PLEASE VIEW THE RELEVANT VIDEO BEFORE ATTENDING THE LAB CLASS
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Example of a Typical Experiment
FAQ: For TLC's, must the sample solvent and eluent solvent be the same?
FAQ: What is the purpose of a sample solvent in the preparation of a TLC plate?
FAQ: How do I determine my sample solvent?
FAQ: In TLC, what is the purpose of using the eluent solvent(s)?
FAQ: How does the eluent solvent(s) work?
FAQ: Do I have to use two solvents for my eluent?
FAQ: Which solvent(s) do you choose for your eluent?
FAQ: When preparing a TLC, why should we choose the least polar solvent to dissolve our sample?
FAQ: What is a "Dichloromethane : Hexane ratio of 3:1"?
FAQ: My TLC spots are too high, i.e., too near the solvent front (or too low, i.e., too near the baseline). What do I do now?
FAQ: How do you properly record information obtained from a TLC?
FAQ: Which is the developing solvent?
Theory: Thin Layer Chromatography (TLC)
During chromatography, a mobile phase (eluent) distributes the compounds present in a mixture over a stationary phase (adsorbent).
Thin Layer Chromatography (TLC) is one type of chromatography, where:
- the mobile phase is a solvent or mixture of solvents (eluent),
- the stationary phase is a solid adsorbent (e.g., silica or alumina) coated on a stationary support such as glass or plastic (TLC plate).
First, the TLC plate is prepared. The unknown solid is applied as a solution by dissolving it in a solvent. Some solution is drawn up into a micropipette, then applied to the adsorbent near one end of the TLC plate (baseline) by allowing the solution to wick out onto the adsorbent. This process is generally referred to as "spotting your TLC plate".
The chromatogram is then developed by placing the TLC plate into a TLC chamber (a beaker containing the mobile phase, i.e., the eluent). The eluent will travel up the adsorbent by capillary action to the top of the TLC plate (known as the solvent front), carrying the sample with it. This process is generally referred to as "running your TLC plate".
Depending on the relative polarity of the sample and the eluent, the sample either adsorbs to the solid phase or is eluted by the mobile phase (eluent). If the eluent is very polar relative to your compound, it will dissolve your sample and the sample will move with the mobile phase.
Overall, the eluent and your sample will compete for a space (an active site) on the adsorbent (stationary phase) coated on the TLC plate. The more polar compound will win this competition and adhere (adsorb) to the adsorbent, while the rest of the mixture will move.
The more polar the compound, the more it will adhere to the adsorbent and the smaller the distance it will travel from the baseline, and the lower its Rf value.
Eluent: the solvent or mixture of solvents (mobile phase) used to develop a TLC chromatogram (plate).
Elution: the overall process of developing a TLC plate.
Rf (retention or retardation factor): is a measure of the distance travelled by the compound spot in relation to the distance travelled by the eluent. A desirable Rf value lies between 0.3 and 0.7, since it is likely that other compounds present in the mixture will be visible on the TLC plate when the Rf is in this range.
Identification of a solid using thin layer chromatography (TLC):
First a TLC plate is prepared by spotting the purified unknown and an authentic sample of each possible compound. Then the TLC plate is developed. For the next step (co-spotting), an authentic sample of the compound closest in Rf value to the unknown is chosen. TLC co-spotting of a second plate allows for preliminary identification of your compound. Three spots are applied to the adsorbent on the baseline of the TLC plate: the purified unknown, an authentic sample, and a co-spot of unknown and authentic sample. If the developed TLC plate shows only one row of spots, it can be concluded that the unknown has been purifed, and that the unknown is possibly the same compound as the authentic sample. However, because Rf values are relative, not absolute, some compounds may have very similar Rf values. A mixed melting point measurement is needed to unambiguously identify the unknown compound.
Purity assessment of a solid using thin layer chromatography (TLC):
Thin Layer Chromatography (TLC) is a separation technique requiring very little sample. It is primarily used to determine the purity of a compound. A pure solid will show only one spot on a developed TLC plate. In addition, tentative identification of the unknown compound can be made through TLC analysis.
