Chromatography
Chapter 4-5
1Dr Gihan Gawish
Dr Gihan Gawish2
Dr Gihan Gawish
(HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry to:
separate, identify, and quantify compounds.
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HPLC
HPLC Instrumentation (Schematic diagram)
Dr Gihan Gawish
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Liquid Mobile Phase
Pump
Injection Valve
Separation Column
Detector
Dr Gihan Gawish
Solvent Reservoirs can be single solvent or multi-solvents
The choice of solvents, additives and gradient depend on: the nature of the stationary phase and the analyte
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HPLC- Mobil phase
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Requirements for HPLC• apply high pressure to force liquid through
the beads faster• pressures to 6000 psi• control flow rate from 0.1 to 10 mL/min
Types of HPLC pumps Reciprocating pumps: most commercial
systems arebased on this design. Syringe pumps
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HPLC- Pumps
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Generally stainless steel and teflon components.
The stationary phase packings are microporous silica 2-10 μm in diameter.
Unmodified silica is very polar.
Some systems use Precolumns to remove impurities from solvent or
sample
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HPLC- Analytical Columns
UV Single wavelength (filter) Variable wavelength (monochromator) Multiple wavelengths (PDA)
Fluorescence Electrochemical Mass Spectrometric
HPLC- Detectors
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The sample to be analyzed is introduced in small volume to the stream of
mobile phase.
The analyte's motion through the column is slowed by specific chemical
or physical interactions with the stationary phase as it traverses the length
of the column.
The amount of retardation depends on the nature of the analyte,
stationary phase and mobile phase composition.
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HPLC- AnalysisAnimation
Retention time varies depending on the interactions between the stationary
phase, the molecules being analyzed, and the solvent (s) used.
Retention time
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Time at which a specific analyte elutes (comes out of the end of the
column) is called the retention time
Retention time under particular conditions is considered a
reasonably unique identifying characteristic of a given analyte.
Dr Gihan Gawish
organic molecules Biomolecules Ions polymers
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HPLC- Analyte
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HPLC results in high resolution (sharp peaks), and rapid separation (minutes to 1 hour).
HPLC can be analytical or preparative.
HPLC can be used for all types of chromatography: size exclusion, ion exchange, reversed phase, and affinity chromatography.
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HPLC- Advantage
Types of HPLC1. Normal phase chromatography
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(NP-HPLC), this method separates analytes based on polarity
NP-HPLC uses a polar stationary phase and a non-polar mobile phase
Adsorption strengths increase with increased analyte polarity, and the interaction between the polar analyte and the polar stationary phase (relative to the mobile phase) increases the elution time.
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Types of HPLC 2. Reverse Phase Chromatography
Dr Gihan Gawish
Reversed phase HPLC (RP-HPLC or RPC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase.
One common stationary phase is a silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group
With these stationary phases, retention time is longer for molecules which are more non-polar
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Practical considerations
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Not all proteins can withstand the pressure of HPLC
All materials must be of the highest quality.
Solvents must be degassed to eliminate formation of
bubbles.
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