Reversed Phase HPLC Mechanisms
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Transcript of Reversed Phase HPLC Mechanisms
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Reversed Phase HPLC Mechanisms
Nicholas H. SnowDepartment of Chemistry
Seton Hall UniversitySouth Orange, NJ 07079
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Reversed Phase HPLC
• Synthesis of RP Packings• RP Column Properties• RP Retention Mechanisms• Important RP parameters• RP Optimization I
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Synthesis of RP Packings
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RP Column Preparation
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Common RP Packings
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RP Column Properties
• Hydrophobic Surface• Particle Size and Shape• Particle Size Distribution• Porosity, Pore Size and Surface Area
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Particle Size
• Columns have a distribution of particle sizes
• Reported “particle diameter” is an average• Broader distribution ---> broader peaks
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Particle SizeDistribution of several column batches
Neue, HPLC Columns Theory, Technology and Practice, Wiley, 1997, p.82
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RP Mechanism (Simple)
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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
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Proposed RP Mechanisms
• Hydrophobic Theory• Partition Theory• Adsorption Theory
See Journal of Chromatography, volume 656.
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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
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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
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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
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None of these can completely explain all of theobserved retention in reversed phase HPLC
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Important Reversed Phase Parameters
• Solvent (mobile phase ) Strength• Choice of Solvent• Mobile Phase pH• Silanol Activity
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Solvent Strength
• Water is “weak” solvent• Increased organic ---> decreased retention• Organic must be miscible with water
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Effect of Solvent
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Solvent Strength
Snyder and Kirkland, Introduction to Modern Liquid Chromatography, Wiley, 1979, p. 286.
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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
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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
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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.
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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
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Common buffersBuffer pKa Values
Phosphate 2, 7
Acetate 4.75
Citrate 3.08, 4.77, 6.40
Useful buffering between pH 2-8.
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Silanol Activity
• RP ligands occupy about 50% of silanols• Others are “active”• Weak acids
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Silica Surface
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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
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Silanol Interactions
• Hydrogen bonding• Dipole-dipole• Ion exchange• Low pH --> silanols protonated• Add basic modifier (TEA) to compete for
sties
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pH Effect on Tailing
Neue, p196
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RP Optimization
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RP Optimization