2008-5435/11/31-12-17 INTERNATIONAL JOURNAL OF OCCUPATIONAL HYGIENE Copyright 2011 by Iranian Occupational Health Association (IOHA) IJOH 3: 12-17, 2011 ORIGINAL ARTICLE Determination of Benzene, Toluene and Xylene (BTX) Concentrations in Air Using HPLC Developed Method Compared to Gas Chromatography ABDULRAHMAN BAHRAMI 1*, HOSIEN MAHJUB 2, MARZIEH SADEGHIAN 1, FARIDEH GOLBABAEI 3 1 Department of Occupational Health, Research Centre for Health Sciences, School of Public Health, Hamedan University of Medical Sciences, Hamedan, Iran; 2 Department of Biostatics and Epidemiology, Research Centre for Health Sciences, School of Public Health, Hamedan University of Medical Sciences, Hamedan, Iran; 3 Department of Occupational Health, Faculty of Health, Tehran University of Medical Sciences, Tehran, Iran. Received May 8, 2010; Revised September 19, 2010; Accepted October 30, 2010 This paper is available on-line at http://ijoh.tums.ac.ir ABSTRACT A new method for analysis of benzene, toluene, and xylene (BTX) using High Performance Liquid Chromatography-UV detection (HPLC-UV) is described and compared to the gas chromatography (GC) method. A charcoal adsorption tube connected to a small pump was used to obtain samples from an atmosphere chamber standard. Samples were extracted with methanol and analyzed by HPLC-UV. Chromatography was isocratic in a mobile phase consisting of water-methanol (30-70). The flow rate was set at 1 ml/min. The analyses were completely separated and were quantified using both methods. The results demonstrated no statistically significant differences between BTX concentrations between the two analytical methods with a correlation coefficient of 0.98-0.99. The GC method provided higher sensitivity than HPLC, but the HPLC determination of BTX were applicable to real samples because its sensivity was lower than the thershold limit recommended by the American Conference of Governmental Industrial Hygienist (ACGIH) for an 8-hour workday. Keywords: Volatile organic compounds, Gas chromatography, Air INTRODUCTION Volatile organic compounds emit from a wide range of sources inside of industrial units and ambient air in urban areas. Anthropogenic volatile organic compounds (VOCs) arise mainly from motor vehicle exhaust, solvent usage, industrial processes, oil refining, petrol storage, and distribution, land filled wastes, food manufacture, and agriculture. They play an important role in the formation of ground-level ozone, photochemical oxidants, and smog episodes, and they are harmful to the ecosystem. Some VOCs are more * Corresponding author: A. R. Bahrami, Email: bahrami@umsha.ac.ir important due to their health effects on humans. Benzene has been shown to cause cancer in both animals and humans, and therefore it is currently classified by the Environmental Protection Agency (EPA), ACGIH, and the International Agency for Research on Cancer (IARC) as a human carcinogen [ 1-9]. Exposure to VOCs in the workplace is subject to regulations and has to be monitored regularly. A large number of methods to date have been reported for the analysis of VOCs in ambient air at the workplace or in ambient air in urban areas by the National Institute for Occupational Safety and Health (NIOSH), the Occupational Safety and Health Administration IJOH January 2011 Vol. 3 No. 1 12-17
Determination of Benzene, Toluene and Xylene (BTX) Concentrations in Air Using HPLC ijoh.tums.ac.ir 13 Fig 1. Schematic diagram of test atmosphere generation system ( a ) ( b ) Fig 2. The chromatograph of HPLC (a) and BTX in GC (b) (OSHA) and Environmental Protection Agency (EPA). Most of these methods of air sampling are done with active or passive samplers and subsequent analysis in a laboratory using gas chromatography with flame ionization detectors or gas chromatography mass spectrometry [ 10-13]. In some methods, VOCs can be monitored by on-line gas chromatography with an FID detector and direct spectroscopic measurement [ 11]. The FID detector is not specific to VOCs and suffers from interference of other hydrocarbons present in ambient air, which can be co-eluted with the VOCs. These limitations have prompted the development of a number of alternative methods such as High Performance Liquid Chromatography (HPLC) for analysis of VOCs. There are a few reports of BTX analysis using an HPLC-Fluorescence detector. Campos-Candel et al. analyzed BTX by HPLC-Fluorescence detector β- cyclodextrin stationary phase and extracted samples with pressured fluid extraction [ 14]. Ghittori et al. used the HPLC-Fluorescence detector to determine benzene concentration in the air. They extracted samples with a mixture of methylene chloride and ethyl acetate [15]. Our objective was to develop a rapid and specific method for simultaneously measuring BTX in ambient air inside of manufactures using HPLC coupled with an UV detector. MATERIALS AND METHODS In order to prepare known concentrations of VOC analyses in the range of interest, a dynamic atmosphere generation system was built in the laboratory. A standard gas-generating device was constructed as
