Liquid Chromatography

DETERMINATION OF ALKYLPHENOLS IN WATER BYDISPERSIVE LIQUID–LIQUID MICROEXTRACTIONBASED ON SOLID FORMATION WITHOUTA DISPERSER

Yu-Hsiang Sung,1 Chih-Hao Liu,2 Mei-I Leong,3 andShang-Da Huang21Head, Forensic Science Section, Hsin-Chu City Police Bureau,Hsinchu, Taiwan2Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan3Food Safety Center of The Civic and Municipal Affairs Bureau (IACM),Macau, China

Dispersive liquid–liquid microextraction based on solid formation without a disperser

combined with high-performance liquid chromatography has been developed for the

determination of 4-tert-butylphenol, 4-n-nonylphenol, and 4-tert-octylphenol. This method

is rapid, easy, and uses only 10 lL of a low toxicity organic solvent (1-hexadecanethiol)

for the extraction solvent and no disperser solvent. The extraction time and centrifugation

time require less than 10min. The linear range was 1–500 ngmL�1 for 4-tert-butylphenol,

2–1000 ngmL�1 for 4-tert-octylphenol, and 5–500 ngmL�1 for 4-n-nonylphenol with

r2� 0.9986. The detection limits were between 0.2 and 1.5 ngmL�1. The recoveries of lake

and river water samples were in the range of 79% to 108%, and the relative standard

deviations were 5% to 10%.

Keywords: 1-Hexadecanethiol; Alkylphenols; Dispersive liquid–liquid microextraction; High-performance

liquid chromatography

INTRODUCTION

Alkylphenol ethoxylates, a class of nonionic surfactants, have been widely usedas detergents for more than fifty years because of their excellent cleaning ability (APEResearch Council 2013). Unfortunately, alkylphenol ethoxylates biodegrade intoalkylphenols, such as 4-tert-butylphenol, 4-tert-octylphenol, and 4-n-nonylphenol.Alkylphenols are more toxic to aquatic life than their parent alkylphenol ethoxylates

Received 22 February 2014; accepted 23 April 2014.

Address correspondence to Shang-Da Huang, Department of Chemistry, National Tsing Hua

University, Hsinchu 300, Taiwan. E-mail: sdhuang@mx.nthu.edu.tw

Color versions of one or more of the figures in the article can be found online at www.tandfonline.

com/lanl.

Analytical Letters, 47: 2643–2654, 2014

Copyright # Taylor & Francis Group, LLC

ISSN: 0003-2719 print=1532-236X online

DOI: 10.1080/00032719.2014.921824

2643

(Manzano et al. 1999) and have been described as endocrine disrupters (EuropeanCommission 2001), representing a serious health and environmental hazard.A number of these phenolic compounds are listed in the U.S. EnvironmentalProtection Agency (EPA) list of priority pollutants (EPA 2009). Also, the EuropeanCommission has included some of these phenols in its Water Framework Directive2000=60=EEC (European Commission 2000) and established that the maximumconcentration of total phenolic compounds in surface water for drinking purposesshould be 1–10 ngmL�1 (European Commission 1975; Bernardo et al. 2010).

Alkylphenols may accumulated into lipid tissues of aquatic organisms(Ahel, McEvoy, and Giger 1993). Para-substituted alkylphenols have been shownto be estrogenic in fish and fish cells (Jobling and Sumpter 1993), rodents and humanMCF-7 breast cancer cells (White et al. 1994; Nagel et al. 1997), and in yeasttwo-hybrid assays (Nishihara et al. 2000). People are exposed to these compoundsin a variety of ways, including through the skin and from contamination ofair and water (Breast Cancer Fund 2012). Consequently, it is necessary to developa simple and rapid method to analyze and determine these toxic compoundsin environmental samples.

