Why compounds Separate from each other in various Chromatography techniques

1. -SUJOY SAHA
BS-MS,
20101095 “Why compounds Separate from each other in Chromatography”
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CHM-332
17-11-2012

2. 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/colorlesscompounds.
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3. 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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4. Basic Principle…
www.m2c3.com/chemistry 4

5. 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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6. 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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7. What are those “Interactive Forces”
Liquid Chromatography
(LC)
(gravity flow)
(adsorption)
(pressure flow)
Gas -solid
Gas -liquid
Column
High performance
Thin layer
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8. Adsorption Chromatography
•Adsorption just on surface
•used mainly in TLC & very small particles in LC
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9. 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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10. 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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11. Molecular Exclusion Chromatography
•Separation based on size
•Small molecules get trapped in pores & take longer to get out
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12. Affinity Chromatography
•Very selective
•Specific binding site is used to concentrate analyte on column
•Used a lot in
biological applications
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13. 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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