1. A L O S Project Workshop IV S OLID- P HASE E XTRACTION

2. 2 ALOS: ROSKILDE-MEETING November 2005 Principles of solid phase extraction Principles of solid phase extraction Features and benefits Features and benefits Silica products and polymers Silica products and polymers Practice Practice Applications Applications Outline

3. 3 ALOS: ROSKILDE-MEETING November 2005 Principles Why do sample preparation? Remove interferences from sample More accurate results Concentrating analytes to improve detection Protecting equipment to reduced costs

4. 4 ALOS: ROSKILDE-MEETING November 2005 SPE is an extraction process whereby an aqueous sample is filtered through a thin bed of sorbent particles, the analytes of interest are removed from the liquid matrix, and concentrated onto the sorbent. Once concentrated, the analytes are removed by an eluting solvent. Principles of SPE

5. 5 ALOS: ROSKILDE-MEETING November 2005 Comparison of LLE vs SPE Disk LLE LLE Uses 200 - 500 ml solvent Uses 200 - 500 ml solvent Shaking / continuous process Shaking / continuous process Forms emulsions Forms emulsions Little selectivity Little selectivity Takes 1 - 2 hours / sample Takes 1 - 2 hours / sample Uses 2 - 20 ml solvent Uses 2 - 20 ml solvent Filtration process Filtration process No emulsions formed No emulsions formed Wide selectivity Wide selectivity (adsorbent) (adsorbent) Takes 10 - 20 min. / sample Takes 10 - 20 min. / sample SPE disk

6. 6 ALOS: ROSKILDE-MEETING November 2005 Problems with the LLE Procedure Tedious and time-consuming Tedious and time-consuming Shaking and separation time Shaking and separation time Evaporation time Evaporation time Expensive-labor and materials Expensive-labor and materials Time factor Time factor Solvent cost and exposure Solvent cost and exposure Solvent disposal Solvent disposal Poor results Poor results Forming of emulsions Forming of emulsions Irreproducible extractions Irreproducible extractions Low recoveries Low recoveries

7. 7 ALOS: ROSKILDE-MEETING November 2005 What are the Benefits of SPE? SPE uses less solvent than LLE SPE uses less solvent than LLE SPE is faster (at least 5 times) SPE is faster (at least 5 times) High capacity High capacity Total SPE costs are considerably less than LLE Total SPE costs are considerably less than LLE High selectivity: broad choice of bonded phases and solvents High selectivity: broad choice of bonded phases and solvents Automation much easier Automation much easier

8. 8 ALOS: ROSKILDE-MEETING November 2005 SPE Column

9. 9 ALOS: ROSKILDE-MEETING November 2005 SPE Column accessories

10. 10 ALOS: ROSKILDE-MEETING November 2005 SPE- Experimental Set-up

11. 11 ALOS: ROSKILDE-MEETING November 2005 Silicagel-Phases Reversed Phase C 18 Adsorption Si-OH Normal Phase NH 2 CN C-OH(OH)

12. 12 ALOS: ROSKILDE-MEETING November 2005 Anion Exchange N + NH 2 Cation Exchange C 6 H 5 -SO 3 H COOH SO 3 H Biochromatography WP PEI (NH 2 ) WP Butyl (C 4 ) WP CBX (COO) Sephadex G-25 Phases

13. 13 ALOS: ROSKILDE-MEETING November 2005 Interactions Non polar: van der Waals ~20 KJ/mole Polar: Dipole / Dipole ~ 40 KJ/mole Hydrogen bond ~40 KJ/mole Electrostatic: Ionic ~600 KJ/mole!

