How Chromatography Separates Mixtures

The stationary phase stays in place. In simple paper chromatography, the paper and the water held within its fibres act as part of this phase. The mobile phase is a solvent that travels through the stationary material.

A small sample is placed near one end of the paper. The paper is then set in solvent with the sample spot above the liquid level. As the solvent rises through capillary action, it carries dissolved substances with it.

Each substance is attracted differently to the solvent and the stationary phase. A component that dissolves well in the mobile phase and interacts weakly with the stationary phase travels farther. A component held more strongly by the stationary phase moves more slowly.

The starting line is drawn in pencil because graphite does not usually dissolve in the solvent. Ink would add extra dyes and create a result that says more about the pen than the sample.

The sample spots should be small and concentrated. Large wet spots spread and overlap. The container is often covered to reduce solvent evaporation and help the atmosphere inside become saturated with solvent vapour.

The paper must be removed before the solvent reaches the top, and the solvent front should be marked immediately. Once the solvent evaporates, its final position may be impossible to see. The separated pattern of spots is called a chromatogram.

A pure substance usually gives one spot under a particular set of conditions, while a mixture may produce several. Matching spot positions can suggest that samples contain the same substance, but colour and position alone do not prove identity.

Scientists can calculate a retention factor by dividing the distance travelled by a substance by the distance travelled by the solvent front. The value can be compared only when conditions such as solvent, stationary phase and temperature are similar.

A reliable procedure includes:

• •Draw the baseline in pencil.
• •Keep sample spots above the solvent.
• •Use small concentrated spots.
• •Mark the solvent front promptly.
• •Compare results under matching conditions.

Paper chromatography is the classroom version of a family of methods that run modern chemistry. Thin-layer chromatography replaces the paper with a coated glass or plastic plate and gives sharper results; it is how a synthetic chemist checks whether a reaction has finished. Gas chromatography pushes a vaporised sample through a long, thin column with an inert gas as the mobile phase, separating substances by boiling point and affinity, and is the standard way to test for drugs in blood, contaminants in food and the chemical make-up of a perfume.

High-performance liquid chromatography does the same job under pressure for substances that cannot be vaporised, including most medicines. All of them rest on the principle in the paper strip: two phases, one moving, and components that spend different fractions of their time in each.

Chromatography began with leaves. In the early twentieth century the Russian botanist Mikhail Tsvet poured an extract of plant pigments into a glass column packed with powdered chalk and washed it through with a solvent. The pigments separated into coloured bands at different heights, including different chlorophylls and the yellow and orange carotenoids. He named the method from Greek words meaning colour writing, and by coincidence his surname means colour in Russian. The idea was largely overlooked for years, until Archer Martin and Richard Synge developed partition chromatography and shared the 1952 Nobel Prize in Chemistry.

Many substances are colourless, so modern chromatography needs ways to reveal what the eye cannot see. Thin-layer plates are often coated with a material that glows under ultraviolet light, and compounds that absorb ultraviolet show up as dark patches against the glowing background. Amino acids can be revealed by spraying the dried plate with ninhydrin, which reacts with them to form purple spots. In instruments, a detector at the end of the column registers each component as it emerges, so the results appear as a series of peaks on a graph rather than as spots.

Retention factors are easiest to understand with numbers. Suppose the solvent front has travelled 8.0 centimetres from the pencil baseline. A blue dye from a sweet coating has moved 2.0 centimetres, giving an Rf of 2.0 divided by 8.0, or 0.25, while a red dye has moved 6.0 centimetres, giving 0.75. Measure to the centre of each spot, and always from the baseline rather than from the edge of the paper. An Rf value can never be greater than 1, because no substance can travel further than the solvent carrying it, so a larger answer signals a measuring mistake.

Chemists use a few tricks to make comparisons more trustworthy. In co-spotting, the unknown and a known reference are placed together on the same starting spot. If they are the same substance, they travel as one neat spot, but if they differ, the spot stretches or splits in two. When substances still overlap, two-way chromatography can help. The sample is run up a square sheet in one solvent, the sheet is dried and turned through ninety degrees, and then it is run again in a different solvent. Spots that overlapped in the first direction often separate in the second, a method developed in the 1940s for mixtures of amino acids.

Which spot travels furthest depends on a contest of attractions, and polarity is usually the deciding factor. Paper is made of cellulose, which holds water and is strongly polar, so polar dyes tend to cling to it, while less polar substances move more readily with a less polar solvent. This is why a permanent marker gives a disappointing result in water: its dyes barely dissolve, and the spot stays on the baseline. Switch the solvent to ethanol or propanone and the colours begin to separate. Changing the solvent changes every Rf value, which is why results must always state the solvent used.

Many modern instruments deliberately turn the arrangement around. In reversed-phase liquid chromatography, the most common form of high-performance liquid chromatography, the stationary phase is silica coated with long hydrocarbon chains, making it oily and non-polar, while the mobile phase is water mixed with a solvent such as methanol or acetonitrile. Now polar compounds pass through quickly and oily ones linger. Chemists often adjust the mixture during a run, gradually raising the proportion of organic solvent to wash out the stubborn, less polar components. By tuning both phases, they can separate substances that differ only slightly in structure.

Chromatography separates mixture components because they move at different rates between a mobile phase and a stationary phase. Their solubility and attractions determine how far they travel. Control the conditions carefully, and a simple strip of paper can reveal substances hidden inside one small spot.