ADSORPTION CHROMATOGRAPHY Muhammad Tanveer Khan

DEFINITION “It is a type of chromatography in which a mobile liquid or gaseous phase is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes.”

PRINCIPLE Principle involves competition of components of sample mixture for active site on adsorbent. These active sites are formed in molecule due to * Cracks * Edges The electrostatic forces present in the molecule, which hold together the crystal lattice, are directed outward. These forces and electrostatic forces of solute molecule cause separation.

PRINCIPLE Separation occurs because of the fact that an equilibrium is established between molecules adsorbed on stationary phase and those which are flowing freely in mobile phase. The more the affinity of the molecule of particular component, less will be its movement.

TYPES

ADSORBENTS “An adsorbent is a substance, usually porous in nature and with a high surface area that can adsorb substances onto its surface by intermolecular forces.”

AN IDEAL ADSORBENT The Ideal adsorbent must fulfill the following requirements: Insoluble in mobile phase Inert to solutes (adsorptive) Colorless especially when work with colored mixtures Suitable particle size enough to give good separation and reasonable flow rate

COMMON ADSORBENTS Hydrated silica gel Silica gel G Silica gel S Silica gel GF254 Silica gel H Silica gel N Silica gel HF254 Silica gel PF254 Instrumentation

COMMON ADSORBENTS Modified silica gel Alumina Kieselghur (Diatomaceous earth) Cellulose MN300 Cellulose microcrystalline

THIN-LAYER CHROMATOGRAPHY “The technique which involves flowing of mobile phase over a thin layer of adsorbent, applied on solid support, where separation of components occur by differential migration which occurs when solvent flows along fine powder spread on glass plates, is called thin –layer chromatography.”

Instrumentation Chromatography jar Capillary tube Thin layer chromatography plate Stationary phase Mobile phase

Instrumentation Chromatography jar: It is made of glass and has a lid on it. Jar maintains proper environment that is required for separation. Capillary tube: It is used to apply sample mixture on TLC plate. Stationary phase: Adsorbents

Instrumentation TLC plate: Borosilicate glass plates are preferred. Most commonly used sizes are; 20 X 20cm 20 X 10cm 20 X 5cm Microscopic slides are also used.

Instrumentation Mobile phase: Mobile phase may be a single liquid or a mixture of liquids. Commonly used mobile phases are; Methanol Ethanol Ethyl acetate Diethyl ether Acetone Chloroform

Procedure Clean and dried chromatography jar is taken. A paper impregnated in the mobile phase is applied to the walls to ensure that atmosphere of the jar is saturated with solvent vapors. Mobile phase is added to the jar at a length of 0.5-1cm from the bottom. Jar is closed. Equilibrium is allowed to be maintained. Base line is marked on adsorbent.

Procedure Sample is applied on TLC plate with help of capillary tube. Sample spot is air dried. TLC plate is put in the chromatography jar and lid is closed. The system is allowed to be static until the solvent move to a proper distance from baseline. TLC plate is taken out and dried.

Location of separated components If the sample is separated into colored components, then the location is dried in ordinary light. But in case of colorless components following are used; Uv lamp Iodine crystals Spraying agents

Documentation Storage of chromatogram for TLC is difficult. It is usually undesirable since plates are employed for repeated use. Various methods for separation include; Rf value in TLC Preservation of chromatogram by peeling off adsorbent. Graphical copying i.e. tracing on transparent paper. Photography

Applications It is used for separation and identification of; Amino acids Peptides and proteins Alkaloids Carbohydrates Fats and fatty acids Antibiotics Narcotic analgesics Glycosides

PARTITION CHROMATOGRAPHY

DEFINITION “This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid.”

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

PRINCIPLE The stationary phase immobilizes the liquid surface layer, which becomes stationary phase. Mobile phase passes over the coated adsorbent and depending upon relative solubility in the coated liquid, separation occurs. The component of sample mixture appear separated because of differences in their partition coefficient.

TYPES

PAPER CHROMATOGRAPHY “.”

Instrumentation Chromatography jar Capillary tube Stationary phase (liquid impregnated paper) Mobile phase

Instrumentation Chromatography jar: It is made of glass and has a lid on it. Jar maintains proper environment that is required for separation. Capillary tube: It is used to apply sample mixture. Stationary phase: liquid impregnated paper

Instrumentation Mobile phase: Mobile phase may be a single liquid or a mixture of liquids. Commonly used mobile phases are; Methanol Ethanol Ethyl acetate Diethyl ether Acetone Chloroform

Procedure Clean and dried chromatography jar is taken. A paper impregnated in the mobile phase is applied to the walls to ensure that atmosphere of the jar is saturated with solvent vapors. Mobile phase is added to the jar at a length of 0.5-1cm from the bottom. Jar is closed. Equilibrium is allowed to be maintained. Base line is marked on adsorbent.

Procedure Sample is applied on paper with help of capillary tube. Sample spot is air dried. Paper is put in the chromatography jar and lid is closed. The system is allowed to be static until the solvent move to a proper distance from baseline. Paper is taken out and dried.

Location of separated components If the sample is separated into colored components, then the location is dried in ordinary light. But in case of colorless components following are used; Uv lamp Iodine crystals Spraying agents

Documentation Storage of chromatogram. Calculating Rf values

Applications It is used for separation and identification of; Amino acids Carbohydrates Tannins Glycosides Alkaloids etc.

ION EXCHANGE CHROMATOGRAPHY

DEFINITION In this type of chromatography, a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. Ion exchange mechanism separates analytes based on their respective charges. Ion exchange chromatography is performed in columns but can also be useful in planar mode.

MECHANISM Ion exchange chromatography uses a charged stationary phase to separate charged compounds including anions, cations, amino acids, peptides, and proteins. In conventional methods the stationary phase is an ion exchange resin that carries charged functional groups which interact with oppositely charged groups of the compound to be retained. Ion exchange chromatography is commonly used to purify proteins.

MECHANISM

SIZE EXCLUSION CHROMATOGRAPHY

DEFINITION It is also known as gel permeation or gel filtration chromatography. This type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allows smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones.

It is generally a low-resolution chromatography technique and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins.

MECHANISM

AFFINITY CHROMATOGRAPHY

DEFINITION This is the most selective type of chromatography. It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. For example, the immobilized molecule may be an antibody to some specific protein. When solute containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic strength or pH.