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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, JayanthPrakhya, 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• Later used to Separate and Identify components of various mixtures • In industrial settings, Paper Chromatography has been replaced by more sophisticated methods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. 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 adsorbtiononto 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

  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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