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Chromatographic Separation

Adam Hasham, Sam Heavenrich, Jayanth Prakhya, Enoch Tieu. Chromatographic Separation. Chromatography. Separates mixtures (physical method) Involves: Stationary Phase; Surface Mobile Phase; Solvent Quantifiable: Retention Factor / Rate of Flow (Rf) Can be:

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Chromatographic Separation

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  1. Adam Hasham, Sam Heavenrich, Jayanth Prakhya, Enoch Tieu Chromatographic Separation

  2. Chromatography • Separates mixtures (physical method) • Involves: • Stationary Phase; Surface • Mobile Phase; Solvent • Quantifiable: • Retention Factor / Rate of Flow (Rf) • Can be: • Preparative: separates mixture for further use • Analytical: measures relative proportions of components • Preparative more easily studied Rf = Distance traveled by solute/Distance traveled by solvent

  3. History • Mikhail SemenovichTsvett • Separate Tints / Types of Chromatography • Trickled mixture through glass tube with Calcium Carbonate powder • Pigments stuck to powder • Different degrees of strength  Coloured bands

  4. Types of Chromatography • Partition • Based on the 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. • Ion Exchange • Resin is used to covalently attach anions or cations by electrostatic forces • Solute Mobile Liquid Phase have opposite charge

  5. Types of Chromatography • Molecular Exclusion • No attraction between stationary phase and solute • Liquid or Gaseous Mobile Phase separates molecules according to size • Affinity • Most selective • Interaction between only 1 component of solute and stationary phase molecule • Only the specific component is attracted to the stationary phase

  6. Adsorption Chromatography • Adsorption: When a fluid solute accumulates on the surface of a solid or liquid • Different amounts of the components of a mixture are adsorbed to the 2 phases • As mobile phase moves through stationery phase, the substances that are easily adsorbed will ‘lag’ • Separation created

  7. Paper Chromatography • Archer JP Martin and Richard LM Synge in 1941 • Strip of porous filter paper substituted for powder • Used to separate proteins • Later used to Separate and Identify components of various mixtures • In industrial settings, Paper Chromatography has been replaced by more sophisticated methods

  8. Common Practice for Paper Chromatography • Place a drop of a mixture on the paper • Dip one edge of the paper into the mobile phase • Through capillary action, adsorbent will move up the paper • Once adsorption occurs and the paper dries, spray-on reagent • Reagent will reveal change in color

  9. Experiment • Purpose: • To predict the composition of various mixtures using Paper Chromatography

  10. Materials

  11. Procedure • Pigment applied to Strip of Chromatography Paper • Strips hung on rack at equal lengths • Adsorbent added (70% Isopropyl Alcohol) • Rack removed once solvent travelled to the top of each strip • The final position of solvent marked • The final position of pigments marked • Distance travelled by the mobile phase and each colour measured

  12. View Demo using the ‘Play’ button after each step. Do not proceed after the Gas Chromatography Demo appears. Use arrow keys for next slide. Procedure

  13. The Mixture involving Red, Yellow, and Blue Food Coloring Pigments Approximate elapsed time 1 minute Observations - Pictures

  14. Observations - Pictures The Mixture involving Red, Yellow, and Blue Food Coloring Pigments Approximate elapsed time 6 minutes

  15. Observations - Pictures The Mixture involving Red, Yellow, and Blue Food Coloring Pigments Approximate elapsed time 18 minutes

  16. Observations - Pictures The Spinach, Pomegranate and Blue Pigments Approximate elapsed time 0 minutes

  17. Observations - Pictures The Spinach, Pomegranate and Blue Pigments Approximate elapsed time 5 minutes

  18. Observations - Pictures The Spinach, Pomegranate and Blue Pigments Approximate elapsed time 13 minutes

  19. Rf • Defined as Rate of Flow or Retention Factor • Calculated by dividing “ Distance travelled by a component” over “ Distance travelled by the mobile phase”

