Column Chromatography: Image taken from http://orgchem.colorado.edu/hndbksupport/colchrom/colchromproc.html

In column chromatography, the vertical column is first packed with silica gel (SiO2) that is mixed with hexane. The gel is loaded above a plug of wool. The wool is for preventing solid materials from contaminating the products. The silica gel in the column is known as the stationary phase (also known as the sorbent). After packing the column with the sorbent and loading the sample, another plug of wool is placed above the sample and the sorbent. This is so that later when pouring the solvent in, there is minimum disturbance to the stationary phase and the sample as disturbance to the surface will result in poor separation.

The basic principle is quite similar to the SPE that I have mentioned in my first post. Except that in the SPE I have mentioned in my first post is use C18 (reversed phase) while the column chromatography I have observed use silica gel (normal phase) thus is based on increasing polarity. This means the least polar component of the mixture will be eluted first and the most polar one will be eluted last.

For column chromatography, a very important factor is the choice of solvent for elution for better separation of components. Solvent to use is determined by Thin Layer Chromatography (TLC).

What is TLC?

TLC is also a method for separation based on polarity but on a much smaller scale than column chromatography thus sample required is also much smaller, TLC is also faster than column chromatography. The stationary phase used in TLC is a thin layer of silica gel on a flat sheet (usually glass). The whole process is similar to paper chromatography (what is that?: Recall from sec chemistry), just that TLC is based on polarity instead of molecular weight.

Thin Layer Chromatography: Image taken from http://www.chemistry.adelaide.edu.au/external/soc-rel/content/tlc.htm

Using a capillary tube, a drop of the loading solvent containing the sample is applied onto the plate. Then, the plate is placed into the solvent submerging only the bottom of the TLC plate (the location where the sample is spotted.) The solvent will then slowly migrate up the plate. It is a matter of trial and error of different kinds of solvents with different ratio to find the ideal solvent to be used for column chromatography. The desired outcome is for the desired spot (the compounds we want) to be as near to the baseline as possible. Baseline is the location where the drop of sample is placed at.

Why is this the desired outcome?

Because on silica gel, the spot nearest to the baseline is the most polar and the spot furthest away is the least polar. So the spot nearest to the baseline in TLC is going to be the last to be eluted in column chromatography (Recall: Elution using silica gel is in increasing polarity). Being the last to be eluted will result in better separation and purification of the compound we want because the desired compound will travel over a longer distance thus is more purified cause more time exposed to the silica gel.

Why in TLC the spot nearest the baseline is the most polar?

Cause there is stronger hydrophilic interaction between the more polar compounds with the silica. Thus the more polar compounds are more attached to the silica (the stationary phase) and the less polar compounds will be carried further away by the solvent.
So in the case of the desired spot being too far away from the baseline, a less polar solvent is used to bring the spot closer to the baseline.

How to get a less polar solvent?

By increasing the ratio of the less polar chemical since usually a mixture of two chemicals are used for the solvent.

If the compounds are colourless, iodine vapour is then used to visualise the spots. This is done by dipping the plate into a bottle of iodine (powder form). Why use iodine in solid form? If recall from secondary school chemistry, pure iodine at room temperature is in solid state, iodine can sublimes at room temperature.

After finding out what solvent to use as the eluent, next we had to check whether the loading solvent is more or less polar than the eluent. If the eluent is more polar than the loading solvent, the eluent can be used directly in the column chromatography to remove the loading solvent. If the eluent is less polar than the loading solvent, a less polar solvent such as hexane is loaded first before loading the eluent. This is because if the loading solvent is more polar than the eluent, it will function as the eluent and push the sample forward, thus the sample will travel over a shorter distance, which will result in less separation.

In column chromatography, since the compounds are colourless, we will not know to collect till which mark, thus the eluent are collected in a series of small tubes. Thus TLC is carried out on each of the tubes with a drop of the desired pure standard beside. A spot that is at the same height as the pure standard with no other spots elsewhere indicates the particular tube where the sample is taken from is pure with only the desired compound in it. Next, all the samples which are pure with only the desired compound in it are combined.
Ta-ta…..pure desired compound is obtained from the mixture.

Feel free to ask questions.

Lim Xin Ni (0607325H)

TG02