agents such as hydrogen peroxide in conjunction with catalysts,
unfortunately, result in low yields and/ or multiple byproducts.12
This article describes a sensitive electrochemical method for
the detection and determination of PCP in contaminated soil. PCP
was oxidized to 1,4-TCBQ by bis(trifluoroacetoxy)iodobenzene
flow thin-layer electrochemical cell (BAS) which was placed
downstream from the injector. The electrochemical cell consisted
of an Ag/ AgCl (3 M NaCl) reference electrode, a stainless steel
auxiliary electrode, and the dual glassy carbon working electrode
containing the glucose oxidase film. The target analyte (1,4-
TCBQ) was reduced to tetrachloro-1,4-hydroquinone (1,4-TCHQ)
in the presence of reduced glucose oxidase and then electro-
chemical detection of the oxidation of 1,4-TCHQ at the electrode
surface was performed at +0.40 V using a CV1B voltammograph
(BAS). Digitization and data acquisition were accomplished using
a DAS-8 A/ D card (MetraByte, Taunton, MA) connected to an
IBM-AT computer with custom software such that peak height
or peak area could be obtained. Note that all lines in contact with
the sample had to be stainless steel to avoid severe peak tailing
due to adsorption of the analyte within the system.
(
BTFAIB) since this high-yield reaction was recently reported for
the oxidation of various chlorinated phenols including PCP under
mild conditions.13,14 A flow injection (FI) electrochemical system
was constructed by immobilization of glucose oxidase on a glassy
carbon electrode. In addition to characterization of the electrode
and the oxidation step, the 1,4-TCBQ was then demonstrated as
an efficient mediator to shuttle electrons between the enzyme and
an electrode when coupled with immobilized glucose oxidase in
the presence of excess glucose. Additional relevant data were
also presented to demonstrate the applicability of the novel
electrochemical scheme for the determination of PCP. To our
knowledge, this is the first demonstration of a hybridized
electrochemical detection system being used in the assay of PCP
in real contaminated soil samples.
Optimization of FI-Mediated Electrochemical System.
The 1,4-TCBQ response with respect to glucose concentration,
flow rate, response time, and buffer pH was tested. The sensitivity
and reproducibility of the 1,4-TCBQ signal were investigated as
well as the stability of the enzyme-based electrode. The optimized
system was then applied to samples of PCP which had been
oxidized by BTFAIB (see below). The substrates and products
of this reaction were tested for possible interferences.
EXPERIMENTAL SECTION
Reagents. Glucose oxidase (EC 1.1.3.4, type X-S, 182 units/
mg), L-tartaric acid, and glutaraldehyde (25% w/ w) were obtained
from Sigma (St. Louis, MO). PCP, BTFAIB, tetrachloro-1,2-
benzoquinone (1,2-TCBQ), 1,4-TCBQ, and Nafion (5% w/ w solu-
P CP Oxidation Using Bis(trifluoroacetoxy)iodobenzene.
Reactions were performed in 0.1 M tartrate buffer (20 mL) using
PCP (10 mM in ethanol) and freshly prepared BTFAIB (250 mM
in ethanol) stock solutions. The reactions were carried out at
room temperature, with light protection for 1 h. At the end of
the reaction, hydrogen peroxide (500 µM) was added to neutralize
any unreacted BTFAIB and concentrated NaOH (8 M, 250 µL)
was added to increase the pH to 3.5. The effect of buffer type,
pH, and the BTFAIB to PCP concentration ratio were optimized.
PCP-spiked tap water samples as well as extracts from soil samples
were oxidized by BTFAIB under the optimized conditions and
tested using the electrochemical detection system.
tion) were purchased from Aldrich (Milwaukee, WI).
D-Glucose
stock solutions (2 M) were allowed to mutarotate for at least 24
h before use. All chemicals were of the highest grade com-
mercially available and used without further purification. Certified
PCP-contaminated soil samples were obtained from Resource
Technology Corp. (Laramie, WY). PCP-contaminated samples
were obtained from a wood-preserving plant in the Montreal
region.
Enzyme-Based Electrode P reparation. A dual glassy car-
bon electrode (BioAnalytical Systems (BAS), West Lafayette, IN)
was polished with 1-µm diamond paste followed by 0.05-µm
alumina slurry (Buehler) and washed with deionized water. The
electrode surface was then cleaned for enzyme immobilization by
sonication for 10 min in a Branson sonicating bath. Glutaralde-
hyde was added to glucose oxidase to initiate cross-linking, and
P reparation of Soil Extracts. Soil samples (2 g) were
extracted with 100 mL of 10 mM NaOH for 4-8 h under light
protection. After centrifugation, the supernatants were collected
and stored in the dark at 4 °C. PCP from soil extracts could be
partially purified and concentrated if necessary by using Sep-Pak
tC18 cartridges (Waters Corp., Milford, MA). Cartridges were
wetted with methanol and then washed with 50 mM phosphate
1
0 µL of this mixture was dropped onto one of the glassy carbon
surfaces. The mixture was dried completely (10-15 min) under
vacuum using water aspiration to form a thin film. A drop (10
µL) of Nafion solution was then dropped onto this film and dried
under vacuum (10-15 min) to form a protective film over the
enzyme layer. The effects of glutaraldehyde, enzyme, and Nafion
concentration on the 1,4-TCBQ response were examined.
FI-Electrochemical Detection Apparatus. The FI-electro-
chemical system consisted of a peristaltic pump (FIA Pump 1000,
Eppendorf North America, Madison, WI) which delivered the
(
pH 8.0). The samples (20-40 mL) were neutralized and passed
through the cartridges. Contaminants were released from the
cartridges using a 50% methanol solution (2 mL). The PCP was
only eluted from the cartridges when the methanol concentration
was increased to 100%. The sample was then diluted back to the
original volume for analysis using HPLC or the electrochemical
system. For samples containing low levels of PCP (<50 ppm),
the concentrated methanol extract could be used directly without
dilution for HPLC analysis since the limit of detection was much
higher than that of the electrochemical system. The sample
preparation technique using the Sep-Pak cartridges resulted in
recoveries of PCP of greater than 95%. It should be noted that
less than 2.5% PCP was recovered in the 50% methanol fraction
when standard PCP at 1 µM was analyzed.
sample and buffer (100 mM L-tartrate (pH 3.5), 40 mM glucose)
at a preset flow rate. Oxygen was removed from the buffer and
sample by constant nitrogen bubbling. A 100-µL sample was
injected into this stream by a motorized injection valve (EVA
injector, Eppendorf). The loading and injection times were
controlled from the EVA injector. The detector was a LC-44 cross-
(
(
(
12) Tang, W.; Huang, C. Chemosphere 1 9 9 6 , 33, 1621-1635.
13) Saby, C.; Male, K. B.; Luong, J. H. T. Anal. Chem. 1 9 9 7 , 69, 4324-4330.
14) Saby, C.; Luong, J. H. T. Chem. Commun. 1 9 9 7 , 1197-1198.
P CP Analysis by HP LC. The HPLC system consisted of a
WISP 710B injector (Waters), a Waters model 590 pump controlled
Analytical Chemistry, Vol. 70, No. 19, October 1, 1998 4135