Chromatography

In a nutshell

Chromatography separates the components of a mixture and lets you identify each one, by comparing how far it travels (an R_f value) or how long it takes to come through (a retention time) with known standards.

Every type works the same way: a mobile phase carries the mixture past a stationary phase, and each component moves at a different rate depending on how strongly it is held back.

Assumed knowledge: Bonding (polarity and intermolecular forces), Organic analysis (mass spectrometry).

Core content

The two phases

Every chromatography method has two phases:

• The stationary phase stays still. It is a solid (such as silica or alumina), or a liquid held on a solid.
• The mobile phase (also called the moving phase) flows past it. It is a liquid solvent, or a gas.

The mixture is put on or into the stationary phase, and the mobile phase carries its components along. Different components travel at different speeds, so they separate.

Three types of chromatography

You need to know three types, and what the stationary and mobile phase is in each.

Column chromatography is set up like this. The mixture is added to the top of the column, solvent is run through, and the separated components are collected one by one as they drip out of the bottom.

Gas chromatography works on a mixture that can be vaporised. An unreactive carrier gas (such as helium or nitrogen) is the mobile phase. It sweeps the vaporised sample through a long, coiled column inside a hot oven, and a detector records each component as it comes out.

What decides how far a component moves

This is the explanation the marks are built on. Separation depends on the balance between solubility in the moving phase and retention by the stationary phase.

• A component that is more soluble in the mobile phase spends more time moving, so it travels further (higher R_f) or comes out sooner (shorter retention time).
• A component that is more strongly retained by (has a greater affinity for) the stationary phase spends more time held still, so it travels less far (lower R_f) or comes out later (longer retention time).

Because each component has a different balance, each moves at a different rate, and the mixture separates.

Still don't get it? · the balance between the two phases

Picture a moving walkway in an airport, lined with shops. Everyone steps on at the same moment.

Some people hardly care about the shops, so they stay on the walkway nearly all the time and reach the end first. Others keep stepping off to browse, then stepping back on. They get to the end much later.

Nobody is moving at a different walkway speed. What differs is how much of their time each person spends on the walkway compared with standing in the shops.

Now map it across, one piece at a time:

1. The walkway is the mobile phase: it carries things along.
2. The shops are the stationary phase: anything stuck there is not moving.
3. Each person is one component of the mixture.
4. A component that "likes" the mobile phase (is more soluble in it) is the shopper who stays on the walkway: it moves furthest, or comes out first.
5. A component that "likes" the stationary phase (is held more strongly by it) keeps stopping: it moves least far, or comes out last.

Exam version: separation depends on the balance between solubility in the moving phase and retention by the stationary phase. Always say both halves: which component is more soluble in the mobile phase, and which is more strongly retained by the stationary phase.

Polarity: predicting which component moves further

In most exam questions the stationary phase is polar (silica and alumina both are) and the solvent is less polar (for example hexane). Then:

• The more polar component is more strongly attracted to the polar stationary phase. It has a lower R_f or a longer retention time.
• The less polar component is more soluble in the less polar mobile phase. It has a higher R_f or a shorter retention time.

For example, with silica and hexane, an alcohol (polar O-H bond, which can hydrogen bond to the silica) is held back more than an alkene or alkane with no polar bond.

Changing the solvent changes the result. If you switch to a more polar solvent, a polar component becomes more soluble in the mobile phase, so it moves further and its R_f increases.

Charge matters too. An ion is attracted to a polar stationary phase more strongly than a neutral molecule, and an ion with a greater charge is attracted more strongly still, so it is retained for longer.

R_f values (TLC)

In TLC, each spot is identified by its R_f value:

$R_f = \frac{\text{distance moved by the spot}}{\text{distance moved by the solvent front}}$

• Both distances are measured from the start line (the pencil baseline), not from the bottom of the plate.
• Measure to the centre of the spot.
• R_f has no units and is always between 0 and 1, because a spot can never move further than the solvent.

Retention times (CC and GC)

In column and gas chromatography, each component is identified by its retention time: the time a component takes to travel through the column, from when the sample is put in to when that component comes out and is detected.

A gas chromatogram plots the detector signal against time. Each peak is one component, and the time at the top of the peak is its retention time.

Identifying a substance: compare with standards

An R_f value or a retention time on its own does not name a compound. You identify a component by comparing it with a standard (a pure sample of a known substance):

• TLC: run the unknown and the standards side by side on the same plate. A spot in the unknown with the same R_f as a standard is likely to be that substance.
• CC and GC: a component with the same retention time as a standard, under the same conditions, is likely to be that substance.

The comparison only works under identical conditions. R_f depends on the stationary phase and the solvent; retention time depends on the column, the temperature and the gas flow rate. Change any of these and the values change.

