CHROMATOGRAPHY IN DETAIL

1. Introduction and definition of
chromatography and paper
chromatography

2. Chromatography:-
• Chromatography was first devised in Russia by the Italian-born scientist
Mikhail Tsvet in 1900. In the first decade of the 20th century, primarily
for the separation of plant pigments.
• Chromatography technique developed substantially as a result of the
work of Archer John Porter Martin and Richard Laurence Millington
Synge during the 1940s and 1950s, for which they won the 1952
Nobel Prize in Chemistry

3. Definition :-
Chromatography is a process for separating components of a mixture. ...
The different components of the mixture travel through the stationary phase
at different speeds, causing them to separate from oneanother.
As a result, he named the technique “chromatography”; “chroma” from the
Greek word for “color”, combined with “graphy”, meaning writing.
In all chromatography there is a mobile phase and a stationary phase. The
stationary phase is the phase that doesn't move and the mobile phase is the
phase that does move. The mobile phase moves through the stationary
phase picking up the compounds to be tested.

4. Paper chromatography
• The discovery of paper chromatography in 1943 by Martin and
Synge provided, for the first time, the means of surveying
constituents of plants and for their separation and identification.
There was an explosion of activity in this field after 1945.
• Paper chromatography, in analytical chemistry, technique for
separating dissolved chemical substances by taking advantage of
their different rates of migration across sheets of paper. It is an
inexpensive but powerful analytical tool that requires very small
quantities of material.

5. Principal of paper chromatography
The principle involved can be partition chromatography or
adsorption chromatography. Partition chromatography
because the substances are partitioned or distributed between
liquid phases. The two phases are water held in pores of the
filter paper and the other phase is a mobile phase which
passes through thepaper.

6. 1-Paper partition chromatography
The principle involved is partition chromatography wherein
the substances are distributed or partitioned between liquid
phases. One phase is the water, which is held in the pores of
the filter paper used; and other is the mobile phase which
moves over the paper. The compounds in the mixture get
separated due to differences in their affinity towards water
(in stationary phase) and mobile phase solvents during the
movement of mobile phase under the capillary action of
pores in thepaper.

7. 2-Paper absorption
Chromatography
The principle can also be adsorption chromatography
between solid and liquid phases, wherein the stationary
phase is the solid surface of the paper and the liquid phase is
of the mobile phase.
But most of the applications of paper chromatography work
on the principle of partition chromatography, i.e., partitioned
between to liquid phases.

8. Application of paper chromatography
• Separating coloredpigments
•Reaction Monitoring
•Isolation and Purification
•Pathology and Forensic Science
• Foods

9. • Analyzing complex mixtures
• Qualitative analysis
Continue…

10. phases of chromatography :
1. Mobile phase:
 The mobile phase is The phase which flow from chromatographic mediaum
Generally the mixture to be separated is desolve in a mobile phase. Also itpasses
through the structure of chromatography Holding the material is called stationary
phase. Differential migrationis the Principal of chromatography.Different
components Of mixture treval at different speed based on the differential
interaction to ward mobile phase or stationary phase.

11.  The mobile phase Chromatography Can be either liquid or gas. For instance Liquid
chromatography is the type of chromatography which use liquid mobile phase. On
the other hand,gas chromatography is the type of chromatography which Use gas
Mobile phase.

12. 2.Stationary phase
 Stationary phase is the type of Phase which is fixed Into a column. Hance a polar
stationary phase Is used in The forward phase Chromatography While non polar is
used in reversephase.
 Stationary phase is over which the mobile phase passes the technique of
chromatography.
 Mobile phase is flows throughcolumn.
 The sample is separated injectedAt the beginning of the column and transported
through the system by mobile phase.

13. Procedure
▪Take a paper which is moist.
▪Side selection as a base and line 1 to 2cm above the edge using
pencil.
▪Take a capillary tube and will spot this particular line.
▪Leave stationary phase which is actually spotted stationary phase
aside to become dry.

14. Continue…
▪Take a chamber and closed the upper part of the chamber
.In this chamber we take a solvent.
▪The solvent is then evaporate and dispersed in the entire
chamber.
▪Take a stationary phase out and find the Rf “retardation
factor”.
▪Calculate the Rf values of the particular component by
using the formula.

15. Formula
Rf = 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑚𝑜𝑣𝑒𝑑 𝑏𝑦𝑠𝑜𝑙𝑢𝑡𝑒
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
Let Suppose
Distance travelled by solvent front =10cm
Distance travelled by component A=2cm
Distance travelled by component B=4cm
Distance travelled by component C=6cm
Distance travelled by component D=8cm

16. Continue…
Rf of component A =2
10 =0.2
Rf of component B = 4
10 =0.4
Rf of component C = 6
10 =0.6
Rf of component D = 8
10 = 0.8

17. Descending paper chromatography
 When the development of the paper is done by allowing the solvent to travel down
the paper it is known as descending techniques.

18. Ascending paper chromatography
 When the development of the paper is done by allowing the solvent to travel
up the paper it is known as ascending techniques.
 Both ascending and descending techniques have been employed for
speration of organic and inorganic substances.
 But the ascending techniques is
Perffered if Rf value of constitution
Are almost same.

19. Ascending descending paper
chromatography
 It is the hybrid of the above two techniques in this techniques the upper part
of the ascending chromatography can be folded over a glass rod allowing
the ascending development to change over into the descending after
crossing the glassrod.

20. Two dimensional chromatography
 In this techniques a square rectangular paper is used .
 The samples is applied to one of the corner.
 The second development is performed at the right angle to the direction of the first
run.
 This type of chromatography can be carried out with identical solvent system in
both the direction or by two solvent.

21. Radial paper chromatography
 This is also known as circular paper chromatography .
 This make use of radialdevelopment.
 In this techniques a circular filter paper is employed.
 Then the various material to be analyzed are placed at the center .
 After drying the spot the paper is fixed horizontally on the petri dish possessing the
solvent so the tounge or the wick.
 When the solvent front has moved through a sufficient large distance the
components get sperated in the from of concentric circular zones.