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Quantitative Chemical Analysis 7e

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the elution solvent. 33. Solvent nomograph showing equivalent solvent ... Gradient Elution. Gradient Elution is the systematic variation 2 or more solvents during HPLC ... – PowerPoint PPT presentation

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Title: Quantitative Chemical Analysis 7e


1
Concentric Type Microdialysis Probe
2
Typical Side by Side Probe
3
Typical 1980s-90s HPLC
4
Particle Size and Solvent Effects
5
Particle Size and Plate Height
6
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7
HPLC Column Construction
8
Silica Packing Beads
9
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10
  • Normal Phase HPLC
  • Bare Silica or Polar Stationary Phase
  • non-polar mobile phase
  • Reverse Phase HPLC
  • Non-polar Stationary Phase
  • Polar mobile phase

11
Bonded Phase Coated Silica Chemistry
Bonded phase silica can be hydrolyzed at
pHslt2. Unprotected silica dissolves at pHs
above 8.
12
Bidentate Protection of Functionalized Silica
13
Hydrolysis Protection of Bonded Phase
14
Monolithic Silica Columns
15
2 micron pore network in monolithic column
16
Monolithic Column Surface has 13 nm pores
17
  • 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

18
Schematic of Dual Piston HPLC pump
19
Typical HPLC injection Port Flow Diagram
20
Flow Cell for UV-Vis Detection
21
Diode Array UV-VIS Detector
22
Anthracene detected at 250 nm with Diode Array
23
Spectral Identification of Peaks with D.A.
24
Refractive Index Detector
25
Evaporative Light-Scattering Detector (requires
non-linear calibration)
26
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28
HPLC Column Selection Flow Chart 1
29
HPLC Column Selection Flow Chart 2
30
  • 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

31
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32
Sample solvent can affect peak shape if it is
different from the elution solvent
33
Solvent nomograph showing equivalent solvent
strengths for three solvents
34
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35
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

36
Isocratic Elution Chromatograms for C18 Separation
37
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44
  • If Dt / tg gt 0.25, use a gradient method
  • If Dt / tg lt 0.25, use an isocratic method

45
System Dwell Time
46
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47
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