Chlorophenols (2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol and 2,4, 6-trichlorophenol) may be presented in natural waters or drinking water as a result of disinfection processes involving chlorination, or as contaminants derived from domestic products, industrial operations and agricultural chemicals. A previous HPLC-UV method for determination of phenol and five chlorophenols in tap water using 4-fluoro-7-nitro-2,1,3-benzoxadiaole as a UV labeling reagent shows limited sensitivity. Here, we present an improved HPLC-fluorescence detection method for simultaneous determination of phenol and the above chlorophenols in tap water after pre-column derivatization with 3-chlorocarbonyl-6,7-dimethoxy-1-methyl-2(1<i>H</i>)-quino- xalinone (DMEQ-COCl), using a short, narrow column (50 × 2.1 mm i.d., packed with 5 μm particles of C18 material) to improve the sensitivity. Standard samples containing the compounds are derivatized with DMEQ-COCl in borate buffer (pH 9.0) at room temperature for 3 mins. The response is linear in the concentration range of 0.01 - 0.05 to 0.5 mg/L with r2 values ≥0.9967 for all compounds. The lower limits of detection are 0.001 to 0.008 mg/L, and the coefficients of variation are less than 8.8%. The recovery values from tap water spiked with standard samples are satisfactory. The present method is suitable for examining whether or not tap water samples are contaminated with phenol and chlorophenols in excess of regulatory values.
In order to confirm the quality and safety of potable water, it is essential to evaluate levels of pollutants. Phenol is a ubiquitous pollutant in the aquatic environment because of its widespread use in the synthesis of dyes and drugs in the chemical industry and its presence in commercial products used in daily life. Five chlorophenols (2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, and 2,4,6-trichlorophenol) are also presented in drinking water as a result of disinfection by means of chlorination, as well as being formed by the reaction of hypochlorite (used as a bleach and disinfectant) with phenolic acids and via the degradation of phenoxy herbicides [
The World Health Organization (WHO) guideline value for 2,4,6-trichlorophenol is 0.2 mg/L and concentrations of chlorophenols in drinking-water are usually less than 0.001 mg/L [
Derivatization with a UV absorber or a fluorescent compound that can react with a functional group of the target compound(s) is one of the most useful strategies to obtain high selectivity and sensitivity. Phenol in river water and wine has been determined by means of HPLC-UV after derivatization with benzoyl chloride, and simultaneous analysis of phenol and chlorophenols in urine has been achieved by means of HPLC with fluorescence detection (FLD) after derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride, though this method requires sample pretreatment [
Since it is very important to quickly check and confirm the quality of water throughout the world, we set out to develop a sensitive and selective method for simultaneous determination of phenol and chlorophenols without any clean-up step. 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) has been used for HPLC-FLD as a fluorescent label reactive with primary and secondary amino groups [
Here, we present a sensitive HPLC-FLD method for simultaneous determination of phenol and five chlorophenols in tap water after pre-column derivatization with 3-chlorocarbonyl-6,7-dimethoxy-1-methyl-2(1H)- quinoxalinone (DMEQ-COCl;
Phenol, 2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 2,4,6-trichlorophenol, DMEQ- COCl, and general reagents were obtained from Wako Pure Chemical Industries (Osaka, Japan). Tap water was collected from the laboratory supply.
The HPLC system comprised a model L-6200 pump (Hitachi, Tokyo, Japan), a Rheodyne injection valve (Cotati. CA, USA) with a 20-μL loop, and a model RF-10AXL fluorescence detector (Shimadzu, Kyoto, Japan) operating at an excitation wavelength of 400 nm and emission wavelength of 500 nm. The HPLC column (Inertsil ODS-4, GL Science Inc., Kyoto) was 50 × 2.1 mm i.d., packed with 5 μm particles of C18 material. Quantification of the peaks was performed using a Chromatopac Model C-R3A integrator (Shimadzu). The mobile phase was prepared by the addition of acetonitrile (320 mL) to a solution of 680 mL containing trifluoroacetic acid (0.1 v/v%) in ultrapure water (Milli-Q water purification system, Simplicity® UV, Millipore Corporation, Bedford, MA, USA). The samples were eluted from the column at room temperature at a flow rate of 0.5 mL/min.
