Tishk I n t e r n a t i o n a l U n i v e r s i t y

FACULTY OF PHARMACY
Department o f Pharmacognosy

PHARMACY

Introduction of THIN LAYER chromatography

(Pharmacognosy-III)
Practical- 1 week-2

G r a d e 4- Fall s e m e s t e r 2020-2021

Dr. Omji P o r w a l
PhD., M.Pharm, PGDPRA , B.Pharm
• Iodine also can be used as a locating agent
 Visualizing Agents

Reagents Compounds
Iodine UV light Aromatic compounds Unsaturated
p-Anisaldehyde compounds Carbohydrate
Bromocresol green carboxylic acid
2,4-dinitrophenylhydrazine Ninhydrin Mainly for aldehydes and ketones Good
Sulfanilic Acid Reagent (Diazotized), for amines
Pauly's Reagent Sulfuric acid phenolic compounds turn orange or
Aniline phthalate yellow with this reagent sprayed on the
Antimony trichloride TLC
Dragendorff’s reagent Sugar
Cardiac glycosides
Alkaloids
Interpreting the Data

The Rf (retention factor) value for each spot should

be calculated.

It is characteristic for any given compound on the

same stationary phase using the same mobile phase
for development of the plates.

Hence, known Rf values can be compared to those of

unknown substances to aid in their identifications.
 Evaluation of chromatogram
• After locating the spots on plates and making their
position and size, they are evaluated either
qualitatively and quantitatively.
• Qualitative:
• In this case, the Rf values of standard or authentic
sample for the same mobile phase is known and is
recorded in literature.
• The Rf value of the sample is calculated and on
comparison with standard we can made
identification of sample.
 Quantitative
• Visual comparison: Spot size, intensity of spot and combination of
two with the known standard spots is made.

• Spot area and weight relationship: area of spot, amount of

substance present is calculated.

• Spot dentiometery: after development of the chromatogram the

plate is sprayed with specfic reagent and color development.

• Direct spectrometry: quantitative measurement s are obtained by

reading the absorption of seperated zones directly on tlc plates at
wavelenth of maximum absorption by spectrometry.

• Spectral reflectance: the spectral reflectance of dyes absorbed on

adsorbent has been investigated as a quantitative technique fir
TLC.
 Applications of TLC
• It is used for separation of all classes of natural
products and is established as an analytical tool in
modern pharmacopoeias.
• - E.g. Acids, alcohols, glycols, alkaloids, amines,
macromolecules like amino acids, proteins and
peptides, 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.

• As a Check on process – checking of distillation fractions and

for checking the progress of molecular distillation.

• Applications of TLC for separation of Inorganic Ions – Used for

separating cationic, anionic, purely covalent species and also
some organic derivatives of the metals.

• Separation of Amino Acids- two dimensional thin – layer

chromatography
• Separation of vitamins – vitamin E, Vitamin D3, vitamin A
THANKS