Reversed Phase HPLC Mechanisms Nicholas H Snow Department
- Slides: 31
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
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 p. H 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% Me. CN 45% Me. OH 70% Water 55% Water 30 x 0. 46 cm C-18, 1. 5 m. L. min, 254 nm, 10 mg each Snyder and Kirkland, introduction to Modern Liquid Chromatography, Wiley, 1979, p. 287.
p. H • Affects ionizable compounds – organic acids – organic bases • In reversed phase we need to suppress ionization as much as possible • May need very precise p. H control
p. H Effect on Retention 1. Salicylic acid 2. Phenobarbitone 3. Phenacetin 4. Nicotine 5. Methylampohetamine 30 x 0. 4 cm C-18, 10 mm, 2 m. L/min, UV 220 nm Snyder and Kirkland, Introduction to Modern Liquid Chromatography, Wiley, 1979, p. 288.
Use of Buffers • 0. 1 p. H unit ---> significant effect on retention • Buffer mobile phase for p. H reproducibility • p. H of buffer should be within 1 p. H unit of p. Ka of acid (best at p. H = p. Ka) • Buffers weak (100 m. M or less) • Check solubility
Common buffers Useful buffering between p. H 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 p. H --> silanols protonated Add basic modifier (TEA) to compete for sties
p. H Effect on Tailing Neue, p 196
RP Optimization
RP Optimization
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