Standard samples of phenol and chlorophenols were dissolved in ultrapure water and acetone, respectively, and adjusted to the concentration of 1 g/L. The standard mixture was diluted with ultrapure water. Borate buffer (0.1 M) was adjusted to pH 9.0 by the addition of NaOH. Borate buffer (100 μL) was added to diluted standard samples (100 μL, containing 0, 0.01, 0.02, 0.05, 0.1, 0.2, or 0.5 mg/L of each compound). DMEQ-COCl solution in acetonitrile (2 mg/mL, 100 μL) was added and vortexed. The mixture was allowed to react for 3 min at room temperature. Saturated L-aspartate solution (100 μL) was added to stop the reaction, and aliquots (10 μL) were injected into the HPLC system.
Water samples spiked with standard samples (lower limit of quantification, 0.05, 0.1, 0.2, or 0.5 mg/L of each compound) were passed through 0.45 μm filters (Cosmonice Filter S, Nacalaitesque) to remove suspended substances and immediately analyzed. Filtration was confirmed to have no effect on the analysis by analyzing a sample spiked with standards before and after filtration; no significant difference in recovery was observed.
Relative recovery was expressed as the ratio of the slope of the calibration curve prepared from a water sample spiked with standard sample to the slope of the standard calibration curve prepared as described above. Relative recovery data were used to assess the accuracy of the method.
For the time course study, the reaction time was set at 2, 3, 5, 10, and 15 min. Phenol and five chlorophenols (100 μL, each 0.1 mg/L), borate buffer (100 μL, pH 9.0)and DMEQ-COCl (100 μL, 2 mg/mL) were mixed as described in Materials and Methods. The maximum peak area or plateau level was reached within 3 mins (
Next, pH dependency (pH 7.5 to 10.0) was examined at the derivatization time of 3 min (
all compounds were stable (more than 95% of the maximum area) in the range of pH 8.5 to pH 9.3.Since, under pH 8.5, it is guessed that a little pH change may effect phenol detection, simultaneous determination of tested compounds is not better at pH 8.5.
In preliminary tests (data not shown), the peak areas of all derivatives obtained with 1 mg/mL of DMEQ- COCl at pH 9.0 and room temperature were less than about 60% of the control. Furthermore, when using 3 mg/mL of DMEQ-COCl, a large blank peak interfered with the peaks of phenol, 2-chlorophenol, and 4-chlo- rophenol. Thus, the derivatization time of 3 min at pH 9.0using 2 mg/mL of DMEQ-COCl was chosen for the assay.
DMEQ-2,6-dichlorophenol, DMEQ-2,4-dichlorophenol, and DMEQ-2,4,6-trichlorophenol were 3.6, 5.7, 7.9, 9.3, 13.6, and 23.9 min, respectively. The running time was 30 mins.
The standard curves of phenol, 2-chlorophenol, 4-chlorophenol, 2,6-dichlorophenol, 2,4-dichlorophenol, and 2,4,6-trichlorophenol were constructed by plotting integrated peak areas of derivatives vs. concentrations of phenolic compounds. The calibration data are summarized in
The method using NBD-F previously developed in our laboratory showed relatively poor sensitivity [
Precision and accuracy for intra-day and inter-day assays of these derivatives are shown in
Compounds | Slope | Intercept | Concentration range | r2 | Lower limit of detection (S/N = 3) |
---|---|---|---|---|---|
Phenol | 12,057 | +50.61 | 0.01 to 0.5 mg/L | 0.9995 | 0.003 mg/L (0.0075*) |
2-Chlorophenol | 8892 | −88.43 | 0.01 to 0.5 mg/L | 0.9982 | 0.002 mg/L (0.0050*) |
4-Chlorophenol | 10,422 | +43.71 | 0.01 to 0.5 mg/L | 0.9984 | 0.001 mg/L (0.0025*) |
2,6-Dichlorophenol | 2799 | +41.28 | 0.02 to 0.5 mg/L | 0.9967 | 0.004 mg/L (0.010*) |
2,4-Dichlorophenol | 5315 | +46.36 | 0.01 to 0.5 mg/L | 0.9990 | 0.002 mg/L (0.0050*) |
2,4,6-Trichlorophenol | 1555 | +18.95 | 0.05 to 0.5 mg/L | 0.9972 | 0.008 mg/L (0.020*) |
*: Data are expressed as absolute amount (ng/20 μL injection).
