In many areas of chemistry, from forensic science to pharmaceutical testing, you will end up with a mixture of compounds. Finding out exactly what is in that mixture requires separating it first.

What you'll learn in this topic:

• The fundamental principles underlying all forms of chromatography.
• How thin-layer (TLC), column (CC), and gas chromatography (GC) work.
• How to calculate and use $R_f$ values and retention times.
• How gas chromatography is combined with mass spectrometry (GC-MS) for absolute identification.

The basic principles of chromatography

All types of chromatography share the same core mechanism: they separate a mixture by passing it over or through another material. To describe this, we use two specific terms.

The components in your mixture separate because they distribute themselves differently between the two phases.

If a component is highly soluble in the mobile phase, it will spend more time moving and travel further or faster. If a component strongly binds or adsorbs to the stationary phase, it will spend less time moving and travel shorter distances or slower.

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Thin-Layer Chromatography (TLC)

Thin-layer chromatography is a quick, inexpensive method used to separate small amounts of mixtures, such as amino acids, analgesics, or transition metal ions. It is the focus of Required Practical 12.

The phases in TLC

In TLC, the stationary phase is a thin layer of a solid (usually silica gel or alumina) coated onto a flat piece of glass, metal, or plastic.

The mobile phase is a liquid solvent.

How TLC works

You start by drawing a pencil line near the bottom of the plate (the baseline). You place a small drop of the mixture on this line and stand the plate vertically in a beaker containing a shallow pool of the solvent.

The solvent must be below the pencil line so the mixture doesn't just dissolve into the bulk liquid. The solvent is then drawn up the plate by capillary action. As the solvent moves up, it carries the components of the mixture with it at different rates.

Once the solvent has almost reached the top, you remove the plate and mark the final height the solvent reached. This line is called the solvent front.

If the spots are colourless (like amino acids), you need a way to see them. You might shine ultraviolet (UV) light on the plate, or spray it with a locating agent like ninhydrin, which turns amino acids purple.

Calculating $R_f$ values

To identify the substances on the plate, you calculate a retention factor, known as the $R_f$ value, for each spot.

$$R_f=\frac{distance\; moved\; by\; substance}{distance\; moved\; by\; solvent}$$

Because you are dividing a distance by a distance, the $R_f$ value has no units.

Once you have calculated the $R_f$ values, you compare them with known standard values measured under the exact same conditions (same solvent, same temperature, same stationary phase) to identify the substances.

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Column Chromatography (CC)

TLC is great for analysis, but what if you actually want to collect the separated chemicals to use later? For bulk separation, we use column chromatography.

• Stationary phase: A solid powder (like silica or alumina) packed into a vertical glass column.
• Mobile phase: A liquid solvent that is poured into the top of the column and moves down through the solid powder.

Instead of the solvent moving up by capillary action, it flows down under gravity or under slight pressure. As the mixture travels down, components with a stronger retention by the stationary phase travel more slowly. The components emerge from the bottom of the column at different times, allowing you to collect them in separate flasks.

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Gas Chromatography (GC)

Gas chromatography is used to separate mixtures of volatile liquids or gases.

• Stationary phase: A solid, or a solid coated by a viscous liquid, packed inside a long, coiled tube (the column).
• Mobile phase: An unreactive "carrier gas" (usually nitrogen or helium).

The mixture is injected into the column, which is housed inside a high-temperature oven. The mixture evaporates and the carrier gas pushes it through.

Here, the separation depends on the differing affinities for the stationary phase, but it also strongly depends on the boiling points of the components. A component with a low boiling point will spend more time as a gas in the mobile phase, so it will travel through the column much faster.

Retention time

In gas chromatography, we do not measure a physical distance like we do in TLC. Instead, we measure the time it takes for a substance to travel all the way through the column and reach the detector.

The detector produces a graph called a chromatogram. Each peak represents a different component. By comparing the retention times of the peaks against the retention times of known pure substances run under the same conditions (temperature, pressure, carrier gas flow rate), you can identify what is in the mixture. The area under each peak tells you the relative amount of each substance.

Gas Chromatography - Mass Spectrometry (GC-MS)

Sometimes, comparing retention times isn't enough to identify a substance, especially if you have a completely unknown compound or two compounds happen to share the same retention time.

To solve this, gas chromatography is frequently coupled with mass spectrometry.

1. The mixture is separated into its individual components by the GC column.
2. As each pure component emerges from the GC column, it is fed directly into a mass spectrometer.
3. The mass spectrometer produces a mass spectrum for each individual component.

You can then use the fragmentation patterns and exact mass peaks in the mass spectra to definitively identify the structures of the components, often by comparing the spectra automatically to a vast computer database.