- SUJOY SAHA

BS-MS,

20101095

“Why compounds Separate from each other in Chromatography”

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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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Basic Principle…

www.m2c3.com/chemistry 4

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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