1. Chromatography 1 Lecture 10 An introduction

2. What is CHROMATOGRAPHY ? Chromato g raphy

3. The Russian botanist Mikhail Tswett coined the term chromatography in 1906 to describe his experiments in separating different colored constituents of leaves by passing an extract of the leaves through a column History of Chromatography

4. WHAT IS CHROMATOGRAPHY ? A modern laboratory technique for separating mixtures into their components in order to analyze, identify, and purify the mixture or components. Chromato g raphy

5. Chromatography is a method of physically separating mixtures of gases, liquids, or dissolved substances. Chromatography can be used to identify drugs, poisons and many other substances. Separation is determined by the molecular size and/or charge. Chromato g raphy

6. Chromatography is a technique used to  separate mixtures of compounds; and to analyze :To examine the mixture or structure by separating it into its parts.  identify unknown compounds:To recognize them and to determine the identity of mixture.  purify compounds: To make them pure by removing substances that are not wanted out of another substances  monitor product formation in the pharmaceutical and biotechnology industries Chromatography is widely used by forensic teams to analyse blood and urine samples for drugs, for paint analysis and testing for the presence of explosives. Forensics ResearchPharmaceutical industry Applications of Chromatography

7. Analytical Analytical technique Determine Chemical composition of a sample Preparative Preparative technique Used to purify sufficient quantities of a substance CHROMATOGRAPHY can be used as

8. General Principles of Chromatography Separation of molecules by distribution between a stationary phase and a mobile phase. – A stationary phase (absorbent) phase the material on which the separation takes place. can be solid, gel, or liquid. Also called matrix, resin, or beads. – The mobile phase is the solvent transports the sample and it is usually a liquid, but may also be a gas. Also called eluting buffer The compounds to be separated are considered solutes

9. ANALOGY…

10. The mechanism that causes the stationary phase to retard the movement of molecules: 1.Sieve mechanism  separation according to size or MW. (molecular sieve = gel filtration = size exclusion = gel permeation). 2.Charge interaction  separation based on net charge. Classification by the Separation mode

11. 3. Solubility characteristics  separation based on polarity Hydrophobic chromatography, reverse-phase chromatography, Adsorption or normal-phase chromatography 4. Biological or Specific interaction  capture any molecule that exhibit such property affinity chromatography, dye-chromatography Antibody-antigen: (Immuno precipitations and other forms) Classification by the Separation mode

12. Classification of Chromatography Gas Chromatography Gas - solid Gas - liquid Liquid chromatography High performance (pressure flow) Thin layer (adsorption) Column (gravity flow) By mobile phase: 1.Liquid chromatography. 2. Gas chromatography.

13. Type of chromatographyMaterial Paper chromatographyFilter paper, cellulose Thin Layer ChromatographySilica gel, alumina, polyamide Gas chromatographySqualene, apezion, carbowax M High Performance Liquid Chromatography C-8, C-18, Licosorb, Silicone

14. Type of chromatographySolvent Paper chromatographyAlcohol Thin Layer Chromatography Hexane, ether petroleum, alcohol. Gas chromatographyHe, Ar, N 2 High Performance Liquid Chromatography Hexane, carbon tetrachloride, ethanol, methanol

15. 15 Types of Chromatography… Paper HPLCGas Thin layer Column

16. 1-Adsorption - for polar non-ionic compounds 2-Ion Exchange - for ionic compounds – Anion - analyte is anion; bonded phase has positive charge – Cation – analyte is cation; bonded phase has negative charge 3-Partition - based on the relative solubility of analyte in mobile and stationary phases – Normal – analyte is nonpolar organic; stationary phase MORE polar than the mobile phase – Reverse – analyte is polar organic; stationary phase LESS polar than the mobile phase 4-Size Exclusion - stationary phase is a porous matrix; sieving 5- Affinity Classification based on Attractive Forces

17. Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that 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. 1.Adsorption Chromatography:

18. 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. 2.Partition Chromatography:

19. In this type of chromatography, the use of 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. 3.Ion Exchange Chromatography:

20. Also known as gel permeation or gel filtration, 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. 4.Molecular Exclusion Chromatography:

22. 5.Affinity Chromatography: