Basic Operations involved in TLC (Thin layer chromatography)



Basic Operations involved in TLC (Thin layer chromatography)

The basic operations which are involved in TLC are the following:
1. Preparation of chromatoplates.
2. Application of samples to ehromatoplates.
3. Choice of adsorbent.
4. Selection of solvent
5. Proper developing system.
6. Location of compounds on chromatoplates.
7. Detection and Identification.
PREPARATION OF PLATES
The size of the plates depends on the type of separation to be carried out
and on the type of chromatographic tank and spreading apparatus available. Most
of the commercial apparatus is designed for plates of 20 x 5 or 20 x 20 cm. and
these are now regarded as “standard“. It is important that the surface of the plate
shall he flat and without irregularities or blemishes. Glass plate are cleaned
thoroughly before use. Washed with water and a detergent, drained and dried. It is
important not to touch the surface of the cleaned plates with the fingers. The first
step is to make the adsorbent into slurry with water, usually in the proportion xg.
have adsorbent and 2x cm of water.

Commercially available applicator has got several specifications with the
help of which the thickness of layers is varied. The most popular thickness used is
0.25 mm. More thinner layers are mostly avoided because thicker layers are used
successfully. After putting the layers on the glass plates,they are activated which
is done by heating in an oven at 100-105 C for about half an hour.

The slurry is thoroughly stirred and spread on the plate by one of the methods
described below:

SEREAQLNG

It is possible to spread the adsorbent in a number of ways,but the main
objective is to produce an absolutely even layer with no lumps or gaps.
Commercial spreaders are in general of two type , in the first case speader is
Moving and in the second case plate is moving,the moving spreader type. the
Glass platest are held in a flat frame and a rectangular hopper containing the slurry
is passed them .in the moving plate apparatus the hopper containing the
fixed and the plates are pushed through under the hopper as indicated by
the diagram:
POURING

This method is only preferable if the adsorbent is very finely divided and
of homogeneous particle size. and if no binder is used, a slurry can be poured on a
plate and allowed to flow over it so that it is evenly covered. Some manual
practice is required to do this properly. Preparation of plates by pouring is
particularly easy with certain types of alumina. Water alone is usually suitable for
making the slurry; a volatile liquid such as ethanol (or an ethanol water mixture)
or ethyl acetate is preferable. The appropriate amount of liquid and solid
adsorbent needed to cover a plate has to be found by trial and error. Good and
even plates can be made by this method but the layer thickness of the layer is not
known.

SPRAYING
In this method slurry on the plates with a spraying gun, But this
method is not very popular due to uneven surface.
DIPPING
In this method small plates can be spread by dipping in slurry of the
adsorbent in chloroform or other volatile liquid. In this method. also, the exact
thickness of the layer is not known. The evenness of the layer may not be very
good. But this is a most convenient method for making a number of plates for
rapid qualitative separation. After spreading the plate is allowed to dry for 5-10
min. and if it has been made with aqueous slurry, it is further dried and activated
by heating at about 110 °C for 30 min. Plates made with volatile organic liquid
may not need this further drying. Then plates are kept in a desicator.

APPLICATION or SAMPLES
The methods for the application of the sample on the chromatoplates are
almost similar to those used in paper chromatography. Still there are few basic
differences. Samples are applied in dilute solutions with a micropipette or a
syringe. The solvent in which the sample is dissolved for spotting should be as
volatile as possible, and also have as low a polarity as possible. Spots should not
be nearer than 1cm centre to centre. A volume of 0.1-0.5 mm should be applied.

DEVELOPMENT

Once the chromatographic plate has been prepared and the samples have
been applied to it. It is placed in a suitable chamber with the lower edge immersed
in the eluent to a depth of 0.5 – l.0 cm. The tank is then closed so that chamber is
fully saturated with the solvent. A number of methods of development have been
used in TLC. Those still commonly employed are detailed below.

ASCENDINGE TECHNIQUE

This is the simplest technique and remains the most popular. In this
technique. The TLC plate is positioned in the development tank after it has come
to equilibrium with the solvent: The application of sample spots should be above
the solvent level. The solvent spreads through the sorbent material by capillary
action. This moves the components to differing extent which determined by
their distribution coefficient,in the direction of flow of the eluent, as shown in the
diagram.

DESCENDING TECHNIQUE

This technique is most commonly used in paper chromatography, but also
used in TLC. The top of the plate, where the spots are located has solvent in a
trough. The solvent flows downward. Some solvent of the same composition is
also placed in the bottom of the tank but the plate is supported above the solvent
level .lt will saturate the chromatographic chamber. Now a number of
modifications have been developed to improve the descending technique.

TWO- DIMENSIONAL DEVELOPMENT

This technique is developed by Martin, which employs a second eluent
system run at right angle to the first. Two-dimensional chromatography is useful
in two situations: (I) when the mixture is so complex that the components are not
all separated by one solvent, and (2) when more positive identification is required
for one or more of the components. The sample is spotted in the normal manner
2 cm. in and up from one comer and the first set of standards is spotted to the
right of the vertical scratch mark, usually 1 cm. apart 2 cm. from the bottom of the
plate. The plate is developed as usual.The plate is not sprayed to develop the
sample spots at this time. The plate is now thoroughly dried and a second set of
standards is spotted in what is now the top left quadrant of the plate. The plate is
turned 90° so the left side of the plate becomes the bottom and the plate is
developed with a second solvent. The plate is dried and sprayed with color-
developing reagents. This technique is used in the case of large groups of similar
chemical structure and properties, such as amino acids., when R,‘ value is very
close together.

