Here are the key principles of the different chromatography techniques covered in the document: - Paper chromatography relies on the differential migration rates of compounds through a stationary phase (filter paper) based on their varying interactions with the mobile phase solvent. Components separate based on differences in solubility and affinity for the mobile vs. stationary phases. - Thin layer chromatography uses a thin stationary phase coating (e.g. silica gel) on a plate. Components separate based on differences in their partitioning behavior between the mobile liquid phase and stationary solid phase during capillary movement. - Column chromatography uses a packed stationary phase within a column. Components separate based on differences in their distribution between the stationary phase adsorbent and percolating mobile phase

1. STK1094 Analytical Chemistry I
Dayang Norafizan binti Awang Che
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
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2. LU 8: Analytical Separation
Techniques
Learning Objectives
At the end of this lesson, students should be able
to:
• Explain the principles of paper chromatography
• Explain the principles of thin layer
chromatography and calculate the retention
factor
• Explain the principles of column chromatography

3. Paper Chromatography
• Method for testing the purity of compounds
and identifying substances.
• Useful technique because it is relatively quick
and requires small quantities of material.
• Substances - distributed between a stationary
phase and a mobile phase.
• The stationary phase: a piece of high quality
filter paper/porous paper.
• The mobile phase: developing solution that
travels up the stationary phase, carrying the
samples with it.
3

4. 4

5. Theory of paper chromatography
A small sample of a mixture is placed on porous
paper which is in contact with the solvent.
The solvent moves through the paper due to
capillary action and dissolves the mixture spot.
Components of the mixture with a stronger
attraction to the paper than to the solvent will
move more slowly. The differences in the rates
with which the components travel along the paper
leads to their separation.
5

6. Principles of Paper Chromatography
 Capillary Action – the movement of liquid within the
spaces of a porous material due to the forces of
adhesion, cohesion, and surface tension. The liquid
is able to move up the filter paper because its
attraction to itself is stronger than the force of
gravity.
 Solubility – the degree to which a material (solute)
dissolves into a solvent. Solutes dissolve into
solvents that have similar properties. This allows
different solutes to be separated by different
combinations of solvents.
 Separation of components depends on both their
solubility in the mobile phase and their differential
affinity to the mobile phase and the stationary
phase.
6

7. Thin Layer Chromatography (TLC)
• Solid-liquid technique.
• Two phases are a solid (stationary phase) and a
liquid (mobile phase).
• Solids most commonly used in chromatography
are silica gel (SiO2) and alumina (Al2O3). Both of
these adsorbents are polar.
• Silica is also acidic. Alumina is available in
neutral, basic, or acidic forms.
7

8. Thin Layer Chromatography (TLC)
 The different components in the mixture move up
the plate at different rates due to differences in
their partitioning behavior between the mobile
liquid phase and the stationary phase (capillary
action).
 The separated spots are visualized with ultraviolet
light or by placing the plate in iodine vapor.
8

9. Stationary Phase: Silica (SiO2)
OH
Si
O
OH
Si
O
O
O
OH
Si
O
O
OH
Si
O
O
OH
Si
O O
O
Si
O
O
Si
O
O
Si
O
O
Si
O O
O
Si
O
O
Si
O
O
Si
O O
O
9

10. Stationary Phase: Alumina
O
Al
O O
Al
O
OH
Al
O
OH
Al
O
OH
Al
OH
O
Acidic: -Al-OH
Neutral: -Al-OH + -Al-O-
Basic: -Al-O-
10

11. Uses of TLC
 To determine how many components there
are in a mixture (is it really pure?)
 To determine the best solvent conditions for
separation on a column.
 To monitor the composition of fractions
collected from column chromatography.
11

12. Advantages of TLC:
• Sensitive, fast, simple and inexpensive
analytical technique.
• It is a micro technique; as little as 10-9g of
material can be detected, although the
sample size is from 1 to 100 x 10-6 g.
12

13. Spotting a TLC plate with sample
Running the TLC plate in
solvent
13

14. Thin Layer Chromatography (TLC)
Visualization
There are various techniques to visualize the
compounds.
1. Sulfuric acid/heat: destructive, leaves charred
blots behind.
2. Iodine: semi-destructive, iodine absorbs onto the
spots, not permanent.
3. UV light: non-destructive, long wavelength
(background green, spots dark), short
wavelength (plate dark, compounds glow).
14