Compounds (mg/L) | Measured (mg/L, Mean ± S.D., n = 5) | C.V. (%) | Recovery (%) |
---|---|---|---|
Phenol | |||
0.02 | 0.0193 ± 0.0015 | 8.0 | 96.5 |
0.1 | 0.0972 ± 0.0052 | 5.4 | 97.2 |
0.5 | 0.516 ± 0.021 | 4.1 | 103.2 |
2-Chlorophenol | |||
0.02 | 0.0204 ± 0.0016 | 7.8 | 102.0 |
0.1 | 0.0998 ± 0.0064 | 6.4 | 99.8 |
0.5 | 0.512 ± 0.019 | 3.7 | 102.4 |
4-Chlorophenol | |||
0.02 | 0.0198 ± 0.0014 | 7.0 | 99.0 |
0.1 | 0.102 ± 0.006 | 5.9 | 102.0 |
0.5 | 0.521 ± 0.020 | 3.8 | 104.2 |
2,6-Chlorophenol | |||
0.02 | 0.0198 ± 0.0015 | 7.6 | 99.0 |
0.1 | 0.0986 ± 0.0043 | 4.4 | 98.6 |
0.5 | 0.504 ± 0.018 | 3.6 | 100.8 |
2,4-Chlorophenol | |||
0.02 | 0.0204 ± 0.0014 | 6.8 | 102.0 |
0.1 | 0.0984 ± 0.0034 | 3.5 | 98.4 |
0.5 | 0.512 ± 0.014 | 2.7 | 102.4 |
2,4,6-Trichlorophenol | |||
0.1 | 0.102 ± 0.007 | 6.9 | 102.0 |
0.5 | 0.498 ± 0.015 | 3.0 | 99.6 |
Compounds (mg/L) | Measured (mg/L, Mean ± S.D., n = 5) | C.V. (%) | Recovery (%) |
---|---|---|---|
Phenol | |||
0.02 | 0.0191 ± 0.0017 | 8.9 | 95.5 |
0.1 | 0.0966 ± 0.0061 | 6.3 | 96.6 |
0.5 | 0.525 ± 0.028 | 5.3 | 105.0 |
2-Chlorophenol | |||
0.02 | 0.0208 ± 0.0017 | 8.2 | 104.0 |
0.1 | 0.0972 ± 0.0070 | 7.2 | 97.2 |
0.5 | 0.482 ± 0.022 | 4.6 | 96.4 |
4-Chlorophenol | |||
0.02 | 0.0206 ± 0.0016 | 7.8 | 103.0 |
0.1 | 0.103 ± 0.007 | 6.8 | 103.0 |
0.5 | 0.486 ± 0.027 | 5.6 | 97.2 |
2,6-Dichlorophenol | |||
0.02 | 0.0198 ± 0.0016 | 8.1 | 99.0 |
0.1 | 0.0976 ± 0.0052 | 5.3 | 97.6 |
0.5 | 0.514 ± 0.022 | 4.3 | 102.8 |
2,4-Dichlorophenol | |||
0.02 | 0.0208 ± 0.0015 | 7.2 | 104.0 |
0.1 | 0.0984 ± 0.0065 | 6.6 | 98.4 |
0.5 | 0.512 ± 0.024 | 4.7 | 102.4 |
2,4,6-Trichlorophenol | |||
0.1 | 0.104 ± 0.009 | 8.7 | 104.0 |
0.5 | 0.493 ± 0.022 | 4.5 | 98.6 |
Tap water samples were collected from the laboratory, and the proposed method was employed to determine phenol and chlorophenols in spiked tap water. As shown in
A simple HPLC-UV method for simultaneous determination of phenol and chlorophenols in water was previously developed using NBD-F or benzoyl chloride as the derivatizing reagent, without complicated sample clean-up [
Compounds | Concentration in tap water sample | Relative recovery (%, mean ± S.D., n = 5) | r2 (Average) |
---|---|---|---|
Phenol | N.D. | 89.8 ± 6.1 | 0.9933 |
2-Chlorophenol | N.D. | 92.6 ± 6.6 | 0.9946 |
4-Chlorophenol | N.D. | 94.2 ± 5.6 | 0.9971 |
2,6-Dichlorophenol | N.D. | 92.4 ± 6.6 | 0.9972 |
2,4-Dichlorophenol | N.D. | 106.8 ± 7.4 | 0.9965 |
2,4,6-Trichlorophenol | N.D. | 111.0 ± 6.9 | 0.9942 |
N.D.: not determined.
phenol, 2,4-dichlorophenol, 2,6-dichlorophenol, and 2,4,6-trichlorophenol (i.e. whether or not these compounds were present in excess of regulatory levels).
YasuhikoHigashi, (2016) Development of Simultaneous HPLC-Fluorescence Assay of Phenol and Chlorophenols in Tap Water after Pre-Column Derivatization with 3-Chlorocarbonyl-6,7-dimethoxy-1- methyl-2(1H)-quinoxalinone. Detection,04,16-24. doi: 10.4236/detection.2016.41003