Chromatography is all about separating a mixture into its constituents by distributing them between two phases – a mobile phase and a stationary phase. The individual substances that make up the mixture will have a greater affinity for one phase over the other.
The mobile phase is a gas or liquid that flows over the stationary phase which can be a solid or a liquid supported on a solid matrix.
In paper chromatography the stationary phase is a piece of filter paper and the mobile phases a solvent (usually water). If we are separating a mixture of inks, some inks will adsorb more strongly to the stationary phase / paper and they will not move far up the paper or chromatogram. Other inks in the mixture will have a greater affinity for the mobile phase / solvent and rise up the chromatogram at a faster rate – hence the mixture is separated out.
Adsorbtion is not the same as absorption!
When a substance adsorbs to the stationary phase it is simply ‘sticking’ to the surface by way of weak intermolecular bonds. Absorption happens when a substance such as water distributes itself throughout a solid such as a sponge.
In thin layer chromatography the stationary phase is silica (SiO2) or alumina (Al2O3) spread on a plastic plate. This is more effective than using paper as a stationary phase as the silica / alumina provides a much higher surface area for adsorption.
You need to be able to describe how to carry out TLC practically and how to analyse the plate (it’s a surprisingly common long answer question) – you can use past papers / mark schemes to develop a perfect answer or use the labels in the diagram below …
The solvent rises up the plate by capillary action with each substance in the mixture moving at a rate determined by its affinity for the solvent vs. the stationary phase, and it appears on the plate as a ‘spot’. The plate must be removed from the solvent before the solvent front reaches the top of the plate (which is marked with a pencil so that Rf values can be calculated) and dried before analysing.
Spots need not be coloured substances – we can look at the plate under UV light to determine the position of spots, or spray with a dye (ninhydrin is commonly used when identifying amino acids which appear as purple spots). Alternatively we can add a few iodine crystals to the bottom of a clean beaker containing the TLC plate and covered with a lid / clingfilm. The iodine vaporises staining the spots more darkly than the rest of the plate.
TLC can be used to qualitatively analyse a mixture, for example to determine whether a particular amino acid is present. In this case we would also spot a sample of the pure amino acid in question onto the pencil starting line and see whether it rises to the same point as one of the the substances in the mixture.
Calculating the retention factor, Rf, for a spot is a more accurate way of analysing the TLC plate since Rf values are characteristic of a particular substance (assuming the type of solvent used and the temperature are standard).
Because the Rf value is a ratio, it doesn’t matter how you measure it – a ruler working in mm is normal.
However, TLC does have a couple of disadvantages as a method of separation: we can only work with very small quantities of a mixture and although it is not impossible to extract a ‘spot’ from the plate, it is not straightforward!
Column chromatography is used to separate a mixture when working with greater quantities and the separate substances are easily collected for further use / analysis (be aware though, it is often a painfully slow technique to carry out 😂).
Chromatography can also be used for the quantitative analysis of complex mixtures in the guise of high performance (pressure) liquid chromatography, HPLC, and gas chromatography, GC.
- HPLC
In HPLC the mobile phase carries the sample mixture through the column packed with the stationary phase at a constant rate (under pressure). The column is connected to a detector that measures the concentration of each substance as it emerges from the column. The resulting chromatogram features a peak for each substance detected and the area under each peak is proportional to the concentration of that substance.
The retention time or elution time, tr, for each substance can be compared with that of a pure sample, once again assuming that the flow rate and temperature are constant.
- GC / GLC
In gas chromatography or gas-liquid chromatography the mobile phase is an inert carrier gas such as nitrogen or helium, and the sample is injected into the stream before it it flows over the stationary phase. The stationary phase consists of a long column (up to 100m) either packed with an inert solid coated in a liquid or the liquid coats the inside of a very thin silica capillary tube. This liquid coating must be stable and non-volatile so that it doesn’t vanish with the carrier gas as it flows past!
Often this non-volatile liquid is a long chain siloxane polymer chemically bonded to the silica tube and as the sample flows through the column the different substances in the mixture are separated out by means of their differing solubilities in the liquid. Since we can easily change the side groups on the polymer chain (and hence influence the types of intermolecular bonding happening between the stationary phase and the substances in the sample mixture), GLC is useful in the separation of a wide range of mixtures.
The column itself is housed in an oven which allows us to keep the temperature stable if we are after a constant flow rate or change the temperature as a means of improving the separation of substances in the mixture. The sample mixture itself must be volatile so that it evaporates on injection into the inert carrier gas and is carried through the column.
Another advantage of GLC is that it is rapid – a mixture of over 100 different compounds such as petrol can be analysed in a couple of hours. If we need to identify the different compounds present rather than simply separate them out and determine the amount of each present, the GLC can be hooked up to a mass spectrometer (GC-MS) which is able to produce a mass spectrum giving us the molecular ion peak and fragmentation pattern for each peak on the gas chromatogram.
Interpreting a gas chromatogram is fairly straightforward …
- we can see that of the two isomers 2-methylbutane and pentane, the branched isomer has a slightly greater affinity for the mobile phase and so has a shorter retention time – it flows through the column at a faster rate than the straight chain isomer
- hydrocarbons with a greater Mr and longer carbon chain such as decane have a greater affinity for the stationary phase and a longer retention time (this makes sense as they will be capable of making stronger intermolecular bonds)
- the area under each peak is proportional to the concentration / amount of each compound in the mixture
- retention times can be used to identify different peaks (substances) by comparing with the retention times of known compounds, assuming that the flow rate / temperature / mobile and stationary phases are standardised.