Thin-layer chromatography is a technique in which a
solute undergoes distribution between two phases, a stationary phase
acting through adsorption and a mobile phase in the form of a liquid. The
adsorbent is a relatively thin, uniform layer of dry finely powdered
material applied to a glass, plastic or metal sheet or plate. Glass plates
are most commonly used. Separation may also be achieved on the basis of
partition or a combination of partition and adsorption, depending on
the particular type of support, its preparation and its use
with different solvent.
Identification can be effected by observation of spots
of identical Rf value and about equal magnitude
obtained, respectively, with an unknown and a reference
sample chromatographed on the same plate. A visual comparison of the
size and intensity of the spots usually serves for
semiquantitative estimation.
Apparatus
(a) Flat glass plates of appropriate dimensions which
allow the application at specified points of the necessary quantities of the
solution being examined and appropriate reference solutions and which
allow accommodation of the specified migration path-length. The plates
are prepared as described below; alternatively,
commercially available pre-coated plates may be used.
(b) An aligning tray or a flat surface on which the
plates can be aligned and rested when the coating substance
is applied.
(c) The adsorbent or coating substance consisting of
finely divided adsorbent materials, normally 5 micron to
40 micron in diameter, suitable for chromatography. It can be
applied directly to the plate or can be bonded to the plate by means
of Plaster of Paris (Hydrated Calcium Sulphate) or with any other suitable
binders. The adsorbent may contain fluorescing material to help in visualising
spots that absorb ultraviolet light.
(d) A spreader which, when moved over the glass plate,
will apply a uniform layer of adsorbent of desired thickness over the
entire surface of the plate.
(e) A storage rack to support the plates during drying
and transportation.
(f) A developing chamber that can accommodate one or
more plates and can be properly closed and sealed. The chamber is
fitted with a plate support rack that supports the plates, back to back,
with the lid of the chamber in place.
(g) Graduated micro-pipettes capable of delivering micro
liter quantities say 10 ml and less.
(h) A reagent sprayer that will emit a fine spray and
will not itself be attacked by the reagent.
(i) An ultraviolet light, suitable for observation at
short (254 nm) and long (365 nm) ultraviolet wavelengths.
Preparation of plates. Unless otherwise specified in the monograph, the
plates are prepared in the following manner. Prepare a suspension of the
coating substance in accordance with the instructions of the supplier and,
using the spreading device designed for the purpose, spread a uniform
layer of the suspension, 0.25 to 0.30 mm thick, on a flat glass plate 20
cm long. Allow the coated plates to dry in air, heat at 100° to
105° for at least 1 hour (except in the case of plates prepared
with cellulose when heating for 10 minutes is normally
sufficient) and allow to cool, protected from moisture. Store the
plates protected from moisture and use within 3 days of
preparation. At the time of use, dry the plates again, if necessary,
as prescribed in the monograph.
Method
Unless unsaturated conditions are prescribed, prepare
the tank by lining the walls with sheets of filter paper; pour
into the tank, saturating the filter paper in the process,
sufficient of the mobile phase to form a layer of solvent 5 to 10mm
deep, close the tank and allow standing for 1hour at room
temperature. Remove a narrow strip of the coating substance, about 5
mm wide, from the vertical sides of the plate. Apply the
solutions being examined in the form of circular spots about 2 to 6 mm
in diameter, or in the form of bands (10 to 20 mm x 2 to 6 mm unless
otherwise specified) on a line parallel with, and 20 mm from, one end of
the plate, and not nearer than 20 mm to the sides; the spots should be 15
mm apart. If necessary, the solutions may be applied in portions, drying
between applications. Mark the sides of the plate 15 cm, or the
distance specified in the monograph, from the starting line. Allow
the solvent to evaporate and place the plate in the tank, ensuring that it
is as nearly vertical as possible and that the spots or bands are above
the level of the mobile phase. Close the tank and allow standing at room
temperature, until the mobile phase has ascended to the marked line.
Remove the plate and dry it.
For two-dimensional chromatography dry the plate after
the first development and carry out the second development in
a direction perpendicular to the first.
When the method prescribed in the monograph
specifies 'protected from light' or 'in subdued light' it is intended
that the entire procedure is carried out under these conditions.
Adjustment of chromatographic conditions
Minor adjustments to the parameters of the test may be
made in order to satisfy the system suitability criteria. These
may be:
Mobile phase. Minor solvent
component of a mixture: ± 30 per cent relative or ± 2 per cent absolute,
whichever is the larger; no other component altered by more than 10 per
cent absolute;
Concentration of
salts. In
the buffer component of the mobile phase; ± 10 per cent; pH of the
aqueous component of the mobile phase. ±
0.2 pH, unless otherwise stated in the monograph, or ± 0.1 pH, when neutral
substances are to be examined;
Volume of solutions
applied: 10-20
per cent of the prescribed volume if plates have fine particles (2-10
micron).
Visualisation
After development, the plate should be examined under
an ultraviolet light having a maximum output at about 254 nm or at
about 365 nm, as the case may be. Alternatively, it may be visualised as
directed in the monograph; where a spraying technique is prescribed it is
essential that the reagent be evenly applied as a fine spray. The
term secondary spot means any spot other than the principal spot.
Similarly, a secondary band is any band other than the principal band.
Semi-quantitative estimation
Identification. The principal spot in the chromatogram obtained
with the test solution is visually compared to the corresponding spot in
the chromatogram obtained with the reference solution in respect of the color,
the size and the Rf of the spots.
Test for Related substances. The secondary spot(s) in the chromatogram
obtained with the test solution is (are) visually compared to either the
corresponding spot(s) in the chromatogram obtained with the reference
solution containing the impurity (ies) or the spot in the chromatogram
obtained with the reference solution prepared from a dilution of the
test solution.
Quantitative measurement
The substances that have been separated after
development of the plate and that respond to UV-Vis irradiation can
be estimated directly on the plate with suitable
instrumentation. Measurement is of the reflectance of the incident light
from the spots by moving the plate or the measuring device. Likewise,
fluorescence may be measured using an appropriate optical system.
Apparatus. The apparatus for direct measurement consist
of: a device for exact positioning and reproducible application of
the amount of solutions onto the plate, a mechanical device for moving the
plate or the measuring device along the x-axis or the y-axis, a
recorder and a suitable integrator or a computer, and a photometer with a
source of light, an optical device for generating monochromatic light and
a photo cell of adequate sensitivity;
for measurement of fluorescence, a suitable filter to prevent light used
for excitation from reaching the detector while permitting emitted light or
a specific portion thereof to pass.
Method. Prepare
the test solution and reference solutions as prescribed in the individual
monograph. Use the same solvent for all the solutions and apply the same
volume of each and develop the plate. Prepare and apply not fewer than 3
reference solutions of the substance under examination,
the concentrations of which span the expected value in the
test solution (about 80 per cent, 100 per cent and 120 per cent).
Treat with the prescribed reagent, if necessary, and
record the reflectance, the transmittance or fluorescence in
the chromatograms obtained with all the solutions. Use the measured
results to calculate the amount of substance in the test
solution. The requirement for resolution and separation are
prescribed in the individual monograph.
