Quantitative Chemical Analysis 7e

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Concentric Type Microdialysis Probe
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Typical Side by Side Probe
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Typical 1980s-90s HPLC
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Particle Size and Solvent Effects
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Particle Size and Plate Height
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HPLC Column Construction
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Silica Packing Beads
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• Normal Phase HPLC
• Bare Silica or Polar Stationary Phase
• non-polar mobile phase
• Reverse Phase HPLC
• Non-polar Stationary Phase
• Polar mobile phase

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Bonded Phase Coated Silica Chemistry
Bonded phase silica can be hydrolyzed at
pHslt2. Unprotected silica dissolves at pHs
above 8.
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Bidentate Protection of Functionalized Silica
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Hydrolysis Protection of Bonded Phase
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Monolithic Silica Columns
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2 micron pore network in monolithic column
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Monolithic Column Surface has 13 nm pores
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• If open tubes work so well in GC, why dont we
  use open tubes in HPLC and get rid of the A
  term in the van Deemter Plot

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Schematic of Dual Piston HPLC pump
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Typical HPLC injection Port Flow Diagram
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Flow Cell for UV-Vis Detection
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Diode Array UV-VIS Detector
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Anthracene detected at 250 nm with Diode Array
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Spectral Identification of Peaks with D.A.
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Refractive Index Detector
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Evaporative Light-Scattering Detector (requires
non-linear calibration)
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HPLC Column Selection Flow Chart 1
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HPLC Column Selection Flow Chart 2
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• Selectivity in HPLC is greatly affected by choice
  of mobile phase
• Strong mobile phases are similar to the
  stationary phase
• Stronger mobile phases result in less retention
  by stationary phase
• Mobile phase strength is different in Normal and
  Reversed phase HPLC

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Sample solvent can affect peak shape if it is
different from the elution solvent
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Solvent nomograph showing equivalent solvent
strengths for three solvents
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Gradient Elution

• Gradient Elution is the systematic variation 2 or
  more solvents during HPLC chromatography
• Gradient mobile phases are used in a manner
  similar to temperature (or pressure) programming
  in GC
• A is the typical designation for water and B
  is the typical designation for organic solvents

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Isocratic Elution Chromatograms for C18 Separation
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• If Dt / tg gt 0.25, use a gradient method
• If Dt / tg lt 0.25, use an isocratic method

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System Dwell Time
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