Transcript Detection of polychlorinated biphenyls (PCBs) by Gas

Detection of polychlorinated biphenyls
(PCBs) by Gas Chromatography
related techniques
Nuwan Balapitiya
Literature Seminar
Louisiana State University
Oct 26, 2009
Objective
Application of Gas Chromatography related techniques to
quantify PCBs in two different real world samples
1. Solid waste samples
2. Human serum samples
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Why PCBs are studied?
Man-made
Persistant organic
pollutants
Many industrial
applications
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Bioaccumulation
Toxicological effects
on animals and
Banned in US 1979
humans
Global ban 2001
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Toxicity
Species
LD50
Fish
Crustaceans (Crab, Lobster)
Rat
3 – 3000 mg / L in water
10 – 2400 mg / L in water
4 – 10 g / Kg B.W.
Rabbit
1 – 3 g / Kg B.W.
Operation of the Prior Informed Consent Procedure for Banned or Severely Restricted Chemicals in International Trade; Decision Guidance
Documents, Polychlorinated Biphenyls; Food and Agriculture Organization of the United Nations, United Nations Environment
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Programme, Rome – Geneva 1992
Structure of PCBs
• PCBs are molecules composed of two benzene rings with 1
– 10 chlorine atoms attached to them
PCB-28
PCB-166
n
n
PCB-209
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General protocol for PCB detection
1) Extraction
– Soxhlet extraction
– Solvent Extraction
– Solid Phase Extraction
2) Separation
– High Performance Liquid Chromatography
– Gas Chromatography
3) Detection
– Electron Capture Detector
– Mass spectrometry
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PCBs detection
Extraction Method
(Solid phase extraction)
Separation
(Gas chromatography)
Detection
Electron Capture Detector
Mass Spectrometry
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Solid phase extraction (SPE)
• SPE is a sample preparation, purification and
pre-concentration technique used with liquid
samples
• Extract volatile and non-volatile analytes
• Used with LC and GC
• Disposable, pre-packed cartridges are available
as columns
– Reverse phase (non-polar)
– Normal phase (polar)
– Ion exchange
http://www.sigmaaldrich.com/Graphics/Supelco/objects/4600/4538.pdf
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SPE process
Conditioning
Inject sample
http://www.sigmaaldrich.com/Graphics/Supelco/objects/4600/4538.pdf
Elution
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Gas Chromatography (GC)
• GC is used to separate volatile and
thermally stable compounds
• Mobile phase is an inert gas (N2, He)
• Stationary phase is either a solid (GSC)
or a liquid-like polymer (GLC)
• Stationary phase is coated inside a
capillary column (Open tubular
columns)
http://www.cseindia.org/html/lab/int_gc.jpg
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GC Instrument
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/gaschrm.htm
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Electron Capture Detector (ECD)
Carrier gas +
Sample inlet
63Ni
Make-up gas inlet
(N2)
Foil
http://www.srigc.com/ECDman.pdf
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Mass Spectrometry (MS)
“MS is an analytical technique that
measures the molecular masses
of individual compounds and
atoms precisely by converting
them into charged ions” *
• Structure of the molecule can be
deduced
• Used as a quantification method
couple with HPLC and GC
http://www.chemguide.co.uk/analy
sis/masspec/masspec.GIF
*Dass, C. (2007). Fundamentals of contemporary mass spectrometry. Hoboken, NJ, John Wiley & Sons, Inc.
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Selected ion monitoring (SIM)
• Ion current from one or few selected ions are recorded
• The ion with the highest abundance is selected
(molecular ion most of the time)
• Gives a significant improvement in detection sensitivity
(1000 fold gain)
• Faster detection because only one or several peaks are
detecting (not the total ion chromatogram)
Dass, C. (2007). Fundamentals of contemporary mass spectrometry. Hoboken, NJ, John Wiley & Sons, Inc.
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Internal Standard
• A known amount of a compound, different from the
analyte, added to the unknown
• Internal Standard,
– should elute near the peaks of interest
– peaks should be well resolved from the analyte peaks
– should be chemically similar to analytes of interest
– should not react with any sample components
– must be available in pure form
• PCB-166, PCB-58 and PCB-186
Miller, J.M.(2005). Chromatography: Concepts and Contrasts. Hoboken, NJ, John Wiley & Sons, Inc.
