Steam Distillation Extraction of 2,4,6- Tribromoanisole (Khairuddin)
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STEAM DISTILLATION EXTRACTION OF
2,4,6-TRIBROMOANISOLE IN MILK SAMPLE
AND ANALYSIS USING GC-MS IN
EI+ MODE
Khairuddin
Jurusan Kimia FMIPA Universitas Sumatera Utara
Jl. Bioteknologi No. 1 Kampus USU Medan
Abstract
The application of a steam distillation extraction method for GC-MS detection of 2,4,6-tribromoanisole in milk product is described. The milk samples were extracted with hexane as the solvent for six hours. Mirex was used as the volumetric standard. The average recovery of the compound were 76-90%. The limit of detection was 1.74 μgL-1.
Keyword : distillation, extraction, milk, hexane and volumetric
INTRODUCTION
The compound of 2,4,6-tribromoanisole (2,4,6-TBA) was found in the environmental samples but the source of this compound in uncertain. It may be a decomposition product from polyhalogenated phenols by microbiological methylation in the environment (Miyazaki.T, S.Kaneko, S.Horii, and T.Yamagishi., 1981). The present of 2,4,6-TBA can also be brought about by biodegradation of tribromophenol used as a sterilant and cleaning agent by the dairy industry.
The fat content in milk sample is regarded as difficult to handle. Several approaches to extraction and clean-up of milk have been reported (Prapamontol, T. and D.Stevenson, 1991). Steam distillation extraction (SDE) is the most applicable to
trace analysis of priority pollutants in environmental samples because SDE provides both the extraction and clean up process in one step. SDE is continuous method for the isolation and concentration of organic compounds for aqueous solution. It can be employed for the isolation and concentration of non-polar and polar relatively non volatile organic substances from water, that are distilled with steam.
In this work, steam distillation extraction has been used to extract 2,4,6-TBA in milk samples with hexane as a solvent. Mirex was used as a volumetric standard. GC-MS in the positive ion impact has been used to determine the 2,4,6-TBA in milk extract. Recovery experiment were performed at μgL
-1
Jurnal Sains Kimia
Vol. 7, No.1, 2003: 11-14
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MATERIAL AND METHOD
Material
Organic solvents, hexane (HPLC-grade) and acetone (analar grade) were obtained from Fisons. 2,4,6-TBA as authentic reference material was purchased from
Aldrich. Mirex (dodecachloropentacyclodecane) was
obtained from British Greyhound (UK). All substances were 99% purity and were used as received.
Stock solution of 100 mgL-1 2,4,6-TBA and 100 mgL-1 mirex were prepared in hexane from stock solution of 2,4,6-TBA at varying concentrations, i.e.0.25, 0.50, 1.00, 5.00, 10.00, 20.00, 60.00, 100.00 µgL-1 with the volumetric standard concentration mirex held constant at 100 µgL-1 . The solutions were analyzed by GC-MS. The calibration graph was obtained by plotting the peak area of 2,4,6-TBA versus mirex by least-squares analysis.
Instrumentation
GC-MS analysis was performed on a Hewlett Packard series II gas chromatography interface to a VG-TRIO 1000 quadrupole mass spectrometer. The GC-MS system was controlled by LAB-BASE data processing system and it was run by an Intel 386 PC 32-bit computer. A fused – silica capillary column DB5-MS (J&W scientific), 15 m long, 0.32 mm internal diameter and 0.25 µm film thickness, was inserted directly into the ion source using helium (CP grade, purity 99.999 %) as a carrier gas.
The GC was operated in the splitless mode with the injector temperature at 270 oC. 1 µL sample was injected manually. The septum purge on-time was 1.0 min. The gas chromatographic oven temperature program
was as follows: initial ramp 50 oC held for 2 min, 30 oC min-1 to 300 oC held for 2 min. The total time per analysis for each sample was 12 min.
The instrument setting were as follow: Ionizing voltage 70 eV, ionizing current 200 µA, ion source temperature 200 oC, interface temperature 250 oC, scan range 50 to 550 u and scan time 0.90 s with interscan 0.10 s for full scan and 0.02 u with 0.08 s dwell time for SIR. Prior to analysis, the mass spectrometer was calibrated in EI+ mode with perfluorotributylamine (PFTBA) calibration compound by monitoring masses 69, 219, 264, and 502. accurate mass data was obtained from full-scan for SIR application.
