Chromatography

Principle, Types and Applications

Chromatography by Dr. Atif

Presented By

Muhammad Atif

PhD Scholar (Pharmaceutical Sciences) AWKUM

Manager QA Decent Pharma Islamabad.

It is a laboratory technique for the separation of a mixture of phyto constituents.
Chromatography derived from two Greek Words

Chroma means “Color”
Graphein mean “ to write”

The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The separation is based on differential partitioning between the mobile and stationary phases.
Chromatography usually consists of mobile phase and stationary phase.

Mobile phase: The mixture of substances to be separated that may be a liquid or a gas that pass through stationary phase.
Stationary phase: a porous solid matrix through which the mobile phase along with sample percolates.

The interaction between the mobile phase and the stationary phase results in the separation of the compounds from the mixture depending upon its partition coefficient between mobile phase and stationary phase.
Due the difference in the partition co-efficient of the compound a different retention time occur on the stationary phase.
Due to this phenomenon various constituents of the mixture travel at different speeds along with mobile phase, causing them to separate.

Chromatographic terms

Chromatograph: equipment that enables a sophisticated separation.
Eluent: Fluid entering column or solvent that carries the analyte.
Eluate: Mobile phase leaving the column.
Stationary phase: Immobilized phase Immobilized on the support particles or on the inner wall of the column tubing.
Mobile phase: Mobile phase moves in a definite direction. It may be a liquid or Gas. The mobile phase moves through the chromatography column i.e the stationary phase where the sample interacts with the stationary phase and is separated.
Retention time: Time takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions.
Analyte: Substance that is to be analyzed in chromatography.

Classification of Chromatography

Classification on the basis of physical state of mobile phase

Liquid chromatography
Gas chromatography
Super critical fluid chromatography

Classification on the basis of chromatographic bed shape

Two dimensional chromatography
Thin Layer Chromatography
Paper Chromatography
Three Dimensional chromatography
Column Chromatography

Classification on the basis of interaction of solute to the stationary phase

Adsorption Chromatography
Partition Chromatography
Ion Exchange Chromatography
Size Exclusion Chromatography

Purpose of chromatography

Preparative purpose

The purpose of preparative chromatography is to separate the components of a mixture for later use, and is thus a form of purification.

Analytical purpose

Analytical chromatography is done normally with smaller amounts of material and is for establishing the presence or measuring the relative proportions of analytes in a mixture.

Adsorption Chromatography

Adsorption Chromatography

Adsorption?
It is a surface phenomenon in which interaction takes place only on the surface of one substance.
Adsorption chromatography is a type of Liquid Chromatography in which chemicals are retained based on their adsorption and desorption at the surface of the support, which also acts as the stationary phase.

Principal

Adsorption Chromatography involves the analytical separation of a chemical mixture based on the interaction of the adsorbate with the adsorbent. The mixture of gas or liquid gets separated when it passes over the adsorbent bed that adsorbs different compounds at different rates.
Adsorbent

A substance which is generally porous in nature with a high surface area to adsorb substances on its surface by intermolecular forces is called adsorbent
Some commonly used adsorbents are Silica gel, cellulose microcrystalline, alumina, modified silica gel.

Adsorbate

In Adsorption the substance whose molecules get adsorbed at the surface is called the adsorbate.

This method is also sometimes referred to as liquid-solid chromatography.
Retention in this method is based on the competition of the analyte with molecules of the mobile phase as both bind to the surface of the support.
The degree of a chemical's retention in adsorption chromatography will depend on the following
Binding strength of this chemical to the support.
Surface area of the support.
Amount of mobile phase displaced from the support by the chemical.
Binding strength of the mobile phase to the support
Electrostatic interactions, hydrogen bonding, dipole-dipole interactions, and dispersive interactions (ie, van der Waals forces) all may affect retention in this type of chromatography.

Types of Adsorption Chromatography

Thin layer Chromatography
Paper Chromatography
Column Chromatography
Gas-Sloid Chromatography

Thin Layer Chromatography

It is a chromatography technique where the mobile phase moves over an adsorbent.
The adsorbent is a thin layer which is applied to a solid support for the separation of components.
The separation takes place through differential migration which occurs when the solvent moves along the powder spread on the glass plates.

Paper chromatography

It is a technique that uses paper sheets or strips as the adsorbent being the stationary phase through which a solution is made to pass is called paper chromatography.
The solid surface of the paper is the stationary phase and the liquid phase is the mobile phase.

Column chromatography

The technique in which the solutes of a solution are entitled to travel down a column where the individual components are adsorbed by the stationary phase.
Based on the affinity towards adsorbent the components take positions on the column. The most strongly adsorbed component is seen at the top of the column.

Gas-Solid chromatography

The principle of separation in GSC is adsorption
It is used for solutes which have less solubility in the stationary phase.
This type of chromatography technique has a very limited number of stationary phases available and therefore GSC is not used widely.

Advantages and Applications

It has a wide range of mobile phases for the separation of compounds.
Adsorption chromatography is an important method to separate many components that are not separated by other techniques.
The complex sample mixtures can be easily separated by this method.
Very few types of apparatus or types of equipment are required for isolation
Adsorption chromatography is used for separation of amino acids.
It is used in the isolation of antibiotics.
It is used in the identification of carbohydrates.
It is used to separate and identify fats and fatty acids.
It is used to isolate and determine the peptides and proteins

Partition Chromatography

Type of Chromatographic technique in which components of a mixture are separated in between the stationary phase and mobile phase.
Developed by Richard Laurence Millington Synge and Archer Martin in 1940.
It is mostly liquid-liquid chromatography as both the stationary and mobile phase are liquid
Some time when the mobile phase is in gaseous state then it is called Gas-Liquid Chromatography.
Stationary phase in both the Liquid-Liquid chromatography and gas-Liquid Chromatography is Liquid.

