Practical chromatography

As with other separation techniques, learners will meet chromatography multiple times through their primary and secondary education. How can we ensure there is a clear progression in the knowledge students develop? How can we maintain their interest and help them develop practical skills? How can we make chromatography in the classroom more engaging?

Progression in ideas and skills

The chemistry behind chromatography involves drawing on concepts studied throughout students’ science education.

The ideas of substance and purity underpin much of the discussion of separation techniques. It is worth revising some of the basic concepts with students before discussing chromatography. Good starting points include:

Developing practical skills

There is a danger students will simply repeat the same chromatography experiment throughout their education. Popular activities with primary-aged children include separating the mixtures found in felt-tip pens and sweets. Between 11–14, students will often separate the mixtures in inks and food colourings. Chromatography of inks and dyes are commonly required in exam specifications.

One of the challenges teachers face is how to develop students’ practical skills and maintain their interest if they are repeating experiments. There are several possible tactics: using different contexts; setting investigative or problem-solving activities; and taking a synoptic approach.

Another challenge with practical work is for students to understand what they are doing and why they are doing it. Getting this correct during lessons will help students answer practical-based exam questions. One useful tactic is to provide plenty of opportunities for students to do more than just follow a given procedure.

Synpotic approach

Chromatography provides a great opportunity for students to research and carry out a practical method, compare observations and measurements with other groups, and make links across the sciences, particularly biology and chemistry.

As preparation work, ask your students to research the pigments found in different leaves. Here are some possible questions to get your students started:

• What is the green pigment in leaves?
• Where have you come across it before?
• Where have you extracted chlorophyll before?
• How did you extract the chlorophyll?
• What techniques did you use?

Then ask them to plan a practical method for analysing a variety of different leaves (e.g. spinach, flowering plant leaves, non-flowering plant leaves etc). You can then give them a standard method for carrying out the practical and have them analyse the similarities and differences between the methods.

Thin-layer chromatography is often used in these standard methods (see image right). Although not commonly used in 11–16 lessons, it is worth, at minimum, discussing with and demonstrating to students. This will allow you to tackle some of the common practical problems encountered with chromatography.

Practical problems and suggested solutions

Chromatography is often seen as a quick and easy activity. However, the chromatograms students see in their textbooks and on exam papers often look nothing at all like the chromatograms from their own experiments; see comparison right.

So, faced with these results we have to work out how to convince students they have carried out a similar process. As well, identify the centre spot is, know what value to use to calculate the R_F value and to compare the R_F values with such big error bars.

For the purple spot in figure B (the textbook diagram), the R_F is 0.8. For the same spot in figure A (typical student results), what is the distance moved by the spot? Where would you draw the line? There will be a very large error in any value calculated.

Improving the results

Paper chromatography doesn’t give very good separation in classroom experiments. You would need a long piece of paper and lengthy elution time to get good separation. However, there are several things you can do to make improvements.

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The effect of changing only the stationary phase: (A) paper (B) thin layer (silica on a plastic backing)

Source: Image courtesy of Kay Stephenson

TLC chromatogram of ink showing the effect of changing only the mobile phase (the eluting solvent): (A) H2O, (B) NaCl (aq) (~2% w/v), (C) NaHCO3 (aq) (~2% w/v), (D) ethanol (aq) (~2% v/v)

Source: Image courtesy of Kay Stephenson

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Changing the stationary phase by using thin layer chromatography (TLC) can lead to better separation. In the gallery, on the first image A and B, TLC gives clear bands with more easily measurable R_F values. The solvent in both cases was a dilute solution of sodium chloride and the same black ink is used in both. Note the difference in the order in which the component dyes move up the stationary phase.

Be explicit with your students that the principles and underlying chemistry of TLC are the same as paper chromatography. The only real difference is the stationary phase, which may be silica on a polyester backing or aluminium oxide coated plates. The mobile phase remains the same but the way the two phases interact with the sample components is different.

Changing the phases is the basis of a good investigation. The activity could be a problem-solving exercise or competition, where students have to work out how to get good separation and then go on to produce the best chromatogram.

Hopefully your students will be able to accept that the chromatograms seen in B and then A–D in the gallery are a little closer to what they might meet in the exam.

Chromatography equipment

Although TLC plates are expensive, a classroom set of small plates can be cut from one standard plate. (Tip: If using right-handed scissors, try cutting with the backing side of the sheet uppermost. This seems to minimise flaking of the fragile coating. Alternatively, use a scalpel, ruler and cutting board.)

An expensive chromatography tank is not necessary to get good chromatograms. There are many methods that give good results. A one-holed boiling tube bung and crocodile clip (figure 6a) is useful for large classes and when volatile solvents (e.g. ethanol or propanone) are needed. You only need small quantities of solvents and can keep fumnes to a minimum.

You can get TLC plates with a fluorescing agent in the coating. These are useful for locating colourless components. Many colourless compounds absorb UV light and appear as darker spots.