Determination of Anilines in Water Samples Using Ionic Liquid-Based Single Drop Microextraction Coupled with High Performance Liquid Chromatography

Cuiqin Wu*, Diyun Chen, Hongmei Deng, Yonghui Liu

School of Environmental Science & Engineering, Guangzhou University

Guangzhou, China *E-mail: wucuiqin@gzhu.edu.cn

Abstract—A novel method for the determination of four anilines (aniline, p-nitroaniline, o–nitroaniline, 2, 4-dinitroaniline) in water samples has been developed using ionic liquid-based single drop microextraction coupled with high performance liquid chromatography. The influence of experimental parameters including extraction solvent, the volume of extraction solvent, extraction time, stirring speed, pH value and NaCl concentration of sample solution, extraction temperature was investigated. The optimal experimental conditions were as follows: 9 μL 1-butyl- 3-methylimidazolium hexafluorophosphate （[C4MIM][PF6]）as extraction drop, 10.0 mL aqueous sample with 0.33 g·mL-1 NaCl, pH 9 was extracted for 25 min, stirring at 600 r·min-1, extraction temperature set at 50℃, then 5 μL sample was injected to HPLC, aniline and o–nitroaniline were detected at 230 nm, p-nitroaniline and 2, 4-dinitroaniline were detected at 360 nm. Under the optimal conditions, good linear relationships were obtained in the concentrations of 0.010-5.000 mg·L-1 of the anilines with the correlation coefficients of 0.9990-0.9999; the detection limits were 0.002-0.005 mg·L-1 (S/N=3); the enrichment factors of [C4MIM][PF6] for the four anilines were from 41.8 to 48.0. The proposed method was applied to the determination of the four anilines in tap, river, lake and waste water and recoveries of the four anilines were in the range of 90.1%-103%; RSD were 3.7%-5.9% (n=5). The method is simple, friendly to environment, sensitive and satisfied to determine real samples.

Keywords- ionic liquid; single drop microextraction (SDME); anilines; high performance liquid chromatography (HPLC)

I. INTRODUCTION

Anilines are widely used in industry for making dyes, cosmetics, medicines, and as intermediates in many chemical syntheses. They are present in the aquatic environmental as a result of industrial discharges from these factories. Most of anilines are proven or suspected to be carcinogenic, and have been implicated in the bladder [1]. So it is important to monitor their levels in environment samples.

High performance liquid chromatography (HPLC) is often used for the determination of anilines, and sample enrichment is usually required. In the past extraction techniques such as liquid-liquid extraction (LLE) [2], solid phase extraction (SPE) [3], microporous membrane liquid-liquid extraction (MMLLE) [4], solid phase microextraction (SPME) [5] and liquid-liquid-liquid microextraction (LLLME) [6] were used for the extraction of anilines. One of the major disadvantages of these methods is the need of using a substantial amount of volatile toxic organic solvents. Therefore, ionic liquids (IL), which are ionic media resulting from the combining of organic cations and

Aiju Zhou School of Chemistry & Chemical Engineering,

Guangzhou University Guangzhou, China

various anions have been proposed as an alternative to these organic solvents in extraction processes, due to their no-detectable vapor pressure, high thermal stability, low toxic, high viscosity and density. Ionic liquid based headspace liquid-phase microextraction (HSLPME) and dispersive liquid–liquid microextraction (DLLME) were used to extract chlorinated anilines in environmental water samples [7, 8].

In the present work, the simultaneous extraction and analysis of aniline, p-nitroaniline, o–nitroaniline, 2, 4- dinitroaniline in environmental water samples was accomplished by single drop microextraction (SDME) using ionic liquid as the extraction liquid drop coupled with high performance liquid chromatography. Different experimental parameters were optimized. The proposed method was friendly to environment with good sensitivity, repeatability and spiked recoveries.

II. MATERIALS AND METHODS

2.1 Chemicals Aniline and p-nitroaniline standard stock solution in

methanol 1000.0 μg·mL-1 were purchased from the Institute for Reference Materials of State Environmental Protection Administration (China). o-nitroaniline was gotten from AccuStandard Company (USA, ≥99.0%). 2, 4- dinitroaniline was gotten from Dr. Ehrenstorfer Company (Germany, ≥99.0%). Acetonitrile of HPLC grade was from Merck Company (Germany). Ionic liquids were purchased from Shanghai Chengjie Chemical Limited Company (China, ≥99%), including 1-butyl-3-methylimidazolium hexafluoro- phosphate ([C4MIM][PF6]), 1-hexyl-3-methylimidazolium hexafluorophosphate ([C6MIM][PF6]), 1-octyl-3-methyl- imidazolium hexafluorophosphate ([C8MIM][PF6]). All other chemicals used were of analytical-reagent grade.

