Determination of phenol pollutants in industrial waste water by MEKC and on-line sweeping technique

Yan Hongyuan¹,Yang Gengliang^{1, 2} , Qiao Fengxia¹, Liu Haiyan¹, Chen Yi²
(¹College of Pharmacy，Hebei University，Baoding, China 071002; ²Center for Molecular Science, Institute of Chemistry, Chinese Academy of Science, Beijing 100080)

Received on  

Abstract An on-line sweeping technique method was developed to the determination of trace phenol、o-nitrophenol and p-nitrophenol in industrial waste water by micellar electrokinetic chromatography (MEKC), in which industrial waste water were simply filtered with 0.45 mm filters, and analyzed with 20 mmol/L Na₂B₄O₇ + 80 mmol/L sodium dodecyl sulfate (SDS)(pH=9.45) as the running buffer and a fused silica capillary tube was loaded with a voltage of 18 kV and detection was performed at UV 214 nm. Under these conditions, 1000-fold enrichment of p-nitrophenol was obtained. the detection limits were 0.03、0.02、0.02 mg/L for phenol, o-nitrophenol , p-nitrophenol respectively when the injected time was 200s. The results show that the method is precise, simple and cost-effective.
Keywords MEKC; on-line sweeping technique; waste water; phenol; nitrophenol

1 INTRODUCTION
The low concentration sensitivity is a disadvantage of capillary electrophoresis techniques and it is due to the extremely small amounts of the sample injected and the limited optical path length for on-capillary photometric detection. So it is difficult to run trace analysis. In order to improve the concentration detection limits of capillary electrophoresis, many methods such as off-line sample concentration (e.g., solid-phase extraction)^[1], increasing the path-length for photometric detector ^[2-3], using high sensitivity detector ^[4-5] and on-line concentration ^[6-9] have been used.
    On-line sample concentration is a solution to solve the problem and the technique is less expensive than using laser induced fluorescence detector or much simpler than preconcentration techniques, e.g., solid phase extraction ^[10] and liquid-liquid extraction ^[11]. Such technique can reduce the error in manual handling and can be used in an automated fashion.
    On-line sweeping is one of on-line sample concentration approach which is defined as a phenomenon where analytes are picked up and concentrated by the pseudostationary phase that enters the sample zone containing no pseudostationary phase in MEKC. It occurs when the sample matrix is void of a charged carrier phase and it does not matter whether the sample matrix has a higher, similar, or lower conductivity compared to the background solution. In theory, it provides for almost unlimited detection sensitivity for analytes that have high affinities toward the pseudostationary phase. 5000-fold improvements have been reported ^[12-13], which is greater than any other reported technique. Because sample sweeping is much efficient, independent of the EOF and works for charged or uncharged solutes, it is becoming an almighty technique.
    In recent decades, analysis of trace phenol pollutants in water was commonly performed by GC or HPLC^[14-16]. Unfortunately these methods are often time-consuming and require derivatization or gradient elution, and the process of sample preparation was very complicated. CE is a promising technique for environmental analysis because of its excellent separation efficiency, high versatility, and low cost ^[17-19]. Because untreated environmental water samples can be injected directly into CE capillaries, considerable savings in analysis time and cost of consumables could be achieved. However, because UV absorption spectrometric detection is the detection technique most widely employed in CE, it often exhibit detection limits that are at least one order of magnitude poorer than those of corresponding GC or HPLC methods. So on-line sweeping concentration is a better solution to solve the problem. Takeda et al. had concentrated bisphenol A and alkylphenols by sweeping with SDS as anionic surfactant^[20] and tetradecyltrimethylammonium bromide as cationic surfactant^[21], respectively, in which 69-, 48-, 55- and 41- fold increases in detection sensitivity were obtained respectively for bisphenol A, 4-tert.-butylphenol, 4-(1,1,3,3-tetramethylbutyl)phenol, and the second peak of 4-nonylphenol isomers by SDS as anionic surfactant. When sweeping with cationic surfactant (tetradecyltrimethylammonium bromide), 56-, 67- and 29-fold increases in detection sensitivity of bisphenol A, 4-tert.-butylphenol and 4-(1,1,3,3-tetramethylbuthl)phenol were obtained, respectively. Monton et al.^[22] got 100 fold enrichment for phenol derivatives by using nonionic micelles of the alkyl polyoxyethylene ether type(Brij35 and Brij58) as the pseudostationary phase. When sweeping with anionic- zwitterionic mixed micelles^[23], 370- and 360- fold improvement in detector response were obtained for some neutral steroids and some phenol derivatives, which were peviously not amenable to sweeping by pure SDS micelles.
    So far, the research of on-line sweeping technique was mainly concentrated on theoretical study, and standard substances were used for validating the sweeping efficiency. Application to real samples was rare. In this work, the determination of trace phenol、o-nitrophenol and p-nitrophenol in industrial waste water was investigated by MEKC with on line sweeping technique. The effect of pH in background solution, the concentration of SDS, injection time on concentration was investigated. By using on line sweeping method, the detector responsive signal could be improved significantly. The developed method allows determination of trace phenol pollutants in wasted water, and the sample clean-up steps that are mandatory for HPLC and GC are avoided.

