Chromatography is a technique used to separate mixtures into individual components. It involves a stationary phase and a mobile phase. Chromatography was first developed in 1900 by Mikhail Tswet to separate plant pigments. There are various types of chromatography based on the phase used, including thin layer chromatography which uses a solid stationary phase and liquid mobile phase. Chromatography has many applications, such as purifying compounds and determining chemical composition.

1. CHROMATOGRAPHY
PRAGATI SINHA
DIPSAR, NEW DELHI

2. CHROMATOGRAPHY
It is Derived from the Greek word Chroma meaning
colour, chromatography provides a way to identify
unknown compounds and separate mixtures a way to
identify unknown compounds and separate mixtures.
Chroma = colour
Graphein = to write
It is a collective term for a set of laboratory technique for
the separation of mixture.

3. CHROMATOGRAPHY
 It was first employed by Mikhail Tswet
(Russian scientist) in 1900. He was worked
primarily for the separation of plant pigments
such as chlorophyll, carotenes. Since, these
components have different colour ( green, orange
respectively) they gave the technique its name.
 Chromatography is the separation of a mixture
into individual components by using some
technique.

4. CHROMATOGRAPH
Y
 It is usually consists of Mobile phase and
Stationary phase.
The mobile phase refer to the mixture of
substances to be dissolve in a liquid or a gas.
The stationary phase is a porous solid matrix
through which the sample contained in the
mobile phase percolates.
The interaction between the mobile phase
and the stationary phase results in the
separation of the compound from the
mixture.

5. CLASSIFICATION OF
CHROMATOGRAPHY
Based upon the nature
of stationary and
mobile phase
Solid
Liquid
Gas
Liquid
Liquid
Gas
Solid liquid chromatography such as HPLC, TLC, Column chromatography
• Solid Gas chromatography such as Gas Solid chromatography
Liquid Liquid chromatography such as Paper partition chromatography
• Liquid Gas chromatography such as Gas liquid chromatography

6. CLASSIFICATION OF
CHROMATOGRAPHY
 Based on the principle of separation
 Stationary phase ( Solid , Liquid)
 Solid stationary phase
 Principle - Absorption chromatography
When the mobile phase containing the dissolved solute
passes over the surface of solid stationary phase
 Then components of mobile phase retained and absorbs
temporary by means of electrostatic forces
 Liquid stationary phase
 Principle- Partition chromatography
When mobile phase containing the solutes passes over the
liquid stationary phase
Then components of mobile phase retained and separated by
the partition coefficient

7. CLASSIFICATION OF
CHROMATOGRAPHY
 Based on Mode of chromatography
A. Normal Phase Chromatography (not
widely used)
Stationary phase – Polar ( Silica gel)
Mobile phase- Non polar
B. Reverse Phase Chromatography
Stationary phase – Non polar ( ODS )
Mobile phase- Polar

8. CLASSIFICATION OF
CHROMATOGRAPHY
 Based on Shape of chromatography
 Planer chromatography
 Column chromatography
 Other types
 Ion exchange chromatography
Stationary phase is Polymeric matrix
 Gel Permeation chromatography (Gel filtration chromatography or
Size exclusion chromatography)
Stationary phase is Porous matrix ( like Cross linked polystyrene,
Agarose Gel )
 Chiral chromatography
 Fast Protein liquid chromatography
 Super critical fluid chromatography

9. Cont...
 Basic Practical Requirement
 Stationary phase
 Mobile phase
 Preparation and activation method
 Development Technique
 Detecting or Visualising agent
 Recorder
Calculation of Rf value (Retardation Factor)
Rf value is calculated for identification the spot (range from
0 to 1)
Rf = Distance travelled by Solute
Distance travelled by Solvent

10. Cont...
Visualisation of spot
Physical method – UV Lamp, Fluorescence Technique
Chemical method – By visualising agent
Compound Reagent Colour
General Iodine vapour Brown
Acids Bromocresol green Yellow
Aldehydes & Ketone 2,4 dinitrophenyl
hydrazine
Yellowish red
Amino acid Ninhydrin Purple
Alkaloid Mercuric nitrate Yellowish brown
Carbohydrate Aniline phthalate Gray black
Lipids Bromothymol blue Light green
Steroid Antimony trichloride Purple
Phenol & Phenolic
compound
Fecl3 Purple

11. APPLICATION OF
CHROMATOGRAPHY
 Separation of mixture of compound
 Purification process
 Purification of photochemical
 Isolation of metabolites
 Estimation of drugs
 Process development
 Purify natural product
 To separate active compound from plant material
 Determine the chemical composition of a sample
 Quantitative and Qualitative analysis of component
 Used in food and beverage industries

12. MCQ
 Chromatography is a physical method that is
used to separate and analyze __________
a) Simple mixtures
b) Complex mixtures
c) Viscous mixtures
d) Metals
 Thin layer chromatography is _____________
chromatography.
a) Partition
b) Absorption
c) Ion exchange
d) Gel permeation

13. MCQ
 In chromatography, which of the following can the
mobile phase be made of?
a) Solid or liquid
b) Liquid or gas
c) Gas only
d) Liquid only
 What is the maximum Rf value for any compound in
Paper chromatography?
a) 2
b) 0.6
c) 1
d) 1.5

14. MCQ
 In Thin layer chromatography, the stationary phase is
made of _________ and the mobile phase is made of
_________
a) Solid, liquid
b) Liquid, liquid
c) Liquid, gas
d) Solid, gas
 In Gas-liquid phase chromatography, the stationary phase
is composed of _________ and the mobile phase is made of
_________
a) Solid, liquid
b) Liquid, liquid
c) Liquid, gas
d) Solid, gas

15. MCQ
 Chromatography also called __________
a) Graph writing
b) Page writing
c) Color writing
d) All of these
 In which chromatography stationary phase more polar
than mobile phase?
a) Reverse phase chromatography
b) Gas chromatography
c) Ion exchange chromatography
d) Radial chromatography