Thin Layer Chromatography Theory

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What is TLC?

TLC stands for Thin Layer Chromatography
  1. It is an analytical technique that can be used to monitor reactions and for the qualitative analysis of complex mixtures and for the identification of unknown compounds.
  2. It is also important for determining the correct solvent system with which to run a column chromatography in.
  3. TLC is composed of two phases, a mobile and a solid phase. The solid phase is a thin solid support that usually consists of Alumina or Silica. The mobile phase is a solvent that moves through capillary action right through the solid phase. In general, the solid phase is usually polar while the mobile solvent is non polar relative to the solid phase. You cannot recover your compound from the TLC plate so keep this in mind if you have small amounts of compound.

How to choose the solvent system?

The choice of the solvent depends on the properties of compounds that you want to separate and the nature of the solid support you are using (ie, is it silica or alumina). For example, if an impurity in a sample is nonpolar, the impurity can be removed using a nonpolar solvent like hexane if a polar Silica solid phase is being used. Likewise, if the compound of interest is polar, then it will have a strong affinity with the polar Silica solid phase and thus, will not be removed in the non polar solvent. The compound can be eluted using a more polar solvent such as ethyl acetate or ethanol. Keep in mind that too high a concentration of methanol can dissolve the silica (~10%).

Visualization Techniques

Some common techniques for visualizing the results of a TLC plate include (if you cannot see it with your eyes):

  1. UV light ( remember to use the UV goggles)
  2. Iodine Staining: is very useful in detecting carbohydrates since it turns black on contact with Iodine
  3. KMnO4 stain (organic molecules)
  4. Ninhydrin Reagent: often used to detect amino acids and proteins

visualizing the results of a T L C plate

Handling TLC

Whenever handling the TLC plate try to use gloves as your fingerprints might give some extra spots. If handling with your hands, hold the TLC plate along the edges.

How to Interpret the TLC?

The behaviour of a compound on a TLC is usually described in terms of its relative mobility or Rf value. Rf or Retention factor is a unique value for each compound under the same conditions. The Rf for a compound is a constant from one experiment to the next only if the chromatography conditions below are also constant:

  1. solvent system
  2. adsorbent
  3. thickness of the adsorbent
  4. amount of material spotted
  5. temperature

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 final results for the TLC should look like a series of dots. If you can visualize the spots then the Rf value can be calculated.

The Rf value is calculated using the following equation:

equation for R F diagram of a T L C plate

Based on the Rf values, the identity and characteristics of the different compounds can be determined. More polar compounds will have smaller Rf values since they will have a stronger affinity for the polar solid phase. This would result in the compound not being carried very far along the TLC plate. Compounds with larger Rf values interact strongly with the less polar mobile phase and thus, tend to be non polar themselves. Sometimes it is impossible to see a good separation on TLC. The picture looks smeared; this may be due to the addition of too much sample or evidence of the presence a lot of impurities. Also, if the side and bottom of the TLC plate is not flat, the solvent front may migrate unevenly.

Limitations of TLC

Although it is a very simple and convenient technique, one of its limitations is that it cannot tell the difference between enantiomers and some isomers. Another disadvantage of TLC is that in order to identify specific compounds, the Rf values for the compounds of interest must be known beforehand.

Steps and Procedure for Running a TLC

  1. First obtain a TLC jar/chamber and add the TLC solvent to it. Add filter paper that is the same size as the TLC plate into the jar to aid equilibration. Close the TLC jar for 5-15minutes to allow for equilibration.
  2. Obtain the sample to be spotted. You must dissolve this sample (even if it is liquid) in an appropriate solvent in order to spot it on a TLC. Use a sample vial, test tube, or small flask to dissolve your sample. Use a solvent with a low boiling point if possible (acetone or methylene chloride for example). This solvent could be easily evaporated.
  3. Obtain a clean TLC plate from the box/desiccators. (Use gloves, oil from hands can contaminate the TLC). Mark a line (using pencil) that is higher than the solvent in the TLC jar and mark down where the sample should be spotted. Use a capillary tube to spot the sample onto the TLC plate. You can spot it several times if the concentration of your sample is not high. Air dry TLC plate after each spotting (use a blow drier to evaporate off higher boiling point solvents). Your sample spot should not be wider when 4-5 mm in diameter. The smaller the diameter of the spot, the better resolution you will observe.
  4. Put the TLC plate into the TLC jar such that the start line is parallel to the TLC solvent. Set the TLC to a slight slant and then close the jar lid. Stop the TLC when the solvent front is close to the top by taking the TLC out of the TLC jar. Mark down the location of the solvent front for determining the Rf values.

diagram of T L C plate