Thin Layer Chromatography
Contents
• Thin
layer chromatography
• History
• Principle
involved
• Applications
of TLC
Practical requirements of TLC
• Stationary
phase
• Glass
plates
• Preparation
and activation of TLC plates
• Application
of sample
Objectives
By the end of this session, students will be able to:
Ø Explain
the principle involved in Thin layer Chromatography
Ø Outline
the components of TLC
Ø Discuss
the preparation and activation of TLC plates
Thin layer
chromatography
History
• In
1938, Izmailov and Shraiber separated plant extracts using 2 mm thick and firm
layer of alumina on glass plate
• In
1944, Consden, Gorden and Martin used filter papers for separating amino acids
• In
1950, Kirchner identified terpenes on filter paper and
• Later
glass fiber paper coated with alumina
• In
1958, Stahl developed standard equipment for analyzing by thin layer
chromatography
Principle
• Principle
of separation is adsorption
• One
or more compounds are spotted on a thin layer of adsorbent coated on a
chromatographic plate
• Mobile
phase solvent flows through because of capillary action (against gravitational
force)
• Components
move according to their affinities towards adsorbent
• Component
with more affinity towards stationary phase moves slower
• Lesser
affinity moves faster
• Components
are separated on a thin layer chromatographic plate based on affinity towards
stationary phase
Advantages
of TLC
• Simple
method and cost of equipment is low
• Rapid
technique and not time consuming like column chromatography
• Separation
of µg of the substance can be achieved
• Any
type of compound can be analysed
• Efficiency
of separation- very small particle size can be used to increase the efficiency
of separation
• Detection
is easy and not tedious
• Capacity
of thin layer can be altered
• Analytical
and preparative separations can be made
• Corrosive
spray reagents can be used without damaging the plates
• Needs
less solvent, stationary phase and time for every separation when compared to
column chromatography
Practical
Requirements
• Stationary
phase
• Glass
plates
• Preparation
and activation of TLC plates
• Application
of sample
• Development
tank
• Mobile
phase
• Development
technique
• Detecting
or visualizing agents
Stationary
Phase
• Several
adsorbents can be used as stationary phase
• Composition
and ratio in which they have to be mixed with water or the solvents to form a
slurry
|
Name |
Composition |
Adsorbent : water ratio |
|
Silica gel H |
Silica gel without binder |
1 : 1.5 |
|
Silica gel G |
Silica gel + calcium sulphate |
1 : 2 |
|
Silica gel GF |
Silica gel + binder + fluorescent indicator |
1 : 2 |
|
Alumina Neutral Basic Acidic |
Al2O3 without binder |
1: 1.1 |
|
Cellulose powder |
Cellulose with binder Cellulose without binder |
1 : 6 1 : 5 |
Glass
Plates
• Specific
dimensions like 20 cm x 20 cm (full plate)
• 20
cm x 10 cm (half-plate)
• 20
cm x 5 cm (quarter plate)
• Width
of commercially available TLC spreader is 20 cm
• Can
prepare plates of different dimensions without using TLC spreader
• Microscopic
slides can also be used for some applications like monitoring the progress of a
chemical reaction
• Development
time is much shorter than 5 minutes
• Glass
plates should be of good quality and should withstand temperatures used for
drying the plates
Preparation
and Activation of TLC Plates
• Slurry
which is a mixture of stationary phase and water is prepared by using the ratio
mentioned earlier
• After
preparing the slurry, TLC plates can be prepared by any of the technique
Pouring
• Slurry
is prepared and poured on the glass plate which is maintained on a levelled
surface
• Slurry
is spread uniformly on the surface of glass plate
• Plates
are dried in an oven
• Drawback
is uniformity in thickness cannot be ensured
Dipping
• Two
plates are dipped in to the slurry and are separated after removing from
slurring and are dried
• Drawback
is large quantity of slurry is required even for preparing fewer plates
Spraying
• Resembles
that of using perfume spray on a cloth
• Suspension
of adsorbent or slurry is sprayed on a glass plate using a sprayer
• Drawback
is layer thickness cannot be maintained uniformly all over plate
Spreading
• Best
technique where a TLC spreader is used
• Glass
plates of specific dimensions (20 cm x 20 cm/ 10 cm / 5 cm) are stacked on a
base plate
• Slurry
after preparation is poured inside the reservoir of TLC spreader
• Thickness
of adsorbent layer is adjusted by using a knob in the spreader
• Normally
a thickness of 0.25 mm is used for analytical purpose
• 2
mm thickness for preparative purpose
• Spreader
is rolled only once on the plates
Activation of Plates
• Plates
are allowed for setting (air drying)
• Done
to avoid cracks on the surface of adsorbent
• Plates
are activated by keeping in an oven at 100 0C to 120 0C
for 1 hour
Activation of TLC
plates
• Nothing
but removing water/ moisture and other adsorbed substances
• By
heating at high temperatures
• Activated
plates can be stored in thermostatically controlled oven or in desiccator
• Used
whenever required
Application of sample
• To
get good spots, concentration of sample or standard solution has to be minimum
• 2-5
µl of a 1% solution is spotted using a capillary tube or micropipette
• Spots
can be placed at random or equidistant from each other by using a template with
markings
• Spots
should be kept atleast 2 cm above the base of the plate
• Spotting
are shouldn’t be immersed in the mobile phase in the development tank
• Atleast
4 spots can be spotted conveniently on a quarter plate (20 cm x 5 cm)
Summary
• In
1958, Stahl developed standard equipment for analyzing by thin layer
chromatography
• Mobile
phase solvent flows through because of capillary action (against gravitational
force)
• Components
move according to their affinities towards adsorbent
• Very
small particle size can be used to increase the efficiency of separation
• Needs
less solvent, stationary phase and time for every separation when compared to
column chromatography
• Glass
plates should be of good quality and should withstand temperatures used for
drying the plates
• Plates
are activated by keeping in an oven at 100 0C to 120 0C
for 1 hour