Recent research has been oriented towards efficient development of economicaland miniaturized extraction methods. Liquid phase microextraction (LPME) is impor-tant pretreatment method for the determination of trace analytes in complex samples.This technique is economical because of minimal solvent usage and the choiceof solvent selected. In 2006, dispersive liquid–liquid microextraction (DLLME) forthe preconcentration of polycyclic aromatic hydrocarbons (PAHs) in water sampleswas developed (Rezaee et al. 2006). The extraction efficiency of DLLME is higher thanmany conventional LPME methods (Jeannot and Cantwell 1996; He and Lee 1997;Pedersen-Bjergaard and Rasmussen 1999; Ma and Cantwell 1999; Jiang and Lee2004). Therefore, DLLME is a popular preconcentration technique (Tsai and Huang2009; Zgoła-Grzeskowiak and Kaczorek 2011; Fari et al. 2007; Rezaee et al. 2010).However, high-density and toxic extraction solvents were used in these experiments,which are typically chlorobenzene, chloroform, or carbon tetrachloride.

Several variations of the DLLME technique have been developed. DLLMEbased on solid formation has been investigated since 2008 (Leong and Huang2008). In DLLME based on solid formation, a low density, low toxicity extractionsolvent was employed with less than 1000 mL of disperser solvent. In 2009, DLLMEwith a very low solvent consumption (Tsai and Huang 2009) was introduced. A smallquantity of a binary mixture of extraction solvent and disperser solvent (total 13 mL)and manual shaking were used. Fine organic droplets were subsequently formed inthe solution by manually shaking the of sample and extraction solvent. The largesurface area of the organic solvent droplets increased the rate of mass transfer fromthe water sample to the extractant, and allowed efficient extraction in a short period.The method used manual shaking for dispersing the solvent in a DLLME based onsolid formation without a disperser combined with high-performance liquid chroma-tography (HPLC) for determination of alkylphenols. Only 10 mL of 1-hexadeca-nethiol as extraction solvent were used in this method and no disperser solventwas used, which precludes the loss of the extraction solvent in the disperser solvent.The extraction solvent was thoroughly mixed with the aqueous sample and theresults had good reproducibility.

2644 Y.-H. SUNG ET AL.

EXPERIMENTAL

Reagents and Samples

The 4-t-Butylphenol (>99%) and 4-t-octylphenol (>97%) were purchased fromSigma-Aldrich (Steinheim, Germany). The 4-n-Nonylphenol (>98%) was obtainedfrom Alfa Aesar (Ward Hill, MA, USA). The 1-Hexadecanethiol and 2-dodecanolwere purchased from Acros (Geel, Belgium). The 1-Dodecanol was obtained fromFluka (Buchs, Switzerland). Dimethoxyether and isopropanol were purchased fromSigma-Aldrich. Tetrahydrofuran and dimethyl sulfoxide were purchased from Tedia(Fairfield, USA). Acetone, acetonitrile, and methanol were obtained from Echo(MiaoLi, Taiwan). Sodium chloride was purchased from Merck (Darmstadt,Germany). Deionized water was prepared with a Milli-Q water purification system(Millipore, Milford, MA, USA).

Stock solutions were prepared by dissolving each analyte in methanol ata concentration of 1000 mgmL�1; these were stored at 4�C. Lake water (Chu Lake,Hsinchu, Taiwan) and river water (Yourou River, Hsinchu, Taiwan) were passedthrough a 0.45-mm membrane filter from Millipore (Bedford, MA, USA) priorto analysis.

Instrumentation

High-performance liquid chromatography was performed on a Waters AcquityUPLC system (Waters, Manchester, UK), equipped with a binary solvent manager,a sample manager, and a photodiode array. A BEH C18 column (2.1mm� 50mmid, 1.7m particle sizes) and a guard column (BEH C18, 1.7m particle size) fromWaters (Manchester, UK) were used at 30�C and the auto-sample manager wasmaintained at 25�C. A detection wavelength of 270 nm was employed for quantifi-cation. The ultrasonic cleaner was a Branson 3510 (Danbury, USA) (100W, 42 kHz).

