Chromatographic techniques | Basics of thin layer chromatography (tlc) and paper chromatography (pc) with principle | Types of paper chromatography | Applications of paper chromatography and TLC.

Chromatography introduction

Before going forward about thin layer chromatography, let’s know first basics about Chromatography. Chromatography is a separation technique whereby the components of a mixture may be separated by allowing the sample or analyte to pass over the material called a stationary phase by a moving phase called a mobile phase.  If the individual components of the mixture move at different rates then the separation will occur and the degree of separation the difference in migration.

Classification of chromatographic Methods

On the basis of the phases involved

The stationary phase may be solid or liquid while the mobile phase may be a liquid or a gas.

On the basis of physical principles involved

Chromatography can be divided into four different groups on the basis of physical properties involved,

Adsorption chromatography

• This type involves surface interactions (adsorption) between two phases.
• Such type of interaction is the result of intermolecular forces between surface atoms of solid and molecules of solute.
• The forces which lead to physical adsorption such as the London forces.
• Electrostatic forces, charge transfer forces, formation of Hydrogen bonds, etc. Are ideally suitable for chromatographic applications.

 Ion-Exchange Chromatography

• In this, a reversible exchange of ions is possible between ions in the liquid phase (mobile phase) and a solid insoluble substance containing ionic sites(solid phase).
• The solid phase has a polymeric matrix. It is permeable and it exchanges ions on a stoichiometric basis.
•  It tends to show a well-defined selectively for one ion over another.

Partition Chromatography(Liquid-Liquid)

•  In this type, the solute molecules are in equilibrium across an interfacial boundary that occurs between the mobile and stationary phases.
• The solute is distributed through the stationary phase and not just at the surface as in adsorption.
• The solubility of the solute in two phases is an important part of Partitioning. If the process was static, in general, it would be identical to solvent extraction.

Exclusion chromatography

• It includes those chromatographic processes in which the separation of sample components takes place according to molecular size.
• Larger molecules pass unhindered through the column, while molecules smaller than critical size will be retarded differently according to their size.

THIN LAYER CHROMATOGRAPHY (Tlc)

Principle of thin layer chromatography (Tlc principle) – What is Tlc?

In the thin layer chromatography technique (TLC) the stationary phase is in form of a thin layer of solid coated on the surface of a glass or metal or plastic plate called chromatophore.  The sample is dissolved in a suitable volatile solvent and applied to the plate.  The sample is then allowed to move along by the thin layer, by introducing the plate in another solvent that acts as a mobile phase. 

The separation of components occurs due to adsorption or partition in a thin layer of solvent present on the solid.  The resolution of the sample depends on the relative movement of individual components.  To compare the relative movement of different solutes term retardation or retention factor is introduced.   It is represented by Rf.  Retardation or retention factor is defined as the ratio of the distance traveled by the solute front from the original line to the distance traveled by the solvent front from the origin line under the given set of conditions.

The practical aspects of TLC are as follows.

Preparation of chromotoplate :-

1. Rectangular glass plates and plastic plates of aluminum foils are used to prepare chromatoplates.
2. The common adsorbents used in TLC are silica gel, alumina, powdered cellulose, etc.
3. The adsorbent is converted into slurry by using solvents such as water, chloroform, etc.
4. A binding agent such as plaster of Paris is incorporated to hold the adsorbent firmly on the plate.
5. The adsorbent is applied on the plate in form of a thin layer in different ways such as by using a commercial spreader, pouring or spraying the slurry, dipping the plate in the slurry, etc.
6. The plate is then dried at 100 to 120oC for about one hour.
7. The sample and the reference are transferred on the plate by using a capillary or micro syringe.
8. The plate is dried before the development.

Preparation and application of sample:-

1. About 0.1 to 1% solution of the sample is prepared in an organic solvent of low B.P.
2. A horizontal line is drawn at the top i.e. solvent front and at the bottom on the plate in such a way that it will remain about 1cm above the solvent level.
3. The spots are applied on the line by means of a capillary or micro syringe.
4. The width of the spot is about 0.5cm and the distance between the spots is about 2cm.
5. The spot should not be nearer to the edge of the plate.
6. The spot is dried in the oven or by hot air or an IR lamp.

Plate development:-

1. A suitable solvent or mixture of solvents is used for development.
2. The chromatogram is developed by ascending technique in a tank in which the plate is immersed in the developing solvent or mixture of solvents to the depth of 0.1 to 0.5cm.
3. The tank is lined with sheets of filter paper which dip into the solvent placed at the bottom of the tank.
4. The tank is fitted with a lid and the space in the tank is saturated with solvent vapors.
5. The chromatoplate is placed at an angle of 45^o.
6. The solvent ascends up the plate and the components of the mixture separate within a few minutes.  The solvent is allowed to run till it covers most of the thin layer.
7. Then the plate is taken out, dried, and subjected to the detection method.

