INTRODUCTION:
In thin-layer chromatography, 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 and aluminum plates being most commonly employed. The separation achieved may be based upon adsorption, partition, or a combination of both effects, depending on the particular type of stationary phase, its preparation, and its use with different solvents.
Apparatus:
Apparatus and materials for thin-layer chromatography consists of the following.
Pre-coated TLC plates.
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.
A template (generally made of plastic) to aid in placing the test spots at definite intervals to mark distances as needed and to aid in labeling the plates.
A suitable manual, semi-automatic, or automatic application device can be used to ensure proper positioning of the plate and proper transfer of the sample, with respect to volume and position, onto the plate. For the proper application of the solutions, micro pipettes, micro syringes, or calibrated disposable capillaries are recommended.
A reagent sprayer that will emit a fine spray and will not itself be attacked by the reagent.
A UV light source suitable for observations under short (254 nm) and long (365 nm) wavelength UV light.
A suitable device for documentation of the visualized chromatographic result.
PROCEDURE :
(1) Select the pre-coated TLC plate as specified in the individual testing procedure.
(2) Prepare solvent system as specified in the individual testing procedure.
(3) Place two filter-paper wicks, 18 cm in height and as wide as the length of the chamber, into the chamber, add about 100 mL of the solvent (sufficient to have a depth of 5 to 10 mm at the bottom of the chamber), seal the cover to the top of the chamber, and allow the system to equilibrate; it is essential that the wicks become completely wet.
(4) Alternatively, the chamber may be completely lined with filter paper. In either case, assure that the filter paper dips into the solvent at the bottom of the chamber.
Note : Where vapor saturation of the chamber by these methods is undesirable, it is so indicated in the individual testing procedure.
Prepare the Test and Standard Solutions as specified in the individual testing procedure.
(6) Apply the solutions in such a way that the diameter of the spots should not exceed 2 to 5 mm (1 to 2 mm on HPTLC plates) or bands of 10 to 20 mm by 1 to 2 mm (5 to 10 mm by 0.5 to 1 mm on HPTLC plates) at an appropriate distance from the lower edge.
(7) During chromatography the application position must be 3 mm (HPTLC) to 5 mm (TLC) above the level of the developing solvent—and from the sides of the plate. Apply the solutions on a line parallel to the lower edge of the plate with an interval of at least 10 mm (5 mm on HPTLC plates) between the centers of spots or 4 mm (2 mm on HPTLC plates) between the edges of bands, and allow to dry.
(8) Avoid physical disturbance of the adsorbent during spotting procedure (by the pipette or other applicator) or when handling the plates. The template will aid in determining the spot points and the 10- to 15- cm distance through which the solvent front should pass. (9) Place a mark 10 to 15 cm above the spot point. Introduce the plate into the developing chamber (test spots towards the bottom). Allow the solvent in the chamber to reach the lower edge of the adsorbent but do not allow the spot points to be immersed.
(10) Put the cover in place, and maintain the system until the solvent ascends to a point 10 to 15 cm above the initial spots.
(11) Remove the plate from the developing chamber, mark the solvent front, air-dry the plates and evaluate the chromatogram as directed in individual testing procedur
Detection:
Observe the dry plate first under short-wavelength UV light (254 nm) and then under long-wavelength UV light (365 nm) or as stated in the individual testing procedure.
If further directed, spray, immerse, or expose the plate to vapors of the specified reagent, heat the plate when required, observe, and compare the test chromatogram with the standard chromatogram.
Document the photocopy of each plate after each observation and started in the self-sealing cover with the respective analysis report.
Measure and record the distance of each spot or zone from the point of origin, and indicate for each spot or zone the wavelength under which it was observed.
Determine the RF values for the principal spots or zones using below mentioned formula.
Rf = Distance traveled by the compound/ Distance traveled by the solvent front
General testing procedure, procedure, chromatography, thin layer chromatography
