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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. Analyze Identify Purify Quantify. Separate. Mixture. Components. Uses of Chromatography.

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What is Chromatography?

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  1. 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. • Analyze • Identify • Purify • Quantify Separate Mixture Components

  2. Uses of Chromatography • Chromatography is used by scientists to: • 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

  3. Applications of Chromatography Forensics Research Pharmaceutical industry

  4. Real-life examples of uses for chromatography: • 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

  5. Milestones in Chromatography • 1903 Tswett - plant pigments separated on chalk columns • 1931 Lederer & Kuhn - LC of carotenoids • 1938 Adams & Holmes -TLC and ion exchange • 1940 Tiselius & 1946 Claesson – Study of properties of solutions in chromatographic process & classify them - • Frontal analysis – Using only sol.n of mixture, no mobile phase is used for development of column • Displacement analysis– Used in adsorption column chromatography using ‘displacer’ • Elution analysis – Common method for column chromatography, using continuous addition of mobile phase over column.

  6. Milestones in Chromatography • 1950 : Reverse phase LC (RP-LC) • 1941 : Martin & Synge – Partition chromatography • 1944 : Martin , Consden & Gorden – Paper chromatography (Nobel Prize in 1954) • 1959 : Gel permeation • 1965 : Instrumental LC (Waters)

  7. Definition of Chromatography • Detailed Definition: • 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. • Terminology: • Differential– showing a difference, distinctive • Affinity – natural attraction or force between things • Mobile Medium – gas or liquid that carries the components (mobile phase) • Stationary Medium – the part of the apparatus that does not move with the sample (stationary phase)

  8. Definition Chromatography is a separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium. Components: mobile phase:a solvent that flows through the supporting medium stationary phase: a layer or coating on the supporting medium that interacts with the analytes supporting medium: a solid surface on which the stationary phase is bound or coated.

  9. Definition of Chromatography • Simplified Definition: • Chromatography separates the components of a mixture by their distinctive attraction to the mobile phase and the stationary phase. • 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.

  10. Types of Chromatography • Liquid Chromatography– separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase) • Gas Chromatography– separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase) • Paper Chromatography– separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase) • Thin-Layer Chromatography– separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)

  11. Types of Chromatography… Thin layer Paper HPLC Gas Column

  12. Types of Chromatography… Paper Column Thin layer

  13. Chromatographic methods classification

  14. (1) Geometry of the system • In column chromatography the stationary phase is contained in a tube called the column. • Planar chromatography: In this geometry the stationary phase is configured as a thin two-dimensional sheet. (i) In paper chromatography a sheet or a narrow strip of paper serves as the stationary phase. (ii) In thin-layer chromatography a thin film of a stationary phase of solid particles bound together for mechanical strength with a binder, such as calcium sulfate, is coated on a glass plate or plastic or metal sheet.

  15. (2) Mode of operation Development chromatography : In terms of operation, in development chromatography the mobile phase flow is stopped before solutes reach the end of the bed of stationary phase. The mobile phase is called the developer, and the movement of the liquid along the bed is referred to as development. Elution chromatography : This method, employed with columns, involves solute migration through the entire system and solute detection as it emerges from the column. The detector continuously monitors the amount of solute in the emerging mobile-phase stream—the eluate—and transduces the signal, most often to a voltage, which is registered as a peak on a strip-chart recorder. Example of planar development chromatography – TLC or PC

  16. (3) Retention mechanism Classification in terms of the retention mechanism is approximate, because the retention actually is a mixture of mechanisms. The main interactions are: • hydrophobic (non-specific) • dipole-dipole (polar) • ionic • special

  17. (4) Phases involved • The primary division of chromatographic techniques is based on the type of mobile phase used in the system: Type of ChromatographyType of Mobile Phase • Gas chromatography (GC) gas • Liquid chromatography (LC) liquid

