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Advances in PCB Analysis 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 and 2Department of Agriculture University of Kentucky Lexington, KY 11 Whole Cell Sensing System Employing Reporter Protein Signal Whole Optical Analytes Cell Transducer Reporter Protein Sugars GFP Metal ions Luciferases Organic pollutants: Alkaline phosphatase PCB metabolites β-Galactosidase 12 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 Systems Analyte Signal Reporter protein 14 Arsenic Poisoning Applications of Arsenic • Agriculture • Treatment for diseases • Industrial uses New Bangladesh Disaster:Wells that Pump Poison...New York Times, November 10, 1998 Long Exposure to Low Doses of Arsenic • Skin Hyperpigmentation and Cancer C&EN November 9, 1998 • Other Cancers • Inhibition of Cellular Enzymes 15 Arsenic contamination in the USA U. S. Geological Survey, Fact Sheet FS 063-00, May 2000 16 Schematic Representation of the Antimonite/Arsenite Pump - - AsO2 SbO2 Cytoplasm ADP ATP AsO - ATP 3- 2 AsO4 ADP ArsA ArsA ArsC Periplasm Membrane ArsB - - AsO2 SbO2 17 Sensory Process in Bacteria-Based Sensing Systems reporter gene Regulatory protein bound to promoter inducer (toxin) reporter gene + Regulatory protein Protein Expression bound to inducer Reporter Enzyme Substrate Product + Signal Signal (analyte, M) 18 Reporter Genes Bacterial Luciferase bacterial luciferase ν(λ Decanal + FMNH2 + O2 Decanoic Acid + FMN + H2O + h max = 490 nm) β−Galactosidase ELECTROCHEMICAL: β−galactosidase p-aminophenyl-β−galactopyranoside p-aminophenol + galactopyranoside + - HO NH2 O NH + 2H + 2e CHEMILUMINESCENCE: β−galactosidase ν(λ AMPGD h max = 463 nm) 19 Dose-Response Curve for Arsenite (β-galactosidase reporter) 60000 E. coli strain AW10 arsR 50000 Induction Time: 10 min ampr pBGD23 40000 lacZ' 30000 20000 10000 Light Intensity (counts) Light Intensity 0 -9.5 -8.5 -7.5 -6.5 -5.5 -4.5 -3.5 log (Arsenite, M) 20 Sensitivity of the Bioluminescence Sensing System 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 ≈ 7 molecules of 8 x 104 Antimonite 21 Comparison of Sensing Systems β-Galactosidase Chemiluminescence hν Electrochemistry Detection limit for antimonite: 10-15 M Detection limit for antimonite: 10-7 M Bacterial Luciferase Bioluminescence hν Detection limit of antimonite : 10-15 M 1011 - fold selective over phosphate, sulfate, arsenate and nitrate 22 4000 Sensing System for Copper Using pYEX-GFPuv 3000 2000 1000 0 Fluorescence Intensity ,(a.u.)u. -6.0 -5.0 -4.0 -3.0 -2.0 250 log (Copper sulfate, M) 200 150 Pcup1 gfpuv 100 50 pYEX-GFPuv (%)(5) Intensity Fluorescence 0 ampR leu2-d Zn2+ Co2+ Fe2+ Ni2+ Cu2+ Cd2+ Ag+ URA-3 23 In vivo Luminescence Emission of Aequorea victoria 24 ampR arsR Calibration Plot for Antimonite pSD10 gfpuv 2.5 × 105 Induction time = 30 min 2.0 × 105 1.5 × 105 1.0 × 105 (counts) 5.0 × 104 0 Fluorescence Intensity -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 log (Potassium antimonyl tartrate, M) 25 Selectivity Studies 80000 10-5M 60000 10-6M 40000 20000 0 