Principle, Procedure, Instrumentation, And Applications Of TLC and HPTLC

Thin-Layer Chromatography

Thin-layer chromatography is a chromatographic technique that is useful for separating organic compounds. TLC was developed by Izmailov in 1938. 

Because of the simplicity and rapidity of TLC, it is often used to monitor the progress of the organic reaction and to check the purity of products.

It is a chromatography technique in which a liquid sample migrates by capillarity through a solid absorbent medium which is arranged as a thin layer on a rigid support.

Principle

Thin-layer chromatography is a solid-liquid technique in which the two phases are a stationary phase (solid) and a mobile phase (liquid).

This 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. 

It is based on the principle of adsorption chromatography or partition chromatography or a combination of both depending on the adsorbent, its treatment, and the nature of solvents employed.

The compounds with higher affinity to the stationary phase will move slowly and the compounds with lower affinity to the stationary phase will move fast. Hence, the separation of the mixture is attained. 

On the completion of the process, the individual components will appear as spots at respective levels on the plates. 


Major Parts of TLC

They are as follows: 

Thin Layer Chromatography Plates 

Plates used are chemically inert and stable. The stationary phase is applied on its surface in the form of a thin layer and the stationary phase on the plate has a fine particle size and also has a uniform thickness.

Specific dimensions of the glass plate are:

Full plate (20×20cm).

Half-plate (20×10cm).

Quarter plates (20×5cm).

These dimensions are used since the width of the commercially available TLC spreader is 20 cm.


Thin Layer Chromatography Chamber 

The chamber is used to develop plates. It is responsible to keep a study environment inside which will help in developing spots also it prevents solvent evaporation and keeps the entire process dust-free. 


Thin Layer Chromatography Mobile Phase 

The 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 the spot is better. 


Thin Layer Chromatography Filter Paper 

It has to be placed inside the chamber. It is saturated in the mobile phase. This helps to develop a uniform raise in a mobile phase over the length of the stationary phase.


TLC Experiment Procedure

The stationary phase is dried and stabilized. 

The sample spots are made at the bottom of the plate with the help of a pencil. 

Apply sample solutions to the marked spots and the mobile phase is applied to them, and this is kept in the TLC chamber. 

And to maintain equal humidity place a moisture filter paper in the mobile phase. 

Place the plate in the TLC chamber and close it tightly so it is kept in such a way that the sample faces the mobile phase. 

Remember to keep the sample spots well above the level of the mobile phase. 

Start and do not emerge it in the solvent. 

Wait till the development of spots once these spots are developed, take out the plate and dry them. 

Then sample spots can be observed under our UV light chamber.  

The sample application is a critical step in achieving good separation in the TLC. 


Selection of Stationary Phase or Adsorbent 

The selection of adsorbent mainly depends on: 

The solubility of compounds for hydrophilic or lipophilic compounds.

Nature of substance to be separated i.e whether it is acidic, basic, or amphoteric.

Adsorbent particle size.

Adsorbent should not adhere to the glass plate 

Reactivity of compounds with the solvent or adsorbent.

The chemical reactivity of compounds with binders.


Adsorbents 

There are several adsorbents which can be used as a stationary phase, their composition and ratio in which they have to mix with water or other solvents to form a slurry for preparing a thin layer of a chromatographic plate are given below,

Inorganic Adsorbents

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Organic Adsorbents 

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Cellulose and its acetylates. 

Charcoal and activated carbon.


Methods for Application of Absorbent

Pouring

Dipping 

Spraying 

Spreading


Pouring 

The adsorbent of finely divided homogeneous particle size is made into a slurry and is poured on a plate and allowed to flow over it, so that the plate is evenly covered with the adsorbent. 


Dipping

This technique is used for small plates.

By keeping the two plates at a time, back to back in a slurry of an absorbent in chloroform or other volatile solvents.

The exact thickness of the layer is not known and the evenness of the layer may not be good. 


Spraying

The slurry is diluted for the operation of the sprayer. But this technique is not used nowadays as it is difficult to get a uniform layer. 


Spreading 

If all the above methods fail to give a thin and uniform layer, then this is used. 

The modern method utilizes the spreading devices for the preparation of uniform layers on glass plates.

The layer thickness will be from 0.2 to 2.0 mm. 


Activation of Plates 

After spreading, the plates are allowed to dry in the air, and further dried and activated by heating at about 100°C for 30minutes. 

The absorbent layer is activated by completely removing the liquids associated with the layer.


Solvent System 

Solvent systems are nothing but the mobile phase. 

The choice of the mobile phase depends on the following factors: 

Nature of the substance to be separated. 

Nature of the stationary phase used. 

