41-95-fold

HPLC-UV Aqueous phase: 5 mL, 20% (w/v) NaCl.LODs: 0.8-1.8 ng/mL

[87]

4 Anilines Stream water Chlorobenzene(8.75 lL)*

Acetone(0.5 mL)*

E.R.: -R.R.: 69-94%E.F.: 212-645-fold

GC-MS Simultaneous derivatization(pentafluorobenzaldehyde). Aqueousphase: 5 mL, pH 4.6. LODs: 0.04-0.09 lg/L

[88]

4 Anilines Tap, river andwastewater

[BMIm][PF6](50 lL)

-(See comments)

E.R.: -R.R.: 93.4-106.4%E.F.: 31-269-fold

HPLC-UV Aqueous solution mixed with IL, then apart is aspirated by a syringe andreinjected in the remaining solution.Aqueous phase: 1.8 mL, pH 7, 12% (w/v)NaCl. LODs: 0.45-2.6 lg/L

[19]

3 Biogenic amines Rice wine 1-octanol(50 lL)


E.R.: -R.R.: 95.42-104.56%E.F.: -

HPLC-FD Pretreatment: addition of trichloroaceticacid, centrifugation, defatting (hexane)and adjust pH 8. Simultaneousderivatization (2,6-dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester). Use ofultrasound-assisted DLLME. Aqueousphase: 600 lL. LODs: 0.02-5 ng/mL

[89]

5 Nitroaromaticcompounds

Well andwastewater


Methanol(0.75 mL)*

E.R.: -R.R.: -E.F.: 202-314-fold

GC-FID Use of experimental design. Aqueousphase: 9 mL, 3% (w/v) NaCl. LODs: 0.09-0.5 lg/L

[90]

3 Mononitrotoluenes Tap, river andwell water



E.R.:R.R.:E.F.: 351-357-fold

GC-FID Aqueous phase: 5 mL. LODs: 0.5 lg/L [91]

Other compounds2 Volatile phenols Red wine Carbon tetrachloride

(50 lL)*Acetone(1 mL)*

E.R.: -R.R.: -E.F.: -

GC-MS Aqueous phase: 5 mL, pH 8. LODs: 28 (4-ethylguaiacol) and 44 (4-ethylphenol)lg/L

[92]

10 Organophosphorusflame retardants andplasticizers

Tap and riverwater andtreated and rawwastewater

1,1,1-trichloroethane(20 lL)

Acetone(0.98 mL)

E.R.: 23-109%R.R.: -E.F.: 190-830-fold

GC-NPD Comparison with SPME. Aqueous phase:10 mL, 20% (w/v) NaCl. LODs: 0.01-0.08 ng/mL

[93]

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4 Trihalomethanes Drinking water Carbon disulfide(20 lL)*

Acetone(0.5 mL)*

E.R.: -R.R.: 92.2-107.8%E.F.: 116-355-fold

GC-ECD Aqueous phase: 5 mL. LODs: 0.005-.040 lg/L

[94]

Butyl and phenyltincompounds

Sea and riverwater

Carbon tetrachloride(11.5 lL)*

Ethanol(0.5 mL)*

E.R.: -R.R.: 82.5-104.7%E.F.: 825-1036-fold

GC-FPD revious derivatization (NaBEt4).queous phase: 5 mL, pH 5. LODs: 0.2-ng/L

[95]

5 Halogenated organiccompounds

Tap and lakewater

2-dodecanol(10 lL)*

Acetone(0.5 mL)*

E.R.:R.R.: 82-101% (GC-ECD) and 81-102% (GC-MS)E.F.: 174-246-fold (GC-ECD) and 228-322-fold(GC-MS)

GC-ECD anGC-MS

ethod based on solidification of floatingrganic drop. Aqueous phase: 5 mL.ODs: 0.005-0.05 lg/L (GC-ECD) and.005-0.047 lg/L (GC-MS)

[8]

Calcium stearate (afterits conversion to stearicacid)

Polymericmatrix (mesh25-60)


- E.R.: 98.5-102%R.R.: -E.F.: -

GC-FID retreatment: extraction (HCl in 2-ropanol). Aqueous phase: 7.5 mL. LOD:5 mg/L

[96]

21 Water solublecomponents (11terpenes and 10terpenoids

Rose water Chloroform(37 lL)

