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Notes on Thin Layer Chromatography


									                          Thin-Layer Chromatography (TLC)

        TLC depends on the absorption of an organic compound on silica gel (SiO2) strips, the
stationary phase, and desorption into a solvent, the mobile phase, that is ascending the strip.
        Molecules that are polar are more strongly attached to the polar, hydrogen-bonded SiO2
than to the less polar solvent, and will desorb less readily than non-polar molecules. Separation of
compounds according to their solubility are readily achieved.

TLC involves:
1. Preparation of the developing jar
2. Application of the compound(s) to the silica gel strip
3. Development of the chromatogram strip in a solvent
4. Visualization of the developed strip
5. Calculation of the Rf (retention factor) value(s)


A. Preparation of the developing jar: Pour the developing solvent in the bottom of the jar to a
   depth of approx. 1 cm. Place a lid on the jar.

B. Spotting the TLC strip: Draw two horizontal pencil lines across the TLC strip, one just over
   1 cm from the bottom, the other 0.5 cm from the top. Mark a dot on the bottom line, and spot
   the strip with your sample using a capillary tube or micropipette. Do not overload the strip
   with your sample, a small spot is enough.
C. Developing the TLC chromatogram: Place the TLC strip in the developing jar, making
   sure the sample spot does not come into contact with the solvent; the line with the spot
   should be above the level of the solvent. Let the strip slant backward and lean against the jar.
   Replace the lid and allow the strip to develop until the solvent front has come to the upper
   line. Remove the strip and let it dry for a few minutes.

D. Visualization: you may not see the spots under visible light; observe them using ultraviolet
   light and lightly circle each spot you observe.


A. Measure the distance each spot you observe has traveled, and the distance the solvent
   traveled. Calculate the Rf value for each spot you observe:

                               Rf = distance traveled by sample
                                    distance traveled by solvent

One vital question remains. What developing solvent should be used? There is no simple
answer, but in general you should use a nonpolar developing solvent for nonpolar substances and
a polar developing solvent for polar substances.

For a given compound, the Rf value (and the distance moved by the spot) will increase as the
polarity of the solvent is increased. This is because the solvent begins to compete more and more
with the silica gel for the polar parts of the molecule, and so "pushes" it up the plate more
effectively. You might expect non-polar compounds to shun a polar moving phase, but it is
important to recognize that no solvent is more polar than the silica gel surface. For this reason even
the most non-polar compound is better off in a polar solvent than it is adsorbed to the surface of the
silica gel. Thin-layer chromatography is best understood by ignoring all but the polar interactions:
it is a competition between the solvent and the silica gel for the polar parts of the molecule.

Common TLC developing solvents:

                                       Hexane, cyclohexane, petroleum ether
                                       Diethyl ether
               INCREASING              Chloroform
                POLARITY               Ethyl acetate
                                       Isopropyl alcohol

Relative polarity of organic compounds:

               INCREASING              Aromatic hydrocarbons
                POLARITY               Ethers, alkyl halides
                                       Aldehydes, ketones, esters
                                       Carboxylic acids

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