Thin layer chromatography (TLC) is a technique used to separate mixtures and identify unknown compounds. In TLC, a sample mixture is applied to a thin silica plate and developed in a solvent tank. The different components of the mixture travel different distances up the plate based on their interactions with the stationary and mobile phases. Compounds can be identified by comparing their retention factor (Rf) values to known standards. TLC is useful for purification, reaction monitoring, and analyzing compound purity. It allows for multiple samples to be run simultaneously and visualized under UV light or after derivatization.

1. Thin Layer Chromatography (TLC)
By: AMIYA KUMAR GHOSH
B.pharm,7th sem,4th year
Roll No. : 27701912002
Registration No. : 122770210002 OF
2012-2013
NSHM Knowledge Campus-Kolkata,
Group of Institutions

2. Chromatography:
Chromatography is the physical separation of a mixture into its individual components.
We can use chromatography to separate the components of
inks and dyes, such as those found in pens, markers,
clothing, and even candy shells. Chromatography can
also be used to separate the colored pigments in
plants or used to determine the chemical composition
of many substances.

3. Gas Chromatography
Used to determine the chemical composition of unknown
substances, such as the different compounds in gasoline
shown by each separate peak in the graph below.
Paper Chromatography
Can be used to separate the components of inks, dyes,
plant compounds (chlorophyll), make-up, and many
other substances
Liquid Chromatography
Used to identify unknown plant pigments & other
compounds.
Thin-Layer Chromatography
Uses thin plastic or glass trays to identify the
composition of pigments, chemicals, and other
unknown substances.
Examples of Chromatography

4. TLC:
• Thin layer chromatography (TLC) is an important technique for
identification and separation of mixtures of organic
compounds.
• In TLC, components of the mixture are partitioned between an
adsorbent (the stationary phase, usually silica gel, SiO2) and a
solvent ( the mobile phase) which flows through the
adsorbent.

5. Why use TLC:
• Identification of components of a mixture (using appropriate standards)
• following the course of a reaction,
• analyzing fractions collected during purification,
• analyzing the purity of a compound.

6. Advantages:
Easy to use
Cheap
Possible multiple analysis
Possible recovery of the products
No sample preparation required
2-dimensional analysis
• Drawbacks:
• Slow (typically 30-60 minutes)
• Limited quality of the separation
• Limited reproducibility
• Evaporation of the mobile phase
(composition varies during the
analysis)

7. Difference between TLC and HPTLC

8. Principle:As the mobile phase rises up the TLC plate by
capillary action, the components dissolve in the
solvent and move up the TLC plate.
Individual components move up at different rates,
depending on intermolecular forces between the
component and the silica gel stationary phase and
the component and the mobile phase.
The stationary phase is SiO2 and is very “polar”.
It is capable of strong dipole-dipole and H-bond
donating and accepting interactions with the
“analytes” (the components being analyzed).
More polar analytes interact more strongly with the
stationary phase in move very slowly up the TLC plate.
By comparison, the mobile phase is relatively
nonpolar and is capable
of interacting with analytes by stronger London forces,
as well as by dipole-dipole and H-bonding.
More nonpolar analytes interact less strongly with the
polar silica gel and more strongly with the less polar
mobile phase and move higher up the TLC plate.

9. STATIONARY PHASE
• Silica is commonly used as stationary phase
• The separation of sample mixture will be depend on the polarity of sample.
• Some modified silica is also used in certain purposes.
Stationery phase Description Application
Silica gel G Silica gel with average particle
size 15µm containing ca 13%
calcium sulfate binding agent
Used in wide range
pharmacopoeial test
Silica gel GF254 Silica gel G with fluorescence
added
Same application with Silica gel
G where visualization is to be
carried out under UV light.
Cellulose Cellulose powder of less than
30µm particle size
Identification of tetracyclines

11. MOBILE PHASE
• The ability of mobile phase to move up is depend on the polarity itself
• Volatile organic solvents is preferably used as as mobile phase.
SOLVENT POLARITY INDEX
Hexane 0
Butanol 3.9
Chloroform 4.1
Methanol 5.1
Ethanol 5.1
Acetonitrile 5.8