Fluorescence Intensity (cps) Fluorescence Intensity - 3- 2- 3- 2- SbO2 As04 Cr2O7 Fe(CN)6 SO4 26 Fluorescent Reporter Proteins in Array Detection Protein Excitation Emission λ λ max max GFP 395 (470) 509 EGFP 488 509 BFP 380 440 GFPuv 395 509 YFP 513 527 CFP 433 475 CobA 357 605 RFP 558 583 27 Production of Fluorescent Porphyrinoid Compounds P H C 3 A A P H3C N HN oxidation NH P A P H C A N 3 A A A A P P NH HN P P UMT H3C N HN Sirohydrochlorin NH HN SAM P H C A NH HN 3 A A A A A UMT P P H3C N HN P P Urogen III P SAM Dihydrosirohydrochlorin (Precorrin-2) NH N A -CH2COOH A CH3 P -CH2CH2COOH A SAM - S-adenosyl-L-methionine P P Trimethylpyrrocorphin UMT- uroporphyrinogen methyltransferase III 28 δ-Aminolevulinic Acid in Urogen Pathway P A P A COOH COOH A P A P COOH NH HN NH HN ALA PBG Urogen III HO O dehydratase deaminase synthase N NH HN NH HN H P A A A H N H N 2 2 ALA PBG A P P P HMB Urogen III ALA - δ-aminolevulinic acid PBG - Porphobilinogen HMB- Hydroxymethylbilane Urogen- Uroporphyrinogen 29 Effect of δ-Aminolevulinic Acid (ALA) 10000 NO ALA NO ALA 8000 1mM ALA 6000 4000 2000 Fluorescence (cps) 0 -2000 024681012 Time (h) 30 Calibration Plot (In the Presence of δ-Aminolevulinic Acid) 45000 O/P 40000 arsR 35000 30000 pSD601 cobA R 25000 amp 20000 15000 Selectivity Study Fluorescence (cps) 10000 40 5000 35 Induction time = 1 h Analyte concentration = 5 x 10-6 M 0 30 -8 -7 -6 -5 -4 -3 25 log (sodium-m-arsenite) 20 15 10 5 Fluorescence (cps) 0 - - - 3- 2- - - Br Cl NO3 PO4 SO4 AsO2 SbO2 31 Chlorinated Organic Compounds OH Cl Cl Cl0-5 Cl0-5 0-5 0-4 OH-PCBs Toxic Effects Polychlorinated biphenyls (PCBs) • Carcinogenic • Neurological OH Cl0-4 OH OH • Estrogenic Cl Cl 0-5 0-3 OH PCB-diols Chlorocatechols 32 Biphenyl and 3-Chlorocatechol Catabolic Pathways Chlorinated biphenyl 3-Chlorocatechol BphA ClcA Dihydrodiol 2-Chloromuconate BphB ClcB 2,3 diOH-ClBP 4-Carboxymethylene- 2-en-4-olide BphC Meta-cleavage compound ClcD Maleylacetic acid BphD Chlorobenzoate Chloro- dihydroxy-benzoate 33 This slide illustrates how the bph and clc pathways can relate to each other. If 3-chlorobenzoate is one of the products of the bph pathway, it can be further degraded to Kreb cycle intermediates via the clc pathway. Not all chlorobenzoates produced by the bph pathway are degraded by the clc pathway, only 3-chlorobenzoates. Other chlorinated benzoates go through other pathways (for example there is a separate pathway for 2-chlorobenzoate). I’m not sure what happens to benzoates that are highly chlorinated. I suspect they must be dechlorinated before they can be degraded, but I’m not sure. P. putida clc Operon ClcA ClcB ClcD O HOOC COOH OH O COOH COOH O COOH Cl OH Cl - + P/O clc R clc A clc B clc D Clc R COOH COOH Cl 34 Regulation of the clc operon The enzymes responsible for 3-chlorocatechol degradation are found on a plasmid isolated from Pseudomonas. The clcABD genes are transcribed as a single message, while clcR is divergently transcribed from a promoter next to the clcABD promoter (P/O).
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