  18. 2) Further divisions can be made based on the type of stationary phase used in the system: • Gas Chromatography • Name of GC MethodType of Stationary Phase • Gas-solid chromatography solid, underivatized support • Gas-liquid chromatography liquid-coated support • Bonded-phase gas • chromatography chemically-derivatized support

  19. Liquid Chromatography • Name of LC MethodType of Stationary Phase • Adsorption chromatography solid, underivatized support • Partition chromatography liquid-coated or derivatized support • Ion-exchange chromatography support containing fixed charges • Size exclusion chromatography porous support • Affinity chromatography support with immobilized ligand

  20. 3) Chromatographic techniques may also be classified based on the type of support material used in the system: - Packed bed (column) chromatography - Open tubular (capillary) chromatography - Open bed (planar) chromatography

  21. Principle of separation

  22. Illustration of Chromatography Stationary Phase Separation 4 3 2 1 Mobile Phase Mixture Components

  23. Types of chromatography on the basis of interaction of the analyte with stationary phase * the interaction determines retention times of analytes • Adsorption – of solute on surface of stationary phase; for polar non-ionic compounds • Ion Exchange – attraction of ions of opposite charges; for ionic compounds anions or cations • Partition - based on the relative solubility of analyte in mobile and stationary phases • Size Exclusion (gel filtration, gel permeation) – separates molecules by size; sieving - not real interaction, small molecules travel longer • Affinity – specific interactions like a particular antibody to protein

  24. (A) uses charge to create the differential affinities among the mixture components for the stationary phase.

  25. (B) usesporesto create the differential affinities among the mixture components for the stationary phase.

  26. C) uses covalent bonds to create the differential affinities among the mixture components for the stationary phase.

  27. i) Adsorption chromatography Chromatography in which separation is based mainly on differences between the adsorption affinities of the sample components for the surface of an active solid.

  28. ii) Partition chromatography Chromatography in which separation is based mainly on differences between the solubility of the sample components in the stationary phase (gas chromatography), or on differences between the solubilities of the components in the mobile and stationary phases (liquid chromatography).

  29. iii) Size-Exclusion Chromatography • Packing contains network of uniform pores into which solute • and solvent can diffuse. • Solute is “trapped” in pore until carried away by solvent. • Residence time in pore related to effective molecular size of solute. • Molecules larger than average pore size are excluded from pore, not retained. • Molecular diameter significantly smaller than pore can penetrate throughout pore, so elute last. • Fractionation of intermediate-sized molecules. Some shape • dependence.

  30. Ion-exchange chromatography Chromatography in which separation is based mainly on differences in the ion-exchange affinities of the sample components. Anions like SO3- or cations like N(CH3)3+ are covalently attached to stationary phase, usually a resin.

  31. Types of SEC : • Gel Filtration Chromatography • Aqueous solvent • Hydrophilic Packings • Gel Permeation Chromatography • Non-polar Organic Solvents • Hydrophobic Packings

  32. Advantages of SEC • Short, well-defined separation times • Narrow bands, good sensitivity • No sample loss since solutes do not interact with stationary phase • Absence of column deactivation • Disadvantages of SEC • Limited number of bands accommodated since short time scale • Not applicable to similar-sized molecules, like isomers

  33. iv) Ion-Exchange Processes in Ion-Exchange Chromatography • Based on exchange equilibria between ions in solution and • ions of like charge on surface of essentially insoluble, high- • molecular weight solid. • Most common cation exchangers: • The strong acid sulfonic acids, –SO3-H+ • The weak acid carboxylic acids, –COOH • Most common anion exchangers: • The strong base ternary amines, -N(CH3)3+OH- • The weak base primary amines, -NH3OH

  34. Mechanism of Ion Chromatography

  35. Comparison of LC and GC Both- - Efficient, highly selective, widely applicable - Only requires small sample - May be nondestructive of sample - May have quantitative analysis Advantages Favorable to LC- - Can separate nonvolatile or thermal unstable samples - Generally applicable to inorganic ions Advantages Favorable to GC- - Simple, less expensive equipment - Rapid - More efficient, higher resolution - Easily interfaced with mass spectrometry

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