14 IJOH January 2011 Vol. 3 No. 1 Bahrami et al. Table 1. The mean of peak area and retention time at different mobile phase for injection of 25 µl of standard solution 25 µg/ml Mobile Phase Compounds Peak area Retention tine (80-20) (70-30) (60-40) (50-50) shown in Fig. 1. All copper and stainless steel tubing connecting the compressed air to the standard gasgenerating device were thoroughly cleaned with solvent, allowed to dry, and then flamed. All air was scrubbed over a bed of a charcoal. Airflow rates into the mixing chamber were controlled with a very fine metering valve maintained at 20 psi head pressure. The 20 L chamber was lined with two consecutive layers of 1/16 in. The configuration of the chamber was suitable for sampling various concentrations by varying the concentration of analyze sample and the flow rate of the pump (Fig. 1). A charcoal adsorption tube (SKC, USA) connected to a small pump was used to collect air samples. The pump was operated at 50-200 ml/minute [ 10, 12]. Two samples were obtained in each condition for analysis with GC and HPLC and 60 samples were obtained. Sample preparation and analysis by gas chromatography The determination of compounds was carried out according to the NIOSH method number 1501 [ 10]. A rotameter was used to adjust the flow. BTX were extracted with carbon disulphide (CS2) from the charcoal. A gas chromatography machine [Model 4600- Unicam Company, England] equipped with FID was used for quantitative measurement. Separation of the compounds was achieved with a glass column 1.5m 4mm i.d. packed with 10% SE 30 on Chromosorb W-AW-DMCS 100-120. This column temperature was programmed at 50 C for 2 minutes then increased to 180 C at a rate of 4 C/minute, and finally kept at a constant temperature of 180 C for 1 minute. Benzene 0.28 5.13 Toluene 0.25 6.95 M&P-Xylene 0.21 9.72 O-Xylene 0.15 12.62 Benzene 1.71 7.3 Toluene 1.64 11.15 M&P-Xylene 1.21 16.60 O-Xylene 0.64 18.32 Benzene 0.33 11.19 Toluene 0.39 19.76 M&P-Xylene 0.24 35.04 O-Xylene 0.13 37.35 Benzene 0.16 19.59 Toluene 0.09 40.43 M&P-Xylene - - O-Xylene - - Sample preparation and analysis by HPLC BTX were extracted with methanol from the charcoal. An HPLC chromatograph equipped with a UV detector (Model K-2600 Knauer) was used for analysis. The UV detector was set at 254 nm. The high performance liquid chromatography column was a C18 Bond Pack 3µm (25 cm 4.6 mm) analytical column. Chromatography was isocratic in a mobile phase consisting of water-methanol (30-70). The flow rate was set at 1 ml/minute. All chemicals and water used were HPLC grade. The figure 2 was shown the peaks of BTX in GC and HPLC chromatograph. A spectrophotometer equipped with scanner (Model UV-1700 SHIMADZU) was used to scan compounds in solution and determine the optimum absorption wavelength in the UV detector. Data analysis was performed using SPSS statistical software for windows. For reliability of the two methods, cornbach alpha intraclass correlation was performed. Comparison between the compound means in two methods repeated analysis was carried out with ANOVA with a P value less than 0.05 considered statistically significant. RESULTS To determine the best absorption of UV detector by HPLC, a solution mixture of 100 µg/ml of each compound was scanned with a spectrophotometer. The results showed that the highest absorption was between 253-254 nm, therefore the absorption of 254 nm was chosen for the UV detector in HPLC. To determine, optimum ratio of methanol-water for the mobile phase of HPLC, different ratios of these solutions were used to analyze VOC and the results were shown in Table 1. As expected, the higher the
