CHROMATOGRAPHY

Chromatography is a technique used for separating or identifying the components in a mixture. It is a powerful method in industry, where it is used on a large scale to separate and purify the intermediates and products in various syntheses. There are several different types of chromatography : 

• Paper chromatography
• Thin layer chromatography (TLC)
• Gas chromatography (GC)
• Liquid chromatography (LC)
• Ion exchange chromatography
• Affinity chromatography

Basic principle

All chromatographic methods require one static part called “the stationary phase” and one moving part “the mobile phase”. The techniques rely on one of the following phenomena: adsorption; partition; ion exchange; or molecular exclusion.

Adsorption

Adsorption chromatography was developed first. It has a solid stationary phase and a liquid or gaseous mobile phase. Each solute has its own equilibrium between adsorption onto the surface of the solid and solubility in the solvent, the least soluble or best adsorbed ones travel more slowly. The result is a separation into bands containing different solutes. Liquid chromatography using a column containing silica gel or alumina is an example of adsorption chromatography.

Eluent:

The solvent that is put into a column is called the eluent.

Eluate:

The liquid that flows out of the end of the column is called the eluate.

Partition

In partition chromatography the stationary phase is a non-volatile liquid which is held as a thin layer on the surface of an inert solid. The mixture to be separated is carried by a gas or a liquid as the mobile phase. The solutes distribute themselves between the moving and the stationary phases, with the more soluble component in the mobile phase reaching the end of the chromatography column first. Paper chromatography is an example of partition chromatography.

Types of chromatography

1. Paper chromatography

In paper chromatography, the sample mixture is applied to a piece of filter paper, the edge of the paper is immersed in a solvent, and the solvent moves up the paper by capillary action. Components of the mixture are carried along with the solvent up the paper to varying degrees, depending on the compound's preference to be adsorbed onto the paper versus being carried along with the solvent. The paper is composed of cellulose to which polar water molecules are adsorbed, while the solvent is less polar, usually consisting of a mixture of water and an organic liquid. The paper is called the stationary phase while the solvent is referred to as the mobile phase. In order to obtain a measure of the extent of movement of a component in a paper chromatography experiment, we can calculate an "Rf value" for each separated component in the developed chromatogram. An Rf value is a number that is defined as: distance traveled by component from application point

 

2. Thin layer chromatography (TLC)

Thin layer chromatography (TLC) is a method for identifying substances and testing the purity of compounds. Separations in TLC involve distributing a mixture of two or more substances between a stationary phase and a mobile phase. The stationary phase is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate. The mobile phase is a developing liquid which travels up the stationary phase, carrying the samples with it. Components of the samples will separate according to how strongly they adsorb on the stationary phase versus how readily they dissolve in the mobile phase.

3. Gas Chromatography:

Gas chromatography makes use of a pressurized gas cylinder and a carrier gas, such as helium, to carry the solute through the column. The most common detectors used in this type of chromatography are thermal conductivity and flame ionization detectors. There are three types of gas chromatography that will be discussed here: gas adsorption, gas-liquid and capillary gas chromatography. Gas adsorption chromatography involves a packed bed comprised of an adsorbent used as the stationary phase. Common adsorbents are zeolite, silica gel and activated alumina. This method is commonly used to separate mixtures of gases.

4. Liquid Chromatography

There are a variety of types of liquid chromatography. There is liquid adsorption chromatography in which an adsorbent is used. This method is used in large-scale applications since adsorbents are relatively inexpensive. There is also liquid- liquid chromatography which is analogous to gas-liquid chromatography. The three types that will be considered here fall under the category of modern liquid chromatography. They are reverse phase, high performance and size exclusion liquid chromatography, along with supercritical fluid chromatography. Reverse phase chromatography is a powerful analytical tool and involves a hydrophobic, low polarity stationary phase which is chemically bonded to an inert solid such as silica. The separation is essentially an extraction operation and is useful for separating non-volatile components.

High performance liquid chromatography (HPLC) is similar to reverse phase, only in this method, the process is conducted at a high velocity and pressure drop. The column is shorter and has a small diameter, but it is equivalent to possessing a large number of equilibrium stages. Size exclusion chromatography, also known as gel permeation or filtration chromatography does not involve any adsorption and is extremely fast. The packing is a porous gel, and is capable of separating large molecules from smaller ones. The larger molecules elute first since they cannot penetrate the pores. This method is common in protein separation and purification.

Supercritical fluid chromatography is a relatively new analytical tool. In this method, the carrier is a supercritical fluid, such as carbon dioxide mixed with a modifier. Compared to liquids, supercritical fluids have solubilities and densities have as large, and they have diffusivities and viscosities quite a bit larger. This type of chromatography has not yet been implemented on a large scale.

5. Ion Exchange Chromatography

Ion exchange chromatography is commonly used in the purification of biological materials. There are two types of exchange: cation exchange in which the stationary phase carries a negative charge, and anion exchange in which the stationary phase carries a positive charge. Charged molecules in the liquid phase pass through the column until a binding site in the stationary phase appears. The molecule will not elute from the column until a solution of varying pH or ionic strength is passed through it. Separation by this method is highly selective. Since the resins are fairly inexpensive and high capacities can be used, this method of separation is applied early in the overall process.

6. Affinity Chromatography

Affinity chromatography involves the use of packing which has been chemically modified by attaching a compound with a specific affinity for the desired molecules, primarily biological compounds. The packing material used, called the affinity matrix, must be inert and easily modified. Agarose is the most common substance used, in spite of its cost. The ligands, or "affinity tails", that are inserted into the matrix can be genetically engineered to possess a specific affinity. In a process similar to ion exchange chromatography, the desired molecules adsorb to the ligands on the matrix until a solution of high salt concentration is passed through the column. This causes desorption of the molecules from the ligands, and they elute from the column. Fouling of the matrix can occur when a large number of impurities are present, therefore, this type of chromatography is usually implemented late in the process.