Chromatography is a laboratory technique used to separate mixtures into individual components. Column chromatography is a type of chromatography that uses a column filled with a solid stationary phase. As a liquid or gas mobile phase passes through the column carrying the sample mixture, the mixture's components separate as they travel through the column at different rates depending on how strongly they interact with the stationary phase. Column chromatography is commonly used to purify compounds by removing impurities based on differences in their adsorption to the stationary phase material in the column.

1. CHROMATOGRAPH
Y
&
COLUMN CHROMATOGRAPHY

2. Chromatography,
The word Chromatography is derived from
two Greek words -
1. chroma which means "color"
1. graphein, which means "to write".
“Color writing”

3. Chromatography
Chromatography is a laboratory separation
technique by which a mixture can be
separated into its individual components

4. TYPES OF CHROMATOGRAPHY
Column chromatography
Paper chromatography
Thin layer chromatography
High performance liquid chromatography
Gas chromatography
Ion exchange chromatography
Gel chromatography
Affinity chromatography

5. Chromatography is a physical method of
separation that distributes components to
separate between two phases, one
stationary (stationary phase), the other
(the mobile phase) moving in a definite
direction.

6. REQUIREMENTS OF CHROMATOGRAPHY
1.Mobile phase (Liquid)
2.Stationary phase
(Liquid/Solid)

7. TECHNIQUES/PRINCIPLES OF CHROMATOGRAPHY
1.Partition chromatography
2.Adsorption chromatography

8. 1.Partition chromatography
The separation of components between two liquid
phases viz original solvent and the film of solvent used
in the column.
The separation of the components from the sample
mixture is carried out by the process of partition of
the components between 2 phases. Both phases are
in liquid form.

9. Partition chromatography
Mobile phase Liquid or Gas
Stationary phase Liquid

10. 2. Adsorption chromatography
Mobile phase Liquid
Stationary phase Solid

11. COLUMN CHROMATOGRAPHY

12. Column chromatography
Column chromatography is a technique which is used to separate
a single chemical compound from a mixture dissolved in a fluid.
It separates substances based on differential adsorption of
compounds to the adsorbent as the compounds move through
the column at different rates which allow them to get separated
in fractions.
This technique can be used on a small scale as well as large
scale to purify materials that can be used in future experiments.
This method is a type of adsorption chromatography technique.

13. Principle
When the mobile phase along with the mixture that needs to be
separated is introduced from the top of the column, the movement
of the individual components of the mixture is at different rates.
The components with lower adsorption and affinity to stationary
phase travel faster when compared to the greater adsorption and
affinity with the stationary phase.
The components that move fast are removed first whereas the
components that move slowly are eluted out last.

15. Procedure
Mobile phase – This phase is made up of solvents and it performs the
following functions:
1. It acts as a solvent – sample mixture can be introduced in the
column.
2. It acts as a developing agent – helps in the separation of
components in the sample to form bands.
3. It acts as an eluting agent – the components that are separated
during the experiment are removed from the column
4. Some examples of solvents used as mobile phase based on their
polarity are – ethanol, acetone, water, acetic acid, pyridine, etc.

16. Stationary phase – It is a solid material which should have good adsorption
property and meet the conditions given below:
1. Shape and size of particle: Particles should have uniform shape and
size in the range of 60 – 200μ in diameter.
2. Stability and inertness of particles: high mechanical stability and
chemically inert. Also, no reaction with acids or bases or any other
solvents used during the experiment.
3. It should be colourless, inexpensive and readily available.
4. Should allow free flow of mobile phase
5. It should be suitable for the separation of mixtures of various
compounds.

17. Applications
● Column Chromatography is used to isolate active
ingredients.
● It is very helpful in Separating compound mixtures.
● It is used to determine drug estimation from drug
formulations
● It is used to remove impurities.
● Used to isolation metabolites from biological fluids.

18. TAPAN KUMAR MAHATO
tapan.mahato@gmail.com