pharmaceutical analysis- TLC INSTRUMENTATION AND DESCRIPTIVE

1. THIN LAYER CHROMATOGRAPHY
What Is Thin Layer Chromatography?
Thin Layer Chromatography is a technique used to isolate non-volatile mixtures. The experiment is
conducted on a sheet of aluminium foil, plastic, or glass which is coated with a thin layer of adsorbent
material. The material usually used is aluminium oxide, cellulose, or silica gel.
On completion of the separation, each component appears as spots separated vertically. Each spot has
a retention factor (Rf) expressed as:
Rf = dist. travelled by sample / dist. travelled by solvent
The factors affecting retardation factor are the solvent system, amount of material spotted, absorbent
and temperature. TLC is one of the fastest, least expensive, simplest and easiest chromatography
technique.
Thin Layer Chromatography Principle
Like other chromatographic techniques, thin-layer chromatography (TLC) depends on the separation
principle. The separation relies on the relative affinity of compounds towards both the phases. The
compounds in the mobile phase move over the surface of the stationary phase. The movement occurs in
such a way that the compounds which have a higher affinity to the stationary phase move slowly while
the other compounds travel fast. Therefore, the separation of the mixture is attained. On completion of
the separation process, the individual components from the mixture appear as spots at respective levels
on the plates. Their character and nature are identified by suitable detection techniques.
Stationary phase:
 The most commonly used stationary phases are adsorbents e.g. fine powders of silica gel,
alumina, Kieselguhr and cellulose, they are specially prepared for TLC.
 Many of these are available with a fluorescent compound e.g. ZnS incorporated inorder to
facilitate the detection of the resolved components of the mixture when viewed under UV light.
 Silica gel is slightly acidic whereas alumina is slightly basic but neutral alumina is also available.
 Silica gel and alumina particles contain hydroxyl groups on their surface which will form hydrogen
bond with polar molecules.
 Alumina is preferred for the separation of weakly polar compounds, but silica gel is preferred for
polar compounds such as amino acid and sugars.
 Other absorbents suitable for special purpose are polyamides, magnesium silicate, calcium
silicate, activated charcoal, modified cellulose with ion exchange properties and the various
forms of organic gel e.g. Sephadex, Bio-Gel P.
 Support used for the stationary phase in TLC is a glass plate , aluminium metal plate or plastic
strip.
 Glass plates can be used in a number of sizes ranging from microscope slides to larger plates
(20cm X 20cm X 20cm).
 Binder is essential for good adherence of the adsorbent to the plate.
 Commonly used binder is gypsum (calcium sulphate) which is incorporated at a level of 10-15%.
 Starch and certain organic polymers are also used as binders.
 Pre-coated plates are advantageous over the prepared plates that have uniform and non-fragile
coating.
BY-SUBHASMITH PRADHAN

2. Mobile phase:
 The solvent used are listed below in order of increasing polarity, it is known as eluotropic series.
 Petroleum ether < n-hexane < carbon tetrachloride < toluene < benzene < chloroform <
dichloromethane < diethyl ether < n-butanol < isopropanol < acetone < methanol < water
 Movement of the solute increases with increasing solvent polarity.
 The solvent is employed in the form of a pool at the bottom of the developing chamber.
 Usually the solvent or eluant is made to ascend on the plate, hence the name ascending TLC.
 The solvent travels through the system by capillary action, thus solvent velocity is determined by
the nature and packing structure of the adsorbent.
 The saturation of the development chamber with solvent vapor has a significant effect on solute
migration.
 Specifically, with under-saturation solvent and solute migrations are slow and not smoothly
ascending.
 This problem can be overcome by lining the developing chamber with a filter paper which is then
saturated with developing solvent to provide uniform chamber saturation.
 Selection of the developing solvent is done by preliminary trial runs using microscopic slides or
small strips.
Thin Layer Chromatography Diagram
Diagram of Thin Layer Chromatography
System Components
TLC system components consist of