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Online Coupling of Bead Injection Lab-On-Valve Analysis to
Gas Chromatography: Application to the Determination of
Trace Levels of Polychlorinated Biphenyls in Solid Waste
Leachates
Anal. Chem. 2009, 81, 4822 – 4830
Jose Quintana, Warunya Boonjob, Manuel Miro*, Victor Cerda**
*University of Santiago de Compostela, Spain
** University of Belaric Islands, Spain
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Experiment
• Three solvents were tested for the elution of PCBs
(isooctane, toluene and ethyl acetate)
• Ethyl acetate was selected as the best solvent, which
gives more than 80 % after one elution
• Four different reversed-phase sorbent materials were
used (Oasis HLB, Lichrolut EN, Bond Elut Plexa,
spherical Upti-Clean C18)
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Multi-Syringe Flow-Injection Bead-Injection Lab-OnValve system coupled to GC
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Samples
• Samples were obtained from urban waste landfill in
Santa Margalida and Mallorca in Spain
• Leachates from urban solid waste landfill (Sample No.1)
• Electronic equipment disposal site (Sample No.2)
• Both samples were spiked at 10 and 50 ng L-1 level
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Chromatogram of landfill leachate sample No.1 spiked at 10 ng /L
level of each PCB congener and 50 ng / L of IS (PCB-166)
mV
850,000
800,000
750,000
700,000
650,000
600,000
550,000
500,000
450,000
400,000
350,000
300,000
250,000
200,000
150,000
100,000
50,000
22
24
26
28
30
32
34
tR (min)
36
38
40
42
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Analytical performance of the online BI-LOV-GC
method for determination of trace level
concentrations of PCBs (PCB-166 IS)
Recovery
(%RSD)
Sample No.1
Recovery
(%RSD)
Sample No.2
Name
LOQ (ng L-1)
10 ng L-1
50 ng L-1
10 ng L-1
50 ng L-1
PCB-28
4.1
107.4 (12.8)
103.1 (6.8)
91.3 (9.3)
92.0 (4.6)
PCB-52
6.1
99.7 (5.7)
87.9 (4.4)
89.5 (10.2)
94.7 (5.0)
PCB-101
2.0
99.1 (5.2)
90.7 (5.9)
81.4 (9.1)
90.0 (4.8)
PCB-118
1.2
100.9 (7.6)
90.3 (5.4)
87.6 (9.4)
97.0 (4.3)
PCB-153
1.4
103.1 (4.2)
88.7 (4.2)
86.4 (2.0)
91.7 (2.3)
PCB-138
1.0
116.2 (11.5)
100.2 (8.8)
98.4 (8.5)
102.2 (7.1)
PCB-180
0.5
112.5 (7.8)
93.9 (4.9)
90.7 (7.9)
99.8 (1.2)
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Sample No.3 – Aroclor 1260
• This is a mixture of PCB-28, PCB-52, PCB-101, PCB118, PCB-153, PCB-138, PCB-180 (still in use in old
transformers and capacitors)
• Aroclor 1260 was spiked with a landfill leachate to get a
more environmentally realistic sample (Sample 3)
• First the mixture was characterized by injecting
individual PCBs
• Then Aroclor 1260 spiked into the leachate sample was
analyzed (Total PCB 500 ng L-1, PCB-166, 50 ng L-1)
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mV
Chromatogram of solid waste leachate sample No.3 spiked
with 500 ng / L of Aroclor 1260 and 50 ng / L of IS (PCB-166)
1,000,000
950,000
900,000
850,000
800,000
750,000
700,000
650,000
600,000
550,000
500,000
450,000
400,000
350,000
300,000
250,000
200,000
150,000
100,000
50,000
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24
26
28
30
32
tR (min)
34
36
38
40
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Comparison of expected and found concentrations of
PCB congeners in a landfill leachate spiked with
Arochlor 1260 at the 500 ng L-1 level
Expected Conc.
(ng L-1)
Found Conc.
(ng L-1)
Name
Mean
SD
Mean
PCB-28
2.1
0.2
<LOQ
PCB-52
2.39
0.09
<LOQ
PCB-101
24.0
1.5
PCB-118
3.7
PCB-153
Student t – test
SD
Cal t
Stat diff.
22.6
0.9
1.37
No
0.6
3.2
0.5
1.21
No
75.5
3.4
76.7
1.7
0.55
No
PCB-138
57.0
3.3
59.8
1.6
1.27
No
PCB-180
59.6
2.9
62.2
3.5
0.97
No
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Summary
• BI-LOV-GC used for the analysis of PCBs in solid waste
leachates
• Two solid waste leachate samples were spiked with 10 and 50
ng L-1 PCBs
• Aroclor 1260 was used as a more environmentally realistic
sample (total PCB concentration 500 ng L-1)
• Limit of quantification is in the range of 0.5 – 6.1 ng L-1
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Analysis of Polychlorinated biphenyls in human serum
by gas chromatography – mass selective detection
operating at high ion source temperature
Journal of Chromatography B, 2009, 877, 1109 – 1116
Buu N. Tran, Li Zhang, Robert Jansing,
Kenneth M.Aldous*
* Wadsworth Center for Laboratiries and Research, New
York State Department of Health, Albany, NY 12201
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Analysis of PCBs in human serum by
GC – MS – SIM
• Human serum is a complex matrix consists of proteins,
lipids and inorganic salts
• Extensive clean-up procedures are required prior to
analysis
• SPE was used for the clean-up
• Agilent 6890N GC was coupled to Agilent 5975B MSD
• MS was used at 70 eV for electron ionization, and
scanning from 35 to 550 amu
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Calibration of GC – MS
