Chromatography

1. Amandeep Singh
Assistant Professor
Department of Biotechnology
GSSDGS Khalsa College
Patiala

2. Partition/Distribution Coefficient
The way in which a given compound partition/distribute itself between 2 immiscible
phases (Stationary phase & Mobile phase)
K= Cs
Cm
Where K= Partition/distribution Coefficient
Cs= Concentration of substance in stationary phase
Cm= Concentration of substance in mobile phase
If K= 0.2 between cellulose (Stationary phase) and CCL4 (mobile phase), it means
concentration of substance in CCL4 is 5 times higher that in cellulose.
As 1 = 0.2
5

3. Techniques
Plane Chromatography
Stationary phase is coated on plane
surface.
Examples:
1. Paper Chromatography
2. Thin Layer Chromatography (TLC)
Column Chromatography
Stationary phase is Packed in
a column.
Examples:
1. Gas Chromatography
2. Ion-Exchange
Chromatography
3. High Performance Liquid
Chromatography (HPLC)
4. Affinity Chromatography
5. Exclusion Chromatography

4. Thin Layer Chromatography
Preparation of Layer
For Preparation of stationary phase for thin layer chromatography, silica gel or
alumina gel is mixed with water to make slurry. The thickness of the slurry on plane
should be 0.25 mm for analytical preparation and 5 mm for preparative preparations.
Silica gel or Alumina gel + Water = Thick Suspension
(Stationary Phase) (Slurry)
For Analytical Separation For Preparative Separation
0.25 mm 5 mm
Reason for covering with watch glass is to make atmosphere
in beaker saturated with solvent vapour.

5. Rf Value
• Rf value in chromatography is known as Retention value or Retention
factor or Retardation factor.
• The Rf values indicate how soluble the particular pigment is in the solvent
by how high the pigment moves on the paper. Two pigments with the
same Rf value are likely to be identical molecules. Small Rf values tend to
indicate larger, less soluble pigments while the highly soluble pigments
have an Rf value near to one.
• Rf = distance traveled by substance/distance traveled by solvent front
OR
• Rf= distance traveled by solute/distance traveled by solvent
A high Rf (i.e. 0.92) would refer to a substance that is very non-polar. It
means that substance moved a 92% of the entire distance the solvent
traveled.
A low Rf value (0.10) would refer to a substance that is very polar.It means
that substance was only able to move 10% of the entire distance the solvent
traveled.
Rf = Distance travelled by solute
Distance travelled by solvent

6. Calculating the Rf Value

7. Detection
1. By Chemicals: By spraying Ninhydrin (reacts with
amino acids to give purple or brown colour spots)
2. Iodine Crystals (Iodine vapours react with spots of the
sample to give brown colour)
3. By Radiation: By using Ultra-Violet Light on the
spots which will show Fluorescence.
The spots that appeared after the chromatography is over are colourless. To
make them visible, any of the detection techniques listed below can be used:

8. How does TLC works?
• Silica gel is a form of Silicon dioxide (SiO2).
• At surface of silica gel, silicon atoms are attached to –OH groups.
• Surface of silica gel is very polar because of –OH groups and can
form H-bond with suitable compound around it.
Main Body of Silica
- O – Si – O – Si – O – Si – O – Si – O -
OH OH OH OH
O O O O

9. • If a compound/solute to be separated is polar, it can interact with polar –
OH groups of silica. It can either make H-bonds (strong) or it can interact
with VanderWall Interactions (weak) with the silica. If a compound make H
bond with the silica then it will travel less with the solvent (non-polar). On
the other hand if the compound make Vanderwall interactions with silica,
then it can travel far with the solvent.
• If the compound/solute is non-polar, then it can not make any bond with
the silica and it will move along with the solvent.
Because of –OH group on surface of silica, solute
can make either these two types of interactions
H-bonding Vander Wall Interactions
Stick Strongly Stick Loosely
Travel Less distance Travel More distance
How does TLC works?