Mode of chromatography (normal-phase or reverse-phase separation to be achieved (analytical or preparative). 


In general, organic solvent mixtures of low polarity are used. 

To minimize the absorption of any component of the solvent mixture the high polar solvents are avoided. 

Water is not used, because it may loosen the addition of the layer on the glass plate. 


Selection of the Solvent is Listed in the Allotropic Series Below 

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Plate Development

Plate development is the process by which the sample is carried upon the plate with the help of the mobile phase. 

The plate is placed in a closed saturated container containing the mobile phase. The mobile phase is grown up to the plate capacity and once it passes the line. 

The samples applied on the plate are carried up by the mobile phase. 

Once the mobile phase has reached approximately two-third of the plate, it is removed and left to dry. 

Sometimes these patches are not possible to be seen so there are a few methods to locate them: 

One such method is to spray iodine on the plate, iodine reacts with organic compounds to give a dark product which is visible on the plate. 

Often a fluorescent material is added to the plate, and under UV light the spot is displayed.


Spotting or Application of the Sample 

A line is drawn on the paper using a ruler and pencil approximately 1 cm from the edge of the plate.

A capillary tube is used to add a spot of the sample to the line. 

The sample should be applied to the same spot three or four times depending on the dilution of the sample.


Development Chambers 

TLC plates are placed vertically in a rectangular chromatography tank as shown in the figure.

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Amruta Sambrekar


Developmental tanks are classified according to the separation technique used. They are:

Tanks for ascending development. 

Tanks for descending development.

Tanks for horizontal development. 

Tanks for thin-layer electrophoresis.

 

Development of Chromatograms

Ascending Development 

The plates after spotting the sample are placed in a chamber containing a solvent at the bottom. 

The flow of the solvent is from bottom to top.

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Amruta Sambrekar

Descending Development

The flow of the solvent from the reservoir to the plate is by means of a filter paper strip. 

The solvent moves from top to bottom of the plate.

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Amruta Sambrekar

Two Dimensional Development

It is used when the components of the mixture are not completely separated by development in a single direction.

The sample spot is applied at the corner of the plate. 

The first development is carried out by the ascending method in one solvent. 

The plate is taken out, the solvent is allowed to evaporate.

The second development is carried out in another solvent by changing the edge of the plate at 90°.

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 Amruta Sambrekar

Detecting Reagents

After the development of a chromatogram, the spots should be visualized.

Colored spots can be detected visually, but for detecting colorless spots anyone of the following methods are used :            

Nonspecific method: This is a method where the number of spots can be detected but not the exact nature of compounds. For example,

Iodine chamber method: where brown or amber spots can be detected or observed when the paper is kept in the tank with few iodine crystals at the bottom.

UV chamber method for fluorescent compounds: when compounds are visible under the UV chamber at 245nm or 365nm fluorescent compounds can be detected.

Specific methods: In this method, specific spray reagents or detecting agents are used to find out the nature of compounds for identification purposes. For example,

Ferric chloride - for phenolic compounds and tannins.

Ninhydrin in acetone - for amino acids.

Dragendroff's reagent - for alkaloids.

3,5-Dinitro benzoic acid - for cardiac glycosides.

2,4-Dinitrophenylhydrazine - for aldehydes and ketones.


Qualitative Analysis of TLC

 Rf value: The Rf value (retardation factor) is calculated for identifying spots o.e in Qualitative Analysis.

 Rf = distance traveled by solute/distance traveled by the solvent

Rf value ranges from 0to1 but ideal values are from 0.3to0.8

Rf value is constant for every compound in a particular combination of stationary phase and mobile phase.

When the Rf value of the sample and reference compound is the same the compound is identified.

Rx value: Rx value is nothing but the ratio of distance traveled by sample and the distance traveled by the standard.

Rx=distance traveled by sample/ distance traveled by standard

Rx value is closer to 1.

Rm value: The Reichert-meissl value is the number of ml of 0.1N aqueous alkali solution required to neutralize the water-soluble, steam volatile fatty acids.

It is a value determined when examining fats. The Reichert value is an indicator of how much volatile fatty acid can be extracted.


Applications

It is a technique used for the analysis of polar compounds like amino acids, sugar, natural products, etc.

Separation of a mixture of drugs of chemical or biological origin, plant extracts, etc.

It is used for the separation of all classes of natural products and is established as an analytical tool in modern pharmacopeias. For example acids, alcohols, glycols, alkaloids, amines, macromolecules like amino acids, proteins, and peptides, and antibiotics.

For checking the purity of the sample.

To identify organic compounds

To purify the sample i.e for the purification purpose.

To evaluate the reaction process by assessment of the intermediate, reaction course, and so forth.