2.2 Instrumentation HPLC was performed using an Agilent 1200 liquid

chromatographic system (Agilent Technologies, USA) including a photodiode array detector (DAD) and a Rheodyne (Cotati, CA, USA) 7725i injector equipped with a 20 μL loop. 25 μL Hamilton gastight syringe (Model 1702 Hamilton BonaduzAG Bonaduz, Switzerland; length: 5.1 cm, i.d.: 0.015 cm). Separation was accomplished using an Agilent Extend-C18 column (250 mm × 4.6 mm, 5 μm) and a mobile phase of acetonitrile-water (35: 65, v/v). The flow-rate of the mobile phase was 1mL·min-1 and the detection wavelengths were set at 230 nm and 360 nm for aniline, o–nitroaniline and p-nitroaniline, 2, 4- dinitroaniline, separately.

Guangdong Natural Science Foundation: 8451009101001037 National Natural Science Foundation of China: 40803026，21001032

978-1-4244-5089-3/11/$26.00 ©2011 IEEE

2.3 Extraction Procedure Ten mL of each working standard solution or sample,

adjusted to pH 9 and containing 0.33 g·mL-1 NaCl was placed in 15 mL glass vials. Magnetic stirring at the speed of 600 r·min-1 was then applied. A 5 mm long polytetrafluoroethylene (PTFE) tube (i.d.: 0.8 mm; o.d.:1.6 mm) was fitted to the blunt needle tip of a 25 μL Hamilton gastight syringe, maximizing the contact area between the drop and the needle tip.

The microsyringe, typically contained 9 μL of the ionic liquid acceptor phase, was clamped above the vial and its needle passed through the Mininert valve until its tip was 1 cm below the surface of the solution. The plunger was depressed and a microdrop of the ionic liquid phase was exposed to the solution at 50℃ for 25 min. It should be mentioned that during extraction, the ionic liquid could be partially solved into the sample, thus reducing the volume of the acceptor phase. Finally, after extraction, 5 μL ionic liquid acceptor phase was retracted into the microsyringe, the PTFE tube was removed, and then the extract was ready for HPLC analysis.

III. RESULTS AND DISCUSSION

3.1 Effect of Extraction Solvents and Drop Volume The physical and chemical properties such as density,

solubility and polarity of extraction solvents have significant influence on the extraction efficiency of the target analytes. Therefore, three ILs including [C4MIM][PF6], [C8MIM][PF6], [C6MIM][PF6] were compared as extraction solvents in this study. The results showed that [C4MIM][PF6] as extraction solvent had the maximum extraction efficiency and minimum interferences.

As for the effect of drop volume, it can be briefly

described as follows. The analytes are rapidly extracted at the surface of the drop, where the rate of extraction is controlled by diffusion to the surface of the extracting medium (i.e. the boundary layer), which is governed by the relative solubility of the analytes (i.e. the partition coefficient). However, once the drop surface becomes saturated, further extraction requires diffusion of the extracted analytes into the bulk solvent within the drop, which is a much slower process. Further increases in drop volume, making surface area increase, will result in increased drop losses as well as extraction time prolonging. In the present study, the effect of drop volume (7-12 μL) on extraction efficiency was investigated (Fig. 1). The volume of 9 μL had the maximum extraction efficiency. Therefore, a

drop of 9 μL [C4MIM][PF6] as extraction solvent was adopted in the following study.

3.2 Effect of Extraction Time and Stirring Speed In the present study, the range of extraction time from 5

to 40 min was investigated [Fig. 2(A)]. Experiments indicated that the peak areas of the four anilines increased with time over the range of 5-25 min. Then, the peak areas decreased with further prolonging of extraction time. This was related to the extraction solvent dissolving.

Stirring can speed the extraction process of the analytes, but high stirring speed can cause drop instability. Therefore, the effect of stirring speed was evaluated in the range of 300- 600 r·min-1 [Fig. 2(B)]. It could be seen that the 600 r·min-1 of stirring speed had the maximum extraction efficiency.

Accordingly, an extraction time of 25 min and a stirring speed of 600 r·min-1 were adopted in the following study.

3.3 Effect of Solution pH and Salt The influence of pH was investigated from pH 4-12 [Fig.

3(A)]. The peak areas of the analytes increased with pH rising, and reached a maximum value at pH 9. Then they decreased slightly. This was related to that the four anilines mostly showed as molecule not be ionized at pH 9 and could be easily extracted by IL.

The effect of the addition of NaCl to the sample on extraction efficiency was tested [Fig. 3(B)]. It was observed that an increase in extraction efficiency in response to the increase in the salt content. The addition of salt to the solution caused the engagement of water molecules in the hydration spheres of the ionic salt. These spheres reduced the concentration of water available to dissolve the anilines [9] and therefore, increased extraction for the anilines from aqueous phase to IL. On the other hand, the addition of NaCl could prevent the dissolution of IL. Accordingly, all subsequent experiments were conducted at 0.33 g·mL-1 NaCl.

