Experiment 5: Chromatography

1. Experiment 5: Chromatography • Reading Assignment: • Experiment 5, pages 42 - 50 • Technique 19, pages 756 – 777 • Technique 20, pages 777 – 792 The following two references provide information on reverse-phase chromatography (C-18 silica) • Technique 21, 794 • Technique 12, pages 689 - 692

2. Experiment 5 • This is a two-day experiment. We will be working in pairs: • Day One: • Part A (Thin-Layer Chromatography) • Part B (Selecting the Correct Solvent for Thin-Layer Chromatography) • Day Two: • Part D (Column Chromatography) • We will skip Part C

3. How Does Chromatography Work? • Chromatography is a method for separating the components of a mixture by differential adsorption between a stationary phase and a mobile (moving) phase

4. Different Kinds of Chromatography • Liquid chromatography (includes column chromatography, thin-layer, and HPLC) • Stationary phase: silica, alumina, etc. • Mobile phase (moving phase): organic solvents • Important properties: polarity • Gas chromatography • Stationary phase: a film of a polymer or a wax. The film must have a high boiling point • Mobile phase: gas (Helium is the usual carrier gas) • Important properties: boiling point

5. Thin-layer chromatography and column chromatography and are different types of liquid chromatography. • The mobile (moving) phase is a liquid. The stationary phase is usually silica or alumina. This phase is very polar. • The principle of operation is the same!

6. Stationary Phase: Silica (SiO2)

7. Stationary Phase: Alumina Acidic: -Al-OH Neutral: -Al-OH + -Al-O- Basic: -Al-O-

8. Thin Layer Chromatography The surface of the plate consists of a very thin layer of silica on a plastic or aluminum backing. The silica is very polar. This is the stationary phase. Spot the material at the origin (bottom) of the TLC plate. Place the plate into a glass jar with a small amount of a solvent in the glass jar. This solvent acts as the moving phase. Remove the plate from the bottle when the solvent is close to the top of the plate. Visualize the spots. Non-polar compounds will be less strongly attracted to the plate and will spend more time in the moving phase. This compound will move faster and will appear closer to the top of the plate. Polar compounds will be more strongly attracted to the plate and will spend less time in the moving phase and appear lower on the plate.

9. s o l v e n t f r o n t o r i g i n Thin-Layer Chromatography: A Two-Component Mixture s o l v e n t f r o n t Less polar! c o m p o n e n t B s o l v e n t f r o n t c o m p o n e n t B More polar! c o m p o n e n t A c o m p o n e n t A mixture o r i g i n o r i g i n Increasing Development Time

10. Important hint! Be sure to remove the TLC plate when it appears that the solvent front isn’t moving! Reason: the solvent is evaporating as it moves up the plate. Results: If you don’t remove the plate all of the spots will appear near the top of the plate!!!!! This isn’t a pretty sight and makes it difficult to get good Rf values!

11. Visualization Method • The previous slide shows colored spots. Most of the time, the spots won’t show unless they are visualized! • Vizualization is a method that is used to render the TLC spots visible. • A visualization method can be: • Ultraviolet light • Iodine vapors to stain spots • Colored reagents to stain spots • Reagents that selectively stain spots while leaving others unaffected.

12. Thin-Layer Chromatography: Determination of Rf Values Rf of component A = dA dS Rf of component B = dB dS The Rf value is a decimal fraction, generally only reported to two decimal places

13. Thin-Layer Chromatography: Qualitative Analysis

14. Part A of Experiment 5 Prepare a TLC plate with five lanes. Spot the plate with pure fluorene, fluorenone and fluorenol as indicated on page 81 of the text in the first three lanes. Spot the plate with an unknown mixture containing the above compound(s) in lane four. Now spot the plate with a reference mixture of the three compounds in lane five. Develop the plate in a screw cap glass bottle using CH2Cl2 Visualize the spots using iodine vapor

15. Which one of these compounds is the least polar? • Which one of these compounds is the most polar? • What would be the relative order of separation on • the TLC plate remembering that CH2Cl2 is not very • polar?

16. Part B of Experiment 5 • In this part of the experiment you will be assigned one of the • mixtures listed in the footnote on page 83. • Your task is to determine which of the three solvents listed • will provide the best separation on the TLC plate. • Benzoin and benzil in acetone, methylene chloride and • hexane • 2) Vanillin and vanillyl alcohol in acetone, 50-50 toluene/ • ethyl acetate and hexane • 3) Diphenylmethanol and benzophenone in acetone, • 70-30 hexane/acetone and hexane • Note carefully: different solvents are used with each • part. Be sure to use the correct solvent!

17. Uses of TLC • To determine how many components there are in a mixture (is it really pure?) • To determine the best solvent conditions for separation on a column • To identify the substances being studied • To monitor the composition of fractions collected from column chromatography • To monitor the progress of a reaction

18. Uses of TLC What solvent should you use to separate the following compounds? • Acetone • Hexane • Acetic acid

19. Uses of TLC A student tries to separate a series of carboxylic acids using hexane. Nothing separates. Why? What should you do next?

20. Uses of TLC A student tries to separate a mixture of aliphatic and aromatic hydrocarbons using acetone. The components all appear at the solvent front on the TLC plate. What was wrong and what should be done next?

21. Column Chromatography The stationary phase (column packing) in the column is very polar! Polar compounds are going to be attracted to the polar column packing by hydrogen bonding or dipole-dipole attractions. Polar compounds are going to move slowly! Non-polar compounds are going to come off the column first, while the polar compounds are going to come off the column last. Usually, one starts will a less polar solvent to remove the less polar compounds, and then you slowly increase the polarity of the solvent to remove the more polar compounds.

22. Principles of Separation on a column

23. Principles of Separation

24. Principles of Separation

25. Principles of Separation

26. Principles of Separation

27. Principles of Separation

28. Principles of Separation

29. REMEMBER… • The stationary phase is POLAR • The more polar component interacts more strongly with the stationary phase • The more polar component moves more slowly. • The non-polar component moves more rapidly.

30. Reverse phase chromatography Silica is alkylated with long chain hydrocarbon groups, using 18 carbons long. This is usually referred to as C-18 silica.

31. Reverse Phase column chromatography • The stationary phase (column packing) is nowNON-POLAR • Non-polar compounds will move more slowly because they are attracted to the column packing. • The more polar component moves more quickly down the column. • Polar solvents, such as water and methanol are used in reverse phase chromatography • Used mainly in columns, such as HPLC

32. How does reverse phase chromatography compare to normal phase chromatography?

33. Normal phase Column Chromatography The column packing in the column is very polar! Polar compounds are going to be attracted to the polar column packing by hydrogen bonding or dipole-dipole attractions. Polar compounds are going to move slowly! Non-polar compounds are going to come off the column first, while the polar compounds are going to come off column last. Usually, one starts will a less polar solvent to remove the less polar compounds, and then you slowly increase the polarity of the solvent to remove the more polar compounds.