This document provides information on different types of chromatography techniques. It discusses planar chromatography, which uses a stationary phase on a plane like paper or glass. Paper chromatography uses cellulose paper as the stationary phase while thin layer chromatography uses silica gel. The document describes the process of each technique and how the mobile phase separates the components of a sample on the stationary phase. It compares the benefits of thin layer chromatography to paper chromatography and provides guidance on performing thin layer chromatography experiments.

1. 1

2. Classification of Chromatography
Chromatography is generally classified on different basis, but the
most popular is on the shape of the
stationary phase.
1. Planar Chromatography.
2. Column Chromatography.
Planar Chromatography is further
divided into
Paper Chromatography and
Thin layer Chromatography. 2

3. Planar Chromatography
Planar Chromatography is a technique in which stationary
phase is present on a plane; which can be paper or a glass of
variant size.
If the plane used is a paper i.e. Cellulose then it is called Paper
Chromatography, where as if glass is used then it is called
Thin Layer Chromatography, popularly TLC. In TLC the
stationary phase is generally Silica gel G.
G in silica gel G stands for Gypsum.
3

4. This Chromatography involves placing a small drop of sample
solution on a paper, the paper serves as a stationary phase.
The paper is placed in a jar containing
small layer of mobile phase at the bottom.
The mobile phase slowly travels along the
paper and carries the sample in the direction
and thus separates components
from the mixture.
Further this chromatography technique
is subdivided into 3 types.
Paper Chromatography
4

5. 5

6. This is a sub type of paper chromatography, where a paper in
a square shape is kept on a paltry dish filled with mobile phase
which travels through the paper from the centre to the
periphery of the circular paper.
A wick made from the same paper is used for the
displacement of mobile phase from
reservoir to paper.
Wick
Circular Chromatography
6

7. In ascending paper chromatography a paper is kept hanging from
a support into a jar filled with mobile phase such that the end of
paper just touches the mobile phase.
The mobile phase runs upward carrying with it the sample
up till a particular height.
All the components in a mixture gets
separated depending upon their polarity.
Analytes are
Separated
According
To polarity Mobile
phase
Ascending Chromatography
7

8. A process with same application as ascending
chromatography is demonstrated here only
with a change in flow direction of mobile
Phase.
Here the reservoir is placed at the top of the
jar and the mobile phase flows upside down.
The sample here gets separate at the bottom of the paper.
In all types of paper chromatography the sample that has
more attraction towards Cellulose takes more time to separate.
Descending Chromatography
8

9. Rf values : Retardation Factor
Calculating the Rf value
V a l u eR
D i s t a n c eF r o n tS o l v e n t
T r a v e l e dS p o tt h a t t h eD i s t a n c e
f=
9

10. Calculate Rf Value
10

11. – The Rf value needs to be between 0.0 and 1.0
• If the value is over 1.0 or less than 0.0, the
calculation is wrong
• If the Rf value is greater than 0.8 or lower
than 0.2 the values are hard to interpret,
thus creating a larger error
• The best Rf values are 0.3 to 0.6
Rf Value
11

12. Rf Value
• What affects the Rf value?
– Temperature
– Solvent
– Thickness of spot
– Concentration of the sample
– impurities in sample
– Type of Paper (material, porosity, etc.)
12

13. Compare to paper chromatography, Thin Layer Chromatography
is more beneficial.
In TLC the mobile phase runs faster than
Paper.
Here the choice of using variety of mobile
phase is easy as silica gel is more inert
than cellulose.
Separation is more better than
Paper chromatography.
Analytes are
Separated
According
To polarity
Mobile
phase
Comparing TLC with Paper
Chromatography
13

14. Component
s
Affinity to Stationary
Phase
Affinity to Mobile
Phase
Blue ---------------- Insoluble in Mobile Phase
Black        
Red       
Yellow          
Mixture Components
Separation
Stationary Phase
Mobile Phase
Similar concept for TLC
14

15. Preparing A TLC???
•Mix Water or suitable solvent to the
absorbent.
•Spread a thin layer of absorbent on an
unreactive hard surface (Glass, plastic,
thick aluminium)
•Heat in oven at 110°C for 30 mins to
activate and dry the plate
•Place a small amount of solvent in a
beaker
•Place a drop of sample solution on the
line 15

16. SolventsSolvents
• Choose a solvent depending on the
polarity of the compound
• Least Polar
• More polar
Petroleum ether
Cyclohexane
Toluene
Chloroform
Acetone
Ethanol
Methanol
16

17. VisualizationVisualization
• Destructive visualization
– Spray plate with H2SO4, and then bake in the oven at
110ºC for 15-20 minutes. Compound is destroyed but all
spots will be visible
• Non-destructive visualization – because of the use
of a UV light the sample will not be destroyed.
Although, not all of the spots on the plate will be
visible.
– Long wave UV
– Short wave UV
– Semi-destructive visualization
17

18. Chromatography is an infinite techno……………………….
Column
Planar
Chromatography
HPLC
UPLC
Paper TLC
GC 18

19. Books for Chromatography
Introduction to modern liquid chromatography
by Llyod R Synder
Principles and Practice of Chromatography
by Raymond P. W. Scott,
Useful e-links
biopharminternational.findpharma.com/...Basics...Chromatography/..
www.youtube.com/watch?v=g3m2OgXmnYM
19

20. Experiment to Separate Silver, Mercury and mixture!!!
Take the solution containing the mixture of these
two metals in form of their corresponding compounds.
Drop a spot on the paper and keep into the mobile
phase and run the water for the given time.
Dry it and then spry with dichromate solution
to make the spots appear clearly.
The three spots will be observed corresponding to the metals….
Calculate the retardation factor value for each…….. 20