Chromatographic Conditions

Acetonitrile (solvent A) and deionized water (solvent B) were used as the mobilephase for liquid chromatography. The flow rate was maintained at 0.4mLmin�1. Theelution program (including lake water samples) including: 0–1min, 30% A; 1–2min,30–50% A; 2–3min, 50–65% A; 3–7min, 65–90% A; and 7–8min, 90–100% A. Theriver water had some interferences, which overlapped with the analyte signals, andthe river water program was altered to 0–1min, 20% A; 1–2min, 20–30% A; 2–8min,30–50% A; 8–15min, 50–90% A; and 15–16min, 90–100% A.

Extraction Procedure

The analytes (100 ngmL�1) were spiked in 5mL of water, followed by additionof 1.5 g of sodium chloride and 10 mL of 1-hexadecanethiol. The solution becamecloudy after vigorous manual shaking for 90 sec. After centrifuging for 2min at5000 rpm, the extraction solvent floated on the surface of the water. The solutionwas immersed in an ice bath for about 5min, and the solidified organic phase(5.5–7 mL) was collected carefully with a customized scoop, 5 mL were transferred

DETERMINATION OF ALKYLPHENOLS IN WATER 2645

into a microsample tube, followed by the addition of dimethylsulfoxide to obtaina final volume of 20 mL. Finally, 10 mL of this mixture was analyzed by HPLC.

RESULTS AND DISCUSSION

To optimize extraction conditions for the determination of alkylphenols,six parameters were investigated. Enrichment factors (EFs) were calculated as theratio of the final concentrations of analytes in the floated droplet after extractionto the initial concentrations of analytes in the aqueous solution. Enrichment factorsserved as the criterion used to evaluate the extraction efficiency.

Selection of Extraction Solvent

For the successful preconcentration of analytes, the selection of extractionsolvent is significant, and a low toxicity compound is preferable. The densities ofthe extraction solvents used in this method were less than the density of water andthe melting points were close to room temperature (10–20�C). Figure 1 shows theextraction solvents used including 1-hexadecanethiol (melting point: 18–20�C),1-dodecanol (melting point: 22–24�C), and 2-dodecanol (melting point: 17–18�C).The 1-Hexadecanethiol, which has relatively high sensitivity for 4-tert-octylphenoland 4-n-nonylphenol, was selected for further testing. The polarity of 1-hexadeca-nethiol is lower than that of the other selected extraction solvents, and hence higherextraction efficiency was obtained for the nonpolar alkylphenols. In addition, this isthe first use of 1-hexadecanethiol as an extraction solvent in DLLME.

Effect of Extraction Solvent Volume

The volume of extraction solvent used was optimized to achieve maximumsensitivity and efficiency. It is not desirable to employ too much 1-hexadecanethiolfor liquid chromatography to prevent peak tailing or damage to the stationary phase

Figure 1. Effect of the extraction solvent (n¼ 3). Samples were spiked with 100 ngmL�1 of each analyte.

Extraction conditions: aqueous sample volume, 5mL; extraction solvent, 10mL; disperser solvent

(tetrahydrofuran) volume, 10mL; 0.5 g sodium chloride; shaking time, 60 s.

2646 Y.-H. SUNG ET AL.

of the column. Figure 2 shows that the enrichment factors increased as extractionsolvent volume was increased from 6 mL to 10 mL; however, 14 mL reduced sensitivitybecause of the dilution effect, and 6 mL of extraction solvent was difficult tooperate (less than 5 mL of the extractant was collected). Therefore, 10 mL of1-hexadecanethiol was employed in subsequent experiments.

Selection of Disperser Solvent

Addition of a disperser solvent causes the extraction solvent to form finedroplets, but too much disperser causes the extraction solvent and analytes todissolve in the samples. Four disperser solvents were tested in this experiment,including acetone, isopropanol, dimethoxyether, and tetrahydrofuran. Isopropanolobtained the highest efficiency and was used in subsequent experiments.