Determination of Benzene, Toluene and Xylene (BTX) Concentrations in Air Using HPLC ijoh.tums.ac.ir 15 Table 2. The mean of peak area analysis of benzene, toluene, BTX standard solutions in methanol and carbon disulfide by HPLC Compound Benzene Toluene M&P-Xylene O-Xylene Standard Solutions Methanol Carbon disulfide Mean Standard deviation Mean Standard deviation 5 0.26 0.02 - - 10 1.21 0.03 - - 30 3.57 0.06 0.55 0.35 50 6.72 1.08 1.29 0.46 100 10.62 1.48 2.81 0.97 5 0.41 0.03 - - 10 1.71 0.04 - - 30 2.91 0.04 - - 50 4.74 0.07 0.54 0.002 100 7.62 1.19 0.57 0.003 5 0.41 0.02 - - 10 1.64 0.04 - - 30 2.62 0.05 0.54 0.01 50 3.42 0.08 1.15 0.02 100 6.62 1.21 1.80 0.01 5 0.41 0.02 - - 10 0.64 0.03 - - 30 1.22 0.06 - - 50 3.23 0.76 0.54 0.01 100 5.62 0.98 0.57 0.01 methanol content of the mobile phase, the lower the retention time became. The results indicate that a methanol-water ratio 70-30 has the best resolution for high peak area and retention time whenever 25 µl of standard solution (25 ng/µl) was injected into HPLC. The flow rate was varied between 0.8 and 1.5 ml/min. Its influence on the resolution was minimal, so 1 ml/min was selected as the optimum flow rate because it provided lower pressure on the column than 1.2 ml/min and shorter analysis than 0.8 ml/min. Standard solutions of 100, 50, 20, 10 and 5 µg/ml of each compound seperately in methanol and carbon disulfide were injected into HPLC. The linearity range and limit of detection (LOD) in each analysis of samples provided from atmospheric generation system are shown in Table 3. As can be seen, as the sensitivity increases, the upper limit of linearity and the LOD decrease for HPLC-UV detection. There was not a significant difference between results obtained from GC and HPLC analysis from samples of the dynamic atmosphere generation system. The P-value for benzene, toluene, m&p-xylenes and o- xylene were 0.89, 0.91, 0.42 and 0.18 respectively. DISCUSSION The result of current study was indicated that HPLC method proposed here can be employed as a alternative for seperating and determining BTX in occupational environments. The results of this study was showed that methanol has high resolution in chromatography than carbon disulfide. As the stationary phase was non-polar in the column for the reversed phase condition in HPLC, polar mobile phase was necessary. The analytes were completely separated and were quantified using both methods. The results obtained also indicate that GC provides a lower detection limit than reversed phase liquid chromatography. In spite of this, the HPLC determination of BTX is applicable to real samples because its sensitivity is lower than that required by threshold limit recommended by ACGIH, NIOSH, and OSHA [ 4]. The results of GC in this research provides lower LOD than the method recommended by the USA National Institute for Occupational Safety and Health based on GC-FID determinations (0.5 µg/sample for benzene and ethylbenzene, 0.7 µg/samples for toluene and p-xylene and 0.8 µg/samples for o-xylene and m- xylene) [ 10]. The HPLC method required the use of methanol as a component of the mobile phase, whereas nitrogen, hydrogen, and air were used in the GC method. As a result, the amount of toxic waste generated and the risk of airborne contamination using the HPLC method may be higher. Campos-Candel et al. compared HPLC-Fluorescence detector and GC-MS for analysis of real samples by using β-cyclodextrin stationary phase and separation of samples with pressured fluid extraction. The HPLC method was unsuccessfully applied to the determination of benzene in real samples because its sensitivity was too low. The sensitivity in current research (0.5) for benzene was less than in that study (4 mg/l) [ 14]. Ghittori et al. used the HPLC-Fluorescence detector to determine low concentrations of benzene in air. They used diffusive personal samplers and analyzed by
16 IJOH January 2011 Vol. 3 No. 1 Bahrami et al. Table 3. Linearity range and detection limits for HPLC-UV detection limit and GC-FID analyses for samples provided from atmospheric generation system Compound Concentration (ppm) Peak area of limit Limit of detection Limit of detection per Realibility* (µg/ml) detection sample ( µg) GC HPLC Benzene 1.23-436.42 13.85 0.1 0.1 0.99 Toluene 0.7-6.11 2.001 0.2 0.2 0.99 M&P-Xylene 1.13-12.27 2.69 0.3 0.3 0.99 O-Xylene 2.03-13.48 2.37 0.5 0.5 0.99 Benzene 1.29-443.14 0.078 0.5 0.5 0.98 Toluene 0.93-6.25 0.098 1 1 0.97 M&P-Xylene 1.96-11.01 0.071 1 1 0.98 O-Xylene 2.45-13.63 0.081 2 1 0.99 desorption with a mixture of methylene chloride and ethyl acetate. The method used did not detect aliphatic or alicyclic hydrocarbons [15]. The HPLC-Fluorescence method is compared with a gas chromatographic method that uses capillary column and flame ionization detection after a collection of an air sample on a Carbotrap 100 tube and following thermal desorption. The current study has some differences with