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.
Chromatography for qualitative analysis
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!
If you are unfamiliar with the technique you can watch Professor Dave here …
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 above ☝️.
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 😂).
Once again if you are unfamiliar with the practical technique of column chromatography, you can watch Professor Dave in action …
Chromatography for quantitative analysis
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.
Practice questions
- A sample of a tripeptide was hydrolysed and thin layer chromatography used to separate and identify the amino acids present.
(a) Suggest a suitable reagent for the hydrolysis of the tripeptide.
The chromatogram is shown below:
(b) Explain how this chromatogram was obtained, including how the amino acid spots were made visible.
(c) Calculate the Rf value for valine.
(d) Explain why each amino acid has a different Rf value.
- A sample of methylbenzene is contaminated with phenylamine. The two compounds can be separated using column chromatography, with silica as the stationary phase and hexane as the mobile phase. Suggest with reason which compound will have the shorter retention time.
- A mixture of organic substances was analysed using gas-liquid chromatography. The gas chromatograph is shown below:
(a) Which substance has the greatest affinity for the mobile phase?
(b) The area underneath each peak is proportional to the mass of the respective compound in the mixture.
| Peak | A | B | C |
| Area / mm2 | 8 | 60 | 42 |
The concentration of B in the mixture is 5.50 × 10-2 g dm-3. Calculate the concentration, in mol dm-3, of substance C in the mixture, given that its Mr is 118 g mol-1.
- A mixture of three organic compounds was separated using gas-liquid chromatography. The amount of each compound in the mixture is proportional to the area under its respective peak in the chromatogram shown below:
(a) What is a suitable substance for the mobile phase and the stationary phase in GLC?
(b) What is meant by the ‘retention time’ for each compound?
(c) Calculate the percentage of compound Y in the mixture.
Answers
- (a) moderately concentrated hydrochloric acid or sodium hydroxide
(b) A pencil line was drawn 1cm from the bottom of the thin layer chromatography plate and the mixture of amino acids acids obtained from the tripeptide hydrolysis was spotted onto the line.
The plate was placed in a beaker containing some solvent (below the pencil line) and a lid / watch glass / clingfilm used to cover the beaker to prevent the solvent from evaporating. The solvent was allowed to rise up the plate and the plate removed before the solvent reached the top. The solvent line was marked with a pencil.
The plate was allowed to dry and then sprayed with ninhydrin solution to show the amino acid spots in purple / uv light shone onto the plate to identify the amino acid spots.
(c) Rf value = (distance travelled by spot / distance travelled by solvent front) = 38mm / 70mm = 0.54
(d) The amino acids have different Rf values because they have different affinities for the mobile phase (the solvent) and the stationary phase (the plate). The R groups of valine and isoleucine are non-polar so will be carried further / faster up the plate as they will have a greater affinity with the mobile phase. Tyrosine has a polar R group / R group capable of making stronger intermolecular bonds with the silica plate so it will have a greater affinity for the stationary phase and not travel as far / have a lower Rf value.
2. Methylbenzene is a non-polar molecule and so will have a greater affinity for the mobile phase compared with the stationary phase. Phenylamine is a polar molecule and so will have a greater affinity for the stationary phase. Methylbenzene will have a shorter retention time than phenylamine.
3. (a) Substance A has the greatest affinity for the mobile phase as it has the shortest retention time.
(b) (42/60) x (5.5 x10-2) = 0.0385 g dm-3; 0.0385 / 118 = 3.26 x 10-4 mol dm-3
4. (a) The mobile phase should be an inert carrier gas such as argon or nitrogen, the stationary phase is non-volatile, non-polar liquid on an inert / silica support.
(b) Retention time is the time that the substance stays in the column.
(c) Area of each peak is (½ x b x h) so peak X has an area of 30, peak Y has an area of 100 and peak Z has an area of 60. % of Y in the mixture is (100/190) x 100 = 52.6%