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.
Experimental
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.