THIN LAYER CHROMATOGRAPHY

Thin layer chromatography is analogous to paper chromatography for identification, segregation and purification of
constituents of a mixture. Here ascending technique is used like the paper chromatography.

• In 1944 Consden,Gorden Martin used filter papers for separating the Amino acids

• In 1950 Kirchner identified terpenes on filter paper

• In 1958 STAHL develop standard equipment for analyzing by THIN LAYER CHROMATOGRAPHY

INTRODUCTION

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 stationaryphase 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.

 Schematic representation of
The free OH groups of silica interacts with
ascending development chamber for
the analyte (materials to be separated)
conventional TLC (side-on view).
Mixture of A and B adsorbed on the stationary phase and free in mobile phase and (B)
schematic representations of the principle of separation.
 Basic Operations Involved in TLC

(1) Methods for production of thin layer plates

(2) Application of sample on chromatoplates

(3) Choice of adsorbent

(4) Choice of solvent

(5) Detecting reagents

(6) Development and Detection

Methods for production of thin layer plates

• The thin layer can be achieved by

• spreading,

• pouring,

• spraying or dipping the thin layer plate to adsorbent.

• Spreading method is highly useful in producing uniform layer.

• Layers can be classified into solid and loose layers.

• Solid layer can be prepared by applying adsorbent on a clean glass plate with the help of applicator.

• Loose layer can be prepared by pouring of suspension plate, dipping plates in suspension, and spraying suspension on
clean glass plate.

Activation It is nothing but removing of water/ moisture other adsorbed substance from the surface of any adsorbent
by heating
 Application of sample on chromatoplates

• 0.1 % sample solution is prepared.

• It is spotted on TLC plate with the help of glass capillary or micro pipette.
• The solvent in which the sample is dissolved is allowed to evaporate.
• The sample solution is spotted in a row at one side of TLC plate at about 2 cm from edge.

Choice of adsorbent

• The most common adsorbent used in TLC is silica gel alumina.

• Kieselguhr, powdered cellulose, and several coating materials are also used.
• The choice of adsorbent is dependent upon its acidity or basicity.
• It is also dependent upon activity, separating mechanism, and can be change according to nature of
compound.
• Normally, a 0.25 mm thick TLC is prepared by spreading method.
 – Thick layer plate can be prepared by mixing silica gel G in water (ratio 25:40), layered on plate then air dried. It
can be activated by heating in an oven for 1 to 2 hours.

– Plaster of Paris is incorporated as binding agent.

– A large number of applicators are available commercially.

– The various methods for preparing layers are as follows.

• Pouring: A measured amount of slurry is pouring onto given size of plate. It is put on leveled surface to

make layer uniform.

• Dipping: Plate is dipped two times into slurry of adsorbent made in Chloroform.

• Spraying: A sprayer is used for uniform distribution of adsorbent on to glass plate.

• Spreading: Slurry is filled in applicator. Either the plate is kept steady or applicator employ slurry in

uniform manner or plate in motion passes beside applicator to make uniform layer.

Choice of solvent

• If the nature of substance is unknown then only method for finding out best solvent is trial and error.
 Detecting reagents

• The chromatogram is generally colorless.

• The spots can be detected by using appropriate detecting reagents.

• Iodine vapour and sulphuric acid (mixed with aromatic Aldehydes or oxidation agents like KMnO4 or Chromic acid)
are common locating agents used in TLC.

• Iodine forms a number of colored loose complexes those are visible in day light.

• Sulphuric acid also can form colored complexes.

• After development the next step is visualization that can be done by using UV light.

• Amino acids can be detected by using spray of ninhydrin.

 Development and Detection

• Chromatoplates are developed once with a single solvent either by following methods.

• Ascending or vertical development: The sample is spotted at one of the plate and then developed by ascending
technique used in paper chromatography. The plates are placed vertical in container and solvent is allow to run
from bottom to top.

• Horizontal development: the sample is placed in the centre of the plated and developed either by slowly
dripping solvent on it from micropipette. This procedure is also known as circular TLC.

• Multiple developments: In this technique, the development is carried out number of times in same direction.

• Stepwise development: It is carried out consecutively with two different solvents but in same direction.

• Gradient development: It is useful when substances change the properties of solvents. This technique is also
known as gradient elution. In this chromatography technique, another more polar solvent is added in solvent
system to modify the polarity of solvent system.

• Continuous development: It is useful method for separation of substances having close Rf values. In this
technique, solvent is forced to run off the edges and collected instead of being left to evaporate.