3. 1. TLC plates, preferably ready-made with a stationary phase: These are stable and
chemically inert plates, where a thin layer of stationary phase is applied on its whole
surface layer. The stationary phase on the plates is of uniform thickness and is in fine
particle size.
2. TLC chamber. This is used for the development of the TLC plate. The chamber maintains
a stable environment inside for proper development of spots. It also prevents the
evaporation of solvents and keeps the process dust-free.
3. Mobile phase. This comprises of a solvent or solvent mixture. The mobile phase used
should be particulate-free and of the highest purity for proper development of TLC spots.
The solvents recommended are chemically inert with the sample, a stationary phase.
4. A filter paper. This is moistened in the mobile phase, to be placed inside the chamber.
This helps develop a uniform rise in a mobile phase over the length of the stationary phase.
Thin Layer Chromatography Procedure
Before starting with the Thin Layer Chromatography Experiment let us understand the different
components required to conduct the procedure along with the phases involved.
1. Thin Layer Chromatography Plates – ready-made plates are used which are chemically inert and
stable. The stationary phase is applied on its surface in the form of a thin layer. The stationary
phase on the plate has a fine particle size and also has a uniform thickness.
2. Thin Layer Chromatography Chamber – Chamber is used to develop plates. It is responsible to
keep a steady environment inside which will help in developing spots. Also, it prevents the
solvent evaporation and keeps the entire process dust-free.
3. Thin Layer Chromatography Mobile phase – Mobile phase is the one that moves and consists of
a solvent mixture or a solvent. This phase should be particulate-free. The higher the quality of
purity the development of spots is better.
4. Thin Layer Chromatography Filter Paper – It has to be placed inside the chamber. It is moistened
in the mobile phase.
Step I: Plate preparation:
 Cleaning – Glass plates must be carefully cleaned with detergent to remove adhering particles
rinsed thoroughly with distilled water, placed in a metal rack and dried in an oven.
 The plate should be handled by the edges or by the under-surface which is not to be coated with
the adsorbent.
 Failure to take the precaution and grease contamination on the glass surface may result in the
formation of poor quality mechanically unstable layer, which is liable to be flaking.
Step II: Adsorbent selection:
Commonly used adsorbents are silica gel, alumina, cellulose and polyamide.
Step III: Slurry preparation:
 Slurry is prepared by the slow addition with stirring of adsorbent, e.g. silica gel or alumina, to a
suitable solvent like water, dichloromethane in a wide mouthed capped bottle.
 Too thick or too thin slurries should be avoided.

4. Step IV: Coating of the TLC plate:
 The supporting plate (glass, metal etc.) should fulfill the following requirements:
 Uniform thickness
 Inert to solvent, solute, stationary phase, identification reagents, procedures
 Sufficient strength to allow vertical development.
 Thin layer may be prepared by pouring, dipping, spraying or spreading the adsorbent slurry over
the plate.
 Generally, the best procedure for the preparation of uniform layers or films is the use of the
commercial spreader.
 However, the best source of uniform plates may be pre-coated plates that are available from
manufacturers and suppliers.
Step V: Activation of TLC plate:
 After the slurry has been spread out evenly the plates are placed horizontally to set for
approximately 10 minutes in a fume cupboard.
 The surplus adsorbent is removed from the glass edged by means of razor blade or glass rod.
 The plate is then activated by heating at 110oC for 1 hour in an oven.
 The drying conditions may vary with the nature of the adsorbent, binder and the solvent.
 After they are dry, the plates should be cooled to room temperature and stored in desiccators
until used.
 Cellulose and polyamide plates are allowed to dry at room temperature and are not normally
heated, then stored in a dust free cabinet.
 After drying, normal layer thickness is in the range of 150mm for analytical and 2mm for
preparative TLC system.
Step VI: Sample preparation:
 The mixture (e.g. a mixture of amino acids) to be analysed is dissolved in a suitable solvent (0.5-
3%).
 The selected solvent should be volatile for rapid evaporation of the solvent is desirable as this
leads to the formation of a small-diameter spot which results in a better separation during
chromatographic development process.
 Reference compounds are similarly prepared and applied to the adsorbent on the same plate
alongside the mixture spot, this helps more ready interpretation of the chromatogram.
Step VII: Sample application on the TLC plate:
 Wipe any excess adsorbent from the back and edges of the plate.
 Sample should normally be applied about 5mm (for small plates) to 10mm (larger plates) from
the edge of the plate.
 However, care should be taken not to immerse the spot in the solvent pool in the development
chamber.
 The spots should be separated from each other by at least 10mm for larger plates.
 Sample application is performed by spotting or streaking the thin layer.
 Analytical plates are usually spotted while preparative plates are streaked.
Step VIII: Spotting:
 Done with a melting point capillary tube or micropipette or microliter syringe.
 The applicator is charged by dipping the capillary end into the solution.
 The solution is then transferred by touching the tip of the capillary onto the adsorbent layer.
 The sample volume is usually in the range of 1-10l.
 The spot must be as small as possible for better separation and minimum tailing.

