THIN LAYER CHROMATOGRAPHY
Introduction
Thin layer chromatography (TLC) 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 liquid.
The adsorbent is relatively thin, uniform layer of dry, finely powdered material applied to glass, plastic or metal sheet. 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 a 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 reference sample chromatograph on the same plate.
Apparatus required
Flat glass plates of appropriate dimensions.
b) An aligning tray or plate surface on which the plates can be aligned and rested when coating substance is applied .
c) An adsorbant or coating substance consisting of finely divided adsorbant material, normally 5 µm to 40 µm in diameter. A variety of coating materials are available, but Silica gel is most frequently used. The adsorbent may contain fluorescing matter to help in visualizing spots that absorb ultraviolet light.
A spreader, which when moved over the glass plate, will apply a uniform layer of adsorbent, of a uniform thickness, usually between 150 to 250 µm.
A storage rack to support the plates during drying and transportation.
The apparatus described above are essentially required for the preparation of TLC plates. Ready to use TLC plates are commercially available, which may be used.
f) A developing chamber that can accommodate one or more plates and can be properly closed .
g) Graduated micro pipettes capable of delivering quantities.
h) A reagent sprayer that will emit a fine spray and will not itself be attacked by the reagent.
i) A viewing cabinet, fitted with ultra-violet light, suitable for observation at short (254 nm) and long (366 nm) ultra-violet wavelengths.
Precautions
The spot must be applied by holding the micro pipette as erect as possible, which avoids undue spreading of the spot and ensures a compact spot, usually 2 to 3 mm in diameter.
The syringes must be cleaned thoroughly, prior to spotting .
Use dedicated syringes, wherever feasible; especially for spotting impurities.
For developing solvents chromatographic grade solvents must be used, which avoids unwanted impurities being introduced on the plate.
All solutions for TLC including the mobile phase must be freshly prepared. TLC solvents may be kept separately to avoid accidental contamination.
If the mobile phase consists of more than two solvents, the solvents must be mixed in the order mentioned, keeping the volume recommended, as accurate as possible.
Unless unsaturated conditions are prescribed the developing chamber must be saturated with the developing solvent, prior to placing the TLC plates in the chamber.
h) The developing chamber is lined with sheet of filter paper which dips into the solvent in the base of the chamber which ensures complete saturation of the chamber with solvent vapour.
i) The developing chamber may be covered by a black cloth or Aluminum foil in case of spotting of light sensitive materials. The developing chamber must be placed on a firm surface, away from turbulence and use of acids; since these factors tend to spoil the plates. The developing chamber must be of good quality, having a flat bottom to ensure uniform flow of mobile phase.
Before and after spotting, the TLC plate must be inspected for any unwanted spots. The edges of the plate may be cut and rounded up for uniform movement of the mobile phase. The TLC plates must always be handled by holding at the edge, to avoid finger prints on the surface. The plates must be placed in an erect fashion in the developing chamber; which must always be covered.
Cutting of the pre-coated full plates into half plates or quarter plates, must be avoided as far as possible. It is a good practice to always use a full plate.
I) A narrow strip of coating substance, about 5 mm wide is usually removed from the vertical side of the TLC plate, to prevent accidental loss of the spot near the edge of the plate. In the case of ready to use plates, the narrow strip of coating substance is already removed.
m) The plates after preparation must be protected from moisture and used within three days of preparation. At the time of usage, the plates may be dried.
n) For drying of applied spots, during spotting a gentle current of air or nitrogen is used. Use of hot air from hair dryers must be avoided as the degradation of the spot, may be inadvertently introduced. The spotting must be carried out at least two cm from the bottom of the plate, to avoid direct contact with the mobile phase.
0) The spraying with reagent for development of the spot must be carried out uniformly over the plate. The spray must never be directed for a long time on a portion of the plate, as it results in localised darkening of the TLC plate.
p) The plates after spotting may be wrapped in Aluminium foil and than placed in a polybag for future reference. It is a good practice to calculate and record the Rf value of experimental spot and standard spot, during the identification test.
Quantitative evaluation -The identification of the raw material is deemed to be satisfactory, if the Rf value of the experimental spot and the standard spot is identical.
In case of degradation products which are denoted by secondary spots; these are compared with main spots of diluted samples (0.5, 1.0 or 2.0 %). These secondary spots are required to be not more intense than the main spot obtained from lower dilution of the parent compound. If more than one secondary spot is observed, the individual intensities of the secondary spots can be compared with the main spots of lower dilutions and can be added to get a rough idea of degradation products or related substances.
Advantages -
The technique is simple and less expensive.
It is one of the most important techniques used in stability indicating methods and gives ready information regarding degradation products.
It is useful technique for identification of the raw material, when compared to an authentic standard.
Disadvantages
The method is not quantitative.
In absence of availability of impurities for spotting, degradation products cannot be identified.
c) The precision and accuracy depends on the technique employed by an individual and hence can vary substantially from person to person.
Note :
Recent changes in the practice of TLC have, however resulted in improved performance both in terms of separation and quantitative measurements. These developments are referred to as High Performance Thin Layer Chromatography (HPTLC), which has combined HPLC techniques with TLC, to give quantitative measurements of high precision.
Schematic drawing of All types granulation