15. Origin Line
Solvent Front Line
Distance traveled
by solvent
Distance traveled by spot
Rf = distance spot traveled from origin line/distance
of solvent front
 Retention Factor (Rf)
15

16. 16

17. Thin Layer Chromatography (TLC)
 The Rf depends on the following parameters:
-solvent system
-absorbent (grain size, thickness)
-amount of material spotted
 Ideally Rf value: 0.2 ≤Rf ≤0.8.
 Adjust the Rf with solvent polarity. Rf increase with
increasing polarity.
17

18. Thin-Layer Chromatography:
A Two-Component Mixture
More polar!
Less polar!
s olvent front
origin mixture
s olvent front
component B
component A
origin
s olvent front
component B
component A
origin
Increasing Development Time
18

19. 19

20. Question: Which samples plotted on the TLC plate
below composed more than one substance?
20

21. Answer :
• Samples 1 and 3 were composed of more
than one substance, as because during
the TLC experiment two or more spots
separated from the original sample.
• Sample 2 may have been composed of
more than one substance, but if it was,
the substances did not separate during
the experiment.
21

22. Column Chromatography
 The stationary phase, a solid adsorbent, is
placed in a vertical glass (usually) column.
 The mobile phase, a liquid, is added to the top
and flows down through the column (by
either gravity or external pressure).
 Column chromatography is generally used as
a purification technique: it isolates desired
compounds from a mixture.
22

23. 23
Packed column

24. Column chromatography
24

25. Column Chromatography
 The mixture to be analyzed by column
chromatography is applied to the top of the
column.
 The stationary phase is held in a narrow tube and
the mobile phase is forced through the tube
under pressure or gravity.
 An equilibrium is established between the solute
adsorbed on the adsorbent and the eluting
solvent flowing down through the column.
25

26. Column Chromatography
• Because the different components in the
mixture have different interactions with the
stationary and mobile phases, they will be
carried along with the mobile phase to
varying degrees and a separation will be
achieved.
• The individual components, or eluents, are
collected as the solvent drips from the
bottom of the column.
26

27. The Adsorbent
Silica gel (SiO2) and alumina (Al2O3) – commonly used as
adsorbents.
Sold in different mesh sizes.
E.g “Silica gel 60” or “silica gel 230-400” - This number
refers to the mesh of the sieve used to size the silica.
Adsorbent particle size affects how the solvent flows
through the column.
.
27

28. The solvent
 The polarity of the solvent which is passed through
the column affects the relative rates at which
compounds move through the column.
 Often a series of increasingly polar solvent systems are
used to elute a column.
 A non-polar solvent is first used to elute a less-polar
compound.
 Once the less-polar compound is off the column, a
more-polar solvent is added to the column to elute
the more-polar compound.
28

29. Column Chromatography
 The stationary phase (column packing) in the column is
very polar.
 Polar compounds are going to be attracted to the polar
column packing by hydrogen bonding or dipole-dipole
attractions. Polar compounds are going to move slowly.
 Non-polar compounds are going to come off the
column first, while the polar compounds are going to
come off the column last.
 Usually, one starts with a less polar solvent to remove
the less polar compounds, and then slowly increase the
polarity of the solvent to remove the more polar
compounds.
29

30. The expected elution order of organic
classes
30

31. • The stationary phase is POLAR.
• The more polar component interacts more strongly with
the stationary phase.
• The more polar, component moves more slowly.
• The non-polar, component moves more rapidly.
Column chromatography
31

32. Reverse Phase Column Chromatography
• The stationary phase (column packing) is now NON-
POLAR
• Non-polar compounds will move more slowly because
they are attracted to the column packing.
• The more polar component moves more quickly down
the column.
• Polar solvents, such as water and methanol are used in
reverse phase chromatography
• Used mainly in columns, such as HPLC
32

33. REFLECTION
•What is the underlying
principle of paper
chromatography?
•What is the underlying
principle of thin layer
chromatography?
•What is the underlying
principle of column
chromatography?