Purpose

To determine the identity of the compounds in an unknown substance via thin-layer chromatography.

Expected Learning Outcomes

• Determine how many compounds are in an unknown sample based on its TLC
• Identify the compounds in an unknown sample by comparing it to known standards using TLC
• Predict which compounds will travel faster on a TLC plate based on its structure and polarity

Introduction

As we saw a few weeks ago in our research experiment, chromatography can be used to separate compounds in a mixture based on their polarity. In this experiment, we will investigate how to use thin-layer chromatography (TLC) in more detail as a way to identify how many compounds are in a sample and what their identities are. TLC is a very common technique in chemistry laboratories that you will learn about in more detail if you take organic chemistry, but you already have the knowledge and skills to understand the basics.

As you learned in the research project lab manual, there are two important physical pieces in chromatography: the mobile phase and the stationary phase. The mobile phase is the part that moves over the stationary phase and carries the compounds of interest with it. In most chromatography (including TLC), the mobile phase is a liquid solvent (the eluent), although it can be a gas (such as in gas chromatography). The stationary phase is always a solid that stays in a “column” (in TLC this “column” is the TLC plate), no matter how much of the mobile phase passes over it. In our research project (and in this TLC lab), our stationary phase was silica gel (for both TLC and column chromatography). Silica gel is essentially made up of microparticles of glass, and because silica is a highly polar compound, it interacts very strongly with other polar compounds (remember that this is also why we get a meniscus in glassware like graduated cylinders since water is highly polar and is attracted to the highly polar silica of the glassware).^[1] Because silica gel is so highly polar, we usually use a fairly non-polar organic solvent for our mobile phase. This means that molecules that are more polar will stick to the polar silica gel more strongly and spend less time dissolved in the non-polar mobile phase, thus they will travel more slowly on the “column”. The opposite is true for non-polar molecules: they interact very weakly with the polar silica gel and spend more time dissolved in the non-polar mobile phase. This means that the faster a compound travels on a TLC plate, the LESS polar it is (because it spends more time in the non-polar mobile phase and less time stuck to the more polar stationary phase).

This also means that we can use chromatography to separate out compounds in a mixture based on their polarity and if we have a known “standard” to compare our sample against, we can identify what our compound is by seeing if it travels at the same rate as one of our standards. This is what we will be doing today. You will be give an unknown sample that contains 1-3 compounds and 4 standards of the compounds that could be in your unknown. Your first task will be to run the standards on multiple TLC plates to ensure that you can effectively use TLC to separate all 4 of your standards. Once you have proven that you can run and interpret a TLC plate, you will have to use TLC to compare the standards to your unknown to figure out which compound(s) is/are in your unknown.

Procedures

Required Special Materials and Chemicals

• 40:60 ethyl acetate:cyclohexane (20 mL)
• TLC plates (6)
• 110 mm diameter filter paper (2)
• forceps (1)
• screw cap vial (1)

Standards:

• A = acetylsalicylic acid
• B = anisole
• C = 2-naphthol
• D = vanillin

TLC of Standards

1. Following the same procedure as for the TLC for the research project, create 2 developing chambers, each with ~5 mL of 40:60 ethyl acetate:cyclohexane as the eluent (you can always add more eluent if you need to).
2. Spot 2 TLC plates: one with standards A, B and C and one with standards B, C and D (singly spot each sample for now and be sure to rinse your spotter with pure ethyl acetate between each sample as specified in the week 2 procedure for the research project). Remember to label the top of each of your TLC plates with your initials, the date and the eluent you will use to develop that plate (40:60 EA:CH in this case)
3. Run each TLC plate in their own developing chamber. Remember to make sure that the places where you spotted your samples are above the level of the eluent in the bottom of the chamber.
4. While waiting for your first two TLC plates to develop, create two more TLC plates with standards C, A and D on one plate and standards A, D and B on the 2nd plate (label the tops of these plates too).
5. Once the first two TLC plates are done running, remove them from the chambers, draw a line where the solvent got to, wait for the solvent to evaporate, then illuminate them with UV light (take pictures as specified in the research project lab manual, but circle all the spots you see in each lane before taking the ambient light picture; make sure it is possible for your instructor to interpret your TLC results based on your images, otherwise you will not earn full-credit).
   • SAFETY NOTE: UV light can damage DNA, so always shine the light down, never up and avoid shining it directly onto your skin or into your eyes. You should also minimize how much UV light gets reflected onto your bare skin and eyes.
6. Make sure that it is possible to distinguish all 4 compounds on the TLC plates. If they aren’t, you will need to re-run the TLC plates in such a way that you can distinguish between all of the compounds (see below for common issues you might see).
   1. If any of the compounds on are too faint to see clearly, double or triple spot those compounds.
   2. If any of the compounds create a such a large spot that they are smearing or overlap with where one of the other compounds is, make a dilution of that sample by adding a few drops of the standard solution to ~0.5 mL of ethyl acetate in a screw cap vial.
   3. If all of the compounds are bunched up near the top or near the bottom of the TLC plates, you probably used the wrong eluent. Dump out the liquid in the TLC chambers into the organic waste beaker and replace it with the 40:60 ethyl acetate:cyclohexane eluent.
   4. If there is a dark smear over the bottom half of the TLC plate, you probably contaminated the eluent with one of the standards. Dump out the liquid in the TLC chamber into the organic waste beaker and replace it with fresh eluent. Make sure that the next TLC plates you run have the places you spotted your samples well above the level of the eluent in the TLC chamber so that the compounds don’t leach out into the eluent.
   5. If you see multiple spots in any of your standard lanes (i.e. lanes with a standard), you probably didn’t rinse your TLC spotter well enough between samples. Rerun the TLC plate the exact same way except rinse your TLC spotter a few more times between each sample. (Note that you might see multiple spots in your unknown lanes and thus seeing multiple spots in a single lane in the next part of the experiment doesn’t necessarily mean that you didn’t rinse your spotter well enough.)
7. Run the 2nd pair of TLC plates you created in step 4 after fixing any issues you found in step 6.
8. If need be, remake your first two TLC plates and rerun them.
9. Draw a picture in your lab notebook of every TLC plate you ran and specify how far from the bottom of the plate everything is, including the origin (where you spotted the samples) and the solvent line.

Once you have 4 TLC plates that all show clear separation between all of the standards, show them to your instructor. If you instructor agrees that there is clear separation between all of the standards on all 4 of the TLC plates, move on to identifying your assigned unknown.

Identifying Your Unknown

1. Create a TLC plate that will compare your unknown to any 2 of the standards (put the unknown in the middle and the standards to either side).
2. Create a 2nd TLC plate to compare your unknown to the OTHER 2 standards.
3. Run the TLC plates in the 2 TLC chambers you created in the previous part of this experiment. Remember to label the TLC plates properly.
4. Repeat until you have sufficient data to definitively identify which compound(s) are in your unknown.
5. Draw a picture in your lab notebook of every TLC plate you ran and specify how far from the bottom of the plate everything is, including the origin (where you spotted the samples) and the solvent line.

Waste Disposal

TLC plates can be thrown in the trash once you have finished with them. Filter paper should be put in the fume hood until all of the solvent has evaporated and then throw it away in the normal trash. Capillary tube spotters should be put into the broken glass receptacle. All other waste should go into the organic waste beaker in the fume hood.