Thin-layer chromatography (TLC) is a separation technique for a mixture of non-volatile compounds monitored by their rate of movement on a thin layer of adsorbent coated a plate or sheet.
There are different types of sheets used in TLC, of which most common are plastic, glass, and aluminum foil while the most common types of coatings (absorbent materials) used are silica gel, cellulose, and aluminum oxide. The adsorbent layer is termed as a stationary phase while the mixture to be separated as a mobile phase.
Principle of Separation Mixture
The separation principle of thin layer chromatography lies in the different travel rates of the compounds present in the mixture being processed. Also, this difference in the rate of travel of the compounds relies on the presence of the attractive forces present between them and the stationary phases (e.g., silica gel, aluminium oxide, and cellulose).
During the process of separation, there is a competition between the solute being separated and the mobile phase used in the process of separation for the binding sites present on the stationary phase. Let us illustrate this with the following example.
We are taking normal phase silica gel as an example as our stationary phase. Since silica gel can be considered as a polar substance, we will describe the separation of a mixture containing a polar compound in it.
Therefore, when the sample mixture moves along with the mobile phase (also known as eluent), the polar compound in the sample would be stronger attracted to the stationary phase and thus making this compound to bind to it firmer.
Whether one works as a biologist or a chemist or a biochemist, he/she can often come across a mixture which should be separated into its constituent compounds. Now there are two ways to do that. One is to undergo chemical assays, which are in their very essence, often hectic and painful.
While on the other hand, one can conveniently opt for thin layer chromatography which not only helps in the separation of the compounds but also aids in their identification. The following are going to be discussed some common and important uses of TLC with appropriate examples where applicable.
The extraction of numerous compounds from various source origins is a common and early step of most of the experimental procedures resulting in getting a mixture of compounds. This mixture can then serve as a starting point for the downstream analyses.
Most often, the primary aim of the extraction is to identify a compound of interest. This is possible only if a standard is already available. This standard is usually a pure form of the compound of interest which has to be identified in the mixture. But, in the absence of the standards, de novo identification would have to be done using some advanced protocols depending on the nature of the compound under investigation.
Let us look at the example of the identification of phytoestrogen from a plant extract. Now the purpose of the experimental design would be to see if some plant contains phytoestrogens or not. The initial steps would involve extracting preparation.
Then, to see whether this extract contains the compound of interest i.e., phytoestrogen, a TLC analysis can be performed running pure phytoestrogen as standard. Identifying the same compounds from different origins.
Just like compound detection, thin layer chromatography can also be used to identify a compound by running samples coming from different sources. In this case, Rf values would be calculated identical values (compounds moved to the same distance) would indicate the presence of identical compounds (see figure 1).
Figure 1. Graphical representation of TLC explaining the identification of the same compound from different samples. The black band at the leftmost position shows the standard while the other two bands on the right show compound moved to the position of standard from different samples. The blue line at the bottom represents a hypothetical start point where the mixture is applied in the experiment.
Identifying the number of compounds
Thin-layer chromatography can also be used to determine the number of compounds present in a mixture. For this purpose, the sample will be run and the number of spots or bands appearing on the plate can be calculated. This can be better understood by looking at figure 2.
Figure 2. graphical representation of a TLC showing how the number of compounds present in a mixture can be calculated using TLC. For example, in the figure, it can be seen that the TLC plate contains 3 spots, so it can be concluded that the sample analyzed contained 3 different compounds.
Reactions’ monitoring via TLC
There are many experimental scenarios in which an experimenter wants to determine the rates of reactions and thereby monitor the underlying progress of the reactions. This can be easily done by doing the TLC of the reaction samples at different time points and monitor the conversion of the reactants to products.
This can be further monitored in two ways:
- To see the appearance of the products on the TLC plate with the passage of time; or
- To observe the disappearance of the reactance (most preferably one of the reactants) with passing time.
This data is then recorded with time and compared to determine the rates of reactions.
Determining a mixture’s or compound’s purity
Thin-layer chromatography is also used to determine the purity of a compound or a mixture of different compounds (for which the components are known.
For example, if some perform purification experiments for a compound of interest and want to determine at the end of the purification experiments whether he/she has got the purified product or not, then TLC can be performed to check the purity with standard run along with. The appearance of a single spot is a key to the successful purification.
On the other hand, if someone prepares a mixture of some compounds and wants to confirm that it contains only the components that he/she has mixed and no impurities, a TLC result showing the number of spots in accordance with the number of compounds mixed is a confirmative indication of no impurities and vice versa.
Monitoring column chromatography separations
Column chromatography is another technique used most often to separate and purify a compound of interest by the experimenters. In this technique, different fractions of the sample being purified are collected which are later on analyzed to see which fractions contain the desired product. One way for this analysis as well as monitoring of the column chromatography fractions is via TLC.