5. Step IX: Developing the chromatogram (Elution)
Tank selection:
 TLC can be developed in wide variety of chambers. For example, microscopic slides are
developed in a small cylindrical glass jar or wide mouthed screw capped glass bottles.
 Larger plates (20cm X 20cm) require a rectangular glass tank of suitable dimension with airtight
lid.
Tank saturation:
 The inside of the development tank is lined with filter paper leaving a gap for viewing the chroma
plate.
 The filter paper is saturated with the selected developing solvent and the requisite amount of the
developing solvent is carefully poured down the side of the tank.
 Then the tank is closed with lid and allowed to stand for about 5 minutes so that the atmosphere
in the tank becomes saturated with solvent vapour.
Insertion of the plate:
 The loaded plate with the origin spots or baseline towards the bottom of the tank is inserted and
tilted.
 Care should be taken that the solvent level is below the origin or baseline.
 The tank is recapped or closed and the solvent is allowed to ascend by capillary action to the
finishing line which is about 0.5mm away from the top edge of the adsorbent layer.
 The time required to complete this development varies greatly with the solvent composition and
the nature of the adsorbent.
 When the solvent reaches the finishing line, the position of the solvent is marked on the
absorbent layer and immediately the plate is removed from the development tank.
 After removal the plate is dried in the fume cupboard.
Step X: Detection of spots/ Visualization of solutes:
 The position of coloured components can be seen in daylight without any difficulty.
 For colourless solutes, visualization or detection techniques are many, such as:
UV light:
 Non-destructive method accomplished by viewing the plate under UV lamp at 254nm (short
wavelength) and 350nm (long wavelength).
 At short wavelength the plate is dark and compounds glow.
 This method is limited to solutes capable of fluorescence.
 At long wavelength, when a fluorescent indicator is incorporated into adsorbent the entire plate
fluoresces green under UV light, and solutes appear as dark spot due to quenching or blocking
effect of the fluorescent background.
Iodine vapor:
 This is a semi-destructive general method for most organic compounds.
 The dried plate is allowed to stand in a closed tank containing iodine crystals scattered over the
tank bottom.
 The spots are revealed as brown stains.
 Their positions should be marked as soon as the plate has been removed from iodine tank since
standing in air for a short while causes the iodine to evaporate and the stained spots to
disappear.

6. Thin Layer Chromatography Experiment
The stationary phase that is applied to the plate is made to dry and stabilize.
 To apply sample spots, thin marks are made at the bottom of the plate with the help of a pencil.
 Apply sample solutions to the marked spots.
 Pour the mobile phase into the TLC chamber and to maintain equal humidity, place a moistened
filter paper in the mobile phase.
 Place the plate in the TLC chamber and close it with a lid. It is kept in such a way that the sample
faces the mobile phase.
 Immerse the plate for development. Remember to keep the sample spots well above the level of
the mobile phase. Do not immerse it in the solvent.
 Wait till the development of spots. Once the spots are developed, take out the plates and dry
them. The sample spots can be observed under a UV light chamber.
Thin Layer Chromatography Applications
 The qualitative testing of Various medicines such as sedatives, local anaesthetics, anticonvulsant
tranquilisers, analgesics, antihistamines, steroids, hypnotics is done by TLC.
 TLC is extremely useful in Biochemical analysis such as separation or isolation of biochemical
metabolites from its blood plasma, urine, body fluids, serum, etc.
 Thin layer chromatography can be used to identify natural products like essential oils or volatile
oil, fixed oil, glycosides, waxes, alkaloids, etc
 It is widely used in separating multicomponent pharmaceutical formulations.
 It is used to purify of any sample and direct comparison is done between the sample and the
authentic sample
 It is used in the food industry, to separate and identify colours, sweetening agent , and
preservatives
 It is used in the cosmetic industry.

7.  It is used to study if a reaction is complete.
Advantages of Thin Layer Chromatography
1. It is a simple process with short development time.
2. It helps with the visualization of separated compound spots easily.
3. The method helps to identify the individual compounds.
4. It helps in isolating of most of the compounds.
5. The separation process is faster and the selectivity for compounds is higher (even
small differences in chemistry is enough for clear separation).
6. The purity standards of the given sample can be assessed easily.
7. It is a cheaper chromatographic technique.
Disadvantages Of Thin Layer Chromatography:
1. Thin Layer Chromatography plates do not have longer stationary phase.
2. When compared to other chromatographic techniques the length of separation is limited.
3. The results generated from TLC are difficult to reproduce.
4. Since TLC operates as an open system, some factors such as humidity and temperature can be
consequences to the final outcome of the chromatogram.
5. The detection limit is high and therefore if you want a lower detection limit, you cannot use TLC.
6. It is only a qualitative analysis technique and not quantitative.