14. 14 ALOS: ROSKILDE-MEETING November 2005 Reversed Phase Principle

15. 15 ALOS: ROSKILDE-MEETING November 2005 Mixed Mode Principle

16. 16 ALOS: ROSKILDE-MEETING November 2005 Polymer- Phase Principle (-CH-CH 2 ) n - N-CH 3 C=O CH 3 R R R

17. 17 ALOS: ROSKILDE-MEETING November 2005 Polymer- Phase Principle (-CH-CH 2 ) n - N-CH 3 C=O CH 3 R R SO 3 H or CH 2 N + R 3

18. 18 ALOS: ROSKILDE-MEETING November 2005 Practice

19. 19 ALOS: ROSKILDE-MEETING November 2005 4 Steps in SPE

20. 20 ALOS: ROSKILDE-MEETING November 2005 Conditioning of silica-based sorbents Add an organic solvent to rinse and activate the alkyl-chains (C 18, C 8 etc.)! Don´t let the column run dry during conditioning!

21. 21 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural dyes in beverages Sample Preparation: 1 mL blackberry-juice is dissolved in 2 mL of distilled water. Column Conditioning: A 3 mL C18 (Baker: 7020-03) SPE cartridge is conditioned with 1 mL methanol followed by one column volume of distilled water. Sample Addition/Wash: The prepared sample is aspirated through the column. A 5 mL distilled water wash is used to remove sugars, sugar colouring and organic acids. Elution: The dye(s) [anthocyanines, flavonoids, tannins and/or alkaloids)] is (are) eluted with a column volume of methanol. Sometimes propanol-2 will be more successful. Analysis: For detailed analysis- an absorption spectrum can be taken from the eluate. - TLC-experiments can be done.

22. 22 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural dyes in beverages: sample load

23. 23 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural dyes in beverages: sample load

24. 24 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural dyes in beverages: washing step

25. 25 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural dyes in beverages: elution step

26. 26 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural + synthetic dyes in beverages:

27. 27 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural + synthetic dyes in beverages: sample load

28. 28 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural + synthetic dyes in beverages: elution step

29. 29 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural + synthetic dyes in beverages

30. 30 ALOS: ROSKILDE-MEETING November 2005 Environmental Environmental Water, Soil Water, Soil Pharmaceutical/clinical Pharmaceutical/clinical Serum, plasma, urine, blood Serum, plasma, urine, blood Food/feed Food/feed Juice, grain, milk Juice, grain, milk Biological/biotech Biological/biotech Water, plasma, urine Water, plasma, urine Application Database

31. 31 ALOS: ROSKILDE-MEETING November 2005 Environmental Environmental - PAH s from water and soil - PAH s from water and soil - PCBs from oil- - PCBs from oil- - Pesticides from water/soil- - Pesticides from water/soil- - etc. - etc. Food/Feed/Beverages Food/Feed/Beverages - Aflatoxine from corn meal - Aflatoxine from corn meal - Caffeine from di-caffeinated diet cola - Caffeine from di-caffeinated diet cola - Vitamin E from juice - Vitamin E from juice - etc. - etc. Pharmaceutical/Clinical/Biological Pharmaceutical/Clinical/Biological - Benzodiazipines from serum - Benzodiazipines from serum - Anabolic Steroids/Urine - Anabolic Steroids/Urine - Aflatoxine from liver - Aflatoxine from liver - etc. - etc.

32. 32 ALOS: ROSKILDE-MEETING November 2005 Thank you for your attention

33. 33 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural dyes in beverages Sample Preparation: 1 mL blackberry-juice is dissolved in 2 mL of distilled water. Column Conditioning: A 3 mL C18 (Baker: 7020-03) SPE cartridge is conditioned with 1 mL methanol followed by one column volume of distilled water. Sample Addition/Wash: The prepared sample is aspirated through the column. A 5 mL distilled water wash is used to remove sugars, sugar colouring and organic acids. Elution: The dye(s) [anthocyanines, flavonoids, tannins and/or alkaloids)] is (are) eluted with a column volume of methanol. Sometimes propanol-2 will be more successful. Analysis: For detailed analysis- an absorption spectrum can be taken from the eluate. - TLC-experiments can be done.

