1. chromatography

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CHROMATOGRAPHY:

CHROMATOGRAPHY Muhammad Tanveer Khan Assistant Professor Faculty of Pharmacy, Lahore Medical and Dental college

CONTENTS:

Introduction Milestones in chromatography What is chromatography? Definition Purpose of chromatography Applications Terms to remember Classification CONTENTS

INTRODUCTION:

Chromatography is a combination of two words; Chromo – Color Graphy – Representation of something INTRODUCTION

MILESTONES IN CHROMATOGRAPHY:

YEAR CONTRIBUTION 1903 Tswett separated plant pigments on chalk columns 1931 Lederer and Kuhn performed liquid chromatography of carotenoids 1938 TLC and ion-exchange chromatography were introduced 1950 Reverse phase liquid chromatography was introduced 1954 Martin and Synge won noble prize 1959 Gel permeation chromatography was introduced 1965 Instrumental liquid chromatography was introduced MILESTONES IN 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. WHAT IS CHROMATOGRAPHY?

PowerPoint Presentation:

Analyze Examine a mixture, its components, and their relations to one another Identify Determine the identity of a mixture or components based on known components Purify Separate components in order to isolate one of interest for further study Quantify Determine the amount of the a mixture and/or the components present in the sample

DEFINITION:

“Chromatography is a method of separating a mixture of components into individual components through equilibrium distribution between two phases .” OR Chromatography is a laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass . DEFINITION

PURPOSE OF CHROMATOGRAPHY:

PURPOSE OF CHROMATOGRAPHY

APPLICATIONS:

APPLICATIONS AREAS EXAMPLES Pharmaceutical Company Determine amount of each chemical found in new product Hospital Detect blood or alcohol levels in a patient’s blood stream Law Enforcement To compare a sample found at a crime scene to samples from suspects Environmental Agency Determine the level of pollutants in the water supply Manufacturing Plant To purify a chemical needed to make a product

TERMS TO REMEMBER:

TERMS TO REMEMBER 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 remains static. It is either covalently bonded to the support particles or to the inside wall of the column tubing.

TERMS TO REMEMBER:

TERMS TO REMEMBER 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. Eluent: It is the solvent that will carry the analyte.

TERMS TO REMEMBER:

TERMS TO REMEMBER 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. Retardation factor ( Rf ): It refers to movement or migration of solute relative to the solvent front.

CLASSIFICATION (based on principle of separation):

CLASSIFICATION (based on principle of separation)

ADSORPTION CHROMATOGRAPHY:

ADSORPTION CHROMATOGRAPHY

Definition:

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 .” Mobile phase: Liquid or gas Stationary phase: Solid

Principle:

Principle Principle involves competition of components of sample mixture for active sites 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:

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.

Adsorbents:

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 .” E xamples include; Hydrated silica gel Silica gel G Silica gel S Silica gel GF 254 Silica gel H Silica gel N

Adsorbents:

Adsorbents Silica gel HF 254 Silica gel PF 254 Modified silica gel Alumina Kieselghur (Diatomaceous earth) Cellulose MN 300 Cellulose microcrystalline

An ideal adsorbent:

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

Types:

Types

PARTITION CHROMATOGRAPHY:

PARTITION CHROMATOGRAPHY

Definition:

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 .” Mobile phase: Liquid or gas Stationary phase: L iquid

Principle:

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 which gives basis for separation .

Principle:

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:

Types

ION EXCHANGE CHROMATOGRAPHY:

ION EXCHANGE CHROMATOGRAPHY

Definition:

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 analyte based on their respective charges .

Mechanism:

Mechanism 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.

Applications:

Ion analysis Separation, purification and analysis of biological substances (peptides, proteins, enzymes, antibodies, nucleotides and oligonucleotides) Separation of ionizable compounds Applications

SIZE EXCLUSION CHROMATOGRAPHY:

SIZE EXCLUSION CHROMATOGRAPHY

Definition :

Size Exclusion Chromatography (SEC) is the separation technique based on the molecular size of the components. Separation is achieved by the differential exclusion from the pores of the packing material, of the sample molecules as they pass through a bed of porous particles. Definition

PowerPoint Presentation:

Gel Filtration: For the separation of biomolecules in aqueous or aqueous/organic mobile phases, SEC is referred to as gel filtration chromatography (GFC ). Gel Permeation: For the separation of organic polymers in non-aqueous mobile phases is called gel permeation chromatography (GPC ).

Applications:

Size exclusion chromatography is used primarily for analytical assays and semi-preparative purifications. It is generally a low-resolution chromatography technique and thus it is often reserved for the final, "polishing" step of a purification. It is useful for determining the tertiary structure and quaternary structure of purified proteins. Applications

AFFINITY CHROMATOGRAPHY:

AFFINITY CHROMATOGRAPHY

PowerPoint Presentation:

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. 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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