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HPLC Columns: Column Configuration Packing Material Bonded Phases

HPLC Columns: Column Configuration Packing Material Bonded Phases. HPLC Column Configuration. Porous frits close the ends of the column to retain the packing particles. HPLC Support Particles. Most often silica or silica gel (hydrated silicon-oxygen).

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HPLC Columns: Column Configuration Packing Material Bonded Phases

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  1. HPLC Columns: • Column Configuration • Packing Material • Bonded Phases

  2. HPLC Column Configuration Porous frits close the ends of the column to retain the packing particles

  3. HPLC Support Particles Most often silica or silica gel (hydrated silicon-oxygen) Less often hydrated aluminum-oxygen polymers (alumina)

  4. HPLC Support Particles Micropellicular Particle: Solid core Thin outer skin of stationary phase 1.5 – 2.5 μm sizes Very low surface area Fast separation, low capacity

  5. HPLC Support Particles Porous Micro-sphere: Most common Large surface area Variety of pore sizes High capacity

  6. HPLC Support Particles

  7. HPLC Support Particles Perfusion Particle: Least common Very large pores (0.5 μm) High flow rate (fast) Macromolecules (proteins)

  8. HPLC Support Particles

  9. HPLC Support Particles

  10. Particle Size Considerations

  11. Particle Size Considerations H  dp2 ΔP  L/ dp2 N ≈ 3500 L / dp L in cm dp in μm

  12. Particle Size Considerations Substituted benzenes

  13. Particle Size Considerations

  14. Solid Stationary Phases (Adsorption – Normal Phase) Liquid-Solid Adsorption Analyte interacts directly with Si-OH groups Silica gel is acidic Irreversible adsorption is likely Slow re-equilibration

  15. Bonded Stationary Phases Use Si-OH reaction Rapid Equilibration No Irreversible Adsorption

  16. Bonded Stationary Phases R group may be: polar (normal phase) non-polar (reversed phase) ionic (ion exchange)

  17. Bonded Stationary Phases Un-reacted Si-OH groups must be shielded or end-capped

  18. Bonded Stationary Phases 1. Histidine 2. Arginine 3. Valine

  19. Bonded Stationary Phases Polycyclic Aromatic Hydrocarbons

  20. Bonded Stationary Phases

  21. Bonded Stationary Phases monomeric C-18 polymeric C-18 end-capping

  22. Bonded Stationary Phases: R groups

  23. Bonded Stationary Phases: R groups

  24. Bonded Stationary Phases: R groups Cross-linked polystyrene ion-exchange resin

  25. Bonded Stationary Phases: R groups

  26. Bonded Stationary Phases: R groups

  27. Bonded Stationary Phases: R groups

  28. Bonded Stationary Phases: R groups Normal Phase R Groups

  29. Bonded Stationary Phases: R groups Normal Phase separation of sugars using an amino bonded stationary phase and 75:25 acetonitrile:water 1. fructose 2. glucose 3. sucrose 4. maltose

  30. Bonded Stationary Phases: R groups Normal Phase R Groups

  31. Bonded Stationary Phases: R groups

  32. Bonded Stationary Phases: R groups

  33. Bonded Stationary Phases: R groups

  34. Bonded Stationary Phases Size Exclusion Stationary phase consists of porous particles with controlled pore diameter Separation based on size (not MW) No chemical interactions involved

  35. Bonded Stationary Phases

  36. Bonded Stationary Phases Each Size Exclusion column has a defined range of separation

  37. Bonded Stationary Phases

  38. Bonded Stationary Phases

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