1. seminar on SOLID PHASE EXTRACTION AND APPLICATION MODH SUDIP C. PA/2010/11 NIPER HYDERABAD

3. Principle

4. Types Solid phases

5. Experimental steps

6. Trace enrichment

7. Solid phase micro extraction (SPME)

8. Comparison with HPLC

9. Application Contents 2

10. What is the solid phase extraction? 3

11. DEFINATION OF SOLID PHASE EXTRACTION (SPE): “A solid phase extraction consists of bringing a liquid or gaseous test sample in contact with a solid phase, whereby the analyte is selectively adsorbed on the surface of the solid phase” Other solvents (liquids or gases)added to remove possible adsorbed matrix components Eluting solvent added to desorb analyte selectively 4

12. SYNONYMS Liquid-solid extraction Column extraction Digital chromatography Bonded phase extraction Selective adsorption techniques 5

14. Retained-while matrix interference are washed through6

15. Retained-while matrix interference are washed through 7

16. Principle of Solid Phase Extraction: Partitioning of compounds between two phases of solid and liquid Must having greater affinity for the solid phase than for the sample matrix Compounds retained on the solid phase can be removed by eluting solvent with a greater affinity for the analytes pH changes can be useful 8

17. In modern SPE the adsorbent is packed between two flitted disks in polypropylene cartridgeand liquid phases are passed through the cartridge either by suction or by positive pressure 9

18. Cartridge 10

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20. OVERVIEW OF SPE 12

21. Solid phases Activated charcoal Alumina Silica gel Magnesium silicate (Florisil) Chemically bonded silica phases and polymers E.g. styrene divinylbenzene 13

23. Polar

24. Ion exchangers It gives different mode of chromatography Other solid supports Polymeric resins, cellulose and zirconia 14

25. Zirconia coated silica as a stationary phase 15

26. Examples of selective stationary phases Reaction of phenobarbitone with pentafluorobenzyl bromide onto the adsorbent Amphetamine by Chiral derivatization of solid Phases Doxorubicin by the metal-loaded phases in which metal cation is loaded onto a reagent-labelled phase Molecularly imprinted polymers synthetic polymeric materials with specific cavities designed for a template molecule 16

27. Technical Data - Solid Phase Extraction (SPE) Media Product Sorbent Abbreviations Description ODS Octadecyl silica 5% carbon load ODS-4 Octadecyl silica 14% carbon load, end capped* ODS-5 Octadecyl silica 18% carbon load, end capped C-8 Octyl silica 8.5% carbon load, end capped FLO Florisil™ Magnesium silicate NH2 Weak anion exchanger Primary amine SAX Strong anion exchanger Quaternary amine (-NR3+) SCX Strong cation exchanger Aromatic benzene sulfonic acid SIL Normal phase silica * End capping masks residual silanol groups, reducing ionic affinity for amines. 17

28. Experimental procedure of five steps: Activation of sorbent by appropriate solvent that conditions the surface of the solid 2. Removal of solvent by liquid similar to the sample matrix 3. Application of sample, the analytes retained by the sorbent 4. Removal of interfering compounds retained in step 3 with a solvent, but shouldn’t remove the analytes (washing step) 5.Elution of the analytes with an appropriate solvent (desorption or elution step) and collecting for analysis 18

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34. Other technique can be used Supercritical fluid Thermal desorption for analytes of high volatility and thermal stability Thermal desorption with GC for occupational hygiene analysis 24

35. Analyte eluted with an organic, relatively volatile solvent is evaporated to dryness Then residue dissolved in appropriate solvent Due to evaporation step, speed with SPE is lost 25

36. IMMUNOAFFINITY PHASES: Highly selective packings of Immunoaffinity phases of specific antibody immobilised on solid support such as agarose or silica Useful for selective extraction of biological importance Substance Diagnosis of cancer ELISA TEST 26

37. IMMUNOAFFINITY PHASES 27

38. TRACE ENRICHMENT WITH SPE Sensitivity depends on Physicochemical properties of the Analyte Detection system Selective clean-up Isolation and concentration step 28

39. Solid phase microextraction: SPME is the technique in which by using special instrument, sampling is possible in a vapour state 29

40. Design of SPME Syringe like instrument Fused silica fiber of a small size and cylindrical shape connected to stainless-steel tube for additional mechanical strength and repeated sampling 30

