Study Set Content:
Developing Chamber Commonly made of () with glass tanks being the preferred material due to their clarity and chemical
inertness.
glass, plastic or
stainless steel
4. Detecting or visualizing agents – Two methods:
Nonspecific Method and Specific Method.
where the number of spots can be detected but not the exact
nature of type of compound.
Non-Specific Method
Specific spray reagents or detecting agent or visualizing agent are
used to find out the nature of compound.
Specific Method
Non-Specific Method Example:
Iodine Chamber Method-
UV Chamber for fluorescent compound
Where brown or amber spots are observed when
the TLC plates kept in a tank with few iodine crystals at the bottom.
Iodine Chamber Method-
When compound are viewed
under UV Chamber at 254 nm or 365 nm fluorescent compound can be
detected.
UV Chamber for fluorescent compound
Specific spray reagents or detecting agent or visualizing agent are
used to find out the nature of compound.
Specific Method
Specific Method Example:
Ferric Chloride
Ninhydrin in Acetone
Dragendroff Reagents
– For phenolic compounds and tannins
Ferric Chloride
For amino acids
Ninhydrin in Acetone
For alkaloids
Dragendroff Reagents
The spots are kept at the bottom portion of the paper and kept in a
chamber with mobile phase solvent at the bottom.
Ascending Development
– In this method solvent moves from top to bottom.
Descending Type
Only length of separation increased, first ascending
takes place followed by descending.
Ascending-Descending Development
The solvent flows through a wick at the center & spreads in all
directions uniformly.
Circular/Radial Development
Uses two solvents and rotates the paper 90 degrees in between. Also known
as two-way chromatography.
Two-dimensional
LIMITATIONS OF PAPER CHROMATOGRAPHY
1. Paper chromatography cannot handle large amounts of samples.
2. Paper chromatography is ineffective in quantitative analysis.
3. Paper chromatography cannot separate complex mixtures.
4. Less accurate than HPLC or HPTLC
APPLICATIONS:
Paper chromatography has evolved over the years and has found widespread applications in separation of
molecules of different polarities. Innumerable applications have been reported in analysis of different
classes of compounds such as:
o Amino acids and organic acids
o Alkaloids
o Polysaccharides
o Proteins and peptides
o Natural and artificial pigments
o Inorganic cations
o Plant extracts
Widely employed laboratory technique and is similar to paper chromatography. However,
instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of
adsorbent like silica gel, alumina, or cellulose.
Thin layer Chromatography