The disperser solvent is used in DLLME for dispersing extraction solvent intothe sample solution. In this experiment, a low volume of extraction solvent was usedand it is easy to disperse the extraction solvent by manual shaking for a few min.Figure 3 shows that the enrichment factors were relatively constant as the dispersersolvent volume was increased from 0 to 40 mL. It is possible because of the lowvolume of the extraction solvent, more of the disperser solvent was used, and moreof the extraction solvent and analytes were dissolved in the aqueous samples.As a result, no disperser solvent was used in the further extractions.

Effect of Salt Addition

The presence of salt increases the ionic strength of the aquatic phase, whichcauses the analytes to partition into the organic phase more easily and increasesthe enrichment factors. Also, increasing the amount of salt decreased the solubilityof the extraction solvent in the aqueous phase, and hence the volume of theextraction phase increases. The results showed that the enrichment factors of4-tert-butylphenol decreased slightly at quantities of sodium chloride over 0.5 g;

Figure 2. Effect of the volume of the extraction solvent (n¼ 3). Samples were spiked with 100 ngmL�1 of

each analyte. Extraction conditions: aqueous sample volume, 5mL; disperser solvent (isopropanol)

volume, 10mL; 0.5 g sodium chloride; shaking time, 60 s.

DETERMINATION OF ALKYLPHENOLS IN WATER 2647

whereas, the EFs of 4-tert-octylphenol and 4-n-nonylphenol increased upon additionof 0 g to 1.5 g of sodium chloride. No significant effect on enrichment factor wasobserved when larger amounts (1.5–2 g) of sodium chloride were added, becausethe solution was saturated. Therefore, 1.5 g of sodium chloride was used insubsequent experiments.

Effect of Manual Shaking Time

Dispersion is the most important factor for successful extraction. Adequatemanual shaking time will form fine droplets of the extraction solvent and result inan excellent cloudy suspension; furthermore, favorable reproducibility is obtained.Different manual shaking periods were investigated in this experiment. The bestresults were obtained when manual shaking times between 90 sec and 120 sec wereemployed, probably because the emulsion is formed quickly and rapidly establisheda large contact surface area between the extraction solvent and the aqueous phase.Therefore, 90 sec was selected as optimum and employed for further experiments.

Comparison Between Manual Shaking and Ultrasound-AssistedEmulsification

The oscillation frequency of ultrasonic-assisted emulsification is higher thanthat of manual shaking. However, ultrasonic-assisted emulsification is not suitablefor low volumes of extraction solvents because it is easy to emulsify the extractionsolvent and lower the efficiency. From observation of the process, the solutionbecame cloudier after manual shaking for 90 sec than after 120 sec of ultrasonic

Figure 3. Effect of the volume of the disperser solvent (n¼ 3). Samples were spiked with 100 ngmL�1 of

each analyte. Extraction conditions: aqueous sample volume, 5mL; extraction solvent (1-hexadecanethiol)

volume, 10mL; 0.5 g sodium chloride; shaking time, 60 s.

2648 Y.-H. SUNG ET AL.

dispersion. Enrichment factors obtained by manual shaking for 90 sec were greaterthan values obtained using ultrasonic-dispersion for 90 sec or 120 sec. 90 s of manualshaking time were selected for subsequent experiments. In 2013, Y. T. Li et al. (2013)compared the extraction droplets produced in manual-assisted emulsification andultrasound-assisted extraction. The manual shaking dispersed analytes well in theemulsion, and that the ultrasound assisted procedure over-emulsified the mixtureand resulted in incomplete phase separation.

Effect of Centrifugation Time

In this method, the extraction time is short and the most time-consuming stepis the centrifugation. The extractant drop is formed quickly after centrifugation at5000 rpm. The effect of centrifugation time was examined in the range 1–10min at5000 rpm, and the centrifugation time had no impact on extraction efficiency after2min. Therefore, 2min was chose for subsequent experiments.

Analytical Performance

Analytical figures of merit are summarized in Table 1. All alkylphenols exhib-ited good linearity with correlation coefficients equal to or exceeding 0.9986. Rela-tive standard deviation values were from 5% to 7% for lake water samples andfrom 6% to 10% for river water. The limits of detection (LODs) were calculatedas three times the standard deviation from seven replicate runs of water fortified witheach analyte. For lake water, the LODs were 0.2 ngmL�1 for 4-tert-butylphenol,0.8 ngmL�1 for 4-tert-octylphenol, and 1.1 ngmL�1 for 4-n-nonylphenol. The LODsranged from 0.2 to 1.5 ngmL�1 for river water samples.