Ethanol(0.42 mL)

E.R.: 72-117%R.R.: -E.F.: 231-378-fold

GC-MS se of experimental design. Aqueoushase: 2 mL. LODs: 0.001-1.121 lg/L

[97]

Cholesterol Milk, egg yolkand olive oil


Ethanol(0.8 mL)*

E.R.: 97.3%R:R.: 95.0-105%E.F.: -

HPLC-UV retreatment: suspend 0.1 g sample inater, centrifuge, take upper phase and

reat with acetonitrile centrifuge and takepper aqueous phase (yolk); centrifugeample, treat with acetonitrile centrifugend take the upper aqueous phase (milk);one (olive oil). Aqueous phase: 4 mL,H 8.5. LOD: 0.01 lg/L

[98]

3 Fatty acids Tap, lake andmodel sea water


Acetone(0.96 mL)

E.R.: -R.R.: 90-110%E.F.: -

GC-FID imultaneous derivatization (ethylhloroformate in ethanol:pyrimidine 4:1/v). Aqueous phase: 4 mL. LODs: 0.67-4.5 lg/L

[99]

Hexanal and heptanal Human blood(serum)

Tetrachloromethane(50 lL)


E.R.: -R.R.: 94-110%E.F.: 63 (hexanal) and73 (heptanal) -fold

HPLC-APCMS

retreatment: centrifugation.imultaneous derivatization (2,4-initrophenylhydrazine). Comparisonith SPE and polymer monolithicroextraction (PMME). Aqueous phase:65 lL, 1.08% (w/v) NaCl, pH 3.0.ODs: 0.17 (hexanal) and 0.076heptanal) nmol/L

[100]


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d MoL0

Pp1Up

PwtusanpScv1

I-MS- PSdwm8L(




ts Reference

5 Personal careproducts

Sea, river andlake water


Methanol(0.62 mL)*

E.R.: 69.4-98.4%R.R.: 60.3-92.9%E.F.: -

GC-MS xperimental design. AqueousmL. LODs: 8-63 ng/L

[101]

EDTA Tap andwastewater


Acetone(0.5 mL)*

E.R.: � 95%R.R.: -E.F.: -

HPLC-DAD phase: 7 mL, pH 2.0. LOD: [102]

Methyl tert-butylether

Distilled, tapand well water



E.R.: -R.R.: 97.4-98.8%E.F.: -

GC-FID phase: 7 mL. LOD: 0.1 lg/L [103]

13 Aromatichydrocarbons

Eluates ofpyrolysis solidresidues


Acetone(0.5 mL)

E.R.: 60.6-113.9%R.R.: -E.F.: -

GC-MS ent: pyrolysis of a mixture ofmass and plastics, extractionmethane) and leaching.son with HS-SPME and HS.n done with HS-GC-MS.phase: 5 mL. LODs: 1.02-

L (benzene was not detected)

[104]

Oleuropein Olive�sprocessingwastewater andolive leavesextract

Water, pH 5.0(40 lL)

Ethyl acetate(1.4 mL)

E.R.: 96.2-109.2%R.R.: -E.F.: 23-42-fold

HPLC-UV ent: ultrasound-assistedn with ethyl acetate (leaves);LE with ethyl acetate

ater). RP-DLLME with 5.3 mL ofane. Use of experimental design.

02 lg/L (wastewater) andmg/kg (leaves)

[63]

Glycyrrhizicacid

Licorice root n-hexanol(140 lL)

Acetone(0.8 mL)

E.R.: 104.1%R.R.: 94.7-99.1%E.F.: 54-fold

HPLC-UV ent: double ultrasound-assistedn with water. Use of extractionighter than water. AqueousmL, pH 1.3 and 0.2 M NaNO3.

