Chromatography, Mechanisms, its Types ,uses.

CHROMATOGRAPHY

Chromatography, Mechanisms, its Types  ,uses.

Chromatography

Mechanisms of Separation in Chromatography

1. Adsorption chromatography 

2. Partition chromatography 

3. Exclusion chromatography

Rf value

Types of Chromatography in Routine Use

1. Paper chromatography 

2. Thin-layer chromatography (TLC) 

3. Ion-exchange chromatography 

4. Gas chromatography 

A gas-chromatograph has six components

Working of chromatography

what is chromatography

Chromatography is a process of separation of a mixture of solutes dissolved in a common solvent.

The separation technique makes use of the differential distribution of the solutes between two phases. the mobile phase and the fixed stationary phase.

• The solvent is the mobile phase which carries the mixture of the solutes through the stationary phase. Depending on the characteristics of the solute molecules, the mobile phase and the stationary phase, there is selective retardation of the solute molecules relative to the moving phase. Under ideal conditions, the resulting different rates of migration bring about complete separation of the solutes.

• The technique of chromatography was originally used for the separation of plant pigments into coloured bands. Chromatography is now rarely used for coloured substances but the original name still remains. Since the mixture contains colourless substances, a technique must be employed for the detection of separated components of the mixture.

• Chromatographic methods can be classified according to the physical state of the solute carrier phase. The two main categories are liquid chromatography in which solute phase is a liquid or solution, and gas chromatography, in which the solute phase is in a gaseous state. Further classification is based on the form of the stationary phase matrix.

•  Liquid chromatography is subdivided into flat and column methods. In flat methods, the stationary phase is supported on a flat surface such as cellulose acetate paper or in a thin layer mechanically supported with glass or plastic. For column methods in liquid and gas chromatography, various types of materials such as ion-exchange resins, diatomaceous earth or internally coated fine glass capillaries may be used. Not all the methods mentioned above are routinely used.

Mechanisms of Separation in Chromatography

There are three mechanisms by which separation occurs in chromatography. They are adsorption, partition and exclusion chromatographies.

     1.Adsorption chromatography  In adsorption chromatography, the components within a sampleare separated because of the differences in their attraction to the stationary phase and the mobile phase. The speed of migration of a component depends on its adsorptive affinity relative to other components. 

• The example of this technique is the thin-layer chromatography (TLC). In ionexchange chromatography, electrostatic forces operate between charged molecules and oppositely charged particles on the ion-exchange resin or modified cellulose or dextran. These electrostatic forces can be altered by changing the pH of the mobile phase. 

2. Partition chromatography 

 Partition chromatography utilises differences in the relative solubility of the solute molecules between the mobile and the stationary phase. The two phases may be liquidliquid or gas-liquid. This methodology is used for gas liquid chromatography (GLC) and for high performance liquid chromatography (HPLC).

3. Exclusion chromatography(Gel-filtration or gel-permeation chromatography) 

When a mixture of small and large particles is allowed to pass over porous solid particles, the smaller molecules or ions pass through the pores of the solid particles. The pore size determines which molecules are able to enter the particle and which are excluded. The particles with large molecular size, such as proteins, are excluded more easily than other smaller particles.

Rf value

Ry value is the ratio of distance travelled by the substance (solute) to that travelled by the mobile phase in the same period. Generally, mobile phase travels faster than the solute components. Therefore Ry value is generally a fraction.

Note R, value is applicable only to thin-layer or paper chromatography.

Types of Chromatography in Routine Use

1. Paper chromatography 

• In paper chromatog. raphy, the stationary phase is a paper, usually cellulose acetate, and the mobile phase is a solvent in which the solutes in the mixture are soluble. Initially, a mixture of substances, e.g., carbohydrates. 

• is spotted at one end of the porous filter paper strip. In ascending type of paper chromatography, the filter paper is hung vertically into the solvent. The solvent moves up through the paper by capillary action. 

• As it passes through the spotted area of solutes, various fractions in the mixture move at different rates. The rate of mobility is affected by relative solubilities and polarities of the solutes and the polarity of the solvent.

• The paper is removed after the separation, dried and sprayed with a chemical for colour development. The spots of the solutes developed at different sites can be quantitated by the area and intensity of the colour.

• Paper chromatography has been employed most commonly for the separation of amino acids and carbohydrates. It is now being replaced by ionexchange, thin layer, HPL or gas chromatography.

2. Thin-layer chromatography (TLC) 

• TLC is very similar to paper chromatography except that the paper is substituted by a thin layer (0.025 mm) of very finely powdered silica gel, alumina, polyacrylamide gel, starch gel or kieselguhr, bound to a glass or plastic plate. 

• The principle of separation is similar to that of paper chromatography. The advantages of TLC over paper chromatography include easier elution of separated spot from the plate by cutting the thin layer, and faster separation of constituents. 

3. Ion-exchange chromatography 

• Ion-exchange resins are suitable polymers which contain ionic groups as a part of their structure. Ion-exchange resins can be, cation exchangers, e.g., with SO or COO" groups; or anion exchangers, e.g.. with N*(NH), group.

• If a suitable ion-exchange resin is used for column packing. its affinities for the members of a group of ionised substances can be used for their separation.

• The ion-exchange resins are extensively used for the separation of amino acids. If a solution containing mixture of amino acids is applied to a column of suitable ion-exchange resin, the amino acids are linked to the resin. The degree of affinity, however, varies with the amino acids. 

• If, after the adsorption, the column is washed or eluted by a series of buffers of varying pH, the individual amino acid can be separated and quantified with ninhydrin. 

4. Gas chromatography 

Gas chromatography is a separation process by which a mixture of compounds in a gaseous state is passed through a gas in a stationary phase. The mobile phase is usually nitrogen, helium or argon. Separation of the mixture is achieved by a difference in the partitioning of the various molecules between the two phases.

• If the stationary phase consists of a thin layer of nonvolatile liquid, the technique is called gas-liquid chromatography (GLC). If the stationary phase is a solid sorbent, then it is called gas-solid chromatography (GSC).

A gas-chromatograph has six components

(1) Pressurised carrier gas with a flow regulator

(2) Sample injection area

(3) A column: gas, liquid or solid

(4) A detector

(5) A recorder

(6) A thermoregulated 

component enclosing the injection area, column and detector. 

Working 

• At a sufficiently high temperature, the components of a mixture to be injected are in a gaseous phase. Each component of the mixture requires specific time to reach the detector after application to the column. 

• The output of the detector is displayed on a recorder. This retention time may be expressed as an absolute time or as a relative retention time with reference to a standard. 

• High performance liquid chromatography (HPLC) In HPLC, the stationary phase is composed of uniform, ultrafine particles which greatly increase its adsorptive area. This stationary phase is packed firmly into a column. 

• The resistance toflow in this column is high, therefore large pressures (500-5000 pounds per square inch) are required to deliver constant flow rates. The elute from the column is monitored by a variety of detectors such as UV or redox-potential electrode detectors.

• HPLC is a very efficient technique for the detection and quantitation of drugs.