Application to Environmental Samples

The reported method was applied for the determination of alkylphenolsin lake (1 mgL�1 4-tert-butylphenol, 5 mgL�1 4-tert-octylphenol, and 5 mgL�1

4-n-nonylphenol) and river water (1 mgL�1 4-tert-butylphenol, 6 mgL�1 4-tert-octylphenol, and 5 mgL�1 4-n-nonylphenol). Chromatograms were obtained from

Table 1. Analytical figures of merit for dispersive liquid–liquid microextraction based on solid formation

without a disperser (n¼ 5)

Analyte

Linear

dynamic

range

(ngmL�1)

Correlation

coefficient (r2)

Lake water River water

Limit of

detection

(ngmL�1)

Relative

standard

deviation (%)a

Limit of

detection

(ngmL�1)

Relative

standard

deviation (%)b

4-tert-butylphenol 1–500 0.9987 0.2 6 0.2 7

4-tert-octylphenol 2–1000 0.9986 0.8 5 1.5 10

4-n-nonylphenol 5–500 0.9987 1.1 7 0.9 6

aLake water sample spiked with 1 ngmL�1 4-tert-butylphenol, 5 ngmL�1 4-tert-octylphenol, and

5 ngmL�1 4-n-nonylphenol.bRiver water sample spiked with 1 ngmL�1 4-tert-butylphenol, 6 ngmL�1 4-tert-octylphenol, and

5 ngmL�1 4-n-nonylphenol.

DETERMINATION OF ALKYLPHENOLS IN WATER 2649

samples fortified with each analyte at the limit of quantification (LOQ). LOQs wereestimated as ten times the RSD of seven replicate runs. A lower gradient was used foranalyzing the river water to avoid overlaps between analytes and matrix interfer-ences. Figure 4 showed results of the river water analysis. No analytes were detectedin unfortified lake and river water. To assess matrix effects, river and lake water werefortified at various concentrations of the analytes to obtain recoveries. As shownin Table 1, the linearity of each analyte was 1–500 ngmL�1 for 4-t-butylphenol,2–1000 ngmL�1 for 4-t-octylphenol, and 5–500 ngmL�1 for 4-n-nonylphenol.The recoveries ranged from 82% to 108% for lake water and from 79% to 107%for river water (Table 2), indicating the feasibility of the reported method forthe determination of alkylphenols in water.

This reported method avoided using the toxic chlorinated solvents employed inconventional DLLME methods (Du et al. 2010; Chen et al. 2008; Farhadi, Matin,

Figure 4. Chromatogram at 270 nm of (A) an unfortified river water sample and (B) river water fortified

with 1 mgL�1 4-tert-butylphenol, 6 mgL�1 4-tert-octylphenol, and 5 mgL�1 4-n-nonylphenol.

Table 2. Recoveries of alkylphenols from field water fortified with the analytes

Analyte Addeda (ngmL�1) Recoverya (%) Addedb (ngmL�1) Recoveryb (%)

4-tert-butylphenol 1 99� 4 1 93� 9

4-tert-octylphenol 5 82� 3 6 79� 3

4-n-nonylphenol 4 108� 6 5 107� 4

aLake water, Chu Lake, Hsinchu, Taiwan.bRiver water, Yourou River, Hsinchu, Taiwan.

2650 Y.-H. SUNG ET AL.

Table

3.Comparisonofthereported

dispersiveliquid–liquid

microextraction(D

LLME)methodwithcomparable

techniques

foralkylphenols

Method

Instrument

Analyte

Extractionsolvent

Disperser

solvent

Lineardynamic

range(ngmL�1)

Lim

itof

detection

(ngmL�1)

Reference

Thismethod

HPLC

Alkylphenols

Hexadecanethiol(10mL

)—

1–1000

0.2–1.5

—

LPME

GC–M

SAlkylphenols,

chlorophenols,

bisphenol-A

Toluene(1.2cm

hollow

fiber)

—2.5–250

0.005–0.015

BasheerandLee

2004

LLLME

HPLC

Alkylphenols,

bisphenol-A

Organic

hydroxidein

ethylene

phase:4-chlorotoluene.