Identification of related compounds in the drugs.

To detect the presence of foreign substances in drugs.

Being a semi-quantitative technique TLC is used for rapid qualitative measurements than for quantitative purposes. But due to its rapidity of results, easy handling, and inexpensive procedure, it finds its application as one of the most widely used chromatography techniques.


HPTLC

Concept of HPTLC

HPTLC or High-Performance Thin Layer Chromatography is a sophisticated and automated form of TLC.

HPTLC is a form of thin-layer chromatography(TLC) that provides superior separation power using optimized coating material, novel procedures for mobile phase feeding, layer conditioning, and improved sample application.


Principle

The principle of separation is similar to that of TLC which is adsorption.


Steps involved in HPTLC

The following steps are involved in HPTLC.


Sample and Standard Preparation 

To avoid interference from impurities and water vapors.

Dry the plates and store them in a dust-free atmosphere.

For normal phase chromatography using silica gel or alumina pre-coated plates, non-polar solvents.

For reverse-phase(RP) chromatography, polar solvents.


Selection of Chromatographic Layer

Depends on the nature of the material to be separated. Commonly used materials are silica gel 60F, alumina, cellulose, etc.


Plates

Generally, plates of 20×20 or 5×7.5cm size having 100-250mm adsorbent thickness are used.

Silica gel 60F²⁵⁴ of pore size 6mm with a fluorescent indicator is a coating material.

The basic difference in TLC and HPTLC plates is the particle size of coated material which is in TLC 10-20micrometer and HPTLC 5-6 micrometer.


Pre-Washing

To remove water vapors, volatile impurities which might get rapid in plates. To avoid these, plates are cleaned by using methanol as a solvent by ascending or descending technique, etc.


Conditioning

Plates are activated by placing them in an oven at 120°C for 15 to 20 minutes.


Sample Application

The sample application of 1.0-1 micrograms /microlitre for HPTLC is the usual concentration range, above this concentration causes poor separation.

Application carried out by Linomat IV (automatic) applicator on the plate which gives uniform safe and standard results.


Preconditioning (Chamber Saturation)

An unsaturated chamber causes a high Rf value.

Saturated chamber by lining with filter paper for 30 minutes before development for uniform distribution of solvent vapors, less solvent for the sample to travel, and lower Rf value.


Selection of Mobile Phase

The selection of appropriate mobile phases is based on trial and error, one's own experience.

For the normal phase, a stationary phase is polar and the mobile phase is nonpolar. Non-polar compounds were eluted first because of lower affinity with the stationary phase. Polar compounds are retained because of higher affinity with the stationary phase.

For the reverse-phase, the stationary phase is nonpolar and the mobile phase is polar. Polar compounds were eluted first because of lower affinity with the stationary phase. Non-polar compounds are retained because of higher affinity with the stationary phase.


Chromatographic Development and Drying

The chromatogram is developed by the following techniques:

Ascending technique. 

Descending technique.

Horizontal technique.

Continuous technique.

Gradient technique.

Multidimensional technique. 

After development, remove the plate and the mobile phase is removed from the plate to avoid contamination of the lab atmosphere.

Dry in a vacuum desiccator, avoid hair dryers because the essential oil components may evaporate.


Common Problems Encountered During Chromatographic Development

Tailing:  It occurs due to the presence of traces of impurities, this can be reduced by buffering the mobile phase.

Diffusion: This is detected as zones on chromatographic plates. This is due to the nonuniformity of the mobile phase.


Detection of Spots

Detection of spots can be done by iodine vapor in an iodine chamber.

Detection under UV light is non-destructive 

spots of fluorescent compounds that can be seen at 254nm (short wavelength) or 366nm (long wavelength).

sports of non-fluorescent compounds can be seen when fluorescent stationary phases are used i.e silica gel GF.


Scanning and Documentation

HPTLC plates are scanned at selected UV regions wavelength by the instrument and the detected spots are seen on the computer in the form of peaks.

The scanner converts bands into peaks and peak height or area is related to the concentration of the substance on the spot.


Comparison Between TLC and HPTLC

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Applications of HPTLC

It is used for the purity control of chemicals, pesticides, steroids, and water analysis.

HPTLC is also widely used for the analysis of vitamins, water-soluble food dyes, pesticides in fruits, vegetables, and other foodstuffs.

It is also applied in pharmaceutical analysis, Biochemistry, and pharmacokinetic studies.


References and Sources

1. SlideShare: Thin Layer Chromatography | By Amruta Sambrekar.

2. Instrumental Method of Chemical Analysis, by B.K.Sharma.

3. Instrumental Method of Chemical Analysis, by Chatwal.

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