Thin Layer Chromatography

THIN LAYER CHROMATOGRAPHY
Introduction
• Thin Layer Chromatography (TLC) is an important technique used for identification and separation of
mixture of chemical compounds into its individual components.
• TLC is a form of liquid chromatography consisting of two phases: A mobile phase (liquid) and A stationary
phase (solid).
• Differences in the interactions between the solutes and stationary and mobile phases enable separation.
• It involves the distribution of components of a mixture to be separated between two phases.
• The components of the mixture are partitioned between an adsorbent (stationary phase), and a solvent (mobile
phase).
• Different compounds will have different solubility and adsorption to the two phases between which they are
to be partitioned.
• Separation of the individual substances is based on their relative affinities towards stationary and mobile
phases.
• Sensitive technique - microgram (0.000001 g) quantities can be analyzed by TLC - and it takes little time for
an analysis (about 5-10 minutes).
Steps in TLC
http://what-when-how.com/organic-chemistry-laboratory-survival-manual/thin-layer-chromatography-tlc-laboratory-manual/
https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Organic_Chemistry_Lab_Techniques_(Nichols)/02%3A_Chromatography/2.03%3A_Thin_Layer
_Chromatography_(TLC)/2.3E%3A_Step-by-Step_Procedures_for_Thin_Layer_Chromatography
Types of TLC/ Development techniques in TLC
1. Ascending TLC
• As per the name, the developing solvent is
found to be moving in an upward direction.
Here, a sufficient quantity of mobile phase is
poured into the development chamber.
• Sample and reference are spotted on the line
drawn a few centimeters from the bottom edge
of the paper suspended from a hook or clip at
the top.
• The ascending technique is preferred if the
Rf values of various constituents are almost
same
2. Descending TLC
• Here, the solvent front travels down the length

of paper suspended from the top inside the
developing chamber. The mobile phase is kept
in a trough in the upper chamber.
• The paper with spotting on the line drawn a few

centimeters from the top is clamped to the top.
Before elution, the jar is covered and
equilibrated with the mobile phase vapor.
• Both ascending and descending techniques have

been employed for separation of organic and
inorganic substances.
3. Ascending – Descending Chromatography
• It is the hybrid of the above
two techniques.
• In this technique, the upper
part of the ascending
chromatography can be
folded over a glass rod
allowing the ascending
development to change
over into the descending
after crossing the glass rod
4. Radial TLC
• This is also known as circular TLC.

• This makes use of radial development.
• In this technique a circular support is employed.
• Then the various materials to be analyzed are placed
at the center.
• After drying the spot the support is fixed horizontally
on the petri-dish possessing the solvent so that the
tongue or the wick.
• When solvent front has moved through a sufficient
large distance, the components get separated in the
form of concentric circular zones.
5. Two-dimensional chromatography
• In this, a square or rectangular paper is used.

• The sample is applied to one of the corners.
• The second development is performed at right angle to the direction of the first run.
• This type of chromatography can be carried out with identical solvent systems in both
the directions or by two solvent systems.
Spray reagents / Methods to locate the spot position
1. Using Conc. H₂SO₄: e.g. Conc. H₂SO₄
2. By using Iodine Chamber: e.g. Solid Iodine crystals
3. Use of specific reagent: e.g. Ninhydrin reagent
4. Use of Fluorescent property: e.g. Using UV Cabinet
5. Other:
• Aluminium chloride For flavonoids Spray plate with a 1% ethanolic solution of aluminium
chloride. Results: Yellow fluorescence in long wavelength UV light (360nm)
• 4-Aminoantipyrine/potassium hexacyanferrate (III) (Emerson reaction) (Emerson reagent) for
the detection of phenols and arylamines
• Ammonium molydbate For detection of phosphoric acid derivatives
• Aniline phthalate For the detection of reducing sugars
• p-Anisaldehyde – sulfuric acid For detection of phenols, sugars, steroids, and terpenes
Applications of TLC
1. Qualitative analysis:
1. Involves the identification of compounds present in the mixture.
2. Identification involves the use of Rf value based on Rf of standard compound.
2. Quantitative analysis:
1. It is done in the TLC or after the removal of the component from the TLC.
2. The latter is generally preferred – the component is cut from the TLC, extracted
by a suitable solvent, measured by colorimeter or UV-Vis spectrophotometer.
3. Alternatively, the extracted solution is evaporated in the vacuum to remove
solvent,
4. Thus, obtained residue is weighed.
3. Preparative Thin Layer chromatography:
1. Operates with large amount (gram quantity) of substances to yield substances enough for
further work in the laboratory.
2. The separated bands are cut, extracted with suitable solvent and filtered.
3. The filtrate is evaporated off in vacuum to yield the residue of the component.
4. Specific application:
1. Includes the separation of many organic and biochemical products.
2. For example, it has been utilized in the determination of indole in whole urine and in the
study of barbiturates, antibiotics, hormones, and amino acids, among others
COMPARISON OR DIFFERENCE BETWEEN HPLC AND TLC
HPLC TLC
Unsuitable when large number of compounds is Suitable even when more compounds are present in
present in the sample the sample
Use of a large number of solvent is inconvenient Large number of solvents can be used at fairly
and costly reasonable cost.
Mixture of solute and solvent is eluted out and Solvent is lost before the detection of a solute and
hence, there is a possibility of interference in the hence, there is no possibility of interference in
measurement of absorbance of a solute measurement of absorbance of solute.
No danger of plate getting spoilt even when the
Very slow eluting compounds can spoil the column. interaction of a solute with stationery phase is
more.
Column is costly Plate is cheaper as compared to column.
COMPARISON OR DIFFERENCE BETWEEN
TLC AND HPTLC
TLC HPTLC
Limited number of surfaces is available commercially Along with silica gel and alumina, C8 and C18 are also
as stationery phase. available commonly.
Particle size (150-200 µm) and pores size of the Particle size (5-10 µm) and pore size of the stationery
stationery phase is bigger and non-uniform. phase is smaller and more uniform.
Thickness of stationery phase is 1-2 min. hence, more Thickness of stationery phase is 0.2nm. Hence,
time is required fro completion of analysis. analysis is complete in a short time period.
Sample capacity is comparatively larger (100µl). Sample capacity is smaller (6 to 20 µl).
All plate development methods are used except for Simultaneous development in two directions along
simultaneous development in two different directions. with usual developmental methods makes possible
analysis of more compounds at the same time.

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THIN LAYER CHROMATOGRAPHY

• Here, the solvent front travels down the length

• The paper with spotting on the line drawn a few

• Both ascending and descending techniques have

• This is also known as circular TLC.

• In this, a square or rectangular paper is used.

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