advances in pcb analysis presented by andy beliveau region 1 usepa office of environmental...
TRANSCRIPT
Advances in PCB Analysis
Presented by Andy Beliveau
Region 1 USEPA Office of Environmental Measurement and Evaluation
November 2002
1
Historical Analysis By Aroclor Pattern Recognition GC/ECD detector with packed and
capillary column. Relies on matching the standard
pattern to the sample pattern. Quantitated only if the pattern matches
(otherwise determined to be a non-detect).
3 to 5 peaks used for quantitation.
2
Limitations to Aroclor Analysis
Mixtures of Aroclor patterns overlap . Which peaks represent which Aroclor? Weathered PCB patterns do not match
the standards. Interpretation is left to the analyst. Resulting quantitation can be
compromised by all of the above.
3
Why use congener analysis
Eliminates interpretation. Weathering is not a problem. It is possible to quantitate all peaks . Sum of the congeners = Total PCB. Congeners can be quantitated individually. Dioxin-like congeners can be quantitated. Non Aroclor PCBs can be identified. Environmentally modified PCBs can be
identifed.
4
Types of Congener Analyses
High resolution GC/ ECD (HRGC)– Long column 60 meters.– Long run time - 60 to 90 minutes .– Requires clean rigorous clean-up of sample.
HRGC/ Low resolution MS (bench top MSD)– Medium column 30 meters.– Medium run time 30-60 minutes. – Eliminates interferences.– Allows for level of chlorination(homologue)
analysis.
5
Type of Congener analysis (cont)
HRGC/ High Resolution MS – Requires a expensive instrument and
highly qualified operator.– Requires a 10,000 resolution.– Allows for wide concentration range.– Medium length column- 60 meters.– Run time - 30 to 60 minutes.– Requires rigorious clean-up of sample
extract for the best results.
6
Congener Analysis by HRGC/ECD
No standard or promulgated EPA method. Can quantitate 125+ congeners (99%). Quantitates and identifies by retention time. Some congeners co-elute depending on
column packing, column length, run time, and GC conditions. May require multiple columns to resolve all congeners.
Requires individual standards or known retention time mixed standards.
7
Congeners by HR/GC/LRMS
No Standardized EPA method. Method #680 not promulgated.
Can quantitate 125+ congeners(99%). Quantitates by retention time and molecular
ions for each chlorination level. Requires individual congener standards. Can achieve HRGC/ECD detection limits. Eliminates non PCB interferences. Can identify non PCB compounds or
environmentally modified PCBs.
8
Congeners by HRGC/HRMS
Method 1668a is not yet promugated but is used by a handful of laboratories.
Can quantitate all 209 congeners in water, soil, or biota.
Has a very wide concentration range ( ppm to ppt in soil and ppb to ppqt in water).
Can quantitate WHO dioxin-like congeners. Can quant total PCB by sum of congeners. Requires rigorous clean-up and sometimes
silica carbon column clean-up.
9
Conclusion: Major uses of congener analysis
Identify congeners in biota and sediments where weathering or metabolizm changes the PCB pattern and Aroclor analysis is unusable.
Confirms the actual concentration of total PCB for cancer risk assessment.
Quantitates WHO doixin-like PCBs for dioxin cancer risks.
Possible to Identify environmentally modified PCBs.