these research concerns in regards to sampling, desorption solvent, mobile phase and detector apparatuses [15]. Analyses of BTX in water, soil and air are usually carried out by gas chromatography using a flame ionization detector (GC-FID) and a stationary phase of polyethylene glycol, although gas chromatography- Mass spectrometry (GC-MS) is becoming increasingly common. However, the use of this stationary phase has the drawback of the impossibility of seperating the m- xylene and p-xylene isomers [ 10, 13,16-19]. CONCLUSIONS The GC method provides a higher sensitivity than HPLC method, but the HPLC determination of benzene, toluene and xylenes are applicable to real samples because its sensivity is lower than standards recommended by ACGIH for the exposure limit value over an 8-hour workday. Although GC-FID is the standard method and the most popular technique recommended by NIOSH, OSHA and EPA for the analysis of BTX in occupational environments, the HPLC method proposed here can be employed as a alternative for seperating and determining BTX in occupational environments. ACKNOWLEDGEMENTS The authors thank the Hamadan University of Medical Sciences for supporting this research. The authors declare that there is no conflict of interests. REFERENCES 1. Leung PL, Harrison RM. Evaluation of personal exposure to monoaromatic hydrocarbons. Occup Environ Med 1998; 55: 249-57. 2. Duarte-Davidson R, Courage C, Rushton L, Levy L. Benzene in the environment: an assessment of the potential risks to the health of the population. Occup and Environ Med 2001; 58: 2-13. 3. Barbara J, Finlayson P, James NP. Tropospheric Air Pollution: Ozone, Airborne Toxics, Polycyclic Aromatic Hydrocarbons, and Particles, Science 1997; 276(5315): 1045 1051. 4. American Conference of Governmental Industrial Hygienists. Threshold limit values for chemical substances and physical agents & biliological exposure Indices. Worldwide. Cincinnati, USA, 2005 5. U.S. EPA. Carcinogenic effects of benzene: an update. Prepared by the National Center for Environmental Health, Office of Research and Development. Washington, DC. USA, EPA/600/P- 97/001F: 1998. 6. Environmental Protection Agency (EPA). Proposed Guidelines for carcinogen risk assessment. Federal Register 1996; 61: 17960-18011. 7. International Agency for Research on Cancer (IARC). IARC monographs on the evaluation of the carcinogenic risk of chemicals to humans. Some industrial chemicals and dyestuffs. Vol. 29. Lyon, France: IARC, 1982. 8. Pyatt D, Hays S. A review of the potential association between childhood leukemia and benzene. Chem Biol Interact 2010; 184(1-2):151-64. 9. Costantini AS, Benvenuti A, Vineis P, Kriebel D, Tumino R, Ramazzotti V. Risk of leukemia and multiple myeloma associated with exposure to benzene and other organic solvents: evidence from the Italian Multicenter Case-control study Am J Ind Med 2008; 51(11): 803-11. 10. P. Eller (Editor), NIOSH Manual of Analytical Methods. US Department of Health and Human Services, Cincinnati, OH, USA,1984. 11. DFG, Analytische Methoden zur Prüfung gesundheitsschädlicher Arbeitsstoffe, Deutsche Forschungsgemeinschaft, Verlag Chemie, Weinheim, 1985. 12. Occupational safety and health administration, Sampling and Analytical Methods, 2008; Available from: http://www.osha.gov/dts/sltc/methods/index.html. 13. Winberry WT, Murphy NT, Riggan RM, Compendium of Methods for the Determination of Toxic Organic Compounds in Ambient Air; US Environmental Protection Agency, Research Triangle Park, NC, EPA/600/4-89/017, Method TO 12 1988. 14. Campos-Candel A, Liobat-Estelles M, Mauri-Aucejo A. Comparative evaluation of liquid chromatography versus gas chromatography using a beta-cyclodextrin stationary phase for the determination of BTX in occupational environments. Talanta 2009; 78(4-5):1286-92. 15. Ghittori S, Maestri L, Fiorentino ML, Imbriani M. HPLC determination of low concentrations of benzene. G Ital Med Lav 1988; 10(4-5):201-5
Determination of Benzene, Toluene and Xylene (BTX) Concentrations in Air Using HPLC ijoh.tums.ac.ir 17 16. W.A. McClenny, in: H.J.T. Bloemen, J. Burn (Editors), Volatile Organic Compounds in the Environment, Blackie, London, 1993. 17. Ciccioli P, Bloemen HJT, Burn J. Volatile Organic Compounds in the Environment, Blackie, London, 1993, 92 174. 18. McClenny WA, Pleil JD, Evans GF, Olivier KD, MW. Holdren, Winberry WD. J Air Waste Manage Assoc 1991; 41: 1308 1318. 19. Allison AM, Yu-Ping C, MacFarlane JK, Gschwend PM. Laboratory Assessment of BTX Soil Flushing. Environ Sci Technol. 1996; 30 (11): 3223 32311.