· 10�7 M

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Commen

Use of ephase: 5

Aqueous1.7 lg/L

Aqueous

Pretreatmpine bio(dichloroCompariValidatioAqueous24.6 ng/Pretreatmextractiodouble L(wastewcyclohexLOD: 0.2 · 10�3

Pretreatmextractiosolvent lphase: 4LOD: 2

Figure 4. Chromatograms of (A) river water and (B) spiked river water at the concentration level of 10.00 lg/L for monochlorophenols,5.00 lg/L for dichlorophenols, 0.200 lg/L for trichlorophenols and 0.100 lg/L for tetrachlorophenols and pentachlorophenol, obtained usingsolid-phase extraction with dispersive liquid-liquid microextraction (SPE-DLLME) combined with gas chromatography with electrochemicaldetection (GC-ECD). Peak identification: (1) 2-chlorophenol, (2) 3-chlorophenol, (3) 4-chlorophenol, (4) 2,6-dichlorophenol, (5) 2,4- or2,5-dichlorophenol, (6) 3,5-dichlorophenol, (7) 2,3-dichlorophenol, (8) 3,4-dichlorophenol, (9) 2,4,6-trichlorophenol, (10) 2,3,6-trichlorophenol,(11) 2,3,5-trichlorophenol, (12) 2,4,5-trichlorophenol, (13) 2,3,4-trichlorophenol, (14) 3,4,5-trichlorophenol, (15) 2,3,5,6-tetrachlorophenol,(16) 2,3,4,6-tetrachlorophenol, (17) 2,3,4,5-tetrachlorophenol, (18) pentachlorophenol, and internal standard (I.S.) 2,4,6-tribromophenol.Concentration of I.S., 1.00 lg/L (Reprinted from [57] with permission from Elsevier).


(15.7:1 v/v)). Optimum extraction parameters were ob-tained with an experimental design. Under these optimumconditions, relative recoveries were 85.5–118% andenrichment factors were 123–175. They also comparedthe method developed with an SDME method. DLLME wasmore efficient with lower LODs (down to 0.2 ng/mL) andhad better extraction recoveries than SDME.

Concerning the application of DLLME to antioxidants,Farajzadeh et al. [84] developed a method to determine

in water Irganox 1010, Irganox 1076 and Irgafos 168(antioxidants used as plastic additives). Some 5 mL ofsolution were extracted with 40 lL of carbon tetrachlo-ride. After centrifugation, the sedimented phase (about30 lL) was evaporated and dissolved in 50 lL ofmethanol. Two optimization approaches (one variable attime and response surface modeling) were compared andthe best results were obtained with the experimentaldesign. Enrichment factors about 200 and recoveries of



nearly 100% were attained in this study. DLLME wascompared with other sample-preparation techniques.LLE provided lower enrichment factors and higher sol-vent consumption, whereas SPE gave higher enrichmentfactors but was also more time consuming. When SPEwas compared with DLLME in terms of consumption oforganic solvents, it was clear that DLLME was better.Later, the same group extended the method to extractIrganox 1010 and Irgafos 168 from polyolefin polymers[62]. In that work, 10 mg of powdered standard polymerwere mixed with 2 mL of acetonitrile and 200 lL ofcarbon tetrachloride. The mixture was heated for 3 h at100�C. After cooling and filtering, 5 mL of water wereinjected in the solution. Once more, a step-by-step ap-proach and an experimental design were used to obtainexperimental conditions, achieving similar values inboth cases.

PAHs have been extracted from waters and fruit juicesby DLLME, as can also be seen in Table 4. Chlorinatedsolvents [85,86], undecanol [11], dodecanol [9], and ILs[66,67] have been used as extractant solvents. Yao et al.[66] developed an IL-DLLME in which the IL was formedin situ by a metathesis reaction. 10 mL of water spikedwith 13 aromatic analytes (biphenyl, 3-tert-butylphenol,a,a,a,6-tetrafluoro-m-toluidiene, 2-nitronaphthalene,naphthalene, acenaphthene, fluorine, phenanthrene,anthracene, fluoranthene, pyrene, and nitrobenzene)were mixed with 38 lL of IL [BMIm][Cl] (as supercooledliquid). The IL was completely dispersed and dissolved inan aqueous solution after gentle shaking. An aliquot ofaqueous LiNTf2 was added and resulted in the formationof a turbid solution. After centrifugation for 5 min, theupper aqueous solution was removed and 10 lL of the IL[BMIm][NTf2] residue was injected into an HPLC system.This method was compared with conventional IL-DLLMEadding 70 lL of [BMIm][NTf2] dissolved in 0.5 mL ofmethanol or acetone into 10 mL of water and also withSDME using 8 lL of IL. In contrast with traditional IL-DLLME, the proposed method did not require the use ofdisperser solvent. Compared to 60-min direct SDMEusing the same extraction IL, a 4.6–8.6-fold increase inanalyte enrichment factors was observed. Using thedeveloped method, LODs were 0.02–0.3 lg/L with RSDsof 3.6–6.9%.

