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
Vant 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 g 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 m, 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