Michael Tswett discovered chromatography in 1901 when he separated chlorophyll, a green pigment found in plants, into five distinct bands or solutes (α-chlorophyll, β-chlorophyll, xanthophyll, viola xanthene, and carotene) using a glass column packed with calcium carbonate (CaCO3) powder and eluting with ethanol. The separation occurred because the solutes migrated down the column at different velocities due to having different degrees of adsorption to the CaCO3 stationary phase, from least adsorption (highest velocity) for carotene to greatest adsorption (lowest velocity) for β-chlorophyll. This demonstrated that chromatography can separate mixtures and defined the basic components and process of column

1. Introduction
to
Chromatography
1
This work is licensed under Creative Commons Attribution-NonCommercial- ShareAlike 4.0 International License
By: Dr. Deepa Verma
VIVA College, Virar (W),
Maharashtra

2. 2
COTTON
PLUG
SLURRY OF CaCO3 POWDER
IN PETROLEUM ETHER
(Stationary Phase)
COTTON PLUG
CONCENTRATED SOLUTION OF
CHLOROPHYLL IN PETROLEUM
ETHER
WASHING/ ELUTION
WITH ETHANOL
 His experiment set up was very simple one.
 A glass column 1-5 cm in diameter and 20-100 cm long was taken.
 The column was packed with the slurry of Calcium Carbonate made in petroleum ether(60-80).
 A concentrated solution of chlorophyll was prepared in petroleum ether(60-80) and a small
volume (~10cm3) was put on the top of the calcium carbonate column in the glass column as a thin
band.
 The solution of chlorophyll was allowed to move down slowly and then the column was washed
with ethyl alcohol for few hours.
Michael Tswett -Russian botanist
 His main interest was to find out the composition of the green plant
pigment, chlorophyll.
 There were two schools of thought on this subject:
 One school thought that chlorophyll was a single solute.
 The other school thought that chlorophyll was a mixture of different
solutes.

3. 3

4. 4
β CHLOROPHYLL
α CHLOROPHYLL
XANTHOPHYLL
VIOLA XANTHINE
CAROTENE
COTTON PLUG
SLURRY OF CaCO3
POWDER IN
PETROLEUM
ETHER
COTTON PLUG
CONCENTRATED SOLUTION OF
CHLOROPHYLL IN PETROLEUM
ETHER
WASHING/ ELUTION
WITH ETHANOL
It was concluded that chlorophyll is a mixture
of five different solutes. α –chlorophyll, β - chlorophyll,
xanthophyll, viola xanthene, and carotene .
Each band was removed from the column and identified.

5. 5
 Michael Tswett’s main problem was resolved and
in the process he discovered a simple, time saving
and relatively less laborious method of separating
complex solutes from a mixture.
 He visualized the coloured bands from
chlorophyll as coloured writing.
 Michael Tswett coined the term “Chromatography for this
new technique of separation (Chromatus = colour,
graphein = to write)
 Today the term chromatography is used for
separations involving colourless solutes also. This
is done to honour the discoverer of the
technique.

6. 6
As a researcher or simply as an inquisitive
person, two questions come to our mind
on the results of Tswett’s experiment.
1. Why do the five solutes separate on
the column?
2. How do the five solutes separate on
the column?

7. Chromatographicseparationasunderstoodfromrunnersrunningarace
7
Initially all the competitors are at a given position. As the race starts, slowly there is
separation between them and one who runs the fastest reaches the goal first and one
who is the slowest reaches the goal last. And there are others in between the two
extremes because of their different speeds.

8. 8
1. Can this concept be applied to the five solutes in
Tswett’s experiment?
YES!!!!
The five solutes have different migration
velocities. Since, carotene has the highest migration
velocity it reaches the other end of the
column first.
 On the other hand β - chlorophyll is the last
solute to come out of the column because of
its slowest migration velocity of the five
solutes.

9. 9
So, we have the answer to the first question.
Migration velocities increase from
β- chlorophyll to carotene.
“The five solutes are separated on the column
because of the differences in their migration
velocities”

10. 10
But why does carotene travel the fastest and β-
chlorophyll the slowest when all the five solute travel
across the column under similar experimental conditions?
Let me give you a hint at this stage.
Although the experimental conditions are similar, the
five solutes travel across a good adsorbent- CaCO3 .

11. 11
We are quite familiar with the phenomena of adsorption. It is
a surface phenomenon in which some solutes (adsorbates) adsorb on
the surface of another solute (adsorbent).
Adsorption process depends on
1. the properties of surface of an adsorbent.
2. the properties of adsorbates.
For example if we consider our palm as a
surface of an adsorbent, then the degree of
adsorption of water and oil (adsorbates) on
the same surface is different.
 Again the same adsorbates may not adsorb to
the same degree on the surface of another
adsorbent, let us say, a cellulose paper.

12. 12
Then we have the answer to the second question.
“The five solutes have different migration
velocities across the column of CaCO3, because
they have different degrees of adsorption on the
surface of CaCO3”
Carotene having least and Chlorophyll –b having
highest degree of adsorption

13. 13
Carotene has the least degree of adsorption on CaCO3
surface and hence the highest migration velocity while β-
chlorophyll has the highest degree of adsorption on
CaCO3 surface and hence the lowest migration velocity
of the five solutes.
We can therefore say that the degree of adsorption on
CaCO3 surface decreases from β- chlorophyll to carotene
and the migration velocity decreases from carotene to β-
chlorophyll.
Therefore what we are now talking about is “Adsorption
column chromatography”.

14. What we studied ?
• Stationary Phase – Definition
• Mobile Phase – Definition
• Setting up of column
• Process of Column chromatography
14