RADIAL DEVELOPMENT
In this method sample spot is applied to the center of the plate and the
solvent is supplied through a hole in the plate with a wick which dips into a
solvent reservoir. As development proceeds the components move out radially
forming circles of increasing diameter. This technique is sometimes referred to as
horizontal chromatography.

SOLVENTS
Generally a solvent or solvent mixture of lowest polarity consistent with a
good separation should be employed. Suitable mixing gives mobile phases of
intermediate eluting power, but it is best to avoid mixtures of more than two
components as much as possible, because more complex mixtures readily undergo
phase changes with changes in temperature. When mixtures are used, great care is
necessary over equilibration. The purity of the solvents is of much greater
importance in thin layer than in most other forms of chromatography because of
the small amounts of material involved.

LOCATION OF SEPARATED SUBSTANCES

Coloured substances are visible as separate spots at the end of the run.
Colourless substances require chemical or physical methods.

CHEMICAL METHODS

This method involves the application of a derivating agent, known as a
locating reagent or chromogenic reagent to the TLC plate. The reagent in a
suitable solvent is applied as a spray to the plate which produces a coloured
derivative. These reagents may be classified as:

(1) NON-SPECIFIC.

They produce coloured spots with a wide range of compounds, such as.
iodine, sulphuric acid. rhodamine and iluorescein.
(2) SPECIFIC.

They only react with compounds containing a particular functional group
such as dinitrophenylhydrazine for carbonyl compounds. Thus, specific
chromogenic reagents could be applied successively to make the spots visible and
also help in the identification of components.

After spraying the plate is heated to accelerate the chemical reaction
between reagent and components in specialized heating chambers.

Sometimes, the TLC plates dip into a solution of the reagent. This method
gives uniform application of reagent to the plate. But samples can be lost from the
plate and spreading of the spots may occur which leads to a loss in resolution and
sensitivity. These methods of location are lo-100 times more sensitive on TLC
than paper.

PHYSICAL METHODS

Physical method of location has advantages that substances on
chromatogram are not converted into other compounds. Physical methods are of
following types:

1. ULTRAVIOLET DETECTION
The most common method of location uses an adsorbent layer
containing a fluorescent indicator. If a compound is naturally fluorescent, it
can be detected by placing the developed plate in a small box and irradiating it
with either short wavelength (254nm) or long wavelength (366nm) UV
radiation. Those compounds that fluoresce produce a variety of colors. Many
other compounds can be made to fluoresce by spraying the coating with acids
or bases. Another fluorescence detection technique involves the use of
fluorescent-coated plates. These are prepared with TLC adsorbents to which
inorganic phosphors have been added. These include manganese-activated
zinc silicate (254 nm). zinc-cadmium sulfide (254-366 nm) and lead-
manganese-activated calcium silicate (254 nm). When a fluorescent-coated
plate is exposcd to ultraviolet radiation, the entire plate glows with a blue
green fluorescence. Those sample compounds that do not fluoresce cover this
background fluorescence and appear as dark spots. Those compounds that
fluoresce naturally add their fluorescence to that coating and produce a
colored spot.
2. DENSITOMETER
Spectrodensitometers or TLC scanners are available for the quantitative
evaluation of thin layer chromatograms based on measuring transmittance.
fluorescence or reflectance intensity. Measurements with these instruments
are precise with standard deviations of 1% being achieved. The instruments
can also be used for the scanning of separations on electrophoretic gels.
i. ABSORPTION MEASUREMENTS
Compounds, which absorb ultraviolet or visible light, can be a quantified for absorption measurements. Absorption measurements can be obtained in either the reflectance or transmission modes.

ii) TRANSMISSION

For transmission measurements the chromatoplate is scanned by a
point light source. The intensity of the beam transmitted through the
adsorbent is taken, as I° while the intensity of the beam transmitted
through adsorbent components spot is It. The transmission value is given
by the Beer Lambert Law:

Wherenc is themcomponent concentration and l is the path length (sorbent thickness). This
deviation is due to (i) considerable scatter of the incident radiation and (ii)
variable thickness and irregularity of the surface.
iii) RFLECTANCE
The instrument components required for reflectance measurements
are similar to those for transmission. There are two standard geometric
configurations for light source and detector, an incidence angle of 0° with.
the detection instrumentation at 45° or vice versa. Reflectance
measurements are not so sensitive to variation in thickness and uniformity
of surface layer and give more precise and accurate measurements.
3. RADIOCHEMICAL DETECTION
Radiolabelled compounds are widely used in radiotracer methods for
following the course of chemical and biochemical reactions. For instance,
the study of metabolic pathways of drugs involves adding a substrate,
analyzing the reaction mixture by taking aliquots at various times,
separating the products by chromatography, then detecting the
radiolabclled compounds by autoradiography,liquid scintillation counting
or in situ measurement of radioactivity.

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