Transcript High Performance Liquid Chromatography 2

HPLC
Chromatography is used to separate mixtures of
substances into their components. All forms of
chromatography work on the same principle.
They all have a stationary phase (a solid, or a liquid
supported on a solid) and a mobile phase (a liquid or
a gas).
The mobile phase flows through the stationary phase
and carries the components of the mixture with it.
Different components travel at different rates.
TLCThin layer chromatography is done exactly as it says - using a
thin, uniform layer of silica gel or alumina coated onto a piece
of glass, metal or rigid plastic.
The silica gel (or the alumina) is the stationary phase. The
stationary phase for thin layer chromatography also often
contains a substance which fluoresces in UV light - for reasons
you will see later. The mobile phase is a suitable liquid solvent
or mixture of solvents.
The diagram shows the plate after the solvent has moved about half way
up it.
The solvent is allowed to rise until it almost reaches the top of the plate.
That will give the maximum separation of the dye components for this
particular combination of solvent and stationary phase
What separates the
compounds as a
chromatogram develops?
What separates the compounds as a
chromatogram develops?
As the solvent begins to soak up the plate, it first dissolves
the compounds in the spot that you have put on the base
line. The compounds present will then tend to get carried
up the chromatography plate as the solvent continues to
move upwards.
How fast the compounds get carried up the
plate depends on two things:
•How soluble the compound is in the solvent. This will
depend on how much attraction there is between the
molecules of the compound and those of the solvent.
•How much the compound sticks to the stationary phase the silica gel, for example. This will depend on how much
attraction there is between the molecules of the compound
and the silica gel
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY - HPLC
High performance liquid chromatography is basically a
highly improved form of column chromatography. Instead
of a solvent being allowed to drip through a column under
gravity, it is forced through under high pressures of up to
400 atmospheres. That makes it much faster.
Fast Protein Liquid Chromatograph (FPLC)
Injector Module
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Column Inlet
3
Detector
Fraction
Collector
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4
• No air bubbles
(Priming)
• Use degassed buffers
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HPLC is a separation technique that involves:
•the injection of a small volume of liquid sample
•into a tube packed with tiny particles (3 to 5 micron (μm) in
diameter called the stationary phase)
•where individual components of the sample are moved down
the packed tube (column) with a liquid (mobile phase)
forced through the column by high pressure delivered
by a pump.
Definitions
􀂙 Stationary phase
-- common name for the column packing material in any type
of chromatography.
-- those samples which have stronger interactions with the
stationary phase than with the mobile phase will elute from
the column less quickly, and thus have a longer retention
time, while the reverse is also true
􀂙 Mobile phase
-- liquid media that continuously flows through the column
and carries the analytes. a carrier for the sample solution
-- normally use mixtures of solvents as mobile phase.
HPLC system
FOUR TYPES OF LIQUID
CHROMATOGRAPHY
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Partition chromatography
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Adsorption, or liquid-solid chromatography
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Ion exchange chromatography
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Size exclusion, or gel, chromatography
WHAT AFFECTS SYSTEM
Column Parameters
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Column Material
Deactivation
Stationary Phase
Coating Material
Instrument Parameters
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Temperature
Flow
Signal
Sample Sensitivity
Detector
WHAT AFFECTS SYSTEM
Sample Parameters
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Concentration
Matrix
Solvent Effect
Sample Effect
Several column types
(can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
Normal phase
• In this column type, the retention is governed by
the interaction of the polar parts of the
stationary phase and solute. For retention to
occur in normal phase, the packing must be
more polar than the mobile phase with respect
to the sample
Reverse phase
• In this column the packing material is relatively
nonpolar and the 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.
Typical stationary phases are nonpolar
hydrocarbons, waxy liquids, or bonded
hydrocarbons (such as C18, C8, etc.) and the
solvents are polar aqueous-organic mixtures
such as methanol-water or acetonitrile-water.
Size exclusion
• In size exclusion the HPLC column is consisted
of substances which have controlled pore sizes
and is able to be filtered in an ordinarily phase
according to its molecular size. Small molecules
penetrate into the pores within the packing while
larger molecules only partially penetrate the
pores. The large molecules elute before the
smaller molecules.
Ion exchange
• In this column type the sample components are
separated based upon attractive ionic forces
between molecules carrying charged groups of
opposite charge to those charges on the
stationary phase. Separations are made between
a polar mobile liquid, usually water containing
salts or small amounts of alcohols, and a
stationary phase containing either acidic or basic
fixed sites.
Types of Detectors
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Absorbance (UV with Filters,
UV with Monochromators)
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IR Absorbance
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Electrochemical
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Fluorescence
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Mass-Spectrometric
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Refractive-Index
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Evaporative Light Scattering
Detector (ELSD)
Photo-Diode Array