1 / 31

Reversed Phase HPLC Mechanisms

Reversed Phase HPLC Mechanisms. Nicholas H. Snow Department of Chemistry Seton Hall University South Orange, NJ 07079 snownich@shu.edu. Reversed Phase HPLC. Synthesis of RP Packings RP Column Properties RP Retention Mechanisms Important RP parameters RP Optimization I.

rainer
Download Presentation

Reversed Phase HPLC Mechanisms

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Reversed Phase HPLC Mechanisms Nicholas H. Snow Department of Chemistry Seton Hall University South Orange, NJ 07079 snownich@shu.edu

  2. Reversed Phase HPLC • Synthesis of RP Packings • RP Column Properties • RP Retention Mechanisms • Important RP parameters • RP Optimization I

  3. Synthesis of RP Packings

  4. RP Column Preparation

  5. Common RP Packings

  6. RP Column Properties • Hydrophobic Surface • Particle Size and Shape • Particle Size Distribution • Porosity, Pore Size and Surface Area

  7. Particle Size • Columns have a distribution of particle sizes • Reported “particle diameter” is an average • Broader distribution ---> broader peaks

  8. Particle SizeDistribution of several column batches Neue, HPLC Columns Theory, Technology and Practice, Wiley, 1997, p.82

  9. RP Mechanism (Simple)

  10. Reversed Phase Mechanisms • Classical measures of retention • capacity factors • partition coefficients • Van’t Hoff Plots • Give bulk properties only - do not give molecular view of separation process

  11. Proposed RP Mechanisms • Hydrophobic Theory • Partition Theory • Adsorption Theory See Journal of Chromatography, volume 656.

  12. Hydrophobic Theory • Chromatography of “cavities” in solvent created by hydrophobic portion of analyte molecule • Surface Tension • Interaction of polar functions with solvent • Stationary phase is passive

  13. Partition Theory • Analyte distributes between aqueous mobile phase and organic stationary phase • Correlation between log P and retention • “organic” phase is attached on one end • Does not explain shape selectivity effects

  14. Adsorption Theory • Analytes “land” on surface - do not penetrate • Non-polar interactions between analyte hydrophobic portion and bonded phase • Weak interactions • dipole-dipole • dipole-induced dipole • induced dipole-induced dipole

  15. None of these can completely explain all of the observed retention in reversed phase HPLC

  16. Important Reversed Phase Parameters • Solvent (mobile phase ) Strength • Choice of Solvent • Mobile Phase pH • Silanol Activity

  17. Solvent Strength • Water is “weak” solvent • Increased organic ---> decreased retention • Organic must be miscible with water

  18. Effect of Solvent

  19. Solvent Strength Snyder and Kirkland, Introduction to Modern Liquid Chromatography, Wiley, 1979, p. 286.

  20. Varying Selectivity 30% MeCN 70% Water 45% MeOH 55% Water 30x0.46 cm C-18, 1.5 mL.min, 254 nm, 10 mg each Snyder and Kirkland, introduction to Modern Liquid Chromatography, Wiley, 1979, p. 287.

  21. pH • Affects ionizable compounds • organic acids • organic bases • In reversed phase we need to suppress ionization as much as possible • May need very precise pH control

  22. pH Effect on Retention 1. Salicylic acid 2. Phenobarbitone 3. Phenacetin 4. Nicotine 5. Methylampohetamine 30x0.4 cm C-18, 10 mm, 2 mL/min, UV 220 nm Snyder and Kirkland, Introduction to Modern Liquid Chromatography, Wiley, 1979, p. 288.

  23. Use of Buffers • 0.1 pH unit ---> significant effect on retention • Buffer mobile phase for pH reproducibility • pH of buffer should be within 1 pH unit of pKa of acid (best at pH = pKa) • Buffers weak (100 mM or less) • Check solubility

  24. Common buffers Useful buffering between pH 2-8.

  25. Silanol Activity • RP ligands occupy about 50% of silanols • Others are “active” • Weak acids

  26. Silica Surface

  27. Dealing with Residual Silanols • Silanols cause peak tailing and excessive retention • Endcapping • bond a smaller group (helps a little) • Pre-treatment of silica • fully hydroxylated best • high purity best

  28. Silanol Interactions • Hydrogen bonding • Dipole-dipole • Ion exchange • Low pH --> silanols protonated • Add basic modifier (TEA) to compete for sties

  29. pH Effect on Tailing Neue, p196

  30. RP Optimization

  31. RP Optimization

More Related