Principal

Separation is based on the differential portioning of the components of the sample mixture between stationary phase and mobile phase.
Immiscible solid support covered by liquid surface on the stationary phase is in the mobile phase.
The liquid surface is immobilized by the stationary solid phase which make it stationary phase.
The mobile phase moves from the stationary phase and components of mixture sample get separated.
Separation of component defends upon the partition coefficient.

Types

Liquid-Liquid Partition Chromatography
Gas-Liquid Partition Chromatography

Liquid-Liquid Partition Chromatography

This type of partition chromatography have both the stationary phase and mobile phase in liquid state.
Paper chromatography
Thin layer Chromatography

Gas-Liquid Partition Chromatography

In this type of partition chromatography the mobile phase is an inert Gas and stationary phase is in liquid state.

Applications

Separation and detection of color mixtures including pigments.
Separation and determination of proteins, lipids, alkaloids, glycosides, and other biomolecules.
Isolation and detection of amino acids and carbohydrates.
Determination of pollutants in food products and beverages.
Isolation of polar and non-polar compounds.
Sequencing of DNA and RNA.
Identification of drugs and impurities.

Adsorption and Partition Chromatography Similarities

Both are types of Chromatography.
Both are used for the purpose of separation.
Both can separate components in all three stages namely gas, liquid and solid.

Adsorption and Partition Chromatography Dissimilarities

Adsorption Chromatography
Separation is based on adsorption
Liquid-liquid extraction
Solid stationary phase
Partition Chromatography
Separation is based on partition
Liquid-Solid extraction
Liquid Stationary Phase

Ion Exchange Chromatography

Ion exchange chromatography retain molecules on the basis of ionic interaction.
The stationary phase in ion exchange chromatography is a resin ion exchanger.
Mobile phase in ion exchange chromatography is an eluent.

Principal

Separation principal of ion exchange chromatography is based upon the reversible exchange of ions in sample with that of the ions present in ion exchange resin.
Ion exchange separations take place in a column covered with an ion exchanger.

Types of Ion exchange Chromatography

Cation Exchange Chromatography

Here cations are retained on negatively charged stationary phase with negatively charged functional groups and is used when the molecule of interest is positively charged.

Anion Exchange Chromatography

Here anions are retained on positively charged stationary phase with positively charged functional groups is used when the molecule of interest is negatively charged.

Separation of Amino Acids Mixture

When amino acid mixture is passed through the cation exchange chromatgraph, individual amino acid can be eluted using buffers of different pH .
A change in pH affects the charge on the particular molecules and, therefore, alters binding.
Bound proteins are eluted out by utilizing a gradient of linearly increasing salt concentration, usually NaCl.
With increasing ionic strength of the buffer, the salt ions will compete with the desired proteins in order to bind to charged groups on the surface of the medium.
This will cause desired proteins to be eluted out of the column.
Proteins that have a low net charge will be eluted out first as the salt concentration increases causing the ionic strength to increase.
Proteins with high net charge will need a higher ionic strength for them to be eluted out of the column.

Applications

Ion exchange chromatography is used for the estimation and separation of molecules with different charges or ions that include proteins, peptides, amino acids, vitamins, carbohydrates, enzymes, etc. either on its own or along with other chromatographic techniques.
It plays an important role in modern drug discovery for isolating natural products.
The nonsolvent extractable natural products can be followed up.
This is also applicable for segregating and purifying organic molecules from natural sources.
Ion exchange chromatography is a powerful and critical tool for pharmaceutical analytical chemists.

Size Exclusion Chromatography

Size-exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC) or gel filtration chromatography.
It is also called as molecular sieving or molecular exclusion chromatography.
Molecules in size exclusion chromatography are separated on the basis of size, shape and molecular weight.
The chromatography column is packed with fine, porous beads.
These beads consist of dextran polymers (Sephadex), agarose (Sepharose), or polyacrylamide (Sephacryl or BioGel P).
These beads pore size works to estimate the size of macromolecules.
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.
Molecules having size larger than the average pore size of the packing are excluded and hence cannot be retained in the coloumn.
The molecules are separated in this way by entrapping or retaining the small molecules and larger molecules pass through the mobile phase without any retention suffering.

Applications

SEC is a widely used technique for the purification and analysis of synthetic and biological polymers, such as protein, polysaccharides and nucleic acid.
SEC is used to purify various species of RNA and viruses using agarose gels.
It is used in copolymerisation studies.
It is also used in molecular weight determination
Separation of sugar, proteins, peptides, rubbers and others on the basis of their size.
SEC technique is used to determine the quaternary structure of purified proteins.

High Performance Liquid Chromatography

A form of column chromatography to separate, identify and quantify different components of a mixture
Developed in 1970
The most widely used analytical technique
HPLC is an extension of conventional liquid chromatography.
Powerful tool in analytical techniques
Columns are tightly packed, and the eluent is forced through the column under high pressure ( up to 5000 psi) by a pump.
Allows to use a very smaller particle size of the column packing material which gives a much greater surface for interactions between the stationary phase and the molecules flowing through it
Allows a much better separation of the component of the mixture.

HPLC technique

Utilize liquid mobile phase to separate the mixture
Analytes are 1^st dissolved in a solvent then through column applying high pressure.
Mixture is resolved into their respective component on the basis of retention time in the column.

Components of HPLC

Pump
Injector
Column
Detector
Data record system

Pump

A pump forces the mobile phase trough the column at a much greater speed than gravity-flow columns
Pumps are designed in order to maintain a stable flow rate.
Flow range 0.01 to 10 ml/min