Chromatography - AN INTRODUCTION

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SEPARATION TECHNIQUES Chromatography بسم الله الرحمن الرحيم Ref. Book : TEITZ text book of Clinical Chemistry and Molecular Diagnostics 4 th Edition

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 components. Described in 1903 by Tswett who used it for the separation of coloured substances From Greek, Chroma – color and -graphy -writing Separate Analyze Identify Purify Quantify

Uses of Chromatography:

Uses of Chromatography Chromatography is used by scientists to: Analyze تحليل – examine a mixture, its components, and their relations to one another Identify تحديد – determine the identity of components of a mixture based on known components Purify طهر – separate components in order to isolate one of interest for further study Quantify كميا – determine the amount of the a mixture and/or the components present in the sample

Uses of Chromatography:

Uses of Chromatography Real-life examples of uses for chromatography: Pharmaceutical Company – determine amount of each chemical found in new product Hospital – detect or estimate analyte levels in a patient’s blood. 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

Definition of Chromatography:

Definition of Chromatography Definition: Chromatography is a laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile phase and a stationary phase. Terminology : Differential – showing a difference, distinctive Affinity – natural attraction or force between things Mobile phase – gas or liquid that carries the components (mobile medium) Stationary phase – the part of the apparatus that does not move with the sample (stationary Medium )

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Explanation Compound is placed on stationary phase Mobile phase passes through the stationary phase Mobile phase solubilizes the components. Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases Illustration of Chromatography

Illustration of Chromatography:

Illustration of Chromatography Components Affinity to Stationary Phase Affinity to Mobile Phase Blue ---------------- Insoluble in Mobile Phase Black         Red        Yellow           Mixture Components Separation Stationary Phase Mobile Phase

Types of Chromatography:

Types of Chromatography

Planar Chromatography:

Planar Chromatography In Paper Chromatography, stationary phase is a layer of water or a polar solvent coated onto the paper fibers In Thin layer Chromatography a thin layer of particles of a material such as silica gel is spread uniformly on glass plate or a plastic sheet. When the thin layer consists of particles with smaller diameters [≈4.5 μ m], the technique is known as high performance thin layer [HPTLC].

Column Chromatography:

Column Chromatography In Column Chromatography, the stationary phase [silica or any other polymer] is packed in a tube or column or is coated onto the inner surface of tube or column. When the mobile phase is a gas, the technique is termed as Gas Chromatography [GC] and the instrument is a G as Chromatograph . When the mobile phase is a liquid, the technique is termed as Liquid Chromatography[LC] and the instrument is a Liquid Chromatograph .

Column Chromatography:

Column Chromatography When the stationary phase in LC consists of small diameter particles, the technique is known as high performance Liquid Chromatography [HPLC]. When a Liquid Chromatograph is connected to a mass spectrometer, the combined techniques is called as Liquid Chromatography / Mass spectrometry [GC/MS]. When a Gas Chromatograph is connected to a mass spectrometer , the combined techniques is called as Gas Chromatography / Mass spectrometry [GC/MS].

Ion exchange Chromatography:

Ion exchange Chromatography Principle : Ion exchange Chromatography is based on an exchange of ions between a charged stationary surface and a mobile phase of the opposite charge. Explanation: Depending on the conditions, the solutes are either positively charged [cations] or negatively charged [anions] and are separated on the bases of difference of their ionic charge and magnitude of charge. The solute ions are selectively eluted by variation in pH, ionic strength or both of mobile phase.

PowerPoint Presentation:

Types of exchangers Anion exchangers Cation Exchangers Applications; Clinical applications like analysis of: Amino acids Peptides Proteins Oligonucleotides Nucleic acids Removal of inorganic ions from aqueous solution [ e.g. water purification in labs]

Partition [ قاطع, تقسيم ] Chromatography:

Partition [ قاطع , تقسيم ] Chromatography Principle : The separation in partition Chromatography is based on the differences in the relative solubility of analyte molecules between the stationary and mobile phases. The stationary phase is a liquid. Examples : Paper chromatography Gas-Liquid chromatography Liquid-Liquid chromatography [LLC] .LLC further divided into: Normal phase : The stationary phase is polar and mobile phase is a relatively non polar liquid. Reverse phase : The stationary phase is non polar and mobile phase is relatively polar.

Adsorption [إمتصاص كيميائي]Chromatography:

Adsorption [ إمتصاص كيميائي ] Chromatography Principle : The separation Adsorption Chromatography is based on the differences in the adsorption and desorption of analyte molecules at the surface of solid particles. The stationary phase is a solid. Electrostatic, hydrogen bonding and dispersive interactions are the physical forces which control this process. Examples : Gas Chromatography

Affinity Chromatography:

Affinity Chromatography Principle : In affinity chromatography, the separation is based on the unique and specific biological interactions of the analyte and ligand .The specificity resulting from enzyme-substrate, hormone-receptor and antigen-antibody interactions are used in this technique.

Affinity Chromatography:

Affinity Chromatography The stationary phase in affinity chromatography is prepared by immobilizing a ligand on particles of a support either directly or via a spacer. A variety of chromatographic support media like agarose, cross linked dextrans, polyacrylamide, cellulose, polystyrene etc are used as beads. Optimum Binding achieved by using specific pH and ionic strength. The elution of the analyte achieved by using excess ligand, competing proteins like substrates or inhibitors, by a pH change of buffer or by using chemicals which break hydrogen bonds like urea etc. Clinical Applications Separation and identification of proteins Separation and identification of antibodies. Separation and identification of lipoproteins Separation of cells

Size Exclusion Chromatography:

Size Exclusion Chromatography Principle : Size Exclusion Chromatography separates analytes on the basis of their molecular sizes . Molecular shape and hydration are also factors in this process. The other names of s ize exclusion chromatography are gel filtration , gel permeation , steric exclusion , molecular exclusion and molecular sieve Chromatography. A variety of materials with different pore sizes are used as stationary phase. The small molecules get temporarily entrapped in the pores while as the large molecules remain entirely in the mobile phase and are eluted rapidly from the column. The molecules that are intermediate in size elute between large and small molecules. Used more for preparative than analytical purposes.

Illustration of Size Exclusion Chromatography:

Illustration of Size Exclusion Chromatography Very small molecules Intermediate size molecules Large molecules Large molecules come out first Small molecules come out later

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