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Jadavpur University

College :- Bengal Institute Of


Pharmaceutical
Sciences

Name :-Kuntal Mitra

Roll No :- 11

Class :- 3rd Yr 6th sem

Subject :-Pharmacognosy (Paper-2)

Topic :-TLC

Course :-B.Pharm (Ayu)

Separation of black ink on a TLC plate


CHROMATOGRAPHY
Introduction:-

Chromatography is the separation of a mixture into individual components using a


stationary phase & a mobile phase.

Types of Chromatography:-

1. Based upon the nature of Stationary phase & mobile phase:

 Gas –Solid Chromatography


 Gas –liquid Chromatography
 Solid –liquid Chromatography
 Liquid –liquid Chromatography

2. Based on the principle of separation:

 Absorption Chromatography
 Partition Chromatography

3. Based on the modes of Chromatography:

 Normal Phase Chromatography


 Reverse Phase Chromatography

4. Other Types of Chromatography:

 Ion-exchange Chromatography
 Gel Permeation Chromatography
 Chiral Chromatography
THIN LAYER
CHROMATOGRAPH
Y
(TLC)

TLC of Kalmegh Leaf extract in time of my WBPPDCL training.


History:-

The history of TLC dates back to 1938 when Izmailov & Shraiber separated plant
extracts using 2mm. thick & firm layer of alumina set on glass plate. In 1944,
Consden, Gorden & Martin used filter papers for separating amino acids. In 1950,
Kirchner identified terpenes on filter paper & later glass fiber paper coated with
alumina. Only in 1958, Sthal developed standard equipment for analyzing by thin
layer chromatography.

Introduction:-

Thin Layer Chromatography (TLC) is a chromatography technique used to


separate chemical compounds. It involves a stationary phase consisting of a thin
layer of adsorbent material, usually silica gel, aluminum oxide, or cellulose
immobilized onto a flat, inert carrier sheet. A liquid phase consisting of the
solution to be separated is then dissolved in an appropriate solvent and is drawn
up the plate via capillary action, separating the experimental solution based on
the polarity of the components of the compound in question.

Principle:-

The basic principle of TLC mainly depends upon the adsorption chromatography.

The most commonly used solvents utilized in this form of


chromatography are silica & alumina. As the components move through the
sorbates their relative rate of migration are affected by their individual affinities
for the sorbents. Separation occurs when one compound is more strongly
absorbed by the sorbents than the other components. When sorbent is silica or
alumina, polar natural products moves slowly compare to non-polar natural
products. Adsorption takes place because of the interaction between the
compound & groups associated with the sorbents. In case of silica, binding occurs
between the compound & the free hydroxyl groups of the sorbates.In this
particular case adsorption involves the hydrogen bonding between compound
functional groups & adsorbent surface hydroxyl group.
Types of Stationary Phase (Silica):-

Name Meaning
G Gypsum
H Containing no foreign matter
R Specially purified
P For preparity work
W Water tolerant
C Channeled
RP Reverse phase
F Fluorescence (60F254)

Retention Factor (Rf):-

The retention factor, or Rf, is defined as the


distance traveled by the compound divided
by the distance traveled by the solvent.

For example, if a compound travels 2.1 cm


and the solvent front travels 2.8 cm, the Rf is
0.75.

The Rf for a compound is a constant from one experiment to the next only if the
chromatography conditions below are also constant:

 solvent system
 adsorbent
 thickness of the adsorbent
 amount of material spotted
 temperature
Practical Requirements:-

 Stationary Phase
 Glass Plates
 Preparation & activation of TLC plates
 Application of sample
 Development Tank
 Mobile Phase
 Development technique
 Detecting or visualizing agent

Plate preparation:-

TLC plates are made by mixing the adsorbent, such as silica gel, with a small
amount of inert binder like calcium sulfate (gypsum) and water. This mixture is
spread as thick slurry on an unreactive carrier sheet, usually glass, thick aluminum
foil, or plastic, and the resultant plate is dried and activated by heating in an oven
for thirty minutes at 110 °C. The thickness of the adsorbent layer is typically
around 0.1–0.25 mm for analytical purposes and around 1–2 mm for preparative
TLC. Every type of chromatography contains a mobile phase and a stationary
phase.

Technique:-

The process is similar to paper chromatography with the advantage of faster runs,
better separations, and the choice between different stationary phases. Because
of its simplicity and speed TLC is often used for monitoring chemical reactions and
for the qualitative analysis of reaction products.

A small spot of solution containing the sample is applied to a plate, about one
centimeter from the base. The plate is then dipped in to a suitable solvent, such
as ethanol or water, and placed in a sealed container. The solvent moves up the
plate by capillary action and meets the sample mixture, which is dissolved and is
carried up the plate by the solvent. Different compounds in the sample mixture
travel at different rates due to differences in solubility in the solvent, and due to
differences in their attraction to the stationary phase. Results also vary depending
on the solvent used. For example, if the
solvent were a 90:10 mixture of hexane to
ethyl acetate, then the solvent would be
mostly non-polar. This means that when
analyzing the TLC, the non-polar parts will
have moved further up the plate. The polar
compounds, in contrast, will not have moved
as much. The reverse is true when using a
solvent that is more polar than non-polar
(10:90 hexane to ethyl acetate). With these
Chromatogram of 10 essential oils colored with vanillin
reagent. Solvents, the polar compounds will
move higher up the plate, while the non-polar
compounds will not move as much.

