Jadavpur University: College
Jadavpur University: College
Jadavpur University: College
Roll No :- 11
Topic :-TLC
Types of Chromatography:-
Absorption Chromatography
Partition Chromatography
Ion-exchange Chromatography
Gel Permeation Chromatography
Chiral Chromatography
THIN LAYER
CHROMATOGRAPH
Y
(TLC)
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:-
Principle:-
The basic principle of TLC mainly depends upon the adsorption chromatography.
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)
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.
Stages of Development:-
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 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.
Likely, the adsorbent was disturbed during the spotting, causing the crescent
shape.
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.
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.
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
References:-