Amines and nitrogenous compounds have also beentarget analytes in DLLME. These applications include theextraction of aromatic amines from waters [19,87,88],biogenic amines from rice wine [89] and nitrotoluenesfrom water samples [19,90,91]. Fan et al. [19] developeda study of the extraction of four aromatic amines(2-methylaniline, 4-chloroaniline, 1-naphthylamine and4-aminobiphenyl) from tap water, river water andwastewater, employing a modification of IL-DLLME, inwhich there was no need for disperser solvent. In thiswork, 50 lL of IL [BMIm][PF6] as extraction solventwere mixed with only 1.8 mL of aqueous sample solution


(12% (w/v) NaCl, pH 7). In order to disperse the IL intothe aqueous phase, 1 mL of the previous mixture wasaspirated into a syringe and reinjected into the remain-ing solution twice. Finally, after centrifugation, the ILphase could be collected at the bottom of the tube anddirectly injected into the HPLC-UV system. The spikedrecoveries were in the range 93.4–106.4% and LODswere 0.45–2.6 lg/L. As occurred in other works, a widepeak corresponding to the IL appeared at the beginningof the chromatogram, but did not interfere at all in thecorrect determination of the four analytes.

Concerning the determination of compounds otherthan those previously mentioned, organophosphorusflame retardants and plasticizers [93], trihalomethanes[94], organotin compounds [95], fatty acids [99], per-sonal-care products [101], volatile phenols [92], EDTA[102], other water-soluble components {e.g., monoter-penes [97]} and methyl tert-butyl ether [103] havebeen extracted from aqueous matrices. Other examplesinclude the extraction of cholesterol from milk, egg yolkand olive oil [98], hexanal and heptanal from humanblood [100], aromatic hydrocarbons from eluates ofpyrolysis solid residues [104], oleuropein from olive-processing wastewater and olive-leaf extract [63], andglycyrrhizic acid from licorice root [7]. In one of theseworks, Hashemi et al. [63] proposed the use of aDLLME methodology with the aim of avoiding evapo-ration and reconstitution of the chlorinated extractionsolvents that are used in conventional DLLME and thatare not compatible with RP-HPLC. In this work, aprevious ultrasound-solvent extraction with ethyl ace-tate was necessary to isolate oleuropein from oliveleaves as well as LLE, also with ethyl acetate, to extractthe target compound from olive-processing wastewater.After applying a central composite experimental designfor the optimization of the microextraction procedure,final conditions were the use of 1.4 mL of the ethyl-acetate supernatant, which acted as the disperser sol-vent, mixed with 40 lL of an aqueous buffer solution atpH 5, which acted as extraction solvent (reversedpolarity). This mixture was then dispersed in 5.3 mL ofcyclohexane. In this way, the aqueous phase was easilycollected after centrifugation and injected into theinstrument, leading to extraction recoveries in therange 96.2–109.2% and enrichment factors of 23–42,depending on the initial matrix treated. Anotherinteresting example is the work carried out by Farinaet al. [92] for the extraction of volatile phenols. Theseauthors developed a method to analyze ‘‘bretty’’ flavorcomponents (4-ethylguaiacol and 4-ethylphenol)caused by the presence of Brettanomyces yeasts in redwines. The DLLME procedure was directly applied to5 mL of wine taken to pH 8, rapidly injecting 1 mL ofacetone (disperser solvent) containing 50 lL of carbontetrachloride (extraction solvent). After centrifugationand collection of the sedimented droplet, it was injected


into the GC-MS system, getting LODs of 28 lg/L for4-ethylguaiacol and 44 lg/L for 4-ethylphenol.

3. Conclusions and future trends

Review of the literature dealing with DLLME of organicanalytes clearly shows that the technique is achievinggreater importance. Most of the applications have beendeveloped for the selective extraction of pesticides andpharmaceuticals in water samples of environmentalinterest. The technique has generally demonstrated avery good performance for aqueous samples (also in thecase of inorganic analytes) but it is desirable to extendthese applications to other analytes and to more complexmatrices. In this sense, a few attempts have shown thatthe combination of DLLME with other techniques (e.g.,solid-liquid extraction or SPE) is also possible, providingconsistent recovery values as well as highly selectiveextractions. We strongly encourage further worksstudying these possible combinations with a widervariety of analytes to demonstrate fully the potential ofthe technique.

