Chromatography separates the components of a mixture through differential interactions between the components and both a mobile phase and a stationary phase. Components migrate through the stationary phase at different rates depending on their varying levels of affinity or "interactive force" for each phase. This differential migration over time allows the components to be separated from one another as the mixture moves through the column. The primary interactive forces that cause separation include adsorption, partition, ion exchange, molecular size exclusion, affinity, and electrophoretic mobility.

1. -SUJOY SAHA
BS-MS,
20101095 “Why compounds Separate from each other in Chromatography”
1
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.
2

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
3

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
5

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.
6

7. What are those “Interactive Forces”
Liquid Chromatography
(LC)
(gravity flow)
(adsorption)
(pressure flow)
Gas -solid
Gas -liquid
Column
High performance
Thin layer
7

8. Adsorption Chromatography
•Adsorption just on surface
•used mainly in TLC & very small particles in LC
8

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
9

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
10

11. Molecular Exclusion Chromatography
•Separation based on size
•Small molecules get trapped in pores & take longer to get out
11

12. Affinity Chromatography
•Very selective
•Specific binding site is used to concentrate analyte on column
•Used a lot in
biological applications
12

13. Gel Electrophoresis
•Separation based on size and charge
•Smaller molecules will migrate further, less tangled
•Used a lot in
biological applications
13

14. 14