Reversed Phase HPLC Mechanisms

Nicholas H. SnowDepartment of Chemistry

Seton Hall UniversitySouth 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 SizeDistribution 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 theobserved 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

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

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 Retention1. 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 ModernLiquid 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 buffersBuffer 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