Reversed Phase HPLC
Mechanisms
Nicholas H. Snow
Department of Chemistry
Seton Hall University
South Orange, NJ 07079
snownich@shu.edu
Reversed Phase HPLC
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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
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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
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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
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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