Chromatography

What is chromatography?

Let's say that we have a deep purple ink. Although it may look like it contains just one colour, with chromatography we can split it up into all its separate pigments - blues, reds, perhaps some yellow and green. This is just one example of chromatography. In this case, it is used to separate a mixture. However chromatography can also be used to analyse mixtures - for example, identifying active ingredients in a drug, or finding out about the products of a reaction.

The method of chromatography

There are a few different types of chromatography and each has a slightly different process. However, the overall method stays the same. Chromatography involves the following steps:

• Take a soluble mixture, known as the solute.
• Add a small amount of the mixture to a static solid, liquid, or gas. This static medium is known as the stationary phase.
• Add some sort of solvent. This is known as the mobile phase.
• The solvent dissolves the mixture and carries it through the stationary phase.
• Different components of the mixture travel through the stationary phase at different speeds. Because of this, they separate out into clear, distinct spots or bands, that we can view on a chromatogram.

One of the simplest forms of chromatography is paper chromatography. It's quite likely that you have carried it out at school before. Here's what the typical setup for paper chromatography would look like:

Fig. 1 - The typical setup for paper chromatography

Principles of chromatography

We introduced you to some key words up above, in particular stationary phase, mobile phase, and chromatogram. These are some of the basic principles of chromatography. Let's now explore exactly what they mean.

Stationary phase

The stationary phase is - as the name suggests - well, stationary. It doesn't move. Examples include paper and silica powder.

Mobile phase

In contrast to the stationary phase, the mobile phase moves. It is a solvent that dissolves the solute you want to analyse or separate, and carries it through the stationary phase.

Chromatograms

Once the chromatography has finished, you'll be left with some evidence of the process. The mixture will have separated out on the stationary phase into different spots or bands. The leftover stationary phase, complete with all of its spots, is called the chromatogram.

For example, in paper chromatography, the stationary phase is a sheet of paper. Once you've finished the experiment, the chromatogram is the paper with its final arrangement of different spots.

Fig. 2 - A chromatogram for paper chromatography

Let's focus on two new terms: relative affinity, and retention factor.

Relative affinity

Components within the solute mixture move at different speeds through the stationary phase. This is all to do with their relative affinities to the two phases.

Components that experience a stronger attraction to the stationary phase are said to have a stronger affinity to the stationary phase. They are less soluble in the solvent and are more attracted to the static medium. The mobile phase can't carry them as easily - this means that the components move more slowly through the stationary phase.

In contrast, components that experience a stronger attraction to the mobile phase are said to have a stronger affinity to the mobile phase. They are more soluble in the solvent and less attracted to the static medium. The mobile phase is really good at carrying them about, so these components travel more quickly through the stationary phase.

What causes these differing relative affinities? As we mentioned, it is all to do with attraction to the two phases.

Let's say that the stationary phase consists of a polar molecule. This means it experiences permanent dipole-dipole forces between itself and any other polar components in the starting mixture. On the other hand, we'd typically use a nonpolar solvent. This means there would only be weak van der Waal forces between the solvent and the components. Polar components therefore experience a much stronger attraction between themselves and the stationary phase, than themselves and the mobile phase. They are more attracted to the stationary phase, are less soluble in the solvent, so we can say that they have a greater affinity to the stationary phase.

Retention factors

We now know that different components travel at different speeds through the stationary phase due to their relative affinities to the two stages. This means that in a given time period, different components will travel different distances. We can see this because they show up as clear, distinct spots.

We use the ratio between the distance travelled by each spot and the total distance travelled by the solvent to calculate things known as retention factors, or Rf values.

Rf values are important because they help us identify components. A particular component always produces the same Rf value under a certain set of conditions - i.e., if things like temperature, mobile phase, and stationary phase are exactly the same. If we calculate the Rf value for a particular component, we can compare it to values in a database to find out the identity of this unknown substance.

To find Rf values, divide the distance travelled by each component by the total distance travelled by the solvent.

$\mathrm{Rf}\mathrm{value}=\frac{\mathrm{distance}\mathrm{travelled}\mathrm{by}\mathrm{solute}}{\mathrm{distance}\mathrm{travelled}\mathrm{by}\mathrm{solvent}}$

Fig. 3 - Calculating Rf values

In the example above, the blue spot has travelled 7.4 cm and the solvent has travelled 9.8 cm. To calculate its Rf value, we use the following equation:

$7.4÷9.8=0.755=0.76$

Rf values have no units and are generally given to two decimal places.

Now that you know about relative affinities, can you predict how Rf values vary between components?

• Components with a greater affinity to the stationary phase travel more slowly through the medium. They move less far in a given time period and so have a lower retention factor.
• Components with a greater affinity to the mobile phase move more quickly through the medium. They move further in a given time period and so have a higher retention factor.

Types of chromatography

There are multiple types of chromatography. They differ in their mobile and stationary phases and their methods, but all follow the principles outlined above. Types include:

• Thin-layer chromatography (TLC).
• Paper chromatography.
• Gas chromatography.
• Column chromatography.
• Partition chromatography.
• High-performance liquid chromatography (HPLC).

Here's a handy table to help you compare them.

Uses of chromatography

We've already discussed some of the uses of chromatography - for example, analysing wastewater. But chromatography has a myriad of different applications. These include:

• Drug detection in urine.
• Analysing metabolites in bodily fluids.
• Extracting pigments from plant extracts.
• Isolating active ingredients in drugs.
• Purifying compounds.
• Separating mixtures of proteins, amino acids, or nucleotides.
• Quality control of alcoholic drinks.