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42. Fused silica fibre coated with thin film of several polymeric stationary phases Reusable and replaceable Small size and cylindrical geometry of fiber Placement into sample or headspace is easy Loading in desorption chamber of GC or Interphase of the HPLC without any modification of Plunger 32

43. Working with SPME Fiber is first drawn into the syringe needle Lowered into the vial by pressing the plunger Fiber cleaned before analysis to remove contaminants Cleaning can be performed in the desorption chamber of HPLC by running solvent Cleaned fiber coating is exposed to a sample matrix for a predetermined, fixed period 33

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45. Limits of detection at the µg/L level with using a flame Ionization detector Limits of detection as low as ng/ L can be reached with an ion-trap mass spectrometer 35

46. Extraction can be performed in two ways 1) Headspace SPME or HS-SPME Fiber is exposed in the vapour phase above a gaseous, liquid, or solid sample 2) Direct immersion or DI-SPME Fiber is directly immersed in liquid samples 36

47. Available SPME Fibres, by Film Type •Absorption Fibres -Polydimethylsiloxane (PDMS) 7, 30, and 100μm Unpolar -Polyacrylate (PA) Polar -Polyethylene glycol (PEG) Polar •Adsorption fibres (with particles) -Carboxen-polydimethylsiloxane (CAR-PDMS) Adsorption -Polydimethylsiloxane- divinylbenzene (PDMS-DVB) Adsorption -Divinylbenzene/ Carboxen-Polydimethylsiloxane (DVB-CAR-PDMS) Adsorption 37

48. SPME applied to liquid, gaseous or heavily contaminated samples chemicals like Substituted benzene compounds Polyaromatic hydrocarbons Nitro- and chlorophenols Naphthols volatile organochlorine compounds polychlorinated biphenyl congeners caffeine Metallic ions 38

49. The SPE process can be performed in a two ways: On-line Off-line In offline SPE eluate from the cartridge is introduced into the chromatograph by means of an injection valve In on-line SPE the extraction cartridge is inserted as part of chromatographic equipment, as loops or high pressure stream of the mobile phase 39

50. Types of online SPE: SPE-GC(SPME-GC) SPE-HPLC 40

51. ONLINE SPE-GC 41

52. ONLINE SPE-HPLC 42

53. 10ppb Nitrosamines in Water: SPME-GC/MS Chromatogram courtesy of J. Clark, Liggett Group, Inc. 43

54. ONLINE SPE-HPLC THT= Tetra hydro thiophene 44

55. Comparison of SPE-HPLC and SPME-GC SPE-HPLC SPME-GC Universality Compounds +++ + Detection Sensitivity ++ +++ Selectivity +++ ++ Identification + +++ Detection limit (µ/ L) 0.05-0.8 0.2-5 Reproducibility (%) 1-15 4-14 Analysis time 90 20 Sample volume (mL) 200 2 Automation +++ +++ Simplicity + +++ 45

56. Comparison of SPE and HPLC Theoretical basis as HPLC Retention and selectivity remain unaffected by particle size Efficiency dependent on: Particle size Column geometry Typical number of plates HPLC ~ 10,000 SPE < 50 Minimum Selectivity(alpha)for Rs=1.2 HPLC 1.06 SPE 3.95 46

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58. Advantages of SPE offers over LLE are Higher selectivity Cleaner extracts More reproducibility The avoidance of emulsion formation 48

59. Application of SPE in various fields Impurity profiling of pharmaceuticals Environmental applications Applications in food chemistry Analysis of wines and other alcoholic beverages Application to biological fluids Hair analysis 49

60. Impurity profiling of pharmaceuticals Residual solvent analysis by USP 467 Involves head space solid phase micro extraction with GC and FID detector 50

61. Application to biological fluids: Simultaneous qualitative and quantitative determination of Drugs of abuse opiates, cocaine, or amphetamines Prescribed drugs tricycle antidepressants,phenotiazines, benzodiazepines in biological fluids was developed Eg. A Weak Cation-Exchange Monolithic SPE Column for Extraction and Analysis of Caffeine and Theophylline in Human Urine 51

62. Urinary Excretion Pattern of Benzophenone-3 and its Metabolite 2,4-Dihydroxybenzophenone in Human Urine 52

63. Hair analysis It is used for the long-term monitoring of drug and alcohol 53

66. THANK YOU All of you 56