Reversed Phase HPLC Mechanisms

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Reversed Phase HPLC Mechanisms

• Nicholas H. Snow
• Department of Chemistry
• Seton Hall University
• South Orange, NJ 07079
• snownich_at_shu.edu

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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 ---gt 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
• Vant 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
the observed 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 ---gt 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
30 MeCN 70 Water
45 MeOH 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.
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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 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.
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Use of Buffers

• 0.1 pH unit ---gt 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 buffers
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 --gt 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