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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.

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Reversed Phase HPLC Mechanisms

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  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

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