1. Lecture 12 • Chromatography Introduction • Ch 7: Thin-Layer Chromatography • Lecture Problem 4 Due • This Week In Lab: • Ch 6: Procedures 2 & 3 • Due: Ch 5 Final Report • Next Week in Lab: • Ch 7 PreLab • Quiz 4

2. Chromatography A separation/purification technique. • Two main types: • Thin-Layer Chromatography (TLC) • Column Chromatography (CC) • Uses: • To separate the components of a mixture - TLC & CC • To determine the purity of a compound - TLC • To see if two compounds are identical - TLC • To monitor the progress of a reaction - TLC • To follow a column chromatography separation - TLC

3. Thin-Layer Chromatography Filter Paper TLC Plate: contains A polar stationary phase (alumina or silica gel) & a very small amount of your sample Mobile Phase: organic solvent(s) of varying polarity TLC Bottle/Chamber

4. Column Chromatography Funnel Mobile Phase: Organic solvent(s) of varying polarity Polar Stationary Phase: alumina or silica gel with your sample loaded onto it - can accommodate a larger amount of sample vs. TLC Small Erlenmeyers to collect fractions A Packed Column

5. Chromatography Basics • How it works: • Your sample is loaded onto the polar stationary phase • Polar compounds will adsorb onto the stationary phase to a greater • extent than non-polar compounds • The mobile phase (eluting phase) helps “push” or elute the • compounds either down a column (for CC) or up a plate (for TLC) • The main concept to consider in chromatography is polarity.

6. Polarity & Intermolecular Attractive Forces • More polar compounds will be more attracted to silica gel than • non-polar compounds due to intermolecular attractive forces - a • dipole-dipole interaction. • The more non-polar compounds will travel more easily and more • quickly through the stationary phase. • The mobile phase helps carry the compounds through the stationary • phase. • Separation of compounds in a mixture is possible because compounds • have different polarities. Non-polar compounds will elute first and • polar compounds will elute last. Silica gel, [SiO2]n

7. Polarity & Intermolecular Attractive Forces Example: Separate a mixture of butyl amine and cyclohexane using TLC Things to consider: Polarity of each compound in the mixture Butyl amine is polar; cyclohexane is non-polar Polarity of stationary phase Silica gel (or alumina) is polar - predict that butyl amine will interact with it more strongly Polarity of the mobile phase - the solvent: you determine what solvent to use Prediction: Cyclohexane will elute first/faster through the stationary phase. Butyl amine will elute last/slower.

8. TLC Separation Example: Separate a mixture of butyl amine and cyclohexane using TLC Mobile Phase: Typically use a mixed solvent system. If the mobile phase is non-polar, cyclohexane will travel along with it, but butyl amine will not as readily. If the mobile phase is polar, both cyclohexane and butyl amine will travel with it, but butyl amine will be slower because it’ll be interacting with silica gel as it’s traveling. Note the separation of spots

9. Chapter 7: TLC Experiment/Separation of Analgesics • A one-day experiment: • Testing and choosing a TLC mobile phase - work in groups. Each person in a group will test two (2) different solvent systems. Pick the solvent system that gives you the best • separation of spots. • TLC analysis on different analgesics (standards). Get Rf • values of these standards. • Using TLC data of the standards, identify analgesics in an “unknown” tablet by comparing Rf values.

10. Chapter 7: TLC Experiment/Separation of Analgesics The Experimental Steps 1. Load sample onto stationary phase/TLC plate (labeled) (a) Dissolve sample in a small amount of organic solvent (b) Use capillary tubes to load on Sample The smaller the spot, the better. Why?

11. TLC plate (labeled) with samples loaded Predict the order of elution for these compounds.

12. 2. Insert TLC plate into TLC chamber (filled with a layer of mobile phase & allow mobile phase to “run up” the TLC plate. Take out when the solvent reaches 1 cm from top of plate (solvent front). Mark the solvent front line with a pencil.

13. 3. Detection: • If the spots are not colored and can’t be seen by the eye, use: • UV lamp for UV-active compounds; most aromatics are • UV-active • If compounds are not UV-active, use an iodine (I2) • chamber • Once you visualize the spots, circle them with a pencil. • 4. Calculate Rf values for each spot/analgesic. • Rf = distance spot traveled from origin line/distance of solvent front • You will obtain Rf values for each analgesic you test. These Rf • values will help you identify analgesics present in an “unknown” tablet. • Make sure to use the same mobile phase as Rf’s will vary with • varying mobile phases.

14. Rf = distance spot traveled from origin line/distance of solvent front

15. Identifying Unknowns via TLC 1. Compare the Rfs of the known analgesics (standards) with the Rfs of the analgesics in your “unknown” tablet. 2. Use the Rfs to identify the analgesics in your tablet. Note: More than one analgesic may be in one tablet. Thus, you may see more than one spot per tablet sample.