10
Sensing Superfund Chemicals with Recombinant Systems
X. Guan,1 W. Luo,2 J. Feliciano,1 R. S. Shetty,1 A. Rothert,1 L. Millner,1 S. K. Deo,1 E. D'Angelo,2 L. G. Bachas,1 and
S. Daunert1*
1Department of Chemistry and2Department of Agriculture
University of KentuckyLexington, KY
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OpticalTransducer
WholeCell
Signal
AnalytesSugars
Metal ions
Organic pollutants:
PCB metabolites
Reporter ProteinGFP
Luciferases
Alkaline phosphatase
-Galactosidase
Whole Cell Sensing System Employing Reporter Protein
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Whole Cell Sensing System Employing Reporter Protein
REPORTER PLASMID
• Reporter Gene
• Regulatory protein controlling the expression of
the reporter gene
• Specificity of the sensing element towards the
analyte
13
•Whole Cell-Based Sensing SystemsWhole Cell-Based Sensing Systems
Reporter Reporter proteinprotein
SignalSignalAnalyteAnalyte
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Arsenic Poisoning
C&EN November 9, 1998
Applications of Arsenic• Agriculture
• Treatment for diseases
• Industrial uses
Long Exposure to Low Doses of Arsenic• Skin Hyperpigmentation and Cancer
• Other Cancers
• Inhibition of Cellular Enzymes
New Bangladesh Disaster:Wells that Pump Poison...New York Times, November 10, 1998
15
Arsenic contamination in the USA
U. S. Geological Survey, Fact Sheet FS 063-00, May 2000
16
ArsAArsA
PeriplasmMembrane
ArsB
AsO43-
AsO2-
AsO2- SbO2
-
AsO2- SbO2
-
ATPATP
Cytoplasm
ADP
ADP
Schematic Representation of the Antimonite/Arsenite Pump
ArsC
17
reporter geneRegulatory proteinbound to promoter inducer (toxin)
reporter gene +
Protein Expression Regulatory proteinbound to inducer
Reporter Enzyme
Substrate Product + Signal
Sig
nal
(analyte, M)
Sensory Process in Bacteria-Based Sensing Systems
18
Bacterial Luciferase
Decanal + FMNH2 + O2 Decanoic Acid + FMN + H2O + hmax = 490 nm)bacterial luciferase
p-aminophenyl-galactopyranosidep-aminophenol + galactopyranoside
GalactosidaseELECTROCHEMICAL:
HO NH2 O NH + 2H+ + 2e-
galactosidase
CHEMILUMINESCENCE:AMPGD hmax = 463 nm)
galactosidase
Reporter Genes
19
log (Arsenite, M)
Lig
ht
Inte
nsi
ty (
cou
nts
)
-3.5-4.5-5.5-6.5-7.5-8.5-9.50
10000
20000
30000
40000
50000
60000
Dose-Response Curve for Arsenite (-galactosidase reporter)
E. coli strain AW10Induction Time: 10 minarsR
lacZ'
ampr
pBGD23
20
1 amol of Antimonite = 6 x 105 molecules
Bacterial density = 8 x 104 bacteria / 100 µL
Therefore, on the average each bacterium detects:
6 x 105
8 x 104≈ 7 molecules of
Antimonite
Sensitivity of the Bioluminescence Sensing System
21
Comparison of Sensing Systems
-Galactosidase
Detection limit for antimonite: 10-15 M
Bacterial Luciferase
Detection limit of antimonite : 10-15 M
BioluminescenceBioluminescence
hh
Chemiluminescence
h
Detection limit for antimonite: 10-7 M
Electrochemistry
1011 - fold selective over phosphate, sulfate, arsenate and nitrate
22
Sensing System for Copper Using pYEX-GFPuv
0
50
100
150
200
250
ZnZn2+2+ FeFe2+2+CoCo2+2+ CuCu2+2+ CdCd2+2+ AgAg++NiNi2+2+F
luo
resc
ence
Inte
nsi
ty (
%)(
5)
log (Copper sulfate, M)
-6.0 -5.0 -4.0 -3.0 -2.0
0
1000
2000
3000
4000F
luo
res
ce
nc
e I
nte
ns
ity
,(a
.u.)
u.