12. Solvent MF
MW
Bp (
o
C)
Density (g/mL)
Hazards* Dipole Elution
Stength
()
Hexane
CH3(CH2)4CH3
C6H14
86.17
68.7
0.659
Flammable
Toxic
0.08 0.01
Toluene
C6H5CH3
C7H8
92.13
110.6
0.867
Flammable
Toxic
0.31 0.22
Diethyl ether
CH3CH2OCH2CH3
C4H10O
74.12
34.6
0.713
Flammable
Toxic, CNS
Depressant
1.15 0.29
Dichloromethane
CH2Cl2
CH2Cl2
84.94
39.8
1.326
Toxic, Irritant
Cancer suspect
1.14 0.32
Ethyl Acetate
CH3CO2CH2CH3
C4H8O2
88.10
77.1
0.901
Flammable
Irritant
1.88 0.45
Acetone
CH3COCH3
C3H6O
58.08
56.3
0.790
Flammable
Irritant
2.69 0.43
Butanone
CH3CH2COCH3
C4H8O
72.10
80.1
0.805
Flammable
Irritant
2.76 0.39
1-Butanol
CH3CH2CH2CH2OH
C4H10O
74.12
117.7
0.810
Flammable
Irritant
1.75 0.47
Propanol
CH3CH2CH2OH
C3H8O
60.09
82.3
0.785
Flammable
Irritant
1.66 0.63
Ethanol
CH3CH2OH
C2H6O
46.07
78.5
0.789
Flammable
Irritant
1.70 0.68
Methanol
CH3OH
CH4O
32.04
64.7
0.791
Flammable
Toxic
1.7 0.73
Water
HOH
H2O
18.02
100.0
0.998
1.87 >1

13. • Elution Strength of Mixed Solvents:
The elution strength of the mixture is assumed to be the weighted average of the elution strengths of the
components:
o
net = o
A (mole % A) + o
B (mole % B)
where: mole % A = (moles A) / (moles A + moles B)
Thus, to determine the o
net of a solvent mixture, the molar ratio of the solvents must first be calculated. For
example, the o
net of a solvent mixture prepared from 1.0 mL of ethyl acetate plus 9.0 mL of hexanes is
calculated as shown below:
o
net = oEtOAc [(moles EtOAc)/(moles EtOAc+moles hexane)] +
ohexane [(moles hexane)/(moles EtOAc+moles hexane)]
where: moles EtOAc = [(volume EtOAc) (density EtOAc)] / [molecular weight of EtOAc]
thus: o
net =
{0.45[(1.0mLEtOAc)(0.902g/mL)/(88.11g/mole)]+0.01[(9.0mLhexane)(0.659g/mL)/86.18g/mole)]}
{(1.0 mLEtOAc)(0.902g/mL)/88.11g/mole) + (9.0 mLhexane)(0.659g/mL)/86.18g/mole)}
and o
net = 0.067

14. Materials :
• TLC plate
• ‘Developing container’
- chamber/ jar/ glass beaker
• Pencil
• Ruler
• Capillary pipe
• Solvents / mobile phase
- organic solvents
• UV lamp

15. Procedure:
• 1.Developing Container Preparation:
Solvent is transferred into the container
with 0.5-1cm in dept from the bottom

16. 2. TLC Plate Preparation
 Commercialy obtained with 5cm x 20cm in
size
 Prepare your size when neccesary
 Line 1 cm from the bottom with a pencil as a
part should be spotted.
Image Notes
1. These were made with lab grade silica gel, on glass slides, with plaster of paris as the binder
2. These were made with silica gel from dessicator packets, prepared in the same way as above but with less suspension to work with
(hence the gaps near the
edge)

17. 3.Spotting’ TLC plates
Make sure that your sample is liquified already.
 stick it using capillary pipe & spott onto the line

18. 4.‘Develop the plate’
after spotting, put the plate inside the chamber in the
ascendant position
Make sure that the depth of solvent doesn’t touch the
spots
Let it develop up to the 1cm from the top of plate
After that, pull out the plate from the chamber and let the
solvent be vaporized

19. 5. Detection of spots
• The color samples are easy to be seen and no need to use UV
lamp to detect them
Chromatogram of 10 essential oils,
Stained with vanillin reagent.

20. Detection :
Compound class Derivatizing agent
General Iodine vapor
General Sulphuric acid (50%)
Acids Bromo cresol green
Aldehyde and ketone 2,4 dinitro phenyl hydrazine
Amines and amino acid ninhydrin
Alkaloids Mercuric nitrate
Barbiturate Diphenylcarbazone
Lipids Bromo thymol blue
Steroids Antimony trichloride
carbohydrate Aniline phthalate
Chromatogram of 10 essential oils,
Stained with vanillin reagent.

