Date: -09-09

SEPERATION AND IDENTIFICATION OF DIGITALIS

GLYCOSIDES BY TLC.

AIM: To carry out the TLC of the given sample of drug (Digitalis powder).

REQUIREMENTS: Beakers, round bottom flask, TLC plate, hot air oven,
5% w/v potassium hydroxide solution, silica gel G, distilled water, methanol
iso-propanol , ethyl acetate, Dichloromethan , heating mantle, stirrers,
conical flask, pipette, measuring cylinder, mortar pestle, funnel, filter paper.

REFERENCE:
(1) “Herbal Drug Technology”, by S.S. Aggarwal, M. Pandhavi,
universities press, pg no 231, 258-262;
(2) “Plant Drug Analysis- a thin layer chromatography atlas”, 2 nd edition
by H. Wagner, pg no 99-113;
(3) “Quality Control of Herbal Drugs- an approach to evaluation of
botanicals” by Dr. Pulok mukherji, pg no 426-482.

THEORY:
Introduction: chromatography is a broad science of physical method meant
to separate and analyse complex molecular mixtures. It depends on the
differential affinities of the solutes in two immiscible phases, a fixed bed
with a large surface area of a fluid which moves through or over the surface
of the fixed on stationary phase.

TLC is a method of analysis in which the stationary phase, a finely divided

solid is spread as a thin layer on a rigid supporting plate and the mobile
phase, a liquid, is allowed to migrate across the surface of the plate by
capillary action.
Adsorbents for TLC: various types of tested adsorbents are available for
TLC. They differ from the usual materials in that their structure is fine
graded i.e. finely divided, with the grain size of the adsorbent lying between
5 to 50 and passing through a number 200 screen.
(1) Silica gel: it is the most extensively used adsorbent. It is employed as
such for adsorbent TLC and muclified for reverse phase
chromatography using substance such as octadecylsilyl to it. It is
suitable for constituents like amino acids, alkaloids, sugars, fatty
acids, lipids, essential oils, steroids and terpenoids.
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(2) Alumina: it has basic surface and is chosen over silica gel for
separation of weakly polar compounds. Similar to silica gel alumina
can be obtained in various forms like alumina and alumina H1 and
alumina F 254 etc. It is suitable for alkaloids, food dyes, phenols,
steroids, vitamins, carotenoids and amino acids.
(3) Kieselguhr: it is a natural adsorbent with low. It is not used to a very
large extent. Other inorganic adsorbents are CaSO4, magnesium
silicate, magnesium oxide, bentonite etc. Kieselguhr is suitable for
sugars, dibasic acids, fatty acids, triglycerides, amino acids and
steroids.
Organic adsorbents: cellulose and its derivatives are used exclusively for
separation of hydrophobic compounds like amino acids, nucleic acids,
carbohydrates and closely related isomers. It is suitable for amino acids,
food dyes and alkaloids.
Polyamides (nylon): nylon is a long chain polymer which because of the
presence of many free amide of carboxylic on its surface is an adsorbent
with strong hydrogen bonding abilities. It is suitable for anthocyanins,
antioxidants, flavonoids and proteins.

Solvent system: the solvent system is chosen by the trial and error method.
The rate of migration of a compound depends on the solvent used. The
simplest systems are mixtures of organic solvent used to separate mono and
bi-functional molecules by adsorptive chromatography on layers of activated
silica gel or alumina. Solvents at the bottom of the series are polar and move
most of the compounds where as those at the top are non-polar and move
few compounds.
If the chemical nature of the solute to be separated is known, a suitable
solvent can be selected using stahl’s triangle.

Selection of supporting plate: the carrier on supporting plate is the backbone

of the entire chromatographic apparatus, and hence it must be stable in
presence of all types of solvents, reactive spray reagents, and also at high
temperatures. Glass plates fulfill these requirements best. Their thickness is
usually chosen between 1.8mm to 5.5mm. Borosilicate glass is also used
when a reaction is carried out at very high temperature.

Preparation of thin uniform layer: the film thickness varies from 0.2mm
generally 0.22mm thickness is used.

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Thin layer can be prepared by the following methods:

1. Dipping: plates are dipped into a slurry of the stationary phase
suspended in a volatile solvent. The solvent is then removed by air
drying or by heating the plates in an oven. The development time for
this method is very short.
2. Spraying: an aerosol spray is used to prepare thin layer, using a slurry
of suitable consistency.
3. Pouring: a known amount of slurry is poured onto the plate. The plate
is then tipped back and forth to spread slurry.
4. Spreading: an application as spreading is used and the layer thickness
can varied as desired.