Pcup1
gfpuv
leu2-dURA-3
ampR
pYEX-GFPuv
23
In vivo Luminescence Emission of Aequorea victoria
24
Calibration Plot for Antimonite
ampR arsR
gfpuv
pSD10
0
5.0 104
1.0 105
1.5 105
2.0 105
2.5 105
-6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0log (Potassium antimonyl tartrate, M)
Induction time = 30 minInduction time = 30 min
Flu
ore
sce
nc
e In
ten
sit
y(c
ou
nts
)
25
0
20000
40000
60000
80000
10-6M
SbO2- As04
3- Cr2O72- Fe(CN)6
3- SO42-
Flu
ore
scen
ce I
nte
nsi
ty (
cps)
Flu
ore
scen
ce I
nte
nsi
ty (
cps)
10-5M
Selectivity StudiesSelectivity Studies
26
Fluorescent Reporter Proteins in Array Detection
Protein Excitation Emission max max
GFP 395 (470) 509EGFP 488 509BFP 380 440GFPuv 395 509YFP 513 527CFP 433 475CobA 357 605RFP 558 583
27
A -CH2COOH
P -CH2CH2COOH
SAM - S-adenosyl-L-methionine
UMT- uroporphyrinogen methyltransferase III
UMT
SAMHN
NH N
N
PA
A
P
A
P
A
P
H3C
H3C
CH3
Trimethylpyrrocorphin
HN
NH N
N
PA
P
A
P
A
P
A
H3C
H3C
Sirohydrochlorin
oxidation
HN
NH
N
PA
P
A
P
A
P
A
H3C
H3C
HN
Dihydrosirohydrochlorin(Precorrin-2)
SAM
UMT
Urogen III
HN
NH
NH
P A
PA
P
A
P
AHN
Production of Fluorescent Porphyrinoid Compounds
28
-Aminolevulinic Acid in Urogen Pathway
ALA
NH2
O
COOH
dehydratase
ALA
ALA - -aminolevulinic acid
HN
NH
NH
P A
PA
P
A
P
AHN
Urogen III
Urogen- Uroporphyrinogen
deaminase
PBG
PBG
NH
COOH
COOH
NH2
PBG - Porphobilinogen
synthase
Urogen III
HMB
HN
NH
NH
P A
PA
A
P
P
AHN
HO
HMB- Hydroxymethylbilane
29
Effect of -Aminolevulinic Acid (ALA)
Flu
ore
sce
nc
e (c
ps)
0 2 4 6 8 10 12-2000
10000
8000
6000
4000
2000
0
Time (h)
NO ALA1mM ALA
NO ALA
30
-8 -7 -6 -5 -4 -30
5000
10000
15000
20000
25000
30000
35000
40000
45000
Flu
ore
scen
ce (
cps)
log (sodium-m-arsenite)
Calibration Plot (In the Presence of -Aminolevulinic Acid)
Selectivity Study
Flu
ore
scen
ce (
cps)
0
5
10
15
20
25
30
35
40
Br- Cl- PO43- SO4
2- AsO2- SbO2
-NO3-
Induction time = 1 hAnalyte concentration = 5 x 10-6 M
ampR
pSD601cobA
O/P
arsR
31
Chlorinated Organic Compounds
OH-PCBs
OHOH
Cl0-4
Chlorocatechols
Cl0-5Cl0-5
OHCl0-4Cl0-5
OH
PCB-diols
OHCl0-3Cl0-5
Polychlorinated biphenyls (PCBs)
Toxic Effects
• Carcinogenic
• Neurological
• Estrogenic
32
Biphenyl and 3-Chlorocatechol Catabolic Pathways
Chlorinated biphenyl
Dihydrodiol
2,3 diOH-ClBP
Meta-cleavagecompound
Chlorobenzoate Chloro-dihydroxy-benzoate
3-Chlorocatechol
2-Chloromuconate
4-Carboxymethylene-2-en-4-olide
Maleylacetic acid
BphA
BphB
BphC
BphD
ClcA
ClcB
ClcD
33
P. putida clc Operon
clc B clc DP/O
Clc R
OH
Cl
COOH
O
OHCOOH
ClcA ClcB ClcD
O
HOOC
O
- +
COOH
COOH
Cl
COOH
COOH
Cl
clc R clc A
34
-10 -9 -8 -7 -6 -5 -4 -3
1.5
1.0
0.5
0
L
igh
t In
ten
sit
y (c
ou
nts
1
06)