  20. Observations – Distance Travelled

  21. Calculations – Rf Values

  22. Analysis – Rf Values • Rf = Degree of Adsorption • Variances caused by Intermolecular Forces • Between Solute and Mobile Phase • Solute carried further by Adsorbent / More adsorption when the forces between them are greater • Forces between Solute and Stationary Phase • More adsorbtion onto Stationary Phase when the forces between them are greater • In Both Cases: • Polarity Major Factor • H-bonding Larger Factor but capability limited • London’s Dispersion Forces Weak

  23. Analysis • Mixture of Food Coloring • Molecular Formulas: • Red: C18H14N2Na2O8S2 • Yellow: C16H10Na2O7S2N2 • Blue: C16H10N2O2 • H-Bonding Pattern • Relatively Similar Polarity • Long Hydrocarbon Chain = Low Polarity • Cellulose Chromatography Paper • Green Coloring • Yellow and Blue Components • Yellow: C16H9N4Na3O9S2 • Blue: C16H10N2O2 • Same Blue Dye; Different Yellow Dye • Higher Polarity Due to Presence of Sodium • Higher Number of Nitrogen Atoms

  24. Analysis • Spinach Extract • Molecular Formulas: • Yellow 1: Carotenes: C40H56 • Yellow 2: Xanthophylls: C40H56O2 • Green: Chlorophyll B: C55H70O6N4Mg • H-Bonding common • Carotenes Not-Polar • Xanthophylls’ O2

  25. Limitations of Paper Chromatography • Pomegranate Analysis • Not Exact / Quantifiable • Not Definite  Colours Subjective • Components found in mixture can’t be re-used • Neither Preparative nor Analytical • Other Types such as Gas Chromatography provide the components in usable form • Prior Knowledge Required of Components • Only Identifiable if known • Incorrect Identifications

  26. Limitations of Paper Chromatography • All Rf Values are Relative • Paper & Some Types of Thin Layer Chromatography • Other Methods • Distribution Constant and Concentration More Reliable • Freundlich Equation for Adsorption • Kovats Retention Index • Van Deemter Equation Below: More sophisticated methods of quantifying chromatography results

  27. Experimental Notes • Safety Concerns • Safety Goggles must be worn at all times • Any contact with the alcohol must be avoided • Alcohol is Very Flammable and requires a well-ventilated area • Modifications • More Sample Pigments • Various Adsorbents • The more data, the more definite the results

  28. Applications of Adsorption Chromatography • Thin-Layer Chromatography • Gas Chromatography (In Column) • Liquid Chromatography

  29. Applications of Adsorption Chromatography • Thin-Layer Chromatography • Mobile Phase: Solution; Stationary Phase: Flat Sheet of Adsorbent (e.g. Silica Gel) • Analyzing organic reactions • Components are separated using TLC plates • They can then be scraped off to be analyzed • Other Applications • assaying radiochemical purity of radiopharmaceuticals • Plant pigments • detection of pesticides or insecticides

  30. Gas Chromatography Click ‘Next Example’ to View Gas Chromatography Demo

  31. Applications of Adsorption Chromatography • Gas Chromatography (In Column) • Mobile Phase: Gas; Stationary Phase: Solid/Liquid • Blood alcohol analysis in forensics • Blood analyzed using capillary columns • Column Pressure 20 psi • Hydrogen is mobile phase • Other Applications • Environmental monitoring • Drug, bomb detection

  32. Applications of Adsorption Chromatography • Liquid Chromatography • Mobile Phase: Liquid; Stationary Phase: Solid/Liquid • HPLC, NPLC • Test for Water Pollution • Analyze metal ions + organic compounds in water • Other Applications • purification of a drug product

  33. Modifications • 2 Dimensional • 2 separation stages • Gas / Liquid Chromatography • Stationary Phase Rotated; 2 Mobile Phases Used • RPLC • Polar Mobile Phase Used • Reverse Rf Analysis

  34. This concludes the presentation. Any Questions?

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