Detection of the separated components:-

1. The components which are coloured can be easily detected.
2. The colorless components can be located by different methods.
3. In the ultraviolet region, most of the organic compounds exhibit fluorescence.
4. When certain reagents such as ninhydrin, conc. H2SO4 etc. is sprayed, and there is the interaction of the components with reagents that can produce coloured or fluorescent spots.
5. When the plate is placed in a chamber containing iodine vapours, brown spots on a yellow background are produced.

Identification and estimation:-

1. Identification of the component is carried out by comparing the Rf value of the sample and the standard.
2. In some cases, the solvent front goes beyond the edges of the plate.
3. Then the movement of any substance is compared with the movement of another substance.
4. Then the ratio is known as Rx value.  It is defined as the ratio of the distance traveled by the solute front from the original line to the distance traveled by the standard substance x.

• The quantitative estimation can be used either by direct method or by Indirect method depending upon the nature of the components

A]Direct Method:- 

Evaluation of components is carried out on the plate directly by any one of the methods given below:-

a. Visual comparison of the spot:-

• A chromatogram of reference solution of known concentration and sample is run on the same plate.
• The spot of the sample is compared for its size and intensity of colour with the spots of reference solutions.

b. Measurement of the area of the spot:-

• When the outline of the spot is well-defined, the area can be measured with a planimeter or graph paper.
• There is a direct relationship between the area of the spot and the amount of the substance.

c. Spot densitometry:-

• After development, the plate is sprayed with conc. H₂SO₄ containing the oxidizing agent and dried for 30 minutes at 180^oC.
• The colour developed is measured with a densitometer to calculate the concentration of the component.

B] Indirect or Elution method:-

The spot of the substance on the chromatogram is eluted in a minimum amount of suitable solvent.  The solution is diluted or concentrated and then analyzed by a suitable instrumental or non-instrumental method.

Application of TLC:–

• In a chemical reaction, paper chromatography is used to study the progress of the reaction.
• Paper chromatography is used for the identification of compounds in drugs, natural products, and biochemical preparations.
• Paper chromatography is used for the identification of a particular compound in a sample.
• Paper chromatography is used to detect the number of components present in the mixture.
• To determine the purity of the sample.
• Flavonoids that occur in plants can be easily separated by using TLC.
• One can detect trace pesticides in water.
• In a forensic laboratory, an analyst can identify the presence of poisons.
• To carry out qualitative and quantitative analysis of biological and metabolic samples which are helpful in the early detection of diseases.

PAPER CHROMATOGRAPHY

What is paper chromatography & What is the principle of paper chromatography?

In paper chromatography, the liquid mobile phase containing solutes is passed over the porous paper, which contains a stationary liquid phase (usually water).

2) The resolution of components occurs due to partitioning between two phases.

3) The rate of distribution and effective separation depends on the partition coefficient of individual solutes between two-phase.

4) The resolution of the sample depends on the relative movement of individual components.  To compare the relative movement of different solutes term retardation or retention factor is introduced.  It is represented by Rf.

Retardation or retention factor (R_f):-

It is defined as the ratio of the distance traveled by the solute front from the origin line to the distance traveled by the solvent front from the origin line under the given set of conditions.

Thus,

The R_f  value of a substance depends upon a number of factors as follows,

• The Solvent used
• The quality of the paper in paper chromatography
• The nature of the mixture
• Temperature
• The size of the vessel etc.

R_f is a function of the partition coefficient. It is a given substance, provided other experimental conditions like temperature, type of paper, duration, direction of development, etc. Are kept constant. Keeping the above factors constant it is possible to compare the Rf value of different substances. For eg. If ds is the distance traveled by solvent front from the original line, and dA and dB are the distance traveled by substances A and B, then their respective R_f values are given by,

This R_f value can be used to identify the component of the mixture.

In some cases, the solvent front runs off the end of filter paper, the movement of a substance in such cases is expressed as R_x but not in R_f value.

The practical aspects of paper chromatography are as follows

Choice of paper

The original work of paper chromatography was carried out by using Whatman filter paper no. 1.  Nowadays papers of different grades that differ in thickness and density are used.  The grade of the paper depends on the loading and rate of flow of solvent.  The paper can be modified in different ways for paper chromatography.

Preparation and application of sample

About 0.1 to 1% solution of the sample is prepared in an organic solvent of low B.P. A paper about 15cm in length and 5cm in breadth is generally used.  A horizontal line is drawn on the paper with a pencil in such a way that it will remain about 1cm above the solvent level.  The spot is applied on the line by means of a capillary or micro syringe. The width of the spot is about 0.5cm and the distance between the spot is about 2cm.  The spots are dried by hot air or an IR lamp.

Solvent system

1) The solvent or a mixture of solvents used as the mobile phase should be saturated with water.    

2) The solvent should be cheap, pure, and not highly volatile.

3) The Rf values of components should be different and between 0.05 and 0.95.

4) The solvent should not react with the sample and the composition should not change with time.

Development of the chromatogram

The development of the chromatogram is carried out in a tank or vessel with a well-fitted lid to prevent the escape of the solvent vapours and to establish equilibrium between liquid and vapours.  The chromatogram can be developed in different ways as given below.

