THIN LIQUID CHROMATOGRAPHY (TLC)

1. Topic outline
● Description of Planer Chromatography
● Description of theoretical principles of Planer
Chromatography
● Degree of retention (Rf)
● Applications
Dr. Khalid Hussain (University College of Pharmacy)
hussain_761@yahoo.com

2. Planer Chromatography
● This chromatography is performed on plane
surface, rather than a column
● It offers a unique advantage of 2-Dimentional
operation
● Selective properties of 2 different solvents can be
used in developing a single chromatogram
● It includes: paper chromatography (PC) and thin
layer chromatography (TLC)/High performance
thin layer chromatography (HPTLC)

3. Theoretical principles
● The principles of column chromatography also apply
here i.e.
1- Separation
is accomplished by successive equilibrations of the
sample components between two phases, the mobile
phase (m) and stationary phase (s).
2- Non-ideal process, similar to column
chromatography, cause zone spreading on the plane
● Degree of retention in plane chromatography is
expressed as retardation factor (Rf)

4. Rf = Distance solute moved
Distance solvent moved
For substances that are very soluble in the liquid,
Rf will be close to ....
For substances that are rather insoluble in the
liquid, Rf will be close to ....
1
0
Hence, Rf lies between 0 and 1
Origin Line
Solvent Front Line
Distance traveled
by solvent
Distance traveled by spot
Google images

5. Distance of the solute moved is measured from the
centre of the spot
K=Cs/CM
In column
chromatography,
distribution coefficient is
expressed by

6. The simple relationship between K and Rf is given
as follows:
Rf = Number of moles of solute in moving phase /
Number of moles of solute in both phases
Rf= CmAm/ CmAm+CsAs
Here Am and As are the cross-sectional areas of the
two phases
Dividing numerator and denominator by CM, we get
Rf =
Am
Am+CsAs/Cm
= Am/Am+KAs
Since, K= Cs/CM

7. Factors effecting Rf
● Rf values are subject to minor influences such as
1- Variations of stationary phase
2- Method of application and development
3- Size of sample
4- Concentration of sample

8. Paper Chromatography
● It is mainly used for qualitative and semi-
quantitative purposes
● It is easy to perform
● Mechanisms involved: liquid-liquid partition,
adsorption, hydrogen bonding, and ion exchange
Nature of the paper
● Made up of highly purified cellulose- a polar
compound- hence has great affinity for water and
polar solvents, hold them through H-bonding
● Paper may be impregnated with alumina, silica
and ion exchange resin etc.

9. Procedure
● Application of sample as a small spot
● Development in a closed chamber saturated with
solvent either by
1- Ascending method:
● Simplest and most popular
● Solvent ascends through the paper by
capillary action
● The rate of ascent is slow and decrease with the
passage of time due to gravity
● Slow rate enhances possibility of achieving partition
equilibrium. Google images

10. 2- Descending method:
● Direction of flow of solvent is downward
● Paper is folded U-shape to prevent rapid
siphoning of solvent
● Solvent descends through gravity and capillary
action
● It is much faster than the ascending method
Google images

11. Detection
● After the development, solute may be detected by a
number of methods briefed as:
– Inherent visible colors
– Derivitization
– UV absorbance, fluorescence
– IR absorbance
– Radioactivity
– Chemical tests
– Bio-autography
Sinensetine 2

12. Applications
● Qualitative (measurement of Rf value)
● Isolation
● Purification
● Semi-quantitative
1-Extract the compound from the paper followed my
spectrophotometry
2- Densitometric determination.

13. Techniques
● Two dimensional development
● Radial development
Sample is spotted at the centre and developed,
components move radially forming circles of
increasing diameters. Fast separation by moving
disc so solvent move by CF and CA
● Reversed-phase chromatography.
Paper is coated with hydrophobic substance, polar
phase is used to elute, separation of non-polar
compounds

14. Thin Layer Chromatography
1- Here the mobile phase is a liquid, flowing past a thin layer of powder
on a solid support
2- Substances that are less attracted to the solid or more soluble in liquid
move faster.
3- And so move further up the plate by the time that the process has been
stopped by taking the plate out of the liquid- larger Rf
Very popular method and often used same as pc. Thin layer of
finely divided adsorbent is supported on glass or plastic plates.
It can be used for qualitative, quantitative and preparative
purposes.

15. Nature of the layer
● Commonly used adsorbents are silica gel, alumina,
diatomaceous earth and powdered cellulose.
● Silica gel which is acidic and has high capacity, is
useful for adsorption and partition chromatography
● Alumina is basic and used primarily for adsorption
chromatography
● Diatomaceous earth is nearly neutral and used for
partition chromatography
● Thickness of the layer varies from 0.15-2.00 mm
● TLC plates can be prepared in Lab or purchased
commercially (much more convenient and more
reproducible than home-made plates)

16. Procedure of development
● Same as paper chromatography, mostly
By ascending method or horizontal method
Isocratic
Gradient
Camag website

17. 3. Detection:
If the spots are not colored and can’t be seen by the eye, use:
• UV lamp for UV-active compounds (most aromatics are
UV-active)
• If compounds are not UV-active, use derivatization
Once you visualize the spots, circle them with a pencil.
4. Calculate Rf values for each spot
Rf = distance spot traveled from origin line/distance of solvent front
Rf values will help to identify an “unknown”
Make sure to use the same mobile phase as Rf’s will vary
with varying mobile phases.

18. Other special techniques
● Modern TLC

19. Modern TLC
● Automatic (sample application, development, detection
and quantification)
● Advantages
1- Cheap and easy
2- More versatile than pc
3- Faster and more reproducible
4- Often used for complex sample separation and
visualization
5- Modern TLC complementary to HPTLC
4- Many samples and standards can be applied on the
same plate

20. Camag website

21. Sinensetine
R

22. Betulinic acid
R

24. Thanks
Google images