2 EXPERIMENTAL
2.1 Apparatus
The experiment was performed on a Waters Quanta 4000E capillary electrophoresis system (Milford, MA, USA) with a built-in 0-30 kV high voltage power supply, a fixed wavelength UV detector near the cathode end and a forced-air cooling system. Uncoated fused capillary (75m m I.D. total length is 60 cm and the effective length is 52 cm) was from Yong Nian Optical Fiber Factory, Hebei Province, China. The UV detector wavelength was set at 214 nm. The temperature remained constant at 25⁰C. Samples were introduced to the capillary by using gravity injection and the injection height was 12 cm. Data processing was carried out with a CKChrom chromatography data system. The sensitivity of the detector is set at 0.005 AUFS.
2.2 Reagents and solutions
Phenol,o-nitrophenol and p-nitrophenol were provided by Tianjin reagent factory. Sodiumdodecyl sulfate (SDS) was purchased from Zhongxi Chemical Plant. The other chemical reagents are all of analytical grade. Stock solution phenols (1.0 mg/ml) were prepared with double-distilled water.
    All of the backgrounds (BGS) were prepared by mixing stock solutions of 200 mM SDS, 100 mM sodium borate with double-distilled water and filtered through 0.45 mm filters (Ruili Separation Instrument Factory, Shanghai, China) prior to use.
2.3 Procedures
The capillary was purged with 1.0 mol/L NaOH (10 min), followed by methanol (10 min), double-distilled water (10 min) and the BGS (5 min) before use. In order to ensure repeatability, the capillary was purged between consecutive analysis with 1.0 mol/L NaOH (3 min) purified water (2 min) and BGS (3 min).

3 RESULTS AND DISCUSSIONS
3.1 Effect of buffer solution pH value
In our experiment, the effects of tris, phosphate and borate as the running buffer were investigated. The results showed that the height of sample peaks and resolution of samples with interferences were better when borate was used as the running buffer. So we chose borate as the running buffer. With the concentration of 40 mmol/L SDS unchanged, different electrolyte systems at different pH values ranging from 5.04 to 11.24 were tested. From the experiment it was found that the heights of sample peaks became higher and the retention time was reduced with the increase of buffer pH value. However, when the pH value was higher than 9.45, the peaks height began to decrease, the ion strength of the buffer is increased and too much Joule heat would be produced. So we choose 9.45 as the optimum pH value.

Fig. 1 Effect of background buffer solution pH on the concentration fold
Experimental conditions: applied voltage: 18 kV; 60 cm/ 75m m; effective length: 52 cm; UV detection wavelength: 214 nm

3.2 Effect of SDS concentration
In this experiment, the effects of SDS concentrations were investigated with the range from 10 to 100mmol/L when the injection time was kept as 100s and the other experiment conditions were the same as above. From the experiment, it showed that the peaks of phenol、o-nitrophenol and p-nitrophenol and other interference could not be separated by ordinary CZE. The reason may be that these compounds have similar velocity. When 20 mM SDS was added as the micellar phase in 20 mM borate electrolyte solution, the separation of phenol、o-nitrophenol and p-nitrophenol was improved. With the increasing of the surfactant concentration, the effect of enrichment improved. But when SDS concentration was above 80 mmol/L, the current was too high to run the separations and much Joule heat is produced, the baseline fluctuated evidently, it is not favorable to separation and determination. So we choose 80 mmol/L as the optimum SDS concentration.

Fig.2 Effect of SDS concentration on the concentration fold
Experimental conditions: buffer solution (20 mmol/L Na₂B₄O₇+different concentration of SDS，pH=9.45); applied voltage: 18 kV; 60 cm/ 75m m; effective length: 52 cm; UV detection wavelength：214 nm

3.3 Effect of the injection time
In the experiment, when the buffer solution (80 mmol/LSDS+20 mmol/L Na₂B₄O_7, pH=9.45) and the other conditions were maintained unchanged, the effect of different injection times on the enrichment of peaks was investigated in the range from 1sec to 900sec. It was shown that the peak increased with the increase of injection time. When the injection time was 900 sec, about 500、550、1000-fold concentration of phenol、o-nitrophenol and p-nitrophenol could be obtained. However, resolution of the sample peaks with each other and impirities decreased and the sample peaks cluttered when the injection time was too long. So we chose 200 sec as the injected time.
3.4 Analysis of samples
In this work, the standard solutions were prepared as the following: the stock solution of phenols were diluted with the double-distilled water to 5.0, 2.0, 1.0, 0.5, 0.2, 0.1 mg/L respectively, then these solutions were analyzed under optimized conditions. The experiment data showed that in the range of 5-0.1 mg/L a good linear relation could be attained for the three phenol pollutants(R>0.997). When the injected time was 200s(S/N=3), the detection limits were 0.03, 0.02, 0.02 mg/L for phenol, o-nitrophenol , p-nitrophenol respectively.

Fig.3 Chromatogram of the standard
buffer: 20 mmol/L Na₂B₄O₇+80 mmol/L SDS, pH=9.45; applied voltage:18 kV; injected time: 200s; UV detection wavelength: 214 nm. Concentrations of standards were 0.4 mg·L^-1: 1.phenol; 2. o-nitrophenol；3.p-nitrophenol.

Table 1 the linear equation and correlation coefficient

    The industrial waste water was filtered through 0.45mm filters then it was directly analyzed under the optimized conditions. Recoveries were determined by the standard addition method. The results were shown in Table 2.

Table 2 Analytical results of the sample

4 CONCLUSIONS
The results obtained above show that the sweeping technique is a good on-line sample concentration approach. It is a fast, effective, and easy way to concentrate neutral and anionic substances inside the capillary. The detector responsive signal can be improved significantly. On-line sweeping method makes it possible for capillary electrophoresis to analyze trace ingredient such as environmental analysis, pesticide analysis of agricultural product and trace drugs in blood, urine, etc.

ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation, P.R. China (Grant No.20375010) and the Natural Science Foundation, Hebei Province, PR China (Grant No.202096, 02245501-D), the Foundation of Ministry of Science and Technology, China (Nos.2002CCA3100, KJCX2-H4), the Excellent Youth Program of Education PRC and the "Bai Ren" Project of Institute of Chemistry, Chinese Academy of Science.

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