Ascending Chromatography:- 

The development of the paper is done by allowing the solvent to travel in an upward direction.

The solvent is placed on the bottom of the tank.

The sample applied should be a few cm from the bottom edge of the paper.

The paper is either suspended from a hook in the solvent or rolled in a cylinder held together by clips and placed insolvent.

The mechanical weakness of the wet paper limits its size of the paper.  Also, there is a limit on the height to which the solvent can rise.

Descending Chromatography     

In Descending Chromatography, the Development of the paper is done by allowing the solvent to travel in a downward direction.

This method is used when R_f values of components of the mixture are very low or very close.

The process is carried out in a well-sealed tank with a through filled with solvent or mobile phase.

The sample is applied to a paper & the paper is introduced into the tank.

The upper end of the paper is immersed in the trough containing solvent.  The flow of the solvent starts with capillary action and is continued by gravity.  The length of the paper regulates the distance moved by the solvent front, hence proper resolution is obtained.

Radial Chromatography

Radial Chromatography is a horizontal method of development.  The process is economical and better separation is achieved.  The sample spot is placed near the center of the circular filter paper.  Two Petri dishes are placed on one another in a tank.  The lower dish is filled with solvent.  The paper is held horizontally and sandwiched between the edges of the Petri dishes.  A radial strip cut to form a wick is connected at the center and the other end of the wick dips in the solvent present in the lower dish.  The solvent is allowed to move radially towards the periphery and the components are spread into a series of concentric bonds.

Two-dimensional chromatography:-

In the case of compounds having simpler chemical constitutions, such as amino acids, the Rf values of components of the mixture are very close, and proper separation is not possible.  In such cases, two-dimensional chromatography devised by Martin is applied.  In this method, a square sheet of paper is used.  The sample is applied at one corner.  The edge of the paper AB is immersed in the solvent and the development is carried out.  This gives partial separation of the components. The sheet is removed, dried turned to 900, and put in another tank with edge AC immersed in another solvent. The development of this solvent gives the separation of the components.

Detection of the separated components

The components which are coloured can be easily detected however in most cases they are colorless hence various methods are used for detection as given below :

• Physical method:- In the ultraviolet region most of the organic compounds exhibit fluorescence. The organic compounds that do not exhibit fluorescence were found to produce it after irradiation with UV light from a high-pressure Hg vapour lamp.

• Chemical Method:- This method depends on the interaction of the components with reagents such as ninhydrin, which can produce coloured or fluorescent spots. The reagent is dissolved in a solvent in which the paper and the components are insoluble and sprayed on the paper to obtain coloured or fluorescent spots.

• Enzymatic and biological method:-  This method is used for the detection of enzymes for which the incubation of the developed chromatogram with necessary reagents is necessary.

• By using radioactive isotopes:- A labeled radioactive element is used for detection and the activity is measured with a GM counter.

Identification and estimation

Identification of the component is carried out by comparing the R_f value of the sample and the standard.  The quantitative

estimation can be either by direct method or by indirect method depending upon the nature of the components.

A).  Direct Method:-  Evaluation of component is carried out on the paper  directly by any one of the methods given below:

1. Visual comparison of the spot:- A chromatogram of reference solution of known concentrations and sample are run on the same paper.  The spot of the sample is compared for its size and intensity of colour with the spots of reference solutions.
2. Measurement of area of the spot:- When the outline of the spot is well defined, the area can be measured with a plan meter or graph paper.  There is a direct relationship between the area of the spot and the amount of the substance.
3. Radiotracer analysis:- A radioelement is used to detect and determine the amount of the substance.  A compound on the chromatogram is subjected to neutron bombardment and the activity is measured by a GM tube to determine the amount of the compound.

B) Indirect or Elution method:-  The spot of the substance on the chromatogram is out and soaked in a minimum amount of suitable solvent. The solution is diluted or concentrated and then analyzed by a suitable instrumental or non-instrumental method.

Advantages of Paper Chromatography

• Simple method
• High efficiency of separation
• Separation on macro or semi-micro or micro scale is possible
• Closely related substances can be separated.

Applications of Paper Chromatography

Paper Chromatography haves the following applications,

• The purity of the sample can be tested by using Paper Chromatography.  The pure compound produces one spot while the impure sample produces more than one spot. Different metal ions with similar or different properties, metal ion complexes, and isomers can be detected.
• In natural products, the number of components can be detected and identified.
• It can be used in the detection and estimation of components such as amino acids, sugars in urine, and other body fluids.
• In research, it can be used to separate and detect components of complex mixtures, and substances with high molecular weight.

Advantages of TLC over paper chromatography:-

1. Simple, rapid method.
2. Separation is sharper.
3. The spots are more discrete, and diffusion is less hence detection method is sensitive.
4. The use of an inorganic reagent eliminates the organic background effect in spectroscopic analysis.
5. More reactive reagents such as conc. H₂SO₄ can be used.
6. The amount of mixture used can be smaller or larger than PC.

Disadvantages:-

1. It is difficult to record and preserve the chromatogram.
2. R_f values may not be reproducible.

FAQs