Dr Arun Kumar
Assistant professor, Department of Homoeopathic Pharmacy
Bakson Homoeopathy Medical College and Hospital, Greater Noida
Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (mobile phase) moves in a definite direction.
TLC is the simplest, fastest, easiest and least expensive of several chromatographic techniques used in qualitative and quantitative analysis to separate organic compounds and to test the purity of compounds.
TLC is a form of liquid chromatography consisting of:
- A mobile phase (developing solvent) and
- A stationary phase (a plate or strip coated with a form of silica gel)
- Analysis is performed on a flat surface under atmospheric pressure and room temperature.
Definition: Thin layer chromatography can be defined as a method of separation or identification of a mixture of components into individual components by using finally divided adsorbent solid/(liquid) spread over a glass plate and liquid as a mobile phase.
Classes of TLC:
- Normal phase: In this, stationary phase is polar (like silica gel) and mobile phase is organic solvent or its mixture which is less polar then stationary phase.
- Reversed phase: Stationary phase is a silica bonded with an organic substance (like long chain aliphatic acid C-18) and mobile phase is a mixture of water and organic solvent which is more polar than stationary phase.
Principle of TLC:
It is based on the principal of adsorption chromatography or partition chromatography or combination of both, depending on adsorbent and nature of solvents employed.
The components with more affinity towards stationary phase travels slower.
Compounds with less affinity towards stationary phase travels faster.
In TLC, a solid phase, the adsorbent, is coated onto a solid support (thin sheet of glass, plastic, and aluminium)as a thin layer (about 0.25mm thick).
The mother preparation to be separated is dissolved in a solvent and the resulting solution is spotted onto the thin layer plate near the bottom.
A solvent, or mixture of solvents, called the eluatant, is allowed to flow up the plate by capillary action. At all times, the solid will adsorb a certain fraction of each component of the mixture and the remainder will be in solution. Any one molecule will spend part of the time setting still on the adsorbent with the remainder moving up the plate with the solvent. A substance that is strongly adsorbed will have a greater fraction of its molecules adsorbed at any one time, and thus any one molecule of it will spend more time sitting still and less time moving and vice-versa.
Separation in TLC involve distributing a mixture of two or more substance between a stationary phase and a mobile phase.
- The stationary phase: is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate.
- The mobile phase: is a developing liquid which travels up the stationary phase, carrying the samples with it।
Components of the samples will separate on the stationary phase according to:
How much they adsorb on the stationary phase versus
how much they dissolve in the mobile phase.
For TLC on silica gel, a mobile phase with as a low a polarity as possible should be used consistent with achieving a satisfactory separation. Polar solvents can themselves become strongly adsorbed thereby producing a partition system, a situation which may not be desirable.
- The solvent should also show a maximum of selectivity in its ability to dissolve or desorbs the substance being separated.
- A more important property of the solvent is its ability to be itself adsorbed on the adsorbent.
The adsorbent is a powdered material applied usually to a glass plate. Silica gel is slightly acidic and, therefore, is best applied to the separation of neutral and acidic substances. Alumina on the other hand is basic and should be used for the separation of basic compounds.
- Uses different migration rates of individual components of mother tincture or very low potencies on thin plate of inert material.
- Solvents such as butanol and methanol are used with migration distance of about 10cm.
- Characteristic bands are shown on layer of silica gel with each band representing a specific component of mother tincture.
1. Thin Layer plate: Place neat and clean plate on aligning tray securely so that it does not slip.
2. Application of adsorbent: Adsorbent and liquid (usually water) are mixed, which when shaken for 30 seconds give a smooth slurry that spreads evenly over plate with spreader.
Adsorbent thickness of Analytical plate – 250-500 micrometre
Adsorbent thickness of Preparative plate – 500-2000 micrometre
Leave plate undisturbed for 15 minutes then dry at 105 degree Celsius for 30 minutes. Store the plate in dessicator.
3. Application of sample: Sample (mother tincture) is applied using suitable micro pipettes as a small spot of 1-2mm about 1cm above the end of plate without disturbing adsorbent.
Mark the starting point near edge of plate and allow it to dry.
4. Placing plate in developing chambers: Place the plate in developing chamber so that solvent reaches lower edge of adsorbent but does not touch the spot point.
Solvent gradually rises in the adsorbent via capillary action. The chamber should be closed until solvent ascends which requires 15-60 minutes.
5. Visualisation: Plate is removed from developing chamber when solvent has moved to about 1cm of top end of adsorbent. Once solvent evaporates, spots are observed under UV light at 254nm (shortwave UV).
6. Calculation of Rf value: Distance travelled by substance relative to distance travelled by solvent depends upon molecular structure of the substance, which is expressed as Retention factor (Rf) value.
Rf value= Distance travelled by solute/ Distance travelled by solvent front
Factors affecting Rf value:
- Nature of adsorbent
- Mobile phase
- Thickness of layer
- Dipping zone
- Chromatographic techniques
Advantages of TLC:
- Low cost
- Short analysis time
- Base of sample preparation
- All spots can be visualised
- Sample cleanup is seldom necessary
- Adaptable to most pharmaceuticals
- Uses small quantities of solvents
- Requires minimal training
- Reliable and quick
- Minimal amount of equipment is needed
- Densitometer can be used to increase accuracy of spot concentration
Solvent system- Chloroform: Methanol = 9:1 (v/v)
Adsorbent- Precoated silica gel plate
Visualization under UV light- Three spots are observed with Rf values: