How to teach post-16 chromatography

In the early 20th century, Mikhail Tsvet invented column chromatography. Since then, scientists have developed many more chromatographic techniques including paper chromatography, thin layer chromatography (TLC), gas chromatography and liquid chromatography.

Chemists can pair many of these techniques with tools to identify the separated components and determine their concentrations. For example, coupling chromatography with mass spectrometry, ultraviolet (UV) spectroscopy, infrared (IR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy.

Chromatography has myriad applications. We use it to look for drugs in blood, to test food for illegal compounds and to monitor pesticide levels, nanoplastics and heavy metals in the environment. Forensic science owes much to chromatography. Scientists also use chromatography to isolate and identify intermediates in multistage chemical reactions, to separate chiral molecules from racemic mixtures, to purify vaccines and to help search for life on Mars.

What students need to know

In chromatography, we dissolve the analyte (or mixture) in a mobile phase which is forced through a stationary phase, sometimes with the help of an inert carrier liquid or gas.

Chromatography technique	Examples of use	Stationary phase examples	Mobile phase examples
Paper	Inks and dyes	Cellulose – can be of different thicknesses and composition	Water, ethanol, pentane, propanone or a mixture of these solvents
Thin layer	Steroids, amino acids, lipids, hydrocarbons, carbohydrates, amines, pesticides and vitamins	Silica or aluminium oxide on an inert surface like glass or plastic	Hexane and ethyl ethanoate mixtures, methanol and dichloromethane mixtures, and butanol and ethanoic acid mixtures
Column	Purines and sugars. To purify drugs and vaccines, for forensics and for food testing	Silica, cellulose, aluminium oxide and magnesium oxide	Hexane, ethyl ethanoate, propane, ethanol, methanol, hydrocarbons, dichloromethane or mixtures of these solvents
Gas	To monitor air quality to detect pollutants. To analyse additives and detect food contaminants	Silica, silicone and polymers such as dimethyl polysiloxane	Helium, nitrogen, argon and hydrogen

Common misconceptions

Students’ most common misconceptions about chromatography include thinking they need to cover the analyte with the mobile phase when doing paper chromatography and TLC, rather than allowing the mobile phase to travel through the analytes from below the baseline. Some learners also believe chromatography has not worked on a pigment if it doesn’t move (when, in fact, that pigment is insoluble in the mobile phase), even though other pigments in the analyte have been separated.

Some students mistakenly believe the pigments move as the mobile phase pushes them along, ie it exerts a force on the pigment. Others only consider the affinity between the mobile phase and component as the reason for separation, rather than the relative interactions between the component and both the stationary and mobile phases.

Ideas for your classroom

Separating mixtures of dyes is a simple but effective way to introduce the power of chromatography. For example, mix one of two different blue dyes with a yellow dye to create a green dye and ask students to identify which blue dye was used in the mixture. They should use chromatography, either TLC or paper, to analyse the green mixture and both blue dyes. This is a GCSE required practical activity you can use to remind students about the application of R_f values.

When doing gas chromatography, get your students to identify the components in a mixture using retention times – often done by comparing retention times with known compounds (Figure 2). Students can identify components A and B by using a standard containing these compounds for comparison. Taking measurements of retention times (time taken for the compound to be seen on the chromatograph, t_rA and t_rB) will help them identify the components.

Task your students to investigate food dyes to help cement chromatography. Anthocyanins – naturally occurring dyes in plants such as blueberry, blackberry, red cabbage and beetroot – often change colour depending on pH. Students can compare chromatograms of anthocyanins using neutral (water), slightly acidic (added citric acid) and alkaline (solution of NaHCO₃ in water) mobile phases. Ask them how chemists could use these methods to enhance the identification of anthocyanins in food dyes?.

Students can also look for the presence of paprika extract (oleoresin) and turmeric (curcumin) in food dyes. These compounds are insoluble in water but will dissolve in oil. Students can use TLC or paper chromatography to hunt for these in food dyes, using a mobile phase of 1:1 mixture of isopropanol and water.

You can use hybrid analyses where chromatography is combined with mass spectrometry, or UV, IR or NMR spectroscopy to give learners a deeper understanding of chromatography applications.

Checking for understanding

Assess students’ knowledge of the different chromatography techniques and applications with recall questions. Understanding is best assessed through application, like the interpretation of a chromatogram.