thin layer chromatography.....

1. THIN LAYER
CHROMATOGRAPHY

2. What is Chromatography
and Thin layer
Chromatography?
Principle of TLC
Follows which specific
principle for
chromatography?
TLC technique
Method and steps of TLC . How TLC
works and generate reults.
Analysis
Analysis of Retention
factor for separtion,
identification and
purification purpose
Limtations & Application
Importance of TLC in
different fields, advantages
of process and its some
limitations.
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Introduction

3. ● TLC is a technique used to isolate non-
volatile mixtures. It is a method for analyzing
the mixtures by separating compounds in the
mixture. TLC is a form of liquid
chromatography consisting of two phases:
A mobile phase (liquid) and
A stationary phase (solid).
• Differences in the interactions between the
solutes
and stationary and mobile phases enable
separation.

4. TLC is based on separation principle i.e. Major
priciple involves in TLC is Adsorption.
• TLC technique involves the distribution of
components of a mixture to be separated between
two phases.
• The components of the mixture are partitioned
between an adsorbent (stationary phase), and a
solvent (mobile phase).
• Different compounds will have different solubility
and adsorption to the two phases between which
they are to be partitioned.
• In TLC separation of the individual substances is
based on their relative affinities towards stationary
and mobile phases.
01

5. The stationary phase
It is a thin layer of
adsorbent . (usually
silica gel or alumina)
coated on a plate.
The mobile phase
It is a developing liquid
which flows through the
stationary phase,
carrying the samples
with it.
Nature
Components with more
affinity towards
stationary phase travels
slower. Components
with less affinity towards
stationary phase travels
faster.

6. Selection of
Adsorbents and
Solvents:
• Adsorbent should not adhere to
glass plate.
• Solvents should be of high
purity.
• Selected based on the nature of
the compound to be separated
(polar or non polar).
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Solvents used as
Mobile Phase:
Inorganic: Silica Gel, Aluminium
Silicate, Bentonite.
Organic: Cellulose & its
acetylates,
Charcoal & activated Charcoal,
Dextran Gel, Polyamides.
Adsorbents used as
Stationary Phase:
Petroleum ether,
Benzene, Carbon
tetrachloride.

7. TLC plate
Preferably ready made with a
stationary phase. These are
stable and chemical inert plates
where a thin layer of stationary
phase is applied on the whole
surface layer.
Mobile Phase
This comprises a solvent or
solvent mixtures. The mobile
phase used should be particulate
–free for proper development of
TLC spots. The solvents
recommended are chemically
inert.
TLC Chamber
This is used for the
development of TLC plate. The
chamber maintains a uniform
environment inside for paper
development of spots.
Filter paper
This is moistened in the mobile
phase to be placed inside the
chamber . This helps develop a
uniform rise in a mobile phase
over the length of stationary
phase.
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3 4
Diagrammatic Representation Of components
System Components

8. Spotting
the sample visualization
The visualization of colored
spots is easy as the colored
spots can be directly seen on
the silica gel plate.however,
most of compounds are
colorless and so visualization
method is required to see the
separate component.
The sample containing
mixture of compounds , which
is to be analyzed is dissolved
in a suitable volatile solvent
to produce a very dilute
solution. A pencil line can be
drawn near the bottom.The
spoting solvent evaporates
quickly leaving behind a small
spot.
Development
Once the spot is dried the
TLC plate is placed
vertically into a tight jar
containing the solvent.this
result depends upon 3
components. Polarity of
the plate ,the polarity of
development solvent and
polarity of components in
spotting sample.
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02

9. .
The Components
visible as separated
spots can be
analyzed by distance
traveled by the
Then
Rf value is
calculated as
shown in
diagram.
component
to that reference
values. once the
solvent reaches the
top the plate is
removed and the
distance travelled by
the component is
measured

10. Different adsorbents will give different values for same
solvents.
Although precise control of temperature is
not necessary , the tank should be kept
away from draughts, sources of heat and
direct sunlight etc.
Nature of adsorbent
Mass of sample
Increasing the mass of sample on the
plate will often increase the Rf of a
drug , especially if it normally tail in
the system.
Temperature Thickness of layer
Standard plates approximately
250micro meter is preferable
thickness of layer. The layers may
be of higher or lower thickness in
individual compounds
Technique
Depending upon the development
technique used i.e. ascending ,
descending , horizontal etc. the Rf value
changes for the same solvent system.
Mobile phase
The purity of solvents and quantity
of solvents mixed should be strictly
controlled.

11.  It is a simple process with a short development
time.
 It helps with visualization of separated
compounds spots easily.
 The methods help to identify the individual
compounds.
 The purify standards of the given sample can
be accessed easily.
 It is a cheaper chromatography technique.
 It is possible to visualize the components
through UV light.
 It is also useful if a small volume of a sample is
available and does not require large volume of
a sample.

12. Do not recogonize the
differences between isomeric
and enantiomeric forms of a
compound
. It gives best results for qualitative
analysis but not for quantitative
analysis.
.
Some of the TLC plates do not have long
stationary phases. In such cases, it poses the
problem of the limitation of the length of
separation for the mixture because greater the
length of the plate, finer would be the
separation of the mixture into individual
components
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Since TLC operates as an open
system, some factors such as
humidity and temperature can
be the consequences of the
final outcome of the
chromatogram
.
TLC is applicable to only non-
volatile compounds, thus limiting its
use.

13. It is widely used in separating multi
components pharmaceutical
applications. It is used in food
industry to separate and identify
colors , sweetening agents and
preservatives.
The qualitative testing of
various medicines such as
sedatives, local anesthetics
and steroids is done by TLC.
TLC can be used to identify
natural products like essential
oils, or volatile oil, fixed oil ,waxes
,alkaloids etc.
separation
Qualitative testing
purification
It is used to purify any sample and
direct comparison is done between
the sample and authentic sample.
Identification

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