21. Resolution
The separation between two analytes on a
chromatogram can be expressed as the resolution,
Rs and can be determined using the following
equation:
Rs = (distance between center of spots)
(average diameter of spots)
In TLC, if the Rs value is greater than 1.0, the analytes
are considered to be resolved.

22. Visualization
Absorption of UV radiation is proportional to concentration
Quantification is possible

23. Rf value:
solvent front
component B
component A
origin
dS
dB
dA
Rf of component A =
dA
dS
Rf of component B =
dB
dS
The Rf value is a decimal
fraction, generally only
reported to two decimal
places
solvent
solute
f
d
d
R 

24. THIN LAYER CHROMATOGRAPHY – Rf’s
Rf values can be used to aid in the identification of a
substance by comparison to standards.
The Rf value is not a physical constant, and comparison
should be made only between spots on the same sheet, run
at the same time.
Two substances that have the same Rf value may be
identical; those with different Rf values are not identical.

25. Mobile Phase Mixture
Composition Number % Ethyl
Acetate
% Hexane
Rf
Salicylic
Acid
Rf
Acetylsalicylic
Acid
1 100 0 0.530 0.545
2 90 10 0.620 0.600
3 70 20 0.255 0.291
4 50 30 0.945 0.291
5 30 50 0.360 0.280
6 20 70 0.270 0.250
7 10 90 0.182 0.164
8 0 100 0 0
Mobile Phase Mixture combinations for the separation of Salicylic Acid (1%) and Acetylsalicylic acid (1%)

26. Time (minutes) Salicylic
acid Rf
Acetylsalicylic
acid Rf
Reaction Mixture Rf Lower
Spot
Reaction Mixture Rf
Upper Spot
15 0.385 0.346 0.385 0.481
30 0.320 0.340 0.360 0.500
45 0.264 0.321 0.321 0.491
Retardation factor (Rf) values for TLC of salicylic acid, acetylsalicylic acid, and reaction mixture in
a 50/50 ethyl acetate/hexane solvent system over time.

27. Some problem:
a. The spot shape is too broad
- Diameter is supposed to be < 1-2mm
b. The movement of solvent
- should be straight up
- unproportionality in stationary phase surface will inhibit the
movement of solvent
c. streaking formation
- caused by too concentrated sample

28. EXAMPLE :
Mobile phase Stationary phase Herbs and Herbal Products
20 volumes of toluene+ 45
volumes
of ethyl acetate,+20 volumes
of glacial acetic acid + 5
volumes of formic acid
silica gel Amalaki
100 volumes of ethyl
acetate+
11 volumes of formic
acid,+11 volumes of acetic
acid +
25 volumes of water.
silica gel Amra
Glacial acetic acid. kieselguhr G. Arachis Oil
A mixture of 1 volume of
hexane and 1 volume
of diethyl ether.
silica gel GF254 Artemisia
Toluene silica gel GF254 Clove Oil
A mixture of 90 volumes of
toluene and
10 volumes of ethyl acetate
silica gel G Eucalyptus Oil
Derivatizing agent
anisaldehyde sulphuric
acid reagent
vanilin sulphuric acid
reagent
starch solution
1 volume of sulphuric
acid and 0.5 volume of
anisaldehyde
anisaldehyde solution,
anisaldehyde solution

29. Reference:
1. R.A.Day, Jr. A.L.Undewood(1987). Analisis Kualititatif. Edisi ke Empat,
ms: 474-529.
2. David G. Watson(2005). Pharmaceutical analysis. Edisi ke-2, ms 315-
331
3. http//orgchem.colorado.edu/handbooksupport/TLC/TLCprocedure.ht
ml
4. Indian pharmacopeia, volume 3, 2007
5. Kapp, Khail. Chapter 7, Thin Layer Chromatography, March 2, 2010
6. Kaine, Mary Ann. Chapter 7, Thin Layer Chromatography, March 2,
2010.
7. Young, Wei. Chapter 7, Thin Layer Chromatography, March 2, 2010.

30. Thank you
Contact: amiyaghosh94@gmail.com

31. • Contact: amiyaghosh94@gmail.com