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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. - PowerPoint PPT Presentation
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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
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