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- SUJOY SAHA
BS-MS,
20101095
“Why compounds Separate from each other in Chromatography”
1
CHM- 332
17-11-2012
Chromatography-
is a technique used to separate and identify the components of a mixture.
Which means ...
The physical separation of a mixture into its individual components.
Initially used for separation of colored substances from plants is now the most extensive technique of separation and purification of colored/colorless compounds.
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Chromatography
• The separation of a mixture by distribution of its components between a mobile and stationary phase over time.
- mobile phase = solvent
- stationary phase = column packing material
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Simply…
Chromatography separates the components of a
mixture by their distinctive “interactive force”
to the mobile phase and the stationary phase.
Compound is placed on stationary phase
Mobile phase passes through the stationary phase
Mobile phase solubilizes the components
Mobile phase carries individual components a certain distance through stationary phase, depending on their affinity to both of the phases
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Why do they separate?
• The differential rates of migration as the
mixture moves over adsorptive materials
provide separation.
• Repeated sorption/ desorption acts that take
place during the movement of the sample over
the stationary bed determine the rates.
• The smaller the affinity a molecule has for the
stationary phase, the shorter the time spent in
a column.
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What are those “Interactive Forces”
Liquid Chromatogr
aphy (LC)
(gravity flow)
(adsorption)
(pressure flow)
Gas - solid
Gas - liquid
Column
High performance
Thin layer
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Adsorption Chromatography
• Adsorption just on
surface
• used mainly in TLC &
very small particles in
LC
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Partition Chromatography
• Used in GC & LC
• Molecules will partition into stationary phase based upon affinity for stationary phase & eventually partition into mobile phase again
• Thin layer is coated onto inside of GC column or on small particles on LC column
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Ion Exchange Chromatography
• Separation of either
cations or anions
• Separation based on
relative strength of ionic
bond
• Anion exchange has
cations on surface
• Used in LC exclusively
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Molecular Exclusion Chromatography
• Separation based on size
• Small molecules get
trapped in pores & take
longer to get out
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Affinity Chromatography
• Very selective
• Specific binding site is
used to concentrate
analyte on column
• Used a lot in
biological applications
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Gel Electrophoresis
• Separation based on size
and charge
• Smaller molecules will
migrate further, less
tangled
• Used a lot in
biological applications
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