Classification of Chromatographic Techniques

 

 

Classification of chromat chromatographic ographic techniques techniques

Chromatography: Chromatography is a set of techniques in which separation of chemical substances takes place place quantitatively as well as quantitativ quantitatively. ely.

Terminologies in chromatography chromatography: Mobile phase: In chromatography the substance which is introduced with or along with the sample and causes the elution of the content of the sample.

Stationary phase: It refers to that part which is present before the introduction of the sample or solute in the column(as in column chromatography) or on a solid support.

Eluent: The substance which separates the component of the mixture in chromatographic techniques. Eluent is that part which brings separation when the solution is passed either from the column or from solid support.

Eluate: The substance which is separated as an individual component of the mixture is eluate.

Classification of chromatograp chromatography hy:   On  

the basis of the interaction of solute to the stationary phase

On the the basis basis of of physical chromatographic bed shape: state of mobile phase

  On

On the basis of interaction of solute to the stationary phase: Adsorption chromatograph chromatography y: Adsorption chromatography is one of the oldest type of chromatography. It utilizes a mobile lliquid iquid or gaseous phase that is adsorbed onto the the surface of a stationary solid phase. The equilibration between the mobile and stationary  phase accounts for the separation of different solutes.

 

 

Principle : Principle of adsorption chromatography involves competition of components of sample mixture for active site on absorbent. These active sites are formed on molecule due to cracks or edges. Separation occur because of the fact that equilibrium is established between molecules adsorbed on stationary phase and those are flowing freely in mobile  phase. The more the affinity of the molecule of particular component, less will be its movement.

Types: Column chromatography Thin layer chromatography Gas solid chromatography

Partition chromatography: chromatography: This form of chromatography is based on a thin film formed on the surface of solid support by a liquid stationary phase. ph ase. Solute equilibrates between mobile  phase and the stationary liquid.

Principle:

 

Separation of components of sample mixture occurs because of partition. Stationary phase is coated with a liquid which is immiscible in mobile phase. Partition of components of sample between sample and liquid\gas stationary  phase retard some components of sample more more as compared to others. This gives basis for separation.

Types: Liquid-liquid chromatography Gas-liquid chromatography

Ion exchange chromatography chromatography: It is a process that allows the separation of ions and polar molecules based on affinity to the ion exchanger. It can be used for almost any kind of charged molecules including large proteins , small nucleotides and amino acids. The solution to be injected is called sample and individually separated components are called analytes. It is often used in protein purification, water analysis, and quality control.

Principle:

The ion exchange chromatography is based on relative retention of the ions during their progress through an ion exchange column which has functional group of opposite charge attached to its surface. The stronger the charge on the ion, the greater is the retention the column.

Types: Cation exchange chromatography Anion exchange chromatography Size exclusion chromatography:

 

It is a chromatographic method in which molecules in a solution are separated  by their size, and in some cases molecular molecular weight.it is usually applied to large molecules or molecular complexes such as proteins and industrial polymers. Typically an aqueous solution is used to transport the sample through column, technique is known as gel filtration chromatography, verses the name gel permeation chromatography, which is used used when an organic solvent is used as mobile phase.

Principle: A mixture of molecule dissolved in liquid( the mobile phase) is applied to a chromatographic column which contains a solid support in the form of microscopic spheres or beads The mass of beads is referred to as column bed. The beads act as traps or sieves and function to filter small molecules which  become temporarily n trapped within within pores. Larger molecules are excluded from beads. Large molecule samples cannot or can only partially penetrate the pores , whereas smaller molecules can access most or all pores. Thus larger molecules elute first, smaller molecules elute later, while molecules that can access all the pores elute last from the column.

On the basis of chromatographic chromatographic bed shape: Two dimensional:

Thin layer chromatography: chromatography: It is useful for separating organic compounds. Because of simplicity and rapidity of TLC, its often used to monitor the progress of organic reactions and to check purity of products.

 

 

Principle: It is also based on the principle of separation. the separation depends on the relative affinity of compounds towards stationary and mobile phase. The compounds under the influence of mobile phase travel over the surface of stationary phase. During this moment, the compounds with higher affinity to stationary phase travel slowly while others travel faster. Thus separation of components in the mixture is achieved. Once separation occurs individual components are visualized as spots at respective level on the plate.

Paper chromatography:  paper chromatography method is an analytical method that is is used to separate coloured chemicals or substances, especially pigments. This can also be used in secondary or primary colours in ink experiments. This method has been largely  by thin layer chromatography, but is still a powerful teaching teaching tool. Double-way  paper chromatography, also called two-dimensional chromatography, chromatography, involves using two solvents and rotating the paper 90⁰ in between. This is useful for separating complex mixtures of compounds having similar polarity, for example, amino acids. The mobile phase is developing solutions that can travel up to the stationary phase carrying the sample along with it.

On the basis of physical state of mobile phase

 

  Liquid

chromatography   Gas chromatography   Super critical fluid chromatography

Liquid chromatography It is a technique used us ed to separate a sample into its individual parts. This separation occurs based on the interactions of the sample with the mobile and stationary phase. Because There are many mobile\stationary combinations that can be employed when separating a mixture, there are several types of chromatography that are based on physical state of those phases. Liquid-solid column chromatography, the most popular chromatography technique, features a liquid mobile phase which slowly filters down through the solid stationary phase bringing the separated components with it.

Gas chromatograp chromatography hy 

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  Gas chromatography is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition.   Typical uses of GC includes testing the purity of a particular substance, or separating the different components of a mixture. In some situations, GC may help in identifying a compound. In    preparative chromatography, GC can be used to prepare prepare pure compounds from a mixture.

 

Supercriticall fluid chromatography Supercritica 

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  It is a form of normal phase chromatography, that is used for the analysis and purification of low to moderate molecular weight, thermally labile molecule.   It can also be used for the separation of chiral compounds. are same to HPLC, however, SFC typically uses carbon   Principles dioxide as the mobile phase; therefore the entire chromatographic flow  path must be pressurized.   The super critical phase represents a state in which liquid and gas  properties converge, supercritical fluid chromatography chromatography is sometimes called “convergent chromatography”.  chromatography”. 

High performance liquid chromatography 

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  It is formally called high-pressure liquid chromatography. It is used to separate the components in a mixture, to identify each component, and to quantify each component.   It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid absorbent material. s ample interacts slightly differently with the   Each component in the sample adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column.