HPLC High Performance/Pressure Liquid Chromatography

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HPLC

• High Performance/Pressure
• Liquid Chromatography

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Advanced Instrumentation
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The System
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Columns
http//www.waters.com/watersdivision/images/resour
ces/ColumnCons.gif
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http//www.esainc.com/images/Cap_cell/HPLC_Col_Sel
.jpg
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Reversed Phase Chromatography

• Packing is nonpolar
• Solvent is polar with respect to the sample.
• Retention is the result of the interaction of the
  nonpolar components of the solutes and the
  nonpolar stationary phase - hydrophobic.

http//www.biotech.iastate.edu/facilities/protein/
hplc.html
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Reversed Phase Chromatography - 2

• Bonded phases made by covalently bonding a
  molecule onto a solid stationary phase like
  silica
• Typical stationary phases are nonpolar
  hydrocarbons, waxy liquids or bonded hydrocarbons
  (such as C18, C8, C4, etc.)
• pH range 2.5 to 7.5 ONLY (or the column degrades)
  
• 60-90 of all analytical LC separations are done
  on bonded phases in the reverse phase mode.

http//www.biotech.iastate.edu/facilities/protein/
hplc.html
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Reversed Phase Chromatography - 3

• Solvents are polar aqueous-organic mixtures such
  as methanol-water or acetonitrile-water.
• Elution can be either
• Isocratic use only one buffer
• Gradient mix in a second buffer

Overlay of the four components trace analysis
chromatograms. (A) is the isocratic elution, (B)
is the gradient elution, shadow line is the
gradient profile from 30 acetonitrile in water
to 65 acetonitrile.
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Capacity

• Analytical ng down to fg
• Semi-prep mg to ug
• Preparative g
• Industrial kg

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Solvents

• Clean
• HPLC grade
• Filtered
• Degassed
• Water
• Polar
• Methanol or
• Acetonitrile
• Maintain pH
• TFA

http//www2.okbu.edu/academics/natsci/chemistry/pe
ople/mrjordan/webtutorials/hplc/hplc.html
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Tubing

• Very small inner diameter
• Consistent i.d.
• Very strong
• Easy to cut
• Fittings available

http//www.microsolvtech.com/catalog/index.asp?pgN
ame19
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Injector
http//www2.okbu.edu/academics/natsci/chemistry/pe
ople/mrjordan/webtutorials/hplc/hplc.html
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Pumps
http//eu.shimadzu.de/products/analytics/chromato/
hplc/lcmodular/pumps/lc10advp.jpg
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Pump schematic
http//hplc.chem.shu.edu/NEW/HPLC_Book/Instrumenta
tion/pump_hed.gif
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Pump cycle
http//hplc.chem.shu.edu/NEW/HPLC_Book/Instrumenta
tion/pump_hed.gif
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Detector
http//www.flowspek.ch/images/k-2600.jpg
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Detector schematic
http//www.oup.com/images/booksites/higson/higson_
fig8.18.jpg
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Fraction collector
http//www.lplc.com/old/instruments/img/cf1big.jpg
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Recorder
http//www2.okbu.edu/academics/natsci/chemistry/pe
ople/mrjordan/webtutorials/hplc/images/recorder.JP
G
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Direct to computer
http//www.unimicrotech.com.cn/images/HPLC-compute
r-007.jpg
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HPLC Data
http//www.mac-mod.com/tr/plumbing-tr.html
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HPLC Data
http//caffeineawareness.org/index.php
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HPLC Data Analysis
Next lecture!