GC-MS: gas chromatography with mass spectrometry

Two different compounds can have very similar retention times, so a retention time alone can be ambiguous. Linking the GC column straight to a mass spectrometer solves this:

1. GC separates the mixture, so the components leave the column one at a time.
2. Each component passes directly into the mass spectrometer as it leaves.
3. The mass spectrum of each component is recorded. Its molecular ion peak gives the M_r, and the spectrum can be compared with a computer database of known spectra to identify it.

The two techniques cover each other's weakness. Two isomers have the same molecular formula, so the same M_r and a molecular ion at the same m/z: the mass spectrometer alone cannot tell them apart. But they usually have different retention times, so GC separates them first.

Required practical 12: separation of species by thin-layer chromatography

Required practical 12 uses TLC to separate a mixture and identify its components (for example, the substances in an analgesic tablet, or transition metal ions in a solution) from their R_f values.

Method, with the reason for each step (the reasons are what the marks are for):

1. Wear gloves and hold the plate by its edges, to prevent contamination from your hands onto the plate.
2. Draw the start line in pencil, not ink. Pencil (graphite) does not dissolve in the solvent; ink would dissolve and move up the plate as extra spots.
3. Use a capillary tube to put a small spot of the mixture, and of each standard, on the line. Small spots stop neighbouring spots spreading and merging. Let them dry.
4. Pour solvent into the beaker to a depth below the start line (no more than about 1 cm). If the solvent is above the line, it dissolves the spots off the plate into the solvent.
5. Stand the plate in the beaker and put the lid on, to stop the solvent evaporating.
6. Remove the plate before the solvent reaches the top, and immediately mark the solvent front in pencil, before the solvent evaporates and the line disappears. (The solvent does not have to reach the top: you can calculate R_f from wherever the front stops.)
7. Dry the plate in a fume cupboard, because the solvent is toxic (hazardous) and volatile.
8. Most spots are colourless, so locate them under UV light or by spraying with a developing agent (for example ninhydrin for amino acids). Draw round each spot in pencil.
9. Measure the distance from the start line to the centre of each spot, and from the start line to the solvent front. Calculate each R_f and compare with the standards.

Worked examples

Worked example 1: calculating an R_f value

On a TLC plate, the start line is drawn 1.0 cm above the bottom edge. After developing, a spot's centre is 2.7 cm above the start line and the solvent front is 7.5 cm above the start line. Calculate the R_f value of the spot.

$R_f = \frac{\text{distance moved by the spot}}{\text{distance moved by the solvent front}}$

$R_f = \frac{2.7\ \text{cm}}{7.5\ \text{cm}}$

$R_f = 0.36$

The centimetres cancel, so R_f = 0.36, with no units. Both distances were given to 2 significant figures, so the answer is given to 2 significant figures.

Where marks are lost: measuring from the bottom edge of the plate instead of the start line. That gives (2.7 + 1.0) / (7.5 + 1.0) = 3.7 / 8.5 = 0.44, which is wrong. Both distances must start at the pencil line.

Worked example 2: identifying the components of a mixture by TLC

A mixture was spotted in lane 1 of a TLC plate, with three pure standards, P, Q and R, in lanes 2 to 4. The plate was developed in one solvent. The chromatogram is shown below (distances measured from the start line). Identify the components of the mixture.

Step 1: read the distances from the chromatogram.

Step 2: calculate each R_f value.

$R_f(\text{mixture, lower}) = \frac{2.0\ \text{cm}}{8.0\ \text{cm}} = 0.25$

$R_f(\text{mixture, upper}) = \frac{5.6\ \text{cm}}{8.0\ \text{cm}} = 0.70$

$R_f(P) = \frac{2.0\ \text{cm}}{8.0\ \text{cm}} = 0.25 \qquad R_f(Q) = \frac{4.0\ \text{cm}}{8.0\ \text{cm}} = 0.50 \qquad R_f(R) = \frac{5.6\ \text{cm}}{8.0\ \text{cm}} = 0.70$

Step 3: match with the standards. The mixture's spots match P (R_f = 0.25) and R (R_f = 0.70). There is no spot at R_f = 0.50, so Q is absent.

Answer: the mixture contains P and R.

The comparison is valid because the mixture and the standards ran on the same plate, in the same solvent.

Worked example 3: identifying components from a gas chromatogram

Three pure alcohols were run separately through a gas chromatograph, under identical conditions, to give these retention times:

An unknown mixture was then run under the same conditions, giving this chromatogram. Identify the components.

1. Count the peaks: two peaks, so two components.
2. Read the retention times: 1.8 min and 4.6 min.
3. Match with the standards: 1.8 min matches ethanol; 4.6 min matches butan-1-ol. There is no peak at 2.9 min, so propan-1-ol is absent.

Answer: the mixture contains ethanol and butan-1-ol.

Confirming with GC-MS: if the column were linked to a mass spectrometer, the component at 1.8 min would show a molecular ion at m/z = 46 (C₂H₅OH, M_r = 46.0) and the one at 4.6 min at m/z = 74 (C₄H₉OH, M_r = 74.0), confirming both identities.

