assignment on thin layer chromatography

1. Chromatography
History:
 Michael Tswett is credited as being the father of liquid chromatography. Tswett
developed his ideas in the early 1900’s.
 In 1950 J. G. Kirchner and his colleagues at the U.S. Department of Agriculture were
working to determine the chemistry of orange and grapefruit flavors.
One day, when one of Kirchner's colleagues was frustrated about a difficult separation,
Kirchner reached across his desk, picked up the abstract of Meinhard and Hall's work,
and said, "Try this". Kirchner and his team found that silicic acid bound with amioca
starch created a satisfactory layer for TLC. He continued his work with sorbent layers on
glass plates and developed TLC essentially as we know it today.
Introduction:
 The word chromatography is derived from two Greek words
 Chroma ….……..color
 Graphos ………..writing
 ‘A technique by which a mixture is separated into its components on the basis of relative
ability of each component to be moved along/through a stationary phase by mobile
phase’
Types of chromatography: according to the packing o the stationary phase
 Thin layer chromatography
 Paper chromatography
 Column chromatography
Thin layer chromatography
Introduction:
TLC is one of the simplest, fastest, easiest and least expensive of several chromatographic
techniques used in qualitative and quantitative analysis to separate organic compounds and to
test the purity of compounds.
Definition:
Thin Layer Chromatography can be defined as a method of separation or identification of
a mixture of components into individual components by using finely divided adsorbent solid /
(liquid) spread over a glass plate and liquid as a mobile phase.

2. Fig : TLC Technic
Synonyms: Drop,spread layer, surface chromatography & open column chromatography.
TLC is a form of liquid chromatography consisting of:
 A mobile phase
 A stationary phase
Analysis is performed on a flat surface under atmospheric pressure and room temperature.
Preparationof TLC chamber:
 A jar with a tight fitting lid added enough of the appropriate developing liquid so
that it is 0.5 to 1cm deep in the bottom of the jar.
 Close the jar tightly.
 It stands for 30minutes so that atmosphere in the jar becomes saturated with
solvent.
Preparationof plates:
Thin-layer chromatography is performed on
 a sheet of glass,
 plastic,
 aluminium foil,
which is coated with a thin layer of adsorbent material,
 Silica gel,
 Aluminium oxide (alumina),
This layer of adsorbent is known as the stationary phase.

3. Glass plates or flexible plates are commonly used for adsorbent. Size used depends on
type of separation to be carried out, the type of chromatographic tank and spreading apparatus
available.
• The standard sizes are 20 x 5 cm.
• The surface should be flat without irregularities.
• The standard film thickness is 250um.
Methods of application of adsorbent:
• Pouring: The adsorbent of finely divided and homogeneous particle size is made
into slurry and is poured on a plate and allowed to flow over it so that it is evenly
covered.
• Dipping: This technique is used for small plates by dipping the two plates at a
time, back to back in a slurry of adsorbent in chloroform.
• Spraying: Slurry is diluted further for the operation of sprayer. But this technique
is not used now a day as it is difficult to get uniform layer.
• Spreading: All the above methods fail to give thin and uniform layers.
Fig: Coater, Hand operated
ACTIVATION OF PLATES:
• After spreading plates are allowed to dry in air and further dried and activated by
heating at 0 about 100 c for 30 min.
• By removing the liquids associated with layer completely, the adsorbent layer is
activated.
Mobile phase:
 Nature of the substance to be separated.

4.  Nature of stationary phase used
 Nature of separation
 Solvent used should be of high purity. Solvents used:
 petroleum ether
 Benzene
 carbon tetrachloride
APPLICATION OF SAMPLE:
• Sample solution in a nonpolar solvent is applied.
• The concentration of a sample or standard solution has to be minimum of a 1%
solution of either standard or test sample is spotted using a capillary tube or
micropipette.
• The area of application should be kept as small as possible for sharper and greater
resolution.
Fig: Sample application
Visualize the spots:
• If there are any colored spots, circle them lightly with a pencil.
• Most samples are not colored and need to be visualized with a UV lamp.
• Hold a UV lamp over the plate and circle any spots you see.
• Make sure you are wearing your goggles and do not look directly into the lamp.
Protect your skin by wearing gloves.
Basic theory:
Rf=
Distance from start to center of substance spot
distance from start to solvent front

5. Factors affecting Rfvalue It depends on following factors:
 Nature adsorbent & Mobile phase
 Activity & Equilibrium
 Thickness of layer & Temperature
 Loading & Dipping zone
 Chromatographic techniques
Applications of TLC:
 It is used for separation of all classes of natural products and is established as an
analytical tool in modern pharmacopoeia
E.g. Acids, alkaloids, amines, macromolecules like proteins and antibiotics.
 for checking the purity of samples
 as a purification process & examination of reaction.
 for identifying organic compounds
 Extensively used as an identification test and test for purity.
 Applications of TLC for separation of Inorganic Ions
 Separation of Amino Acids
 Separation of vitamins – vitamin E, Vitamin D3, vitamin A
 Application of TLC in quantitative analysis
Conclusion:
TLC shows”overloading" due to too much sample but shows good separation. Almost is
not enough compound But Ok.
REFERENCE:
 A text book of Pharmaceutical analysis by Dr. A.V.Kasture. Dr.K.R.Mahadik,
Dr.Swadodkar, Dr.H.N.More, Volume – II, 19 th edition, June 2010, page no:18-27
 Pharmaceutical drug analysis, by Ashutosh Kar, 2001 edition, pg no- 515.