Reversed Phase HPLC Mechanisms Nicholas H Snow Department

  • Slides: 31
Download presentation
Reversed Phase HPLC Mechanisms Nicholas H. Snow Department of Chemistry Seton Hall University South

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

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

Synthesis of RP Packings

Synthesis of RP Packings

RP Column Preparation

RP Column Preparation

Common RP Packings

Common RP Packings

RP Column Properties • • Hydrophobic Surface Particle Size and Shape Particle Size Distribution

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”

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,

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

RP Mechanism (Simple)

RP Mechanism (Simple)

Reversed Phase Mechanisms • Classical measures of retention – capacity factors – partition coefficients

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

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

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 •

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

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

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

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 •

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

Effect of Solvent

Effect of Solvent

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

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

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

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

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

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.

Common buffers Useful buffering between p. H 2 -8.

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

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

Silica Surface

Silica Surface

Dealing with Residual Silanols • Silanols cause peak tailing and excessive retention • Endcapping

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 -->

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

p. H Effect on Tailing Neue, p 196

RP Optimization

RP Optimization

RP Optimization

RP Optimization