Joseph K. Myers
3-9-04
Diasteromers have different physical properties. Therefore, it is possible to use the previous technique of thin-layer chromatography to separate them.
Similarly, the technique of column chromatography may be used for separation. Somewhat larger samples can be obtained through this technique.
For example, cis and trans 1,2-cyclohexanediols are less polar (cis) and more polar (trans). This is caused by the orientation of the OH groups on the rings, and by their intramolecular interactions. The trans diastereomer is relatively more likely to interact with the stationary phase, and moves more slowly through the column.
The concepts are largely the same as other chromatographic techniques. See [2] for further discussion.
None.
| Compound | MW | mp(C) | bp | d(g/cm3) |
|---|---|---|---|---|
| cis-1,2,-cyclohexanediol | 116.158 | 100 | 120 | 1.0297 |
| trans-1,4-cyclohexanediol | 116.158 | 100 | not given | 1.18 |
| benzyl alcohol | 108.138 | -15.4 | 205.31 | 1.0419 |
| methyl benzoate | 136.149 | -12.4 | 199 | 1.0837 |
For TLC separation:
Obtain a solution of 5% cis and trans 1,2-cyclohexanediols.
Prepare a mixture of 75% petroleum ether and 25% 2-propanol by volume.
Place small spots of solution onto the thin-layer plate.
Develop the plate in manner described in the last experiment.
Notes: this process was not performed, by the directions given to the class on the morning of the experiments.
For column chromatography:
Prepare the solvent with an 80:20 mixture of hexanes and ethyl acetate.
Prepare the column for chromatography by using a half-inch of sea sand on the bottom, followed by enough solvent to fill most of the column. Carefully add the silica to the column until it is somewhat more than half full. Allow it to settle, tapping the sides of the column. Carefully add a final one-half inch of sea sand, being sure not to let it mix with the silica below it. Rinse the sand down with more solvent.
Prepare the 50/50 mixture of methyl benzoate and benzyl alcohol, using 0.05 g of each, dissolved together in a total of 0.5 mL of the solvent.
Let the column drip about once every second, and pour in the mixture of benzyl alcohol and methyl benzoate which are to be separated.
The methyl benzoate, which is less polar, will come first. Monitor the product of the column drip by using an ultraviolet lamp and a TLC plate. As soon as the drip makes a dark spot on the plate, start to gather the methyl benzoate fraction.
Maintain the level of solvent in the column above the top of the sea sand, during the entire time that the mixture is separating through the silica.
When a satisfactory amount of methyl benzoate fraction has dripped, an intermediate container may be used if desired, to avoid contaminating the benzoate with the beginning of the benzyl alcohol fraction.
About five minutes after the methyl benzoate fraction has started, continue taking samples of drops onto a TLC plate and inspecting them under the lamp. When the drops start to lighten, the intermediate level betweewn the two fractions has been reached. As soon as the drops appear to redarken, begin to receive the benzyl alcohol fraction into a new container.
A great amount of trouble is caused by some groups finishing their experiment and using the ultraviolet light for a long period of time to record results, while other students need to use it to monitor the progress of their column.
It is possible to use one TLC strip for quite a few analysis drops under the light, if they are small drops. However, it is not usually advisable to run more than one separation on a single TLC strip.
Depending on the instructions, students may not have to carry out the TLC separation of the diastereomers of 1,2-cyclohexanediol.
| compound | run length | spot height | Rf value |
|---|---|---|---|
| methyl benzoate | 7.35 | 3.1 | .422 |
| benzyl alcohol | 6.61 | 1.3 | .197 |
We may have been more successful in separating the methyl benzoate and benzyl alcohol because we did not run our column as quickly as the others.
We prepared our mixtures extremely accurately by evenly creating larger portions (1 mL of each), rather than trying to inaccurately weigh 0.05 g. We then mixed this together in the proper amount with the solvent.
Our class did not perform the cis and trans portion of the experiment.
1. Gilbert JC, Martin SF. Experimental Organic Chemistry. 2002. 3rd Edition. p198-204,175-182.
2. Myers JK. Gas Chromatography. 2004. 2nd Printing. p1.
A classmate said that the cis diastereomer went up higher in the separation.