Acceptor

phase:0.2M

tetraethylammonium

glycol.

—0.05–200

0.017–0.0048

Lin

etal.2011

DLLME

HPLC

Alkylphenols,

short-chain

ethoxylates

Trichloroethylene

(50mL

)

Acetone

1.5mL

7–3000

0.02–0.1

Wuet

al.2012

DLLME

GC

Alkylphenols

Trichloroethylene

(15mL

)

Acetone

(1mL)

0.5–8

0.07–0.17

Bernardoet

al.

2010

DLLME

LC–M

S–M

SAlkylphenols,

bisphenol-A

1-O

ctan

ol(100mL

)—

0.09–50

0.001–0.01

Salgueiro-G

onzalez

etal.2013

MA-H

S-SPME

GC–M

SAlkylphenols

——

0.02–5

0.002–0.02

Zgo

ła-G

rzeskowiak

2010

MASE

LC–M

S–M

SAlkylphenols

Hexane(500mL

)—

0.01–20

0.0005–0.003

Salgueiro-G

onzalez

etal.2012

HPLC,High-Perform

ance

Liquid

Chromatography;LPME,Liquid

Phase

Microextraction;GC–M

S,GasChromatography–Mass

Spectrometry;LLLME,

Liquid–Liquid–Liquid

Microextraction;DLLME,DispersiveLiquid–Liquid

Microextraction;LC–M

S–M

S,Liquid

Chromatography–Tandem

Mass

Spectrometry;

MA-H

S-SPME,Microwave-AssistedHeadspace

Solid-Phase

Microextraction;MASE,Mem

braneAssistedSolventExtraction.

2651

and Hashemi 2009; L. H. Li et al. 2012; Qiao et al. 2010). Comparison of this methodwith other techniques for alkylphenols is shown in Table 3. The reported methodemployed the lowest volume of extraction solvent. The extraction time of thismethod was shorter than that of LPME (Basheer and Lee 2004), liquid–liquid–liquidmicroextraction (LLLME) (Lin, Fuh, and Huang 2011), microwave-assistedheadspace solid-phase microextraction (MA-HS-SPME) (Zgoła-Grzeskowiak 2010),and membrane assisted solvent extraction (MASE) (Salgueiro-Gonzalez et al. 2012).The extraction time was quite similar to that of DLLME methods (Bernardo et al.2010; Wu, Wang, and Ding 2012; Salgueiro-Gonzalez et al. 2013). The linearity of thismethod was better than LPME (Basheer and Lee 2004). However, the linearity andLODs of this method are higher than the other methods because of the low volumeof extraction solvent and the use of different instruments. In addition, combiningother techniques with the reported method for analysis of aqueous samples maybe more effective and useful. Manual shaking in this method may be improvedwith a more efficient shaker (Ku et al. 2013). Using a better solvent collection systemmay enhance the separation of water and organic solvent in the collected extractantdrop (Chang, Wie, and Huang 2011).

CONCLUSIONS

An environmentally-friendly DLLME based on solid formation withouta disperser method was developed for the extraction of alkylphenols at low concen-tration levels in water. This method used a new and low toxicity extraction solvent(1-hexadecanethiol). No disperser solvent was used. The amount of organic solventwas minimized and the extraction time of this method was shorter (90 sec) thanconventional LPME methods. The apparatus of the method was simple and inexpen-sive. For environmental samples, the results obtained by DLLME based on solidformation without a disperser showed that it could be successfully applied for theseparation and preconcentration of alkylphenols in water.

FUNDING

This work was supported by the National Science Council of Taiwan (NSC99-2113-M-007-004-MY3).

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