1. Introduction to a 2-step analysis for persistent organic pollutants Peggy Krahn National Oceanic and Atmospheric Administration National Marine Fisheries Service

2. Peggy Krahn directs the National Marine Fisheries Environmental Conservation Division Laboratory in Seattle

3. Peggy gave us a tour of the 2-step process she uses to look for persistent organic pollutants. If you have downloaded RealPlayer you can listen to Peggy’s tour when you click on her picture. We’ve also included brief text descriptions. RealPlayer

4. The reason for a 2-step process is to start looking for contaminants with a less expensive approach. If the first “rapid analysis” stage shows that there are contaminants, then the second stage can provide more complete information. Let’s start with the rapid analysis step.

5. Parts of Rapid Analysis Tissue extraction with solvent Sample precleanup HPLCAutosampler Automated Instrumental Analysis Cosmosil PYE column PDA PDA Detector 10 Chromatogram

6. Compounds the rapid analysis step can identify and measure these contaminants u Pesticides: HCB, o,p’-DDT, p,p’-DDT, o,p’-DDD, p,p’- DDD,p,p’-DDE u Specific types of PCBs (“congeners”) 77, 101*, 105, 110, 114, 118, 126, 128, 138, 153, 156, 157, 169, 170/190, 180, 189, 200, total PCBs

7. The first step is to get rid of the water in the sample and mix it with gasoline-like solvents.

8. The next step is to separate the solids from the liquids. You do this with a centrifuge that spins the mixture very rapidly. Before using the centrifuge, you have to make sure it is balanced. Otherwise it will walk off the table with your sample! The centrifuge itself is behind Darr’s arm.

9. There is now too much solvent in the sample. Darr here is boiling the solvents off under a hood that keeps the fumes away from her.

10. Next, Darr is using a column to separate natural materials in the sample from the contaminants The black band in the glass column is the natural materials being captured, allowing the contaminants to go through to the bottle at the bottom

11. Back to the hood to boil off solvents

12. These are the actual instruments that measure the contaminants

13. The computer calculates a graph with peaks that show amounts of specific contaminants measured by the instruments in the previous slide

14. Sample wt (g) 0.26 ± 0.042 – 3 SelectedRapid AnalysisNIST CB Congenersn= 99(published) † = coeluting CB congeners 163, 164 ng/g, wet wt 10590 ± 19 88.9 ± 13 118247 ± 34 267 ± 25 138500 ± 78 664 ± 8 † 153 823 ± 111870 ± 9 15629 ± 4 38 ± 1 180495 ± 69 483 ± 9 Analyses of NIST whale blubber control material by rapid analysis

15. Parts of Detailed Analysis Tissue extraction with solvent Sample precleanup Automated Instrumental Analysis 10 Chromatogram MSD GC/MS HPLC

16. Contaminants measured in detailed analysis  Pesticides: HCB,  -HCH, ß-HCH, lindane, aldrin, dieldrin, endosulfan I & II & sulfate, HPE, heptaclor, heptaclor epoxide, oxychlordane,  -chlordane,  -chlordane, trans- nonachlor, cis-nonachlor, o,p’-DDT, p,p’-DDT, o,p’- DDD, p,p’-DDD,o,p’-DDE, p,p’-DDE u PCBs: 17, 18, 28, 31, 33, 44, 49, 52, 66, 70, 74, 82, 87, 95, 99, 101/90, 105, 110, 118, 128, 138/163/164, 149, 151, 153/132, 156, 158, 170/190, 171, 177, 180, 183, 187/159/182, 191, 194, 195, 199, 205, 206, 208, 209 u Toxaphenes u PAHs

17. A marine mammal blubber sample (1-2 g) is weighed.

18. The sample is ground for 5 minutes

19. Balancing the sample for the centrifuge to separate the solid from the liquid

20. Pouring the liquid off through a column to take out the natural materials and leave the contaminants in the liquid

21. Brown jars with all the liquid Tubes of liquid being heated to boil off the extra liquid

22. The High Performance Liquid Chromatograph (HPLC) to get rid of the rest of the lipids (fats)

23. We use this column filled with sand with a coating on it to separate the lipids from the contaminants

24. We boil off solvent again. Then we go further by putting it in the vial and use nitrogen gas get rid of more solvent. Then we can see contaminant peaks on the instrument.

25. A chromotgram from the Mass Spectrometer showing great detail

26. Jenny (like her blue hair?) and Doug figured out a way to quickly transfer data from the Mass Spec to a computer database

27. SelectedNIST AnalytesGC/MS (certified) Sample wt (g) ~ 1.52 – 3 ng/g, wet wt ng/g, wet wt HCB 29.6 ± 1.3 32.9 ± 1.7 -chlordane 50.0 ± 7.3 46.9 ± 2.8  -chlordane 50.0 ± 7.3 46.9 ± 2.8 p,p’-DDE 526 ± 33 445 ± 37 10527.0 ± 1.6 30.1 ± 2.3 118 77.4 ± 3.2 74.6 ± 5.1 153/132 236 ± 11 213 ± 19 15611.4 ± 0.910.3 ± 1.1 170/190 41.2 ± 4.840.6 ± 2.6 Comparison of Results of Detailed Analysis to NIST whale blubber Standard Reference Material

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