Chromatography

Dr. Tahir Ali RIPS

Riphah International University

For more detail follow the link: https://www.youtube.com/watch?v=e8i374hrWQg

What is Chromatography?

Chromatography, literally "color writing", was first employed by Russian scientist Mikhail Tsvet in 1900.

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 .

What is Chromatography?

What is Chromatography?

Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components.

Separate

• Analyze• Identify• Purify• Quantify

ComponentsMixture

Applications of Chromatography

Forensics

Research Pharmaceutical industry

Types of Chromatography…

Paper

HPLC Gas

Thin layer

Column

CLASSIFICATION

According to mechanism of separation

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

ION-EXCHANGE CHROMATOGRAPHY

DEFINITION:

• Ion-exchange chromatography (or ion chromatography) is a chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids.

• For more detail visit the following links:– https://www.youtube.com/watch?v=efUrl_djzQ0– https://www.youtube.com/watch?v=q3fMqgT1do8

• If the stationary phase is represented by R− or R+

and the sample by X+ and X−, retention in IEC can be represented as

X+ + R−K+ X+R− + K+ (cation exchange) X- + R+Cl- X-R+ + Cl- (anion exchange)

APPLICATIONS:

It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.1. Protein purification2. Water analysis3. Quality control

AFFINITY CHROMATOGRAPHY

• It is a method of separating biochemical mixtures based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.

APPLICATIONS:

• Purify and concentrate an enzyme solution

• Purification of recombinant proteins• Purification of antibodies

SIZE-EXCLUSION CHROMATOGRAPHY

• 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 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. This causes the larger molecules to pass through the column at a faster rate than the smaller ones

SIZE-EXCLUSION CHROMATOGRAPHY

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.

ADSORPTION CHROMATOGRAPHY

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

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

Adsorption chromatography

Column chromatography

Thin layer chromatography

Gas solidchromatography

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 phaseInert to solutes (adsorptive)Colorless especially when work with colored mixturesSuitable particle size enough to give good separation and reasonable flow rate

COMMON ADSORBENTS

Hydrated silica gelSilica gel GSilica gel SSilica gel GF254

Silica gel HSilica gel NSilica gel HF254

Silica gel PF254

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.”

InstrumentationChromatography jarCapillary tubeThin layer chromatography plateStationary phaseMobile 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

usedsizes are;• 20 X 20cm• 20 X 10cm• 20 X 5cm

Microscopic slides are also used.

InstrumentationMobile 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.

ProcedureSample 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, thenthe location is dried in ordinary light. But in case ofcolorless 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 documentation include;

Rf value in TLCPreservation of chromatogram by peeling off adsorbent.Graphical copying i.e. tracing on transparent paper.Photography

ApplicationsIt is used for separation and identification of;

Amino acidsPeptides and proteinsAlkaloidsCarbohydratesFats and fatty acidsAntibioticsNarcotic analgesicsGlycosides

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.

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

Partition chromatography

Liquid-liquidchromatography

Gas-liquidchromatography

PAPER CHROMATOGRAPHY

Instrumentation

Chromatography jarCapillary tubeStationary 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 ofliquids.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, thenthe location is dried in ordinary light. But in case ofcolorless 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 acidsCarbohydratesTannins GlycosidesAlkaloids etc.