Monitoring the progress of a reaction:
The progress of a reaction can be monitored by Thin Layer Chromatography. At various times during the experiment, a TLC plate is spotted three times, then developed and viewed under UV light:
- At position 1 is an authentic sample of the starting compound.
- At position 2 is the reaction mixture.
- At position 3 is the authentic sample of the product.
The reaction has gone to completion, when the TLC plate shows that there is no more starting material left in the reaction mixture.
Earl N. Meyer wanted to determine the purity of the benzoic acid which he had obtained from Chemical Recycling. He decided to check the purity by chromatographic analysis using a silica gel TLC plate. On the TLC plate he marked the baseline with a pencil, indicating the spotting position with a tick. Earl dissolved about 20 mg of the benzoic acid in 1 ml of methanol. Then he dipped a micropipette into the solution and lightly touched the TLC plate at the tick marked on the baseline.
Next, Earl prepared the developing chamber. He cut a filter paper to line a 400 ml beaker. He then prepared the eluent: 10 ml of a mixture of dichloromethane and methanol in a ratio of 10 : 1. He added the eluent to the beaker, swirling it onto the filter paper to completely wet it. Earl placed the TLC plate in the developing chamber using tweezers and watched the eluent travel up the plate by capillary action. When the eluent was near the top of the plate, he used tweezers to remove the plate from the chamber, taking care to immediately mark the solvent front with a pencil.
When the TLC plate was dry, Earl viewed it using long wavelength, ultraviolet light. He observed a single blue spot on a light green background. His TLC indicated that the sample tested contained a single component and that his benzoic acid was pure. He used a pencil to circle the spot and a ruler to make measurements needed for calculation of the retention factor.
First, prepare a TLC plate: dissolve a very small amount of your unknown sample in a volatile solvent, then apply the solution i.e., spot the adsorbent at the baseline.
Next, prepare the TLC chamber: Cut the portion of the filter paper that rises above the beaker edge.
Prepare the eluent using a mixture of the suggested solvents. Use a total of ~10mL eluent in a 250 mL beaker.
When measuring liquids, read from the bottom of the meniscus, i.e., look at the lowest part of the curve at the liquid's surface.
Swirl the eluent to wet the filter paper and cover the beaker with a lid. This will saturate the chamber with eluent vapors and prevent evaporation of the eluent.
Develop the TLC plate, that is, allow the eluent to wick up the TLC plate until it is about 1 cm from the top of the plate. Immediately after removing the TLC plate from the beaker, carefully draw a line along the solvent front. Be quick, because the eluent will evaporate within less than a minute.
Visualize the developed plate using an ultraviolet lamp or other visualization method:
- If the spot is observed to be too close to the baseline, your eluent is not polar enough.
- If the spot is observed to be too close to the solvent front, your eluent is too polar in relation to your compound.
The best Rf (retention or retardation factor) lies between 0.3 and 0.7.
If you want the Rf of your TLC spot to be smaller, i.e., the spot to be lower down on the plate, you must decrease the eluent polarity. Either choose a different eluent (solvent) or adjust the solvent ratio by increasing the percentage of the nonpolar solvent relative to the polar solvent in the eluent. The reverse holds true for the Rf of a spot that you want to be larger, i.e., the spot to be further up on the plate.
Q: For TLC's, must the sample solvent and eluent solvent be the same?
No. Sometimes sample solvent and eluent solvent(s) are the same, but this is not necessary. If you are wondering why think about the different functions that each solvent plays in the TLC process.
Q: What is the purpose of a sample solvent in the preparation of a TLC plate?
Before you can develop your TLC plate, you must transfer some sample onto it. The simplest method to do this requires that you dissolve your sample in a solvent. Spotting the TLC plate using a micropipet and waiting for the solvent to evaporate will leave the sample on the TLC plate.
Q: How do I determine my sample solvent?
As a rule of thumb, your sample solvent should: 1) dissolve your sample when cold, 2) have a low boiling point (so it will evaporate quickly) and 3) be as non-polar as possible (e.g., dichloromethane), because if some sample solvent should remain on a developing TLC plate, it will not greatly change the polarity of the eluent(s) you will be using.
