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Reversed Phase HPLC Mechanisms Nicholas H. Snow Department of Chemistry Seton Hall University South Orange, NJ 07079 [email protected] Reversed Phase HPLC • • • • • Synthesis of RP Packings RP Column Properties RP Retention Mechanisms Important RP parameters RP Optimization I Synthesis of RP Packings RP Column Preparation Common RP Packings RP Column Properties • • • • Hydrophobic Surface Particle Size and Shape Particle Size Distribution Porosity, Pore Size and Surface Area Particle Size • Columns have a distribution of particle sizes • Reported “particle diameter” is an average • Broader distribution ---> broader peaks Particle Size Distribution of several column batches Neue, HPLC Columns Theory, Technology and Practice, Wiley, 1997, p.82 RP Mechanism (Simple) 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 Proposed RP Mechanisms • Hydrophobic Theory • Partition Theory • Adsorption Theory See Journal of Chromatography, volume 656. 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 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 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 None of these can completely explain all of the observed retention in reversed phase HPLC Important Reversed Phase Parameters • • • • Solvent (mobile phase ) Strength Choice of Solvent Mobile Phase pH Silanol Activity Solvent Strength • Water is “weak” solvent • Increased organic ---> decreased retention • Organic must be miscible with water Effect of Solvent Solvent Strength Snyder and Kirkland, Introduction to Modern Liquid Chromatography, Wiley, 1979, p. 286. Varying Selectivity 30% MeCN 45% MeOH 70% Water 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. 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 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. 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 Common buffers Buffer pKa Values Phosphate 2, 7 Acetate 4.75 Citrate 3.08, 4.77, 6.40 Useful buffering between pH 2-8. Silanol Activity • RP ligands occupy about 50% of silanols • Others are “active” • Weak acids Silica Surface 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 Silanol Interactions • • • • • Hydrogen bonding Dipole-dipole Ion exchange Low pH --> silanols protonated Add basic modifier (TEA) to compete for sties pH Effect on Tailing Neue, p196 RP Optimization RP Optimization
