Thin layer chromatography (TLC) is a technique utilized in chromatography for the separation of mixtures.
This process is conducted on a substrate made of glass, plastic, or aluminum foil, which is covered with a thin layer of adsorbent material, typically silica gel, aluminum oxide, or cellulose (blotter paper).
The adsorbent layer is referred to as the stationary phase. Once the sample is applied to the plate, a solvent or a mixture of solvents (termed the mobile phase) is drawn up the plate through capillary action.
Separation occurs because various analytes travel up the TLC plate at differing rates.
TLC Chamber:
The TLC plate is positioned in a shallow solvent pool within a developing chamber, ensuring that only the very bottom of the plate is submerged in the liquid.
TLC Solvents Selection:
To identify the most suitable solvent or combination of solvents (referred to as a “solvent system”) for developing a TLC plate or chromatography column containing an unknown mixture, it is essential to vary the polarity of the solvent through multiple trial runs, employing a method of trial and error. Diligently observe and document the outcomes of the chromatography for each solvent system. You will notice that increasing the polarity of the solvent system results in all components of the mixture moving more rapidly (and conversely, decreasing the polarity slows their movement). The optimal solvent system is defined as the one that provides the best separation.
TLC elution patterns typically correspond to those observed in column chromatography. Given that TLC is significantly quicker than column chromatography, it is frequently employed to identify the most suitable solvent system for column chromatography. For example, when establishing the solvent system for a flash chromatography process, the optimal system is one that transports the target component of the mixture to a TLC Rf value between 0.25 and 0.35, while also ensuring that this component is separated from its closest neighbor by a difference in TLC Rf values of no less than 0.20. Consequently, a mixture is evaluated using TLC to ascertain the most effective solvent(s) for a flash chromatography procedure.
Beginners frequently find themselves uncertain about where to begin: Which solvents should they select from the shelf for eluting a TLC plate? Due to concerns regarding toxicity, cost, and flammability, the commonly used solvents include hexanes (or petroleum ethers/ligroin) and ethyl acetate (an ester). While diethyl ether is an option, it is highly flammable and volatile. Alcohols such as methanol and ethanol are also viable choices. Acetic acid (a carboxylic acid) can be utilized, typically as a minor component of the system, as it is corrosive, non-volatile, very polar, and produces irritating vapors. Acetone (a ketone) is another solvent that can be employed. Methylene chloride and chloroform (halogenated hydrocarbons) are effective solvents, but they are toxic and should be avoided whenever feasible. In cases where two solvents exhibit equal performance and toxicity, the more volatile solvent is favored in chromatography, as it will be simpler to eliminate from the desired compound following isolation from a column chromatography process.
Inquire with the laboratory instructor regarding the available and recommended solvents. Subsequently, combine a non-polar solvent (such as hexanes, which is a blend of 6-carbon alkanes) with a polar solvent (like ethyl acetate or acetone) in different percentage mixtures to create solvent systems with varying degrees of polarity. The charts provided below will assist you in selecting the appropriate solvents.
Interactions between the Compound and the Adsorbent
The degree to which an organic compound adheres to an adsorbent is influenced by the strength of various types of interactions, including ion-dipole, dipole-dipole, hydrogen bonding, dipole-induced dipole, and van der Waals forces.
In the case of silica gel, the primary interactive forces between the adsorbent and the substances being separated are predominantly of the dipole-dipole nature. Highly polar molecules exhibit relatively strong interactions with the polar SiOH groups present on the surface of these adsorbents, leading them to adhere or adsorb onto the fine particles of the adsorbent, whereas weakly polar molecules are held less firmly.
Typically, weakly polar molecules tend to traverse the adsorbent more swiftly than their polar counterparts. In general, the compounds adhere to the elution order as described above.
The Rf value
The retention factor, or Rf, is defined as the distance traveled by the compound divided by the distance traveled by the solvent.
For example, if a compound travels 2.1 cm and the solvent front travels 2.8 cm, the Rf is 0.75:
The Rf for a compound is a constant from one experiment to the next only if the chromatography conditions below are also constant:
- solvent system
- adsorbent
- thickness of the adsorbent
- amount of material spotted
- temperature
Due to the challenges in maintaining consistency across experiments, relative Rf values are typically utilized. The term “Relative Rf” refers to values that are expressed in relation to a standard, or it indicates a comparison of the Rf values of compounds analyzed on the same plate simultaneously.
The greater the Rf value of a compound, the farther it moves on the TLC plate. When assessing two distinct compounds subjected to the same chromatography conditions, the compound exhibiting a higher Rf is less polar, as it interacts with the polar adsorbent on the TLC plate to a lesser extent. In contrast, if the structures of the compounds within a mixture are known, one can infer that a compound with low polarity will possess a higher Rf value compared to a polar compound analyzed on the same plate. The Rf value can serve as supporting evidence regarding the identity of a compound. If there is a suspicion about the identity of a compound that has not yet been confirmed, an authentic sample of the compound, or a standard, is applied and run on a TLC plate alongside (or overlapping) the compound in question. If two substances exhibit identical Rf values, they are likely (though not definitively) the same compound. Conversely, if their Rf values differ, they are certainly distinct compounds. It is important to note that this identity verification must occur on a single plate, as replicating all the variables that affect Rf precisely from one experiment to another is challenging.