Sample Extraction
Steam Distillation Extraction of 2,4,6- Tribromoanisole (Khairuddin)
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Figure 1. Steam distillation apparatus; modified design Nielsen-Kryger
Recovery Studies
Spiking solutions were prepared in acetone. Four different spike solutions of 2,4,6-TBA were used for spike experiments. Each 250 mL of milk sample was spiked with 1 mL of 0.25, 5.00, 15.00, 50.00 μgL-1 of 2,4,6-TBA spike solution giving an equivalent concentration of 1, 20, 60, and 200 ngL-1 respectively in the milk samples . Each flask was slowly shaken manually to prevent the formation of emulsion and then was allowed to equilibrate overnight in a refrigerator before extraction. The samples were brought up to room temperature before proceeding with the sample extraction
RESULTS AND DISCUSSION
Representative mass spectrum and mass chromatograms for the 2,4,6-TBA are shown in Figure 2.
Figure 2.Mass spectrum and mass chromatograms for 2,4,6-TBA (retention time 5,70 min) in spiked milk after sample enrichment by SDE
The extraction of pollutants in milk can be length, labour intensive and costly because of the fat content. Several approaches to extraction and clean up of pollutant in milk(4,6), including steam distillation extraction[7] have been reported. Steam distillation of milk reduces the cost of the analysis, because it requires smaller volumes of solvent (25 mL hexane). Also the extracts from different kinds of milk were obtained clean and ready after evaporation to 1 mL for direct injection into GC-MS without further treatment.
The calibration graph was linear over the concentration range 0.25 to 100 µgL-1 with correlation coefficient R : 0.99615, regression line equation y = 0.172x + 0.023 and standard deviation of y and x, Sx/y =
0.099. The limit of detection (LOD) was calculated from the slope and intercept of the regression line. The LOD is defined as concentration yielding a signal exceeding the background ion signal by three standard deviation SD, The background being given by intercept and standard deviation Sx/y. The
Jurnal Sains Kimia
Vol. 7, No.1, 2003: 11-14
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mL extract and the analytical method detection limit for milk was 6.92 ngL-1 by extrapolation with the original volume 250 mL. standard calibration were measured under SIR mode because of the low levels of 2,4,6-TBA detected.
The absolute recovery of 2,4,6-TBA by the steam distillation extraction procedure was determined by using concentration in the calibration range (0.25-100.00 µgL-1). Recoveries of 2,4,6-TBA using this procedure were evaluated at the four spiked levels (0.25-100.00 µgL-1) because these were expected concentration range for 2,4,6-TBA in milk samples.
The results (Table 1) show average recoveries of 76% (0.25 µgL-1), 87% (5.00 µgL-1), 90% (15.00 µgL-1), 89% (50.00 µgL
-1
) with the standard deviation of 14.5, 10.8, 8.1, and 7.5%, respectively.
Table 1. Percentage recoveries of 2,4,6-TBA from spiked milk extracted by steam distillation extraction and analyzed by GC-MS a
No. Spike level, µgL-1
Mean % Recovery ± SD
1 2 3 4
0.25 5.00 15.00 50.00
76 ± 14.5 87 ± 10.8 90 ± 8.1 89 ± 7.5
a
All experiment were performed in triplicate, n = 3
CONCLUSION
Steam distillation is simple, clean, consumes only small amounts of solvent, and provides sufficient sample volume for identification by GC-MS. 2,4,6-TBA in milk samples could be extracted quantitatively without the need for the removal of total fat. The main disadvantages of using steam distillation for the separation of 2,4,6-TBA is that this method is very-time consuming to liquid-liquid extraction or ultrasonic extraction.
REFERENCES
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JenkinsE.H and P.J.Baugh, 1993, Anal. Proc., 30, 441-442.
Mines.J, G.Font and Y.Piro, 1993, J. Chromatogr., 642, 195-204
Miyazaki.T, S.Kaneko, S.Horii, and T.Yamagishi., 1981, Bull. Environm. Contain. Toxicol., 26, 577- 584.
Prapamontol.T and D.Stevenson, 1991, J. Chromatogr., 552, 249-257
Watanabe.I, T.Cashimoto, and R.Tatsukawa., 1983, Arch. Environ. Contam. Toxicol., 12, 615-620. Wittlinger.R and K.Ballscbmiter, Fresenius, 1990, J.
