chromatography

What is Chromatography? What are the uses of Chromatography? Types of of Chromatography?Column chromatography,Paper chromatography,Partition principle

Partition principle:

When a solute is allowed to equalibrate itself between two equals volumes of two immiscible liquids the ratio of the concentration of the solute in the two phases at equilibrium at a given temperature is called the partition coefficient.
A mixture of substance with different partitions coefficients can be quantitatively separated by a technique known as counter current distribution, first developed by l.c. Craig in which many repetitive partition steps takes place.
The place principle of partition is exploited in gaseous or liquid chromatography technique also separation depend upon the partition of the solute molecules between 2 liquid, supported on a suitable solid, and the gas flowing through the system.

Partition coefficient:-


Partition coefficient also known as distribution coefficient is a definitive term normally used to describe the way in which a given compound distributes or partition it self between two immiscible phases the stationary and mobile phase.
The solute upon entering a chromatographic system immediately distributes it’s a between the stationary and the mobile phases.
The mobile phase flow is stopped the compound will be in equilibrium between the stationary phase and the stopped mobile phase.
The concentration of the compound in each of the phases is described by the partition coefficient which is expressed as follows.
Cs and cm are the concentration of compound in the stationary and the mobile phase respectively.
Cs and cm are the concentration of compound in the stationary and the mobile phase respectively.
Partition coefficient is the basic principle of all chromatographic methods.

Nature of partition forces:-

The distribution of a solute between the stationary and the mobile phases is a result of the balance of forces between and the molecules of each phase .
The attractive or repulsive interactions are accompanied by a release or consumption of energy.
The amount of interaction energy provides a measure of the strength of the interaction and serves to classify the interaction as physical or chemical in nature.
Dispersion forces and electrostatic forces are the typical physical interactions which contributes most to partitioning of the solute between two phases.
Dispersion {london} interactions- this interaction is of a non polar nature.
London’s dispersion interaction is the only force between two molecules.
Nonpolar molecules do not possess any permanent dipole moment.
Polar interactions it is a dipole-dipole interaction
The solvents whose molecules have permanent dipole exhibits much more intermolecular attraction as compared to the non polar molecules.
Another type of polar interactions which is very important in chromatography is hydrogen bonding.

Paper chromatography:-

The paper commonly used consists of a highly purified cellulose.
Cellulose a homopolysaccharide of glucose contains several thousand anhydro glucose units linked through oxygen atoms.
Cellulose fibres in the paper hold moisture tightly through formation of hydrogen bonds.
The cellulose itself take negative charges in the company of water.

Apparatus:-

The apparatus required for paper chromatography consists of support for paper a solvent trough, and an tight chamber in which the chromatogram is developed.
The sample is applied to the paper and a small spot.
This is done before dipping the paper into the eluting solvent.
Generally used device or platinum loop or capillary tube.
There are two main techniques which may be employed for the development of paper chromatogram-ascending or descending technique.
In both cases the solvent is placed in the base of a sealed tank to allow the chamber to become saturated with the solvent vapour.
After equilibration of the chamber is achieved, the development of the chromatogram may be started.
In ascending technique the paper is allowed to hang in aniline {or} is suspended in a manner that the base of paper is in the contact with the solvent at the base of the chamber.
By capillary action the solvent moves upwards along with its samples also separated in the descending technique separation of the sample is achieved as the solvent moves downward under gravity.

Solvent system:-

Usually in paper chromatography the stationary phase is water. Since it is very well absorbed by cellulose.
The mobile phase which is less polar stationary phase.
The mobile phase is usually a mixture of various solvents such as alcohols, acids, phenols and hydrocarbons.

Detection:-

If the sample components are coloured the analysis becomes simple.
When the components are colourless they can be imparted colour by spraying the paper with colour producing reagents.
In case of detection of amino acids ninhydrin reagent sprayed on paper reacts with amino acid to form blue or purple colour.
Thin layer chromatography:-
Thin layer chromatography may be either carried out by the adsorption principle or partition principle.

Preparation of layers:-

The glass plate on which the thin layer is prepared should be even and is thoroughly washed and dried before layer application.
Modern thin layer chromatography kits provide plastic or glass plate. The material of which the thin is to be made of silicon gel.
While preparation stationary phase for adsorption chromatography a binder such as calcium sulphate is mixed with the slury. Is adsorption chromatography is to be performed the thin layer is activated by heating at 1100c for several hours.

Plate development:-

The choice of solvent and the method of elusion are much the same as per paper chromatography.
The procedure must be conducted in a closed chamber to prevent evaporation of the solvent.
One of the greatest advantages of tlc is a speed at which the separation is achieved.

Detection:-

Several detection methods are available. Those specific for tlc are:
Spraing the plate with 25 to 50% of sulphuric acid in ethanol and heating.
This result in changing of most of the compounds which show up as brown spots.
Iodine vapours are used extensively as a universal reagent for organic compounds.
The iodine spot disappears rapidly but can be made more permanent by spraying with 0.5% benzene solution in absolute ethanol.
Advantages and applications:-
Thin layer is more versatile faster and more reproducible it is used as pilot technique to quickly determine the complexity of a mixture.
Thin layer technique has often been used to identify drugs contaminants.
It has also been widely used to resolve plant extracts and many other biochemical preparations.

Column chromatography:-

Column’s:-
The columns are usually made up of glass or polyacrylate plastic. Different commons differ in their dimensions.
Laboratory column’s usually have a diameter of 2-70 mm and a length of 15 to 150 cm.
The columns are provided with a inlet and outlet.
The inlet which may be simple or filtered with a ground glass adopter provides for the elluring solvent to enter the column.
Column chromatography
Introducing of sample:-
It is necessary that the sample to be applied reaches the surface of the column below the top layer of the solvent.
The solvent is then added to the column to a height of 5 to 10 cm.
The column and is then connected to a suitable reservoir which contain more solvent.
Techniques of elusion:-
Column development:-
Continuous passage of suitable elluant {mobile phases} through the packed column the separate the components of the sample applied to the column.
This process is known as column development.

There are two main techniques of elution:-

{1} isocratic elution
{2} gradient elution.
M when a single solvent is used as an eluant during development the process is known as “isocratic separation” the process where the composition of the mobile prices changed giving rise to a gradient is known as “gradient” elution.

Flow rate:-

Fc is expressed as the volume of mobile phase per unit time.
For a satisfactory resolution it is absolutely necessary that the elluant floor should be maintained at a stable rate.
Column packing also influence flow rate. Unevenly packed column leads to distortion of the flow leading to unsatisfactory resolution more densely packed column and retard the flow of the mobile phase and decrease the flow rate.

Analysis and collecting of effluent:-

Effluent as it emerges from the column outlet is analysed.
There are two approaches to analysis of solutes in the effluent.
In modern approach to the monitary equipment is programmed to read the inherent property of the desired compound such as the ultraviolet absorption or radioactivity.

Various monitary equipment are:-

Fluorescence detector
Polarimeter
Voltameters
Refractometer detector
Conductivity detector

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