In most works, optimization of experimentalparameters influencing the extraction is normallycarried out using a step-by-step approach, in whicheach factor is varied sequentially, and in only a fewworks have experimental designs been used. The rel-atively large number of influencing factors and thepossible interaction between them clearly suggest thatthe use of experimental designs is highly recommendedin order to achieve the best extraction conditionsquickly in a relatively small number of experiments.The use of chemometrics, in particular, experimental-design methodology, brings about the possibility tovary each factor at the same time in a more pro-grammed and coherent way, so that results obtainedcan be interpreted following a more rational andfruitful approach and optimal analytical conditions canbe reached faster, considerably reducing the number ofexperiments.

Although it has not been specifically indicated in thecorresponding publications, it is also clear that DLLMEcan somehow be used as clean-up procedure or solventexchanger. The injection of the mixture of ‘‘extractionand dispersion’’ solvents, which already contain thetarget analytes in an aqueous solution, has been devel-oped with success (recovery values are maintained andcleaner extracts are obtained). This is also another use ofthe technique that should also be taken into account andthat has not been widely extended or applied in the lit-erature. Also of particular interest is its application forreversing solvent polarity, which has been recentlyintroduced.

One of the main disadvantages of DLLME is that itsefficiency is restricted by solvent selection to systems

capable of forming a dispersive phase, somewhat limit-ing its range of application by sample. However, theintroduction of new solvents (e.g., ILs, which arenumerous) may provide new insight.

It is also interesting to emphasize the fact that fewauthors calculate absolute extraction recoveries. Inmany applications, DLLME is not totally quantitativeand recovery values are not too high but consistent.Instead of this approach, we prefer to give final resultsin terms of relative recoveries or even as enrichmentfactors.

Though not fully explored, DLLME offers severalinteresting variations {e.g., the final collection of afloating drop (for lower density extraction solvents), thecollection of a solid drop (when the extraction solventcan be solidified at temperatures close to 0�C), the aid oftemperature (without disperser solvent) or ultrasoundto start dispersion, the use of co-solvents, or even car-rying out a simultaneous derivatization reaction}. Allthese factors clearly extend the applicability of thetechnique.

It is highly desirable to continue direct comparison ofthe method performance (with or without variations)with other techniques, involving the same analytes andmatrices in order to bring about rigorous discussion ofextraction efficiency, among other aspects.

So far, DLLME has not been applied prior to capillaryelectrophoresis (CE), in which nL of the sample areintroduced into the separation capillaries. When DLLMEmethod development is analyzed, it is clear that it is verynecessary to evaporate the extraction solvent and toreconstitute it in a suitable media to avoid currentbreakdown at the beginning of CE analysis. In CE, con-ductivity of the sample is also of particular importance inorder to carry out on-line sample preconcentrationtechniques. Furthermore, when ILs are used, it is clearthat direct injection is only possible in LC instruments,while GC systems require suitable modification of theinjector (as has already been proposed [105]). However,in CE, the high viscosity of IL would surely make thedirect injection difficult – up to the point of blocking thecapillary.

Automation of DLLME seems to be very difficult andhas not yet been achieved, although an attempt wasmade for the analysis of inorganic species [106].Automation is also a research area in which westrongly encourage new developments, although itseems that important advances in this direction are notto come because most of the process is genuinelymanual.

All these considerations and our detailed review of thecurrent literature clearly suggest that DLLME will con-tinue to be used and that there will be interesting andchallenging applications. Its clear advantages over othersample-pretreatment procedures are valuable and desir-able, and surely encourage a promising future.



AcknowledgementsA.V.H.H. and M.A.R. wish to thank the Spanish Ministryof Education for the FPU Grant at the University of LaLaguna. J.H.B. also thanks the Spanish Ministry ofScience and Innovation for the Ramon y Cajal contract atthe University of La Laguna. This work has beensupported by the Spanish Ministry of Science and Inno-vation (Projects AGL2008-00990/ALI and AGL2009-07884/ALI).

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dispersive liquid-liquid microextraction for determination of organic analytes

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