TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick answer as to how many components are in a mixture. TLC is also used to support the identity of a compound in a mixture when the Rf of a compound is compared with the Rf of a known compound (preferably both run on the same TLC plate).
A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina). A small amount of the mixture to be analyzed is spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid. This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary action.
As the solvent moves past the spot that was applied, an equilibrium is established for each component of the mixture between the molecules of that component which are adsorbed on the solid and the molecules which are in solution. In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others. When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried, and the separated components of the mixture are visualized. If the compounds are colored, visualization is straightforward. Usually the compounds are not colored, so a UV lamp is used to visualize the plates. (The plate itself contains a fluorescent dye which glows everywhere except where an organic compound is on the plate.)
When you need to determine the best solvent or mixture of solvents (a "solvent system") to develop a TLC plate or chromatography column loaded with an unknown mixture, vary the polarity of the solvent in several trial runs: a process of trial and error. Carefully observe and record the results of the chromatography in each solvent system. You will find that as you increase the polarity of the solvent system, all the components of the mixture move faster (and vice versa with lowering the polarity). The ideal solvent system is simply the system that gives the best separation.
TLC elution patterns usually carry over to column chromatography elution patterns. Since TLC is a much faster procedure than column chromatography, TLC is often used to determine the best solvent system for column chromatography. For instance, in determining the solvent system for a flash chromatography procedure, the ideal system is the one that moves the desired component of the mixture to a TLC Rf of 0.25-0.35 and will separate this component from its nearest neighbor by difference in TLC Rf values of at least 0.20. Therefore a mixture is analyzed by TLC to determine the ideal solvent(s) for a flash chromatography procedure.
Beginners often do not know where to start: What solvents should they pull off the shelf to use to elute a TLC plate? Because of toxicity, cost, and flammability concerns, the common solvents are hexanes (or petroleum ethers/ligroin) and ethyl acetate (an ester). Diethyl ether can be used, but it is very flammable and volatile. Alcohols (methanol, ethanol) can be used. Acetic acid (a carboxylic acid) can be used, usually as a small percentage component of the system, since it is corrosive, non-volatile, very polar, and has irritating vapors. Acetone (a ketone) can be used. Methylene chloride or and chloroform (halogenated hydrocarbons) are good solvents, but are toxic and should be avoided whenever possible. If two solvents are equal in performance and toxicity, the more volatile solvent is preferred in chromatography because it will be easier to remove from the desired compound after isolation from a column chromatography procedure.
Ask the lab instructor what solvents are available and advisable. Then, mix a non-polar solvent (hexanes, a mixture of 6-carbon alkanes) with a polar solvent (ethyl acetate or acetone) in varying percent combinations to make solvent systems of greater and lesser polarity. The charts below should help you in your solvent selection. You can also download this pdf chart of elution order.
The strength with which an organic compound binds to an adsorbent depends on the strength of the following types of interactions: ion-dipole, dipole-dipole, hydrogen bonding, dipole induced dipole, and van der Waals forces. With silica gel, the dominant interactive forces between the adsorbent and the materials to be separated are of the dipole-dipole type. Highly polar molecules interact fairly strongly with the polar SiOH groups at the surface of these adsorbents, and will tend to stick or adsorb onto the fine particles of the adsorbent while weakly polar molecules are held less tightly. Weakly polar molecules generally tend to move through the adsorbent more rapidly than the polar species. Roughly, the compounds follow the elution order given above.
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:
Since these factors are difficult to keep constant from experiment to experiment, relative Rf values are generally considered. "Relative Rf" means that the values are reported relative to a standard, or it means that you compare the Rf values of compounds run on the same plate at the same time.
The larger an Rf of a compound, the larger the distance it travels on the TLC plate. When comparing two different compounds run under identical chromatography conditions, the compound with the larger Rf is less polar because it interacts less strongly with the polar adsorbent on the TLC plate. Conversely, if you know the structures of the compounds in a mixture, you can predict that a compound of low polarity will have a larger Rf value than a polar compound run on the same plate.
The Rf can provide corroborative evidence as to the identity of a compound. If the identity of a compound is suspected but not yet proven, an authentic sample of the compound, or standard, is spotted and run on a TLC plate side by side (or on top of each other) with the compound in question. If two substances have the same Rf value, they are likely (but not necessarily) the same compound. If they have different Rf values, they are definitely different compounds. Note that this identity check must be performed on a single plate, because it is difficult to duplicate all the factors which influence Rf exactly from experiment to experiment.
All of the above (including the procedure page) might sound like TLC is quite an easy procedure. But what about the first time you run a TLC, and see spots everywhere and blurred, streaked spots? As with any technique, with practice you get better. Examples of common problems encountered in TLC:
See how well you understand TLC by taking the online TLC Technique Quiz!
Original content © University of Colorado at Boulder, Department of Chemistry and Biochemistry.
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