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An introduction to chromatography Objective of chromatographic methods To identify the compounds of a mixture = qualitative analysis To quantify these compounds To retrieve the separated solutes (preparative techniques) Spectroscopic methods (UV-vis, IR, RMN, MS) can also be used directly for mixtures of compounds but are limited to 2 or 3 analytes at a time. The objective of chromatographic methods is to separate the compounds present in a mixture. It is then possible to identify and quantify them. The three steps of chromatography Introduction of the sample in the separative device (the sample must have been prepared for this purpose) Separation with stationary and mobile phases Detection of the separated species getting out of the chromatographic system The three steps of chromatography A chromatogram is produced: Solute 1 Solute 2 Analysis time The three steps of chromatography Basic principle of chromatographic techniques: Different affinity of the solutes for two non-miscible phases Stationary phase (SP): Solid or Liquid Mobile phase (MP): Gaseous, Liquid or Supercritical The three steps of chromatography Pressure (MPa) Supercritical fluid 9 8 31°C * 7.3 MPa 7 Liquid 6 Gas 10 20 30 40 Temperature °C State diagram for carbon dioxide Homogeneous phase separation methods NON CHROMATOGRAPHY Driving force Method Centrifugal force Centrifugation Electrical Electrophoresis Electrodialysis Concentration gradient Dialysis, Osmosis Pressure Microfiltration, Ultrafiltration Reversed osmosis Field Flow Fractionation Principles of separation used in chromatography Property Technique Example Application Molecular size Size Exclusion Chromatography Polymers Volatility Gas Phase Chromatography Perfumes Charge Ion Exchange Chromatography Amino acids Solubility Partition Liquid Chromatography Any solute Polarity Adsorption Liquid Chromatography Any solute Thin Layer Chromatography Plant extracts Specific affinity Affinity Chromatography Proteins Chirality Chiral chromatography Drugs, perfumes