• Decafluorotriphenylphosphine
(DFTPP) was used as the reference
material to evaluate the
performance of the GC – MS
system (EPA)

MS spectrum of DFTPP was studied at ion source
temperatures 230 0C and 300 0C

Base peak is m/z 198

Molecular ion peak is m/z 442
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Total Ion Chromatogram of DFTPP
Abundance
28000
26000
24000
22000
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
198.0
230 0C
40
60
80
100
120 140
442.1
160
180
200
220 240
50000
260 280
300
320
340
360 380
400 420 440
198.0
45000
442.1
300 0C
40000
35000
30000
25000
20000
15000
10000
5000
0
40
60
80
100
120 140
160
180 200
220
m/Z
240
260 280 300
320 340
3 60 380
400 420 440
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Total Ion Chromatogram of PCB-209
Abundance
900
497.7
230 0C
800
700
600
500
400
300
200
100
0
50
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
100
150
200
250
300
350
100
450
500
497.7
300 0C
50
400
150
200
250
m/Z
300
350
400
450
500
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High ion source temperature (+ / –)
•
•
•
•
Enhance the molecular mass responses
Reduce signals of fragment peaks
No increase in the background
Higher sensitivity
• Shorten the lifetime of the source
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Comparison of precision, accuracy, instrumental detection
limits (IDLs) of PCB mixture at two ion source temperatures
230 0C
PCB
300 0C
Congener
%Mean
recovery
RSD
IDL
(ng/mL)
%Mean
recovery
RSD
IDL
(ng/mL)
44
96
14.4
0.4
94
3.5
0.1
101
89
13.8
0.3
84
2.5
0.1
153
88
12.3
0.3
82
3.1
0.1
105
81
14.5
0.3
81
3.2
0.1
138
86
26.1
0.6
87
2.6
0.1
187
93
27.0
0.7
86
3.2
0.1
128
79
11.6
0.3
82
3.4
0.1
180
95
26.3
0.7
78
3.6
0.1
170
95
22.2
0.6
79
3.0
0.1
206
96
20.3
0.6
79
4.8
0.1
209
120
23.0
0.8
80
5.0
0.1
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Method development
• Newborn calf serum (NCS) was used for the method
development
• PCB-58 and PCB-186 were selected as IS,
– Less than 1% weight in aroclors
– Present in small quantities in the environment
– Do not co-elute with any of the target analytes
• NCS samples were spiked with 0.5 ng/g of serum per each
PCBs to find the Method Detection Limit (MDL)
• Three NCS samples were spiked with 1, 10 and 50 ng/g
serum were used for the method validation
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Determination of Method Detection Limit
PCB
0.5 ng/g
MDL
Congener
% Recovery
RSD
(ng/g)
44
122
6.0
0.1
101
112
7.2
0.1
153
108
13.4
0.2
105
116
7.6
0.1
138
122
11.0
0.2
187
108
8.9
0.1
128
108
7.2
0.1
180
108
5.3
0.1
170
110
5.5
0.1
206
104
8.0
0.1
209
84
7.7
0.1
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Method development
• Newborn calf serum (NCS) was used for the method
development
• PCB-58 and PCB-186 were selected as IS,
– Less than 1% weight in aroclors
– Present in small quantities in the environment
– Do not co-elute with any of the target analytes
• NCS samples were spiked with 0.5 ng/g of PCBs to find
the Method Detection Limit (MDL)
• Three NCS samples were spiked with 1, 10 and 50 ng/g
serum were used for the method validation
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GC/MSD chromatogram of NCS spiked PCBs at 10
ng/g serum aquired in SIM mode
Abundance
58 (IS)
186 (IS)
5500
5000
4500
4000
3500
38
3000
28
44
77
2500
52
2000
1500
170
195
206 209
1000
500
20.00
30.00
40.00
Elution time (min)
50.00
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Method validation: Precision accuracy for
matrix spike PCBs at 1, 10, 50 ng/g serum
PCB
1 ng/g
10 ng/g
50 ng/g
Congener
Recovery
%
RSD
Recovery
%
RSD
Recovery
%
RSD
44
106
4.6
83
5.5
97
2.6
101
110
4.3
79
4.3
90
2.5
153
111
5.2
84
6.5
89
4.3
105
111
4.6
89
7.8
106
5.5
138
113
7.6
105
6
112
2.9
187
117
8.6
89
5.8
94
3.6
128
112
4.5
92
6.8
108
4.6
180
113
7.6
90
8
98
5.2
170
111
4.2
98
8.1
106
6.1
206
105
4.8
80
11.4
80
8.2
209
82
8.2
64
11.3
65
8.5
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GC/MSD chromatogram of human serum extract acquired in
SIM mode
Abundance
58 (IS)
186 (IS)
11000
10000
9000
8000
118
7000
5000
138
153
6000
4000
180
3000
2000
1000
20.00
40.00
Elution Time (min)
38
Summary
• PCBs were analyzed using GC – MS – SIM method
• Two ion source temperatures was tested (230 0C and 300 0C)
• NCS was used for the method development
• Method was tested for the human serum
• Limit of detection was 0.1 ng/g of serum (100 ng L-1)
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Critique
• Detection limit was lower than the EPA approved level 500 ng
L-1
• Recovery % changes from experiment to experiment
• Recovery % is over 100% for some PCBs
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Acknowledgements
• Prof. Robin L. McCarley
• McCarley Research Group
• Audience
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Questions?
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