Chemistry 121

Lab 6: Thin layer chromatography of various functional groups

Objective: Use thin layer chromatography to separate and characterize the intermolecular forces of a mixture of benzene derivatives.

Introduction: The most widely used methods of separating components of organic chemical mixtures involve some form of chromatography. At its simplest, chromatography is the separation of two or more compounds by distributing them unequally between two phases, one of which is moving (called the “mobile phase”) and the other of which is stationary. The unequal distribution comes about due to the different solubilities and adsorptivities these compounds have for the two phases.

In thin layer chromatography (TLC), the stationary phase is adsorbent silica bound to a thin flexible plastic sheet, called a TLC plate. Silica (silicon dioxide) is considered to be a polar substance and its oxygen atoms have two lone pairs of electrons each. The mobile phase is an organic solvent system (in other words, a mixture of one or more organic solvents), which, by capillary action, will move up the stationary phase. Solvent systems are considered to be non-polar compared to the silica, though there are degrees of non-polarity.

The sample mixture is applied as a small spot near one edge of the TLC plate; this is called “spotting”. The plate is then put vertically into a solvent system reservoir such that the spotted edge is placed down (but keeping the spots above the level of the reservoir) and the solvent system will ascend the plate. As the solvent system goes up, the compounds in the sample mixture will (ideally) separate; some of the compounds should stick to the stationary phase and some should dissolve and be carried up the plate along with the mobile phase. This process is called “developing” the TLC plate.

To get an idea of why compounds in the mixture separate, consider a mixture that contains both a polar and a non-polar compound. The polar compound will favor the adsorbent silica (the stationary phase) because the silica is highly polar (following the rule of “like dissolves like”). The non-polar compound will favor dissolving in the non-polar solvent system and travel upward with the solvent front. Thus, each compound will ascend the plate at different rates, with more polar compounds tending not to rise quickly. Separation is achieved!

In this experiment, you will examine the TLC mobility of various benzene derivatives.

Materials and methods

• 5% (w/v) solutions of benzaldehyde, benzoic acid, benzyl alcohol, methyl benzoate and benzamide in methylene chloride

• 0.5% (v/v) glacial acetic acid in ethyl acetate

• hexane

• one 2 by 4 inch standard silica TLC plates

• capillary tubes

• two 10 mL graduated cylinders

• ultraviolet viewing boxes

• developing jars

• pencil, ruler, forceps, transfer pipets

Procedure

1. Obtain a 2-inch wide TLC plate and six capillary tubes for spotting the plate. Handle the plate by its edges, taking care not to bend the plate excessively or to otherwise cause the silica to flake off.

2. Using a pencil (not a pen!) and a ruler, draw a straight line lightly across the width of the plate, about 1 cm from what will be the bottom of the plate. Along this line, draw lightly five evenly-spaced tick marks; this is where you will be spotting the solutions.

3. Dip the end of a capillary tube into one of the five solutions (note which one) and watch it fill (it won’t fill to the top). Spot the plate by lightly touching it to the TLC plate at one of the tick marks. Note the spreading circle of moistness as the capillary empties; make sure this spot does not exceed 2 mm in diameter (if it seems to be getting bigger, merely lift the capillary tube off of the plate, wait for the wet spot to dry and respot the plate at the same location – repeat this until the tube is empty).

4. Be sure to record in your notebook which tick mark corresponds to which compound you have spotted. In fact, drawing an exact replica sketch of your TLC plate is an excellent visual aid, especially since you can sketch in what the TLC plate looks like after developing. It is usual to orient your sketch with the line at the bottom.

5. Repeat step 3 with the four other solutions, taking care to use a fresh capillary tube for each different solution.

6. Prepare the development chamber by obtaining a jar. Make the solvent system in the glassware provided: a 50/50 mixture of hexane and 0.5% acetic acid in ethyl acetate. 10 mL should be plenty of solvent; carefully pour the solvent into the jar. Right and cap the jar; note the level of solvent at the bottom and make sure that when the TLC plate is put in the jar that the spots are above the level of solvent.

7. Develop the plate by placing it in the jar such that it leans across the jar. Make sure the spots are not immersed in solvent and make sure that the top of the plate does not touch any of the saturated filter paper. Cap the jar and let the solvent rise (you can see an ascending solvent front as time goes on).

8. When the solvent front is about 2 to 3 cm from the top of the plate, remove the plate from the jar and quickly mark the solvent front with the pencil. Let the plate dry in the hood.

9. Place the dry TLC plate in the UV (ultraviolet) box; the compounds should appear as dark purple spots. Use a pencil to outline all the spots. Make a sketch of the plate in your notebook, preserving all of the different distances.

10. Generate a data table with the following headings:

TLC plate lane number / Distance traveled by compound (mm) / Distance traveled by solvent front (mm) / Rf value

Give yourself enough rows to accommodate all five compounds.

11. Measure the distance from the tick mark to the solvent front in each lane and the distance from the tick mark to the compound spot in each lane; enter the measurements (in millimeters) in the appropriate row in the table.

12. Calculate the retention factor (Rf) value by dividing the distance traveled by the compound by the distance traveled by the solvent front.

Questions

1. a. Predict the order of the total intermolecular forces of the five different benzene derivatives, from lowest to highest. Indicate why you think the order is so.

2. Were there differences between your predicted order and the measured order?

3. If you answered yes to question 2, what else could be going on here? Come up with a new ranking based on your answer. Does this match the TLC results?

4. Benzoic acid and benzamide are solids at room temperature, while the rest of the benzene derivatives are liquids. Is the Rf value of a compound (which you know is already affected by the compound’s intermolecular forces) affected by whether it is a liquid or solid at room temperature?

5. Using observations from the lab, explain your answer to question 4.