Column Chromatography and TLC

Column Chromatography and TLC

Written by Lyudamila


The method used for purification of chemical compounds or establishing how many different compounds a mixture carries or determining a chemical mixture’s polarity is widely known as chromatography. The basic principle of chromatography is to separate certain compounds based on their structure, ability to hydrogen bond and polarity between the stationary phase and mobile phase. There are different types of chromatography devices used and various techniques of separation that are applied to separate chemical mixtures. Here, we present data from using Column Chromatography and Thin-Layer Chromatography to separate fluorene from 9-fluoreneone and to determine the best solvent mixture for a separation.

Table of Physical Constants:

Chemical Name Chemical Formula MW: (g/mol) Boiling Point (°C) Melting Point (°C) Refractive Index (nD)
Hexane C6H14 86.18 69 1.375
Silicon Dioxide SiO2 60.1 1600-1725
Fluorene C13H10 166.22 295
9-Fluorenone C13H8O 180.19 342 1.6309
Acetone C3H6O 58.08 57 1.35900

Safety Information: Hexane and Acetone are flammable.

Materials & Methods:

Column Chromatography:

To pack the column, silica gel was mixed with 14mL of a non-polar solvent, hexane and transferred inside the column. 1mm of sand was delivered to the column so that it would sit atop the silica gel bed. To load the column, about .3mL of the mixture of fluorine and 9-fluorenone was delivered which was visible by a yellow band. Following this, 4 elutions were collected in test tubes as hexane was continuously being added to the column. The solvent system was changed to a mixture of hexane and acetone (70:30) and fractions of the mixture were collected into separate test tubes until the yellow band eluted off the column.

Thin-Layer Chromatography:

The 4 elutions from the hexane-only solvent system were collected and concentrated to approximately 1/4th of the original volume and the fractions of the mixture from the hexane-acetone solvent system were collected and concentrated to ½ of the original volume. A sample (sample #1) from the hexane elutions and a sample(sample #2) from the hexane-acetone fractions were marked separately away from each other via a microcapillary tube onto the thin layer or adsorbent coated on the TLC plate. A third sample of the original mixture (sample #3) that was loaded at the top of the column was also marked on the TLC plate. TLC solvent was used to soak onto the TLC plate to allow nonpolar substances to move up the plate most rapidly and to allow polar substances to move up the plate at a slower rate or not at all. The TLC plate was removed as soon as the solvent traveled up the plate until it was 1cm from the top. The retention factor was then calculated.


The solvent systems separated fluorene and 9-fluorenone based on their difference in structure and polarity. The hexane solvent system was helpful in washing away anything that was hydrophobic since it’s nonpolar, and it essentially washed off most of the fluorene since fluorene is not as polar as 9-fluorenone. The solvent system with a mixture between acetone and hexane  allowed the yellow band to be eluted off the column because polar solvents such as acetone are helpful in moving chemical compounds down the column that tend to have a higher polarity, such as 9-fluorenone.

Structurally, fluorene does not have a carbonyl functional group and 9-fluorenone does. For this difference, the oxygen that sticks out from 9-fluorenone was able to hydrogen bond to the silica gel beads which allowed it to be held tighter in the column than fluorene. Because 9-fluorenone was held tighter in the silica gel beads in the column, it did not go down the column as fast as fluorene did. In principle, the chemical compound that flows through the column at a faster speed is more non-polar; therefore, in this case fluorene was more non-polar than 9-fluorenone.

The retention factors were calculated to find the distances the samples of the compounds being tested moved up the plate relative to the distances moved by the solvent front. The plate showed that fluorene moved up the plate at a higher level than 9-fluorenone. The Rf=.8cm for fluorene and .67cm for 9-fluorenone. Fluorene was visible only under UV light because it is a colorless compound, unlike 9-fluorenone which is yellow. It was hard to measure accurately the Rf for the original mixture that held the combination of both chemical compounds but visibly, there was a yellow mark on the TLC plate that was parallel to the yellow mark of 9-fluorenone from sample 2, and there was a colorless mark on the TLC plate that was visible under UV lighting that was around the same length area of fluorene from sample 1.


By analyzing how strongly the two compounds were attracted to the stationary phase of the silica gel in the column, we can understand why the compounds moved down the column at the rate that they did. Separation of the compounds resulted from their difference in migration rates which were affected by the compounds’ difference in structure and polarity. In column chromatography, non-polar compounds move down the column at a faster rate than polar compounds but when carrying out a TLC protocol, we can see that non-polar molecules move up the plate more rapidly than polar compounds that move up the plate at a slower rate or don’t move up at all.
Overall, my TLC plate could have shown better results had the length of my silica gel been higher than 4cm. If I had done the procedure in a better way, the separation between the two compounds would have been purer and my retention factors would have changed.


  1. CHEM 345-Majors Organic Chemistry I Lab
  2. Mohrig, et.al. 2010. Techniques in Organic Chemistry.221-256
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