Analytical Chemistry

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Analytical Chemistry
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Chapter 25High Performance Liquid
Chromatography (HPLC)
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HPLC Instrument
Mobile Phase liquid (aqueous or organic solvent)
Stationary Phase solid or liquid (bonded
stationary phase)
Analyte semivolatile or nonvolatile compounds
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HPLC Column
System requires high pressure!
100 to 400 atm
Several types of column/solvent combinations are
used.
1) Normal Phase
Polar stationary phase/nonpolar mobile phase
2) Reverse Phase
Nonpolar stationary phase/polar mobile phae
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Stationary Phase
Microporous Silica Particles
Sponge-like Structure
Aggregate of Particles
H-Bonded Vicinal Silanol Groups
Isolated Silanol Groups
Particles are permeable to solvent.
102 m2/g of particles
Only use for pH lt 8.0
Use polystyrene particles for pH 8-12
Geminal Silanol Groups
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Bonded Stationary Phase
Surface Silanol Group
Surface Bonded Functional Group
Common Functional Groups
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Elution Process
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Elution Process
Eluent Strength is a measure of the solvent
adsorption energy
Adsorption of pentane on bare silica is defined
as a value of zero.
Elutropic Series rank solvents by their
relative abilities to displace solutes from a
given adsorbent.
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Isocratic v.s Gradient Elution
Isocratic elution elution is performed with a
single solvent.
Gradient elution elution is performed by
gradually changing the solvent composition.
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Gradient vs. Isocratic
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UV-Vis Detection
Important to choose solvent that does not
significantly absorb at the detection wavelength.
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HPLC Injection Loop
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Selecting Separation Mode