Thin Layer

Chromatography
 Thin layer chromatography (TLC)
is a method for identifying substances and
testing the purity of compounds.

TLC is a useful technique because it is

relatively quick and requires small
quantities of material.
Separations in TLC involve distributing a mixture of two
or more substances between a stationary phase and a
mobile phase.

The stationary phase:

is a thin layer of adsorbent (usually silica gel or alumina)
coated on a plate.

The mobile phase:

is a developing liquid which travels up the stationary
phase, carrying the samples with it.
Components of the samples will separate on the
stationary phase according to
how much they adsorbed on the stationary phase versus
how much they dissolved in the mobile phase.
 STATIONARY PHASE

Silica is commonly used as stationary

phase
The separation of sample mixture will be

depent on the polarity of sample.

Some modified silica is also used in

certain purposes.
prof. aza 5
 Stationery phase Description Application

Silica gel G Silica gel with average Used in wide range

particle size 15m pharmacopoeial test
containing ca 13%
calcium sulfate binding
agent

Silica gel G254 Silica gel G with Same application with

fluorescence added Silica gel G where
visualization is to be
carried out under UV
light.

Cellulose Cellulose powder of Identification of

less than 30m particle tetracyclines
size.
prof. aza 7
 MOBILE PHASE

The ability of mobile phase to move up is

depent on the polarity itself

Volatile organic solvents is preferably used as
as mobile phase.
 MOBILE PHASE

SOLVENT POLARITY INDEX

Heksana 0

Butanol 3.9

Chloroform 4.1

Methanol 5.1

Ethanol 5.1

Acetonitrile 5.8

Air 9.0
MATERIALS
TLC plate
Developing container
- chamber/ jar/ glass beaker
Pencil
Ruler
Capillary pipe
Solvents / mobile phase
- organic solvents
UV lamp
METHOD
Thin Layer Chromatography (TLC)
 1.Developing Container
Preparation

Solvent is transferred
into the container with
0.5-1cm in dept from the
bottom
 2. TLC Plate Preparation
Commercialy obtained with
5cm x 20cm in size
Prepare your size when
neccesary
Line 1 cm from the bottom
with a pencil as a part should
be spotted.
 3.Spotting TLC plates

Make sure that your sample is

liquified already.
stick it using capillary pipe &
spott onto the line you have made

 4.Develop the plate
after spotting, put the plate inside
the chamber in the ascendant
position
Make sure that the dept of solvent
doesntb touch the spots
Let it develop up to the 0,5-1cm
from the top of plate
After that, pull out the plate from
the chamber and let the solvent be
vaporized
 5. Detection of spots
The color samples are -
easy to be seen and no
need to use UV lamp to
detect them
Iodination-put the plate in which the spots face to (1
the iodine crystall and see what is the spot color
changing
Ninhydrin: (2

-spesific identification of amino acid compounds.

- Ninhydrin solution will show a purple spot when it
is sprayed to the amino acid spot.
KMnO4 (3
used to identify a reducing agent such as glucose,
fructose, vitamin C and others.
Alkaline tetrazolium blue (4
specificaly used for corticosteroid identification
 Iodine vapour
The plate is put into a tank containing iodine
crystals. This treatment will produce brown
spots with many organic compounds; the
staining is reversible, so that if it is
necessary to recover the compound once it
has been located, the iodine may be allowed to
evaporate by exposing the plate to air and
then the marked spot containing the
compound of interest may be scraped off the
plate

prof. aza 19
 If a permanent record of the plate is
required it has to be covered to prevent
the iodine evaporating or the iodine
spots may be sprayed with starch
solution in order to stain them
permanently. Iodine is used as a location
agent in pharmacopoeial TLC tests of
fixed oils and of cetrimide.

prof. aza 20
 Potassium permanganate
Potassium permanganate provides a

method for the detection of sugars and

sugar- like molecules, and drugs with
aliphatic double bonds.
It is used in TLC identity checks for the
antibacterial agents clindamycin and
lincomycin and in a check for related
substances in spectinomycin.

prof. aza 21
 Ninhydrin solution
This reagent gives pink spots with primary

amines and yellow spots with tertiary amines.

It is used in pharmacopoeial identity tests for
some of the aminoglycoside antibiotics such
as gentamycin, in a limit test for aminobutanol
in ethambutol and can be used as a general
screen for nitrogen-containing drugs in
conjunction with Dragendorff reagent.

22
 Dragendorff reagent will produce
orange spots with tertiary amines and
may be used to overspray plates which
have been sprayed in the first instance
with ninhydrin.

prof. aza 23
 Alkaline tetrazolium blue
This reagent is quite specific for
corticosteroids producing blue spots on
a white background. The tetrazolium
spray is used in a test for related
foreign steroids in fluclorolone
acetonide.

prof. aza 24
 Ethanol/sulphuric acid 20%

This reagent is used to produce

fluorescent spots from corticosteroids
such as dexamethasone or prednisolone
by spraying the plate, heating to 120C
and then observing the plate under UV
light at 365 nm.

prof. aza 25
 Visualizing Agents
Alkaloids: Dragendorffs reagent

Cardiac glycosides: Antimony trichloride

Sugar: Aniline phthalate

Amino acids: Ninhydrin

Identification unknown drugs using standard
Reference
TLC
 The use of Rf as separation
parameter
- The distance taken through by the solvent to move up will be assigned as
solfent front
The distance taken trrough by the sample to move up will be assign as -
sample front
Rf value is obtained by dividing the sample front toward solvent front -

Rf = sample front
solvent front

-
 Thin-Layer Chromatography:
Determination of Rf Values
solvent front

Rf of component A =
dA component B
dS
Less polar!
dS
dB
Rf of component B =
dB
component A
dS
More polar!
dA
The Rf value is a decimal
fraction, generally only origin
reported to two decimal
places
6. Quantitative determination of known sample

Done by scratching the spot using spatula, and -

extract the compound using the suitable
solvent
The liquid extract can be determined its -
content using other method such as
spectroscopy.
 Problems commonly occur in TLC and
how to solve
a. The spot shape is too broad
- Diameter is supposed to be < 1-2mm
b. The movement of solvent
- should be straight up
- unproportionality in stationary phase
surface will inhibit the movement of solvent
c. streaking formation
- caused by too concentrated sample
 TLC Compared to Paper
Chromatography

1. Precise and effective

2. More stable toward various organic solvents
 Advantages
Cheap

Simple

The developing can be monitored visually

Able to use various chemical as a detector

RF VALUE Depends on number of factors :

Nature of Adsorbent: Different adsorbents

will give different Rf value for same solvent.
The Mobile phase: The purity of solvents and
quantity of solvent mixed also affect Rf value.
Activity: Temperature and storage conditions
of adsorbent also affects Rf value.
Thickness of layer: The layers may be of
higher or lower thickness which also varies Rf
value.
RF VALUE Depends on number of factors :

Equilibrium: Equilibrium of chamber used for

development is more important in TLC than in
paper chromatography and hence saturation of
atmosphere with the solvent vapour is
important.
The Temperature: Generally constant

temperature must be maintain to carry out

separation in order to avoid changes in solvent
composition.
RF VALUE Depends on number of factors :

Loading: Loading of sample must be small and

appropriate about 10micro gram. If loading is more
the spreading of spot and tailing may occur.
Dipping Zone: Distance of starting point from the
solvent surface is also a important factor.
Chromatographic Technique: Rf value is also

affected by different techniques applied for

chromatogaphy.
These are some of the possible factors which affects
Retention Factor (Rf ) Value.
Question: What is wrong with the plate shown
below?
THANK YOU