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 BAHRAMI1*, HOSIEN MAHJUB2, MARZIEH SADEGHIAN1, FARIDEH GOLBABAEI3

1
Department of Occupational Health, Research Centre for Health Sciences, School of Public Health,
Hamedan University of Medical Sciences, Hamedan, Iran; 2Department of Biostatics and Epidemiology,
Research Centre for Health Sciences, School of Public Health, Hamedan University of Medical Sciences,
Hamedan, Iran; 3Department 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 important due to their health effects on humans.

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,
bahrami@umsha.ac.ir
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
Email: 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 -
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 l of standard solution 25 g/ml
Mobile Phase Compounds Peak area Retention tine
Benzene 0.28 5.13
Methanol-Water Toluene 0.25 6.95
(80-20) M&P-Xylene 0.21 9.72
O-Xylene 0.15 12.62
Benzene 1.71 7.3
Methanol-Water Toluene 1.64 11.15
(70-30) M&P-Xylene 1.21 16.60
O-Xylene 0.64 18.32
Benzene 0.33 11.19
Methanol-Water Toluene 0.39 19.76
(60-40) M&P-Xylene 0.24 35.04
O-Xylene 0.13 37.35
Benzene 0.16 19.59
Methanol-Water Toluene 0.09 40.43
(50-50) M&P-Xylene - -
O-Xylene - -

shown in Fig. 1. All copper and stainless steel tubing
connecting the compressed air to the standard gas-
generating 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
1.5m4mm i.d. packed with 10% SE 30 on Chromosorb
W-AW-DMCS 100-120.
This column temperature was programmed at 50C
for 2 minutes then increased to 180C at a rate of
4C/minute, and finally kept at a constant temperature
of 180C for 1 minute.
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 3m (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 g/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
Methanol Carbon disulfide
Compound Standard Solutions
Mean Standard deviation Mean Standard deviation
5 0.26 0.02 - -
10 1.21 0.03 - -
Benzene 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 - -
Toluene 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 - -
M&P-Xylene 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 - -
O-Xylene 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 l of
standard solution (25 ng/l) 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 g/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 g/sample for
benzene and ethylbenzene, 0.7 g/samples for toluene
and p-xylene and 0.8 g/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 -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*
(g/ml)
detection sample ( g)
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
GC
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
HPLC
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 Prfung
gesundheitsschdlicher 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 TO12 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, 92174.
18. McClenny WA, Pleil JD, Evans GF, Olivier KD, MW. Holdren,
Winberry WD. J Air Waste Manage Assoc 1991; 41: 13081318.
19. Allison AM, Yu-Ping C, MacFarlane JK, Gschwend PM.
Laboratory Assessment of BTX Soil Flushing. Environ Sci
Technol. 1996; 30 (11): 322332311.