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

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  m, 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