• Two dimensional development: It is resemble to two dimensional paper chromatography. Square plates are
used. The sample is spotted at the corners of the plate and allows to run in both directions with same or different
solvents.
 Thin Layer Chromatography Applications:

• The qualitative testing of various medicines such as sedatives, local anesthetics, anticonvulsant
tranquilisers, analgesics, antihistamines, steroids, and 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.

• It is used to study if a reaction is complete.

 Summary

• In general, the adsorptivity of compounds increases with increased polarity (i.e. the more polar the
compound then the stronger it binds to the adsorbent).

• The eluting power of solvents increases with polarity. Therefore, low polarity compounds can be eluted
with low polarity solvents, while higher polarity compounds require solvents of higher polarity.

• The stronger a compound is bound to the adsorbent , the slower it moves up the TLC plate.

• Non-polar compounds move up the plate most rapidly (higher Rf value), whereas polar substances travel
up the TLC plate slowly or not at all (lower Rf value).
 During ninhydrin reaction amino groups of proteins react with
ninhydrin to form blue-violet compound called Rhumann’s purple.
STRATEGIC PLANNING
 Percolated TLC Plates Supports for stationary phases (glass, aluminum, and plastic)
• Glass has been found to be a very robust support. It is rigid and transparent, and has high chemical
resistance and good heat stability. The glass backing is economical (reusable). However, glass plates are
relatively heavy and thick. They cannot be easily cut to desired size (see steps for handling and cutting
TLC plates in the Basic Protocol, below). Because glass backing is fragile and highly susceptible to
breakage, there is also a potential safety issue.

• Aluminum foil is preferable to all other materials for TLC plates. Compared with glass plates, foil plates
are thin, lightweight, and easy to handle. They can easily be cut to desired dimensions with scissors and
can be stored in a laboratory notebook. Moreover, aluminum plates have strong adsorbent layer
adherence and are good for use with eluents containing a high concentration of water. However, they are
not as chemically resistant as glass to reagents that contain strong acids, concentrated ammonia, or
iodine (i.e., they do not tolerate long treatments in an iodine chamber).

• Plastic—polyethylene terephthalate (PET) film—plates are becoming less frequently used. Their
advantages (thin, lightweight, easy to handle, can be easily cut, etc.) are similar to aluminum-foil plates,
but their flexibility (adsorbent layer may be more susceptible to cracking) and considerably inferior heat
stability are very marked disadvantages.
 Adsorbent layers and stationary phases
• The standard silica coating (silica 60 with a mean pore diameter of 60 A° ) is the most commonly used
adsorbent in TLC, although for some very sensitive substances less active adsorbents such as aluminum
oxide are preferred to prevent sample decomposition.

• Moreover, in the early days, the use of cellulose, polyamide, and Florisil (magnesium silicate) as
adsorbent agents was also described.

• For selection of an adsorbent, one considers the properties of the compounds to be separated: first, the
solubility of the sample compounds (hydrophilic or hydrophobic); then, whether the compounds can
chemically react with the adsorbent or the Eluent.

• Based on these considerations it is recommended that:

• for lipophilic substances: silica, aluminum oxide, acetylated cellulose, polyamide

should be used;

• for hydrophilic substances: cellulose, cellulose ion exchangers, polyamide, and

reversed-phase silica should be used.
• Several different types of TLC stationary phases are listed according to polarity in Figure 3. Figure 4
shows affinity of common functional groups for silica gel (approximate).

• Assuming that a polar adsorbent (silica gel) is used, the more polar compounds will be eluted more
slowly and the more nonpolar compounds will be eluted more rapidly.

• The charts depicted in these figures are very useful to help predict the order of elution; however, the
functional groups should always be viewed and considered within the context of a whole molecule.
 Solvent System (Mobile Phase)

• Finding a suitable solvent system is usually the most difficult part of TLC experiments, and solvent system is the
factor with the greatest influence on TLC.

• Only in a few cases does the solvent consist of only one component, and mixtures of up to five components are
commonly used.

• No matter how many components are present, the prepared solvent system must be a homogenous system with
no sign of cloudiness.

• Three criteria are usually considered for choosing a solvent system: solubility, affinity, and resolution.

• The first step in solvent selection is to determine the solubility of the sample.

• The desired mobile phase will be able to provide the greatest solubility while balancing the sample affinity for
the solvent and the stationary phase to achieve separation.

• Resolution is improved by optimizing the affinity between sample, solvent, and stationary phase.

• Most TLC solvent systems contain a polar solvent and a chromatographically less polar solvent.

• Figure lists some common mobile phase solvents according to their polarities and elution power with silica 60
as the stationary phase (Halpaap’s eluotropic series, Halpaap and Ripphahn, 1976; Hahn-Deinstrop, 2006).

• With these solvents, there are some common combinations for organic molecules with silica gel as the stationary
phase