34. 34 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural dyes in beverages: sample load

35. 35 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural dyes in beverages: sample load

36. 36 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural dyes in beverages: washing step

37. 37 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural dyes in beverages: elution step

38. 38 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural + synthetic dyes in beverages:

39. 39 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural + synthetic dyes in beverages: sample load

40. 40 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural + synthetic dyes in beverages: elution step

41. 41 ALOS: ROSKILDE-MEETING November 2005 Experiment 1: Rapid Extraction of natural + synthetic dyes in beverages

42. 42 ALOS: ROSKILDE-MEETING November 2005 Experiment 2: Rapid Extraction of iron-ions from water samples or waste water Sample Preparation: Dissolve 100 mg Fe(NO 3 ) 3 or FeCl 3 in 10 mL of distilled water or use an amount of approximately 100mL waste water, which contains iron (III) ions. Column Conditioning: A 3 mL sulfonic acid (Baker: 7090-03) SPE column is conditioned with 2 mL methanol followed by one column volume of distilled water. Sample Addition/Wash: 2 mL of the sample is aspirated through the column. The column is washed with 2 mL of distilled water. Elution: Fe 3+ -ions are eluted in 1-2 mL hydrochlorid acid (c=0,1 M). The eluate is coloured yellow. Analysis: For detailed analysis - add NH 4 SCN to the eluate. The colour changes to deep red.

43. 43 ALOS: ROSKILDE-MEETING November 2005 Experiment 2: Rapid Extraction of iron-ions from water samples or waste water

44. 44 ALOS: ROSKILDE-MEETING November 2005 Experiment 2: Rapid Extraction of iron-ions from water samples or waste water

45. 45 ALOS: ROSKILDE-MEETING November 2005 Experiment 2: Rapid Extraction of iron-ions from water samples or waste water

46. 46 ALOS: ROSKILDE-MEETING November 2005 Experiment 2: Rapid Extraction of iron-ions from water samples or waste water

47. 47 ALOS: ROSKILDE-MEETING November 2005 Experiment 3: Rapid Extraction of sugars (glucose) Sample Preparation: Dissolve 100 mg glucose in 10 mL water. Column Conditioning: A 3 mL Amino (Baker: 7088-03) SPE column is conditioned with 2 mL ethanol. Sample Addition/Wash: 2 mL of the prepared sample is aspirated through the column. Elution: Sugar is eluted with 2 mL water. Analysis: For detailed analysis - use few drops of Fehling-solutions. For detailed analysis - an absorption spectrum can be taken from the eluate.- TLC-experiments can be done.

48. 48 ALOS: ROSKILDE-MEETING November 2005 Experiment 4: Rapid Extraction of ß-carotine from Multivitamine-juice or carot-juice Sample Preparation: 5 mL of Multivitamine-juice or carot-juice is filtered or centrifuged. Column Conditioning: A 3 mL C18 (Baker: 7020-03) SPE cartridge is conditioned with methanol followed by one column volume of distilled water. Sample Addition/Wash: The prepared sample is aspirated through the column. A 2 mL distilled water wash is used to remove all the other compounds. Elution: ß-carotine is eluted with about 2 mL heptane-acetone mixture (8:2,v,v). the eluate is coloured yellow. Analysis: For detailed analysis - an absorption spectrum can be taken from the eluate. - TLC-experiments can be performed.

49. 49 ALOS: ROSKILDE-MEETING November 2005 Experiment 4: Rapid Extraction of ß-carotine

50. 50 ALOS: ROSKILDE-MEETING November 2005 Experiment 4: Rapid Extraction of ß-carotine

51. 51 ALOS: ROSKILDE-MEETING November 2005 Experiment 4: Rapid Extraction of ß-carotine

52. 52 ALOS: ROSKILDE-MEETING November 2005 Students-Experiment: Rapid Extraction of Haribos wine gum dyes

53. 53 ALOS: ROSKILDE-MEETING November 2005 Students-Experiment: Rapid Extraction of Haribos wine gum dyes

54. 54 ALOS: ROSKILDE-MEETING November 2005 Students-Experiment: Rapid Extraction of Haribos wine gum dyes