log (concentration, M)
3-chlorocatechol
4-chlorocatechol
4-chlorobiphenyl
Selectivity Studies
plasmid courtesy of S.M. McFall & A.M. Chakrabarty
clcR
clcA
clcB
lacZampr
pSMM50R-B
CatecholBiphenyl2-chlorophenol4-chlorophenol
35
-10 -9 -8 -7 -6 -5 -4 -3 -2
5
4
3
2
1
0
Lig
ht
Inte
ns
ity
(co
un
ts
10
5)
log (concentration, M)
Response to Catechol and Chlorocatechols
Time of induction: 5 min
3-chlorocatechol
catechol
4-chlorocatechol
36
Analysis of Chlorocatechols in Simulated Fresh Water (SFW) and Simulated Seawater (SSW)
0.5
9.60 0.5210
SSW 1.03 0.041.0
0.48 0.030.49 0.020.5
9.70 0.3510
SFW 0.96 0.061.0
0.51 0.050.50 0.01
Bacterial SensorHPLCvalue (mgL-1 )
Experimental valueSD(mgL-1)TheoreticalSample
37
Analysis of Chlorocatechols in Soils
1.73 0.28 2.0Not detectable2.0
0.43 0.090.5Not detectable0.5Organic Potting Soil
2.24 0.30 2.0Not detectable2.0 0.43 0.080.5Not detectable0.5Maury 54.0 4.550
9.55 0.7510
1.90 0.182.0Not detectable2.0
0.52 0.040.5Not detectable0.5Woolper
52.0 2.650
9.75 0.5510
1.95 0.082.02.03 0.042.0
0.51 0.020.50.49 0.020.5Sand
Experimental SD(mgkg-1)
Theoretical Value(mgkg-1)
Experimental SD(mgkg-1)
TheoreticalValue(mgkg-1)
Bacterial SensorHPLC
Sample
38
Lig
ht
Inte
ns
ity
(co
un
ts)
0
2105
4105
6105
8105
4-chlorocatechol 3,4-PCB-diol 4-chloro-PCB-diol biphenyl 4-chlorobiphenyl 3-chloro-benzoic acid 2-chlorophenol or 4-chlorophenol
Selectivity Study
39
Field Challenges
Background Signal
Freeze Drying
Liquid Drying
Vials (luciferase)
Strips (-galactosidase)
Viability of the cells
Field-kits
Conventional Analytical Techniques
40
Response to Arsenite Using -galactosidase
Test Strips
0 0.1 0.2 0.5 1Arsenite (M)
41
Bacteria with Induced
BioluminescenceCollected by Fiber
Dialysis Membrane
Analyte
BioluminescenceGenetically Engineered
Cells
Tip of OpticalFiber
Fiber Optic Sensor
42
Microfluidic Platform
Prototype CD for Six Simultaneous Analyses
43
Calibration Plot for Antimonitein the Microfluidic Set-up
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
-5.5 -5.0 -4.5 -4.0
log (Potassium antimonyl tartrate, M)
Flu
ore
scen
ce
(A.U
.)ampampRR
arsRarsR
gfpuv pSD10pSD10
44
Whole Cell Sensing Systems
• Feasible to sense Superfund chemicals with recombinant systems• Employ these whole cell systems in detection of water and soil samples• Design methods that are amenable to field studies:
• Handeling of liquid and freeze dried reagents• Incorporate whole cells in a microfluidics platform
• Remote sensing• Incoporation of whole cell sensing systems in fiber optic set-ups
45
Collaborators
Dr. Marc J. MadouDr. Barry RosenDr. Jan Roelof van der Meer
46
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