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Chromatography is the separation of a mixture in to a individual components using a
stationary phase and a mobile phase .There are two general types of chromatographic
techniques, planar and column systems. In planar chromatographic methods,
separation is usually terminated after a fixed time and the different distances moved
by each zone are measured. In column technique, the path length for each compound
in a mixture is the same and it is the time taken to travel this distance, which is

Thin Layer Chromatography

A thin layer of sorbent, such as alumina, silica gel, cellulose or crosslinked dextran, is
uniformly coated on a glass or plastic plate. Each sample to be analyzed is applied as
a spot near one edge of the plate. The mobile phase is usually placed in a closed
container until the atmosphere is saturated with solvent vapors . One edge of the plate
is placed in the solvent. The solvent migrates up the thin layer by capillary action,
dissolving and carrying sample molecule. Separation depending on the sorbent and
solvent chosen. After the solvent reaches a predetermined height, the plate is removed
and dried. Sample components are identified by comparison with standards on the
same plate.
The distance a component migrates, compared with the distance the solvent front

moves, is called the retention factor Rf. Rf value is a valuable diagnostic measure for

the identification of components.

Rf     =       Distance leading edge of component moves

               Total distance solvent front moves
TLC most commonly used as a semi quantitative screening test. Technique
refinement has resulted in the development of semi-automated equipment and the
ability to quantitate the separated compounds. Absorbance at each developed spot is
measure using a densitometer, and the concentration is calculated by comparison with
a reference standard chromatographic under identical condition.

Advantages of TLC
           Simple and cost effective method
           Rapid technique and not time consuming like column chromatography
           Any type of compound can be analyzed
           Separation of microgram of the substance can be achieved
           Detection is easy and not tedious
           Capacity of the thin layer can be altered and 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

Applications of TLC
The applications are wider and there is no limitation to the compounds that can be
analyzed by TLC. Different types of applications are,
              For checking the purity of samples
              For identifying organic compounds
              Separation     of   carbohydrates,   vitamins,     antibiotics,   proteins,
                alkaloids, glycosides etc.
              Examination of reaction

High Performance Thin Layer Chromatography (HPTLC)
It is useful in qualitative and quantitative analysis of pharmaceuticals. HPTLC is a
major advancement of TLC principle requiring shorter time and better resolution. The
basic difference between conventional TLC and HPTLC is in particle and pore size of
adsorbents and the sample application and development. The HPTLC plates are
similar to conventional TLC plates but they differ in particle size, uniformity, and
layer thickness of stationary phase. The use of smaller particle size helps in greater
resolution and sensitivity. About 3-6 cm solvent front migration is sufficient to effect
proper separation. Sample preparation in HPTLC needs high concentrated solution, as
very less amount of sample needed be applied. The size of the sample spot must not
exceed1 mm in diameter. There are different techniques for the spotting of samples;
the linear development method is most familiar technique in HPTLC. Here the plate is
placed vertically in solvent system in a suitable container. The solvent is usually fed
by capillary action and chromatogram can be developed from both sides. Circular
development, anti-circular device, and multiple developments are some of the other
methods which are used for development of plates. HPTLC is rapidly gaining
importance in biochemistry of natural products and in analysis of bio fluids the infield
of pharmacokinetics. HPTLC is now days applied to obtain “Finger-Print” patterns of
herbal formulations, quantification of active ingredients and detection of adulteration.
The following are the advantages of HPTLC over simple TLC technique.

              The use of pre coated plates with stationary phase particle size of
                 less than 10 electrons in diameter.
              Wide choice of stationary phases like silica gel, for normal phase and
                 C18, C8 etc. for reverse phase mode
              Auto sampler instead of manual spotting and streaking for
                 preparative purpose
              New types of development chambers, which requires fewer amounts
                 of solvents for developing
              More efficiency because of smaller and uniform size of adsorbents
              The use of UV/Visible/ Fluorescence scanner, which scans the entire
              Improved Data processing capabilities by the use of computers

The technique of high performance liquid chromatography is so called because of its
improved performance when compared to classical column chromatography. It is also
called as High Pressure Liquid Chromatography. Since high pressure is used when
compared to classical column chromatography. The development of HPLC from
classical column chromatography can be attributed to the development of smaller size
particles. Smaller particle size is important since they offer more surface area cover
the conventional larger particle sizes. The porous particle of 5 microns offers a
surface area of 100-860 sq.meters/gram with an average of 400 m2/g. These offer very
high pate counts up to 1,00,000/meter. Depending up on the mechanism of separation
and materials used for stationary phase and mobile phase HPLC can be classified in to
two types of systems.

The technique of HPLC is so called because of its improved performance when
compared to the classical chromatography. It is also called high pressure liquid
chromatography since high pressure is used.

The development of HPLC from classical column chromatography can be attributed
to the development of smaller particle sizes. Smaller particle size is important since
they offer more surface area over the conventional larger size particles.

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