Sample preparation and application: the dried are conditioned if necessary,

in a controlled humidity chamber. Samples ranging from a few micrograms
are dissolved in 10-100ml of volatile solvent. They are then applied as apots
or as thin streaks with a capillary tube or a micro line syringe. The solvent
maybe evaporated between successive applications. Area of sample
application should be as small as possible.

Development:--linear
--radial
--extended
--gradient
--temperature controlled

Chromato plates are placed inclined at about 45 degree in the tank. The tank
is closed with a lid and the solvent is allowed to move up the plate from the
point of application. When the mobile phase has traveled two-third of the
plate, the plate is removed from the chamber, the solvent front is marked and
allowed to dry. The various development techniques are:

1. Linear development: the TLC plates are developed linearly in the

ascending, descending or horizontal mode. In ascending mode, the
sample is spotted at one end of the plate and then developed in the
ascending direction. It is known as vertical development. In the
descending mode the solvent is allowed to flow from the reservoir to
the plate through a strip of filter paper. It has no advantage in terms of
efficiency of separation and speed analysis.

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2. Radial development: it is also known as circular TLC. It is performed

on horizontal chromate plates.
3. Extended development: it consists of the following techniques:
Continuous development technique: it is used to achieve
complete separation of compounds which resolve with
small differences in Rf value.
Multiple development technique: in this repeated
development is carried out with the same solvent in the
same direction, each time after drying.
Two-dimensional technique: it is used to separate
complex mixtures. The plates are developed in one axis
first and then, after drying they are developed in the other
axis with the same solvent or different solvent.
4. Gradient development: in this two different adsorbents are applied
simultaneously. Eg: temperature controlled development, in this
development is carried out at definite temperature.

Detection: before starting the procedure to detect various solutes, the

chromatogram is allowed to dry. This is done either at room temperature or
in an oven. Application of visualizing agent to help in detection is done in a
number of ways
- exposure of the chromatogram to vapours
- dipping the plate in reagent solution
- spraying or impregnating the plate with reagent
- allowing the solution to develop as in the normal TLC

The visualization procedures maybe classified, for convenience into

following:
1. Non-destructive method: UV method for fluoroscent compounds under
UV chamber at 254 & 365nm.
Iodine vapour treatment also widely used wherein some phyto constituents
show brown, amber or yellow zones after exposure to iodine vapour.

2. Reagents causing irreversible reaction: charring is widely used technique

for the detection of carbon containing compounds. This involves spraying
the plates with sulphuric acid and then heating in an oven at 110 degrees for
10-30mins. The organic compounds are destroyed and a dark point of carbon
remains.

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Analysis: in TLC, qualitative analysis of unknown compound is done by

comparing Rf values with standard values.
As solutes should never travel the full length of the stationary phase in TLC
all the Rf values are less than 1. The Rf value depends on the amount of
stationary phase, the humidity, layer thickness, solvent quality, saturation of
the chamber, development distance, temperature, amount of substance added
and presence of impurities.

Rf =distance from origin to point of maximum intensity

Distance from origin to solvent front.

Rf = retention time

PROCEDURE:

(a) Preparation of Extracts:-

 Weighed 2gm (>1% total cardenolides) or 10gm (<0.1% total
cardenolides) of powder drug.
 Extracted the drug by heating with 30ml 50% ethanol and 10ml
10% lead acetate solution under reflux for 15 minutes.
 Cool and filtered the extract and collected the filterate.
 Filterate was extracted with Dichloromethane & Isopropanol (3:2)
for 3 times. (Each time 15ml mixture was taken).[note:-care
should be taken to avoid emulsion formation by gental shaking].
 Combined the lower phase after sepration and is filtered over
anhydrous sodium sulphate.
 Evaporated the filterate to dryness and collected the residue after
evaporation.
 Residue was dissolved in 1ml Dicholoromethane & Isopropanol
mixture (3:2) and used for chromatographic sampling.

(b) Preparation of standard Solution:-

 5mg of Digoxin was dissolved in 2ml methanol.
(c) Adsorbents:- Silicagel G
(d) Solvent system:-
 Ethylacetate:Methanol:Water (81:11:08)
(e) Spraying reagent:- Kadde’s reagent.
 Sample solution + Kadde’s reagent blue/violet colour.

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 Kadde’s reagent (3,5-di nitro benzoic acid in methanol + 5% w/v

solution of KOH).

RESULT:

CONCLUSION:

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