UNIT –III
Thin layer chromatography
Introduction, Principle, Methodology,
Rf values, advantages, disadvantages
and applications
Dr. Nisha Sharma,
Associate Professor
School of Pharmaceutical Sciences
C.S.J.M. University, Kanpur
1
THIN LAYER CHROMATOGRAPHY
• M. Tsvet- Father of Liq. Chrom. 1900’s
• Introduced by Izmailov and Shraiber in 1938-
separation of plant extracts
• Kirchner: 1950- coated plates- ident. terpenes
• E. Stahl in 1958 Designed equipment
• Also known as surface, strip, open column,
spread layer chromatography
2
PRINCIPLE
• Based on separation by adsorption
• Depends on the relative affinity of components
towards stationary and mobile phase
• Suppose, affinity of component A has strong
towards adsorbent, then the movement will be
less. Hence the different constituents separate.
• Liquid – solid chromatography
3
PRINCIPLE
• Competition b/w molecules of analyte &
solvent
• Both binds with the adsorbent surface
• Degree of retention depends upon:
a. Binding strength of analyte with support
b. Surface area of support
c. Amount of mobile phase displaced by the
analyte
d. Binding strength of solvent phase to adsorbent
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PRINCIPLE
The retention may be affected by
• Electrostatic interactions
• Hydrogen bonding
• Dipole dipole interactions
• Van der-waals forces
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Methodology
• Almost similar to paper chromatography
1. Coating material:
Adsorbent Nature Activity Mechanism Substances
Silica gel Acidic Active Adsorption/ Acidic / neutral
Partition
Alumina Basic Active Adsorption/ Basic & neutral
partition
Keisulguhr Neutral Inactive Partition St. hydrophilic
subst
Cellulose Neutral None Partition Water soluble
powder compounds
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Methodology
• Coating material: other examples: CaPO 4, Mg
Trisilicate, silica gel alumina (1:1), acetylated
cellulose etc.
• Adsorbent : must adhere to plate
• Binders: CaSO4, starch, hydrated silica O
• Gypsum- widely used binder
• Ex: Silica gel G, Alumina G--- G stands for
Gypsum
• Zinc silicate- inert fluorescent indicator
• Ex. Silica Gel GF 7
2. PREPARATION OF TLC PLATES
• Suspension of slurry is prepared
• Methods to prepare:
a) Pouring method: back & forth
b) Dipping: Peifer (1962)- 2 plates at a time- CHCl 3 or
CHCl3— MeOH
c) Spraying: Reitsema– Sprayer
d) Spreading- applicator- aligning tray, spreader,
developed by DESAGA (west G)
0.1-0.5 mm thick for analytical work
0.5-2 mm – preparative work
e) Precoated plates- ready to use- 0.1-0.2mm
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3. Activation of Adsorbent: To remove liq. from
TLC, Dry for 30min. in air, & then in oven at
110°C fro 30 min, For very active plates-
150°C- 4 hrs
4. Purification of silica gel G layers: To remove
Iron as impurity, run plates in MeOH:HCl::
9:1 v/v, Fe gets migrated to solvent front,
again activate at 110°C, but if CaSO 4
dissolves reused by adding Suitable binder
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5. Sample application: Microsyringe
Sample solution: non polar/ volatile solvent
6. Developing tank: tank + lid, saturation of
tank is must, or lack of reproducibility in
Rf
7. Solvent system: Stahl’s triangle: better
separation-mix. of solvents
Ex: Ethyl acetate : methanol:: 99:1
Trial error basis – best solvent system
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Stahl’s triangle
Pola
active r
Elu
vity
ti
ng
acti
so
lve
ent
S
nt
orb
Ad s
a cti ve Non
in pola
M r
ilic
Hy
dro
h
ph
op
ilic
Lip
Mixtu
re
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8. Development methods: Solvent allowed to rise at
ht of 15-18cm on 20 cm plate, 20-40 minutes
• Solvent front marked and dried
• 9. Determination of components:
• Colorless--- U.V.
• Visualizing reagent
• Corrosive reagents – chromic acid, H2SO4
10. Evaluation:
• Qualitative (R.f)
• Quantitative : Direct and indirect methods
• Direct: Visual, spot areas, densitometry, direct
spectrometry
• Indirect: Elution- Instrumental 12
After After
a b
5 min 15 min
sample
THIN LAYER CHROMATOGRAPHY
a. Solvent front i.e. distance travelled by solvent
b. Distance travelled by Sample
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Saturation of TLC Tank
Cover plate
Thin layer plate
Solvent
wick for
saturation
Solvent mixture
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Advantages
• Combines advantages of PC & CC
• Equipment simple
• Short development time compared to PC
& CC
• Separation is fairly good on inorganic
adsorbent material
• Wide choice of stationary phase-
adsorption, partition, ion exchange
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Advantages
• Fast recovery of separated components –
powdery coating of plate, scrapping, spot/
zone, quantitative removal, dissolve,
spect./colorimeter
• Easy visualization of separated components,
florescent compds. Detected easily under U.V.
because inorganic background don’t produce
fluorescence
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Advantages
• Sensitivity: Delineated, sharp spots, 10 to 100
folds as compared to PC
• Variable thickness of layers: Thin layers for
qualitative and thicker layers for preparative
• Chemically inert stationary phase- application
of strong heat or corrosive reagent like
sulfuric acid
• Cost effective
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Disadvantages
• Reproducible results are difficult
• Not automated procedure
• Only non volatile substance or subst. with low
volatility can be separated
• Length of plate is limited
• Separation is in open system- humidity, temp.
• Quality of separation is limited
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Applications
• Used for separation of all types of natural
compounds
• To check the purity of samples
• As a purification process of isolated compd
• To examine the reaction: intermediate
products
• To identify organic compds.
• As a check on process- progress of
procedure
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Applications
• Used for separation of inorganic ions-
cationic, anionic, covalent species. organic
deriv. of metals
• Sep. of vitamins
• Amino acids
• Sep. of alcohols, glycols, alkaloids, amines,
proteins, antibiotics
• Used in all type of Industries, Medicine,
Forensic science etc
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References
• Jeffry G.H., J. Mendham et al. Vogel’s Text book of
Quantitative Chemical Analysis, 5th Edition, 1989,
Longman Scientific & Technical, Bath Press, Great
Britain.
• G.R. Chatwal, SK Anand. Instrumental methods of
chemical analysis (Analytical Chemistry), ed. 1995,
Himalaya Publishing House, Bombay.
• A.H. Beckett & J.B. Stenlake. Practical Pharmaceutcal
Chemistry, Part Two, ed. 4th, 1997, (reprint 2003) CBS
Publishers & Distributors, New Delhi.
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