Steam Distillation Extraction Of 2,4,6- Tribromoanisole In Milk Sample And Analysis Using Gc-Ms In Ei+ Mode
Steam Distillation Extraction of 2,4,6- Tribromoanisole
(Khairuddin)
STEAM DISTILLATION EXTRACTION OF 2,4,6TRIBROMOANISOLE 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 2,4,6tribromoanisole 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 μgL1
levels in spiked milk samples
11
Jurnal Sains Kimia
Vol. 7, No.1, 2003: 11-14
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 LABBASE 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
12
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
The milk samples were obtained from a
Dairy source. 250 mL of milk sample was
diluted to 1 litre with distilled water and
extracted with 25 mL hexane using a
modified Nielsen-Kryger steam distillation
extraction system[5] for six hours (Figure 1).
The extract solution was dried with
anhydrous sodium sulphate . The volume of
the solvent extract was reduced to about 10
mL using a rotary evaporator. It was placed
in 1.0 mL graduated conical vials and
concentrated
by use of a oxygen-free
nitrogen blow down to a final volume of
approximately 0.5 mL. The vial was rinsed
several times with hexane before addition of
the appropriate amount of volumetric
standard, mirex. The volume of extract was
adjusted to 1.0 mL with hexane prior to GCMS analysis.
Steam Distillation Extraction of 2,4,6- Tribromoanisole
(Khairuddin)
Figure 1. Steam distillation apparatus; modified
design Nielsen-Kryger
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
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.
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
peak area ratio at LOD is 0.023 + 3(0.099).
The LOD of 2,4,6-TBA was 1.72 µgL-1 for 1
13
Jurnal Sains Kimia
Vol. 7, No.1, 2003: 11-14
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 2,4,6TBA 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 µgL1
) 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.
1
2
3
4
a
Spike level,
µgL-1
0.25
5.00
15.00
50.00
Mean % Recovery ± SD
76 ± 14.5
87 ± 10.8
90 ± 8.1
89 ± 7.5
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.
14
REFERENCES
Filek.G, M.Bergamini, and W.Lindner, 1995, J.
Chromatogr.A, 712, 355-364.
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,
Chromatogr., 552, 249-257
1991,
J.
Watanabe.I, T.Cashimoto, and R.Tatsukawa., 1983,
Arch. Environ. Contam. Toxicol., 12, 615-620.
Wittlinger.R and K.Ballscbmiter, Fresenius, 1990, J.
Anal. Chem.,336, 193-200.
(Khairuddin)
STEAM DISTILLATION EXTRACTION OF 2,4,6TRIBROMOANISOLE 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 2,4,6tribromoanisole 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 μgL1
levels in spiked milk samples
11
Jurnal Sains Kimia
Vol. 7, No.1, 2003: 11-14
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 LABBASE 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
12
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
The milk samples were obtained from a
Dairy source. 250 mL of milk sample was
diluted to 1 litre with distilled water and
extracted with 25 mL hexane using a
modified Nielsen-Kryger steam distillation
extraction system[5] for six hours (Figure 1).
The extract solution was dried with
anhydrous sodium sulphate . The volume of
the solvent extract was reduced to about 10
mL using a rotary evaporator. It was placed
in 1.0 mL graduated conical vials and
concentrated
by use of a oxygen-free
nitrogen blow down to a final volume of
approximately 0.5 mL. The vial was rinsed
several times with hexane before addition of
the appropriate amount of volumetric
standard, mirex. The volume of extract was
adjusted to 1.0 mL with hexane prior to GCMS analysis.
Steam Distillation Extraction of 2,4,6- Tribromoanisole
(Khairuddin)
Figure 1. Steam distillation apparatus; modified
design Nielsen-Kryger
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
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.
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
peak area ratio at LOD is 0.023 + 3(0.099).
The LOD of 2,4,6-TBA was 1.72 µgL-1 for 1
13
Jurnal Sains Kimia
Vol. 7, No.1, 2003: 11-14
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 2,4,6TBA 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 µgL1
) 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.
1
2
3
4
a
Spike level,
µgL-1
0.25
5.00
15.00
50.00
Mean % Recovery ± SD
76 ± 14.5
87 ± 10.8
90 ± 8.1
89 ± 7.5
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.
14
REFERENCES
Filek.G, M.Bergamini, and W.Lindner, 1995, J.
Chromatogr.A, 712, 355-364.
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,
Chromatogr., 552, 249-257
1991,
J.
Watanabe.I, T.Cashimoto, and R.Tatsukawa., 1983,
Arch. Environ. Contam. Toxicol., 12, 615-620.
Wittlinger.R and K.Ballscbmiter, Fresenius, 1990, J.
Anal. Chem.,336, 193-200.