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Figure 1. Effect of volume of extraction solvent on extraction efficiency

1. aniline, 2. p-nitroaniline, 3. o-nitroaniline, 4. 2, 4-dinitroaniline

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Figure 2. Effects of extraction time (A) and stirring speed (B) on

extraction efficiency The numbers from 1 to 4 are the same as in Fig.1

3.4 Effect of Extraction Temperature

High extraction temperature can accelerate the diffusion of the analytes from aqueous phase to IL and however, it also can increase the evaporation of the analytes and the dissolution of IL. Therefore, the effect of extraction temperature on extraction efficiency was tested (Table 1). When the extraction temperature was set at 50℃, it got the best extraction efficiency. Accordingly, the extraction temperature of 50℃ was adopted in the following studies.

3.5 Analytical Performance Quality parameters of the proposed method were

evaluated under optimized conditions. A calibration study was performed by employing standard solutions of the four target analytes over concentration range of 0.010-5.000 mg·L-1. Each analyte exhibited good linearity with correlation coefficient r≥0.9990 in the studied range. Repeatability was studied by performing five repetitive determinations of 0.2 mg·L-1 standard and the relative standard deviation (RSD) values were not more than 4.7%. The limits of detections (LOD) based on signal-to-noise ration of 3 (S/N=3) were in the range of 0.002-0.005 mg·L-1. The enrichment factors of the proposed procedure were defined as the ratio CIL/Ca, where CIL was the concentration of the analytes in the IL phase after extraction and Ca was the original concentration of the analytes in the aqueous phase, it ranged from 41.8 for 2,4- dinitroaniline to 48.0 for o- nitroaniline. Table 2 summarized the analytical data.

3.6 Analysis of Real Samples To test the applicability and accuracy of the proposed

method in real samples, such as tap water, lake water, river water and waste water, were analyzed, respectively. Before use, all the water samples were filtered through 0.45 μm micropore membranes. The results indicated that the contents of the four anilines in the four samples were under their detection limits. The results of the spiked recoveries were shown in Table 3. A typical chromatogram was shown in Fig. 4.

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Figure 3. Effects of pH value (A) and NaCl concentration (B) of solution

on extraction efficiency The numbers from 1 to 4 are the same as in Fig.1

TABLE 1. THE EFFECT OF TEMPERATURE ON EXTRACTION EFFICIENCY

Extraction

temperature (℃)

Peak area

aniline p-nitroaniline o–nitroaniline 2, 4-dinitroaniline

30 187.5 219.9 337.5 159.6

40 204.5 229.9 354.8 172.9

50 265.3 300.4 403.5 211.6

60 87.9 93.6 157.4 74.3

TABLE 2. ANALYTICAL PERFORMANCE OF THE PROPOSED METHOD

Analytes Linear range / (mg·L-1)

Correlation coefficient, r

RSD (%, n=5)

Detection limit / (mg·L-1)

Enrichment factor

aniline 0.010-5.000 0.9992 4.1 0.005 45.3 p-nitroaniline 0.010-5.000 0.9990 4.5 0.002 44.0 o–nitroaniline 0.010-5.000 0.9999 4.7 0.002 48.0 2,4- dinitroaniline 0.010-5.000 0.9992 3.6 0.005 41.8

TABLE 3. RECOVERIES AND RSD OF ANILINES IN SPIKED WATER SAMPLES (%) (n=5)

Sample Spiked

concentration/ (mg·L-1)

aniline p-nitroaniline o–nitroaniline 2,4- dinitroaniline

Recovery RSD Recovery RSD Recovery RSD Recovery RSD

Tap water 0.1 96.4 5.8 93.5 4.2 95.6 4.3 98.3 5.0 0.2 99.9 5.3 94.8 4.1 97.5 4.0 99.0 4.8 2 98.6 5.0 95.1 3.7 96.5 3.9 101.1 4.3

Lake water 0.1 94.8 5.5 91.5 5.9 92.5 5.7 90.3 5.8 0.2 97.1 5.1 93.2 5.1 95.3 5.5 91.8 5.5 2 98.5 4.7 95.4 5.0 95.7 5.2 92.5 4.9

River water 0.1 90.2 5.6 93.6 5.8 92.1 5.7 92.6 5.9 0.2 93.0 5.3 97.3 5.6 95.3 5.4 94.6 5.6 2 95.3 5.2 98.1 5.0 96.7 4.9 95.0 5.4

Waste water 0.1 97.8 5.6 90.1 5.7 93.6 5.5 91.9 5.5 0.2 100 5.1 91.3 5.5 94.4 5.2 93.6 5.6 2 103 4.9 93.5 5.1 95.1 4.8 95.1 5.3

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t/min t/min

Figure 4. Chromatogram of anilines extracted by [C4MIN][PF6] from waste water and spiked waste water a. Chromatogram of waste water b. chromatogram of waste water contained 0.2 mg·L-1 mixed standard

The numbers from 1 to 4 are the same as in Fig.1