Determination of Nitrobenzene in Water Using Ionic Liquid-Based Liquid-Phase Microextraction Coupled With HPLC

Nitrobenzene is widely used as an intermediary in the chemical industry. The release of nitrobenzene into the environment is of great concern because of its toxicity, persistence, and accumulation in food.1,2 It is therefore critical to monitor nitrobenzene in the environment.

Many studies have been reported for the determination of nitrobenzene in samples,1,3,4 and HPLC is among the techniques used.3,4 However, because of the low concentration of nitrobenzene and the complexity of environmental samples, it is difficult to distinguish traces of nitrobenzene from various interfering substances in the matrix and obtain accurate results. Therefore, sample pretreatment is usually needed prior to instrumental analysis.

In general, liquid–liquid extraction (LLE) is the most commonly used technique for the isolation and enrichment of nitrobenzene in environmental samples. However, the method requires an appreciable amount of toxic solvents, which are hazardous both to operators and the environment. Moreover, the method often uses evaporative concentration procedures, which are very time-consuming. Therefore, a variety of microextraction techniques that require no or minimal amounts of solvent have been developed in recent years. Among them, solid-phase microextraction (SPME) and liquid-phase microextraction (LPME) are the two most promising extraction techniques for the analysis of nitrobenzene.

SPME, which was developed by Pawliszyn and co-workers,5 is a rapid, simple, solvent-free, and easily automated technique for the isolation of organic compounds from gaseous, liquid, and solid samples. Some publications have reported the use of SPME for the analysis of nitrobenzene in water.1,3,6 The drawbacks of SPME are limited lifetime, fragility of fibers, and possibility of sample carryover. In addition, it is very difficult for SPME to extract some polar compounds, such as nitrobenzene, without derivatization.7,8

LPME9 is an attractive alternative for sample preparation. The advantage of LPME is that it is inexpensive and offers users the freedom to select appropriate solvent for the extraction of target analytes. Carbon tetrachloride10 and 1-octanol11 are the most widely used extraction solvents in LPME. However, the low viscosity of the extraction solvents often results in the relatively small drop volume (typically 1 µL) and thus low sensitivity in chromatography. Furthermore, the repeatability is often unsatisfactory because of the volatility of the extraction solvent, and the solvents commonly used in LPME are incompatible with reversed-phase HPLC.

A typical ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate ([C4MIM][PF6]) reportedly has considerable viscosity, low miscibility with water, and good dissolvability for nitrobenzene.12 Therefore, it should be possible to stably suspend a large-volume drop on the needle of a microsyringe for efficient LPME and thereby provide high sensitivity for HPLC determination. Another advantage of [C4MIM] [PF6] for LPME is its compatibility with HPLC and the fact that it does not harm the HPLC column. However, to the best of the authors' knowledge, use of [C4MIM] [PF6] as an extraction solvent for the LPME of nitrobenzene directly in a water sample prior to HPLC has not been reported thus far. Hence, it is important to investigate the possibility of using [C4MIM][PF6] as an extraction solvent in LPME for the analysis of nitrobenzene in water with high selectivity and a high enrichment factor.

A method for the determination of nitrobenzene in water was developed using HPLC preceded by LPME in headspace mode based on [C4MIM][PF6]. Experimental parameters affecting the extraction of nitrobenzene were optimized; these included extraction temperature and time, acidity, ionic strength, and sample volume. Under the optimized experimental conditions, the repeatability, recovery, detection limit, and linearity of the proposed method were then evaluated. Finally, the procedure was applied to detect nitrobenzene in the water sample from the authors' laboratory.


Reagents and samples

Nitrobenzene was obtained from the Shanghai Reagent Plant (Shanghai, China). Standard stock solution (10 g/L) of nitrobenzene was prepared in methanol. Working solutions were prepared by appropriate dilution of the stock solution with water. [C4MIM][PF6] (99%) was obtained from Chengjie Co. (Shanghai, China). HPLC-grade methanol was purchased from Tedia Co. (Fairfield, OH). All other chemicals were analytical-grade reagents from Beijing Chemicals (Beijing, China); ultrapure water (Molecular Science and Technology, Chongqing, China) was used throughout the experiments.

Experimental wastewater from the authors' laboratory was collected for nitrobenzene measurement. The water sample was collected and filtered through 0.45-µm filter membranes. The sample was then stored in the refrigerator at 4 °C prior to extraction.

Extraction procedure

Figure 1 - Schematic of ionic liquid-based liquidphase microextraction: 1) stir bar,

2) sample solution,3) ionic liquid drop, 4) septum, 5) microsyringe.

Extraction was performed as shown in Figure 1 according to Ref. 9, with slight modification. Briefly, 3 µL of [C4MIM][PF6] was injected into a 50-µL microsyringe (Anting, Shanghai, China). Next, the stainless steel needle of the microsyringe was pushed through the septum of a vial containing 8 mL of solution, and the plunger of the microsyringe was depressed to expose a 3-µL [C4MIM][PF6] drop. The vial was then placed into a thermostable bath under constant stirring speed, and the microsyringe was clamped into place so that the needle of the syringe was exposed to the headspace of the 8-mL sample solution held in the vial. After extraction, the [C4MIM][PF6] drop was retracted into the microsyringe and collected in a vial containing 50 µL of methanol before injection into the HPLC system for analysis.

HPLC determination

The HPLC equipment used throughout the experiment was an L-7000 system (Hitachi, Tokyo, Japan), which included an L-7100 pump, L-7300 column oven, and L-7455 diode array detector set at 280 nm. A personal computer (Founder Science and Technology, Beijing, China) equipped with a D-7000 HPLC system manager program for HPLC (Hitachi) was used to record and process the chromatographic data and control the HPLC equipment hardware. A manual injection valve with a 20-µL loop (Hitachi) was used for injection. A C18 column (250 mm × 4.6 mm, particle size 5 µm) (Elite, Dalian, China) was used to separate the analyte enriched in the [C4MIM][PF6] drop. The mobile phase was a mixture of methanol and water (70:30, v/v) delivered at a flow rate of 0.5 mL/min. Retention time was used for identification. Quantifications were achieved using the peak height of the chromatograms. Each analysis was done in triplicate, and the average of the quantification results was reported.

Results and discussion

Optimization of extraction parameters

Figure 2 Effect of temperature on the extraction of nitrobenzene. LPME conditions: 3.0 μL of [C4MIM][PF6], 8.0 mL of aqueous solution, pH 7.00, 30 min; no salt was added. HPLC conditions: C18 column (250 mm × 4.6 mm × 5 μm), 0.5 mL/min methanol-water (70:30, v/v), 30 °C, 280 nm, 20 μL.

1. Effect of temperature. An increase in temperature usually improves the evaporation of target compounds from the sample matrix to the headspace. In this experiment, the effect of temperature (30–50 °C) on extraction efficiency was investigated. The temperature had a marked effect on the extraction (Figure 2). With the increase in temperature, the extraction efficiency improved gradually, as expected. However, high temperature leads to low viscosity of the ionic liquid, which may shorten the lifetime of the drop. Therefore, an extraction temperature of 40 °C was used in the following experiments.