The appropriate solvent in context of thin layer chromatography


will be one which differs from the stationary phase material in polarity. If polar
solvent is used to dissolve the sample and spot is applied over polar stationary
phase TLC, the sample spot will grow radially due to capillary action, which is not
advisable as one spot may mix with the other. Hence, to restrict the radial growth
of sample-spot, the solvent used for dissolving samples in order to apply them on
plates should be as non-polar or semi-polar as possible when the stationary phase
is polar, and vice-versa.

Visualizing the Spots:-

As the chemicals being separated may be colorless, several methods exist to


visualize the spots:

 Often a small amount of a fluorescent compound, usually Manganese-


activated Zinc Silicate, is added to the adsorbent that allows the
visualization of spots under a backlight (UV254). The adsorbent layer will
thus fluoresce light green by itself, but spots of analyte quench this
fluorescence.
 Iodine vapors are a general unspecific color reagent
 Specific color reagents exist into which the TLC plate is dipped or which are
sprayed onto the plate.

Stages of Development:-

Chromatography of an extract of green leaves (for example spinach) in 7 stages of


development. Carotene elutes quickly and is only visible until step 2. Chlorophyll
A and B are halfway in the final step and lutein the first compound staining
yellow.

Step 1 Step 2 Step 3 Step 4

Step 5 Step 6 Step 7


Factors influencing TLC:-

1. Polarity or nature of Stationary Phase


2. Polarity or nature of mobile Phase
3. Relative Humidity
4. Saturation Chamber
5. Particle size of the adsorbent used in stationary Phase
6. Flow of the mobile Phase
7. Temperature
8. pH of the mobile phase
9. Sample volume
10. Thickness of solvent of the stationary Phase
11. Solvent used in sample
12. Migration distance
13. Impurities in the mobile phase
14. Rf value
15. Spot size on application

Advantages of TLC over other Chromatographic techniques:-

 Simple equipment
 Short development time
 Wide choice of Stationary Phase
 Early recovery of separated components
 Easy visualization of separated vaporizes
Troubleshooting TLC:-

All of the above (including the procedure page) might sound like TLC is quite an
easy procedure. But what about the first time you run a TLC, and see spots
everywhere and blurred, streaked spots? As with any technique, with practice you
get better. One thing you have to be careful Examples of common problems
encountered in TLC:

 The compound runs as a streak rather than a spot

The sample was overloaded. Run the TLC again after diluting your sample. Or,
your sample might just contain many components, creating many spots which run
together and appear as a streak. Perhaps, the experiment did not go as well as
expected.

 The sample runs as a smear or a upward crescent.

Compounds which possess strongly acidic or basic groups (amines or carboxylic


acids) sometimes show up on a TLC plate with this behavior. Add a few drops of
ammonium hydroxide (amines) or acetic acid (carboxylic acids) to the eluting
solvent to obtain clearer plates.

 The sample runs as a downward crescent.

Likely, the adsorbent was disturbed during the spotting, causing the crescent
shape.

 The plate solvent front runs crookedly.

Either the adsorbent has flaked off the sides of the plate or the sides of the plate
are touching the sides of the container (or the paper used to saturate the
container) as the plate develops. Crookedly run plates make it harder to measure
Rf values accurately.
 Many, random spots are seen on the plate.

Make sure that you do not accidentally drop any organic compound on the plate.
If get a TLC plate and leave it lying on your workbench as you do the experiment,
you might drop or splash an organic compound on the plate.

 No spots are seen on the plate.

You might not have spotted enough compounds, perhaps because the solution of
the compound is too dilute. Try concentrating the solution, or, spot it several
times in one place, allowing the solvent to dry between applications. Some
compounds do not show up under UV light; try another method of visualizing the
plate. Or, perhaps you do not have any compound because your experiment did
not go as well as planned.

If the solvent level in the developing jar is deeper than the origin (spotting line) of
the TLC plate, the solvent will dissolve the compounds into the solvent reservoir
instead of allowing them to move up the plate by capillary action. Thus, you will
not see spots after the plate is developed.

 You see a blur of blue spots on the plate as it develops.

Perhaps, you used an ink pen instead of a pencil to mark the origin.

Application of TLC:-

1. (a) For separating of essential oils containing drug with the help of mobile
phase (Benzene:ethylacetate=95:5) or (Benzene:ethylacetate=9:1), can be
separated like Eucalyptus Oil.
TLC of Halud in time of my WBPPDCL training

(b) With the help of solvent system CHCl3:C2H5OH:Glacial CH3COOH


=94:5:1, we can separate the pigments from Curcuma rhizome.

2. In case of alkaloidal drugs we can use the solvent system like


CHCl3:diethylamine =9:1 for separation.
3. For the separation of cardiac
glycoside (digitalis,digitoxin)
with the help of
CHCl3:CH3OH:H2O =64:50:10
4. For the separation of flavonoids
we can use the solvent system
or mobile phase
CHCl3:ethylacetate =6:4.
5. When we can not separate the
mixture of components by the
Column Chromatography then
the preparative TLC is
performed to obtain the each
compounds ,from the Rf of the
spot or sample.

Preparative TLC of Kalmegh to isolate


andrographolide in time of my WBPPDCL training
6. Determination of the pigments a plant contains
7. Detection of pesticides or insecticides in food
8. Analyzing the dye composition of fibers in forensics,

9. Identifying compounds present in a given substance .

References:-

 Vogel's Textbook of Practical Organic Chemistry


 Text book of Pharmaceutical Analysis
 Pictures are taken from my training in WBPPDCL Drug Testing Lab.
 Internet

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