Worked example 4: explaining an order of separation (model answer)

A mixture of butan-1-ol and 1-chlorobutane is separated by TLC on a silica plate, using hexane as the solvent. Explain which compound has the greater R_f value.

1. 1-chlorobutane is less polar than butan-1-ol (butan-1-ol has a polar O-H group and can form hydrogen bonds).
2. 1-chlorobutane is more soluble in the (non-polar) hexane mobile phase / butan-1-ol is more strongly attracted to the polar silica stationary phase.
3. So 1-chlorobutane travels further and has the greater R_f value.

Notice the order: (1) compare polarity, (2) link it to the phases, (3) conclude. A conclusion with no comparison of polarity, or a polarity statement with no link to a phase, loses marks.

Common exam mistakes

• Giving only half of the separation principle. "It depends on solubility in the solvent" is not enough. Examiners require both parts: solubility in the moving phase and retention by the stationary phase.
• Measuring R_f distances from the bottom of the plate. Both distances are measured from the start line.
• Giving R_f a unit, or getting a value greater than 1 by dividing the wrong way round. R_f = spot distance ÷ solvent front distance, with no units.
• Circular answers about retention time. Examiners report answers like "the retention time is shorter because the retention time is shorter". Give the reason: compare polarity, then link it to attraction to the stationary phase or solubility in the mobile phase.
• Getting the cause and effect of polarity backwards. A compound is not polar because it hydrogen bonds; it has a polar O-H bond, which is why it can hydrogen bond. And an alkene is not polar because of its C=C bond.
• Thinking M_r decides the retention time in column chromatography. In these questions it is polarity and affinity for each phase that matter.
• Saying the substances rise up a column. In column chromatography the solvent moves down the column; in TLC it moves up the plate.
• Describing paper chromatography when the question asks about TLC or column chromatography, or writing general theory without naming the type you would use and applying it to the compounds in the question.
• Forgetting to compare with a standard. You identify a substance by comparing its R_f or retention time with a known standard under the same conditions; an R_f on its own identifies nothing.
• Writing "for safety" for a practical step. Each step needs its specific reason: gloves prevent contamination from your hands; solvent below the start line or the spots dissolve into it; fume cupboard because the solvent is toxic.
• Claiming the solvent must reach the top of the plate. It does not: R_f can be calculated from wherever the solvent front stops, as long as you mark it.
• Explaining polarity when asked to deduce it. If a question asks which isomer is more polar from the R_f values, deduce it from the data and link it to the phases; do not write a paragraph about why the molecule is polar.
• Expecting mass spectrometry alone to identify isomers. Isomers have the same M_r, so the same m/z for the molecular ion. It is the different retention times in GC that separate them.

Key definitions

• Stationary phase: the phase that does not move (a solid, or a liquid held on a solid); components are retained by it.
• Mobile (moving) phase: the liquid solvent or gas that moves through or over the stationary phase, carrying the components.
• Principle of separation: separation depends on the balance between solubility in the moving phase and retention by the stationary phase.
• R_f value: the distance moved by a spot divided by the distance moved by the solvent front, both measured from the start line.
• Retention time: the time taken for a component to pass through the column, from injection of the sample to detection of that component.
• Thin-layer chromatography (TLC): a plate is coated with a solid and a solvent moves up the plate.
• Column chromatography (CC): a column is packed with a solid and a solvent moves down the column.
• Gas chromatography (GC): a column is packed with a solid, or with a solid coated by a liquid, and a gas is passed through the column under pressure at high temperature.

Specification

• I can state that chromatography can be used to separate and identify the components in a mixture.
• I can describe thin-layer chromatography: a plate coated with a solid, with a solvent moving up the plate.
• I can describe column chromatography: a column packed with a solid, with a solvent moving down the column.
• I can describe gas chromatography: a column packed with a solid or a solid coated by a liquid, with a gas passed through under pressure at high temperature.
• I can explain that separation depends on the balance between solubility in the moving phase and retention by the stationary phase, and use polarity to predict which component moves further.
• I can explain how retention times and R_f values are used to identify different substances.
• I can explain the use of mass spectrometry to analyse the components separated by GC (GC-MS).
• I can calculate R_f values from a chromatogram.
• I can compare retention times and R_f values with standards to identify different substances.
• I can carry out Required practical 12: separation of species by thin-layer chromatography, and justify each step of the method.

Related notes

• Bonding (polarity, permanent dipoles and hydrogen bonding, which decide the order of separation)
• Organic analysis (mass spectrometry and the molecular ion, used in GC-MS)
• Atomic structure (how a mass spectrometer works)
• Amino acids, proteins and DNA (3.3.13): separating amino acids by TLC and locating them with ninhydrin or UV light
• Nuclear magnetic resonance spectroscopy (3.3.15): another technique for identifying a compound once it has been separated
• Organic synthesis (3.3.14): checking the purity of a product