Lecture: Chromatography

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CHROMATOGRAPHY

INTRODUCTION

Chromatography is a combination of two words;

* Chromo – Meaning color

* Graphy – representation of something on paper

DEFINITION

“ It is a physical separation method in which the components of a mixture are separated by differences in their distribution between two phases, one of which is stationary (stationary phase) while the other (mobile phase) moves through it in a definite direction . The substances must interact with the stationary phase to be retained and separated by it .

CHROMATOGRAPHY TERMS Chromatogram:

It is the visual output of the chromatograph.

Chromatograph:

It is equipment that enables a sophisticated Separation.

Stationary phase (bounded phase):

It is a phase that is covalently bonded to the support particles or to the inside wall of the column tubing.

Mobile phase:

It is the phase which moves in a definite direction.

Analyte (Sample):

It is the substance to be separated during chromatography.

Eluate:

It is the mobile phase leaving the column.

Retention time:

It is the characteristic time it takes for a particular analyte to pass through the system(from the column inlet to the detector) under set conditions.

Eluent:

It is the solvent that will carry the analyte.

Retardation factor ( R ):

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Lecture: Chromatography

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Fraction of an analyte in the mobile phase of a chromatographic system.

CLASSIFICATION

According to mechanism of separation

1) Ion-exchange chromatography2) Affinity chromatography3) Size-exclusion chromatography4) Adsorption chromatography5) Partition chromatography

ION-EXCHANGE CHROMATOGRAPHY

It is a process that allows the separation of ions and polar molecules based on their charge.

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 chromatography is performed in columns but can also be useful in planar mode.

PRINCIPLE:

Ion - exchange chromatography retains sample molecules on the column based on ionic interactions.The surface of stationary phase displays ionic functional groups (R-X) that interact with analyte ions of opposite charge.

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.

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Lecture: Chromatography

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TYPES:

Cation exchange chromatography:

Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group.

Anion exchange chromatography:

Anion exchange chromatography retains anions using positively charged functional group.

APPLICATIONS:

It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.

Protein purification Water analysis Quality control

AFFINITY CHROMATOGRAPHYThis is the most selective type of chromatography.The method was discovered and developed by Pedro Cuatrecasas and Meir Wilchek for which the Wolf Prize in Medicine was awarded in 1987 .It is a method of separating biochemical mixtures and based on a highly specific biological interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.

DEFINITION:

“ 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.

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Lecture: Chromatography

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PRINCIPLE:

The stationary phase is typically a gel matrix (often agarose) . The molecule of interest has a known and defined property. The process is an entrapment in which the target molecule becomes trapped on stationary phase. The Stationary phase can then be removed from the mixture, washed and then target molecule is released from the entrapment.

APPLICATIONS:

Purify and concentrate an enzyme solution Purification of recombinant proteins Purification of antibodies

SIZE-EXCLUSION CHROMATOGRAPHY

The technique was invented by Grant Henry Lathe and Colin R Ruthven. They later received the John Scott Award for this invention.It is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight.It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers.

DEFINITION:

It is also known as gel permeation or gel filtration chromatography.

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Lecture: Chromatography

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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.

PRINCIPLE:

Smaller molecules are able to enter the pores of the media and, therefore, molecules are trapped and removed from the flow of the mobile phase . The average residence time in the pores depends upon the effective size of the analyte molecules . However, molecules that are larger than the average pore size of the packing are excluded.

APPLICATIONS:

Purification and analysis of synthetic and biological polymers, such as; Proteins, Polysaccharides, Nucleic acids.

It is also useful for determining the tertiary structure and quaternary structure of purified proteins.

It is generally a low-resolution chromatography technique and thus it is often reserved for the final, "polishing" step of a purification.

ADSORPTION CHROMATOGRAPHYDEFINITION:

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Lecture: Chromatography

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“ 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* EdgesThe 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. 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)

Adsorption chromatography

Column chromatography

Thin layer chromatography

Gas solidchromatography

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Lecture: Chromatography

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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 GF 254 Silica gel H Silica gel N Silica gel HF 254 Silica gel PF 254 Modified silica gel Alumina Kieselghur (Diatomaceous earth) Cellulose MN 300 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

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

TLC plate:Borosilicate glass plates are preferred. Most commonly used

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Lecture: Chromatography

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sizes are; 20 X 20cm 20 X 10cm 20 X 5cm

Microscopic slides are also used.

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 crystal Spraying agents

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Lecture: Chromatography

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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. 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,

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Lecture: Chromatography

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separation occurs. The components of sample mixture appear separated because of differences in their partition coefficient.

TYPES:

PAPER CHROMATOGRAPHYInstrumentation:

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

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

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.

Partition chromatography

Liquid-liquidchromatography

Gas-liquidchromatography

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Lecture: Chromatography

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Base line is marked on adsorbent. 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.

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