Q: In TLC, what is the purpose of using the eluent solvent(s)?
Eluents are the mobile phase in chromatography, i.e., the solvent in the developing tank.
During the development of the chromatogram, the eluent distributes the sample you transferred to the TLC plate over the adsorbent on the plate.
Q: How does the eluent solvent(s) work?
The compound sample you spotted onto the stationary phase on the TLC plate has two choices: adsorb to the solid phase or being eluted by the mobile phase.
If your eluent(s) is very polar relative to your compound, it will dissolve your sample and carry it in the mobile phase.
Overall, the eluent solvent(s) (also called the mobile phase) will compete with your sample for a space on the adsorbent (stationary phase) coated on the TLC plate. The more polar compound will win this competition and adhere (ad sorb) to the adsorbent, while the rest of the mixture will move with the mobile phase [upwards].
An eluent that is very polar in relation to the sample, will result in the compound spot being observed with a larger Rf, i.e., closer to the solvent front. On the other hand, if you eluents are too non-polar, the compound spot will be observed with a smaller Rf, i.e., closer to the baseline.
Q: Do I have to use two solvents for my eluent?
Two solvents are not always necessary. Sometimes one solvent will be appropriate. However, two solvents allow you to adjust the eluent polarity easily when a developed TLC plate does not give an appropriate result.
A suitable eluent is one that gives a retardation factor (Rf, also called retention factor) close to 0.5 upon developing the TLC plate.
Is your Rf too high or too low, then you can readily adjust the polarity if the eluent was prepared by mixing two miscible solvents by changing the relative proprotions within the eluent mixture (e.g., from 1:1 to 3:1).
If only one solvent was used, it will be necessary to switch to a solvent with a different polarity.
Q: Which solvent(s) do you choose for your eluent?
The eluent solvent(s) you use is usually determined experimentally.
A two-solvent eluent should contain one polar solvent and one non-polar solvent; BUT it is important that these two solvents are miscible with (i.e., dissolve in) each other. A good bet is to start with a ratio of 1:1, and then adjust the ratio depending on results obtained from developed chromatograms.
If you use a one-solvent eluent, you should start with a solvent which is not highly polar as a first choice.
Q: When preparing a TLC, why should we choose the least polar solvent to dissolve our sample?
A solvent that is not very polar will affect the eluent(s) to a lesser degree if the solvent does not fully evaporate after spotting the TLC.
Q: What is a "Dichloromethane : Hexane ratio of 3:1"?
A ratio tells you something about the relationship of the given components in a mixture. In this case, three units (e.g., 3 mL) of Dichloromethane should be mixed with one unit (e.g., 1 mL) of Hexane to produce a total of 4 units (here: 4 mL) of the mixture.
Q: My TLC spots are too high, i.e., too near the solvent front (or too low, i.e., too near the baseline). What do I do now?
When your sample produces an Rf value between 0.3 and 0.7 you do not need to adjust your eluents.
However, if your spot is too close to either the solvent front or the baseline, you need to adjust the polarity of your eluent system.
Spot is too close to the baseline:
This means that your eluent is, in relation to your sample, not polar enough. Increase the polarity of your eluent either by increasing the ratio of the more polar solvent of your eluent or by switching to a more polar solvent.
Spot is too close to the solvent front:
Your eluent is, in relation to your sample, too polar. You have to decrease the polarity of your eluent, either by increasing the ratio of the less polar solvent of your eluent system or by switching to a less polar solvent.
Q: How do you properly record information obtained from a TLC?
Use a pencil (not a pen) to lightly indicate the solvent front and to outline the spots that you see upon visualizing the developed TLC plate with either the UV lamp or with the iodine stain.
Once you have outlined your TLC plate, calculate the Rf value for each component, and sketch a replica of the TLC in your lab notebook.
Q: Which is the developing solvent?
The developing solvent is the solvent that is placed into the developing tank used to develop your TLC plate. It is the "mobile phase" for the chromatography and may also be referred to as the eluent, eluting solvent, or solvent system.