We throw a lot of things down the plughole, from food scraps tipped down the sink to wipes and tissue flushed down the toilet. Between them, they contain all manner of substances - preservatives, disinfectants, stabilisers, bleaching agents, and more. If the levels of any of these chemicals get too high, they can damage the fragile ecosystems of our rivers, lakes, and other waterways. So how can we accurately measure the concentration of these potentially harmful substances in wastewater? One such method is chromatography.
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Jetzt kostenlos anmeldenWe throw a lot of things down the plughole, from food scraps tipped down the sink to wipes and tissue flushed down the toilet. Between them, they contain all manner of substances - preservatives, disinfectants, stabilisers, bleaching agents, and more. If the levels of any of these chemicals get too high, they can damage the fragile ecosystems of our rivers, lakes, and other waterways. So how can we accurately measure the concentration of these potentially harmful substances in wastewater? One such method is chromatography.
Chromatography is a separation and analytical technique used to split a soluble mixture into its component parts.
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.
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:
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
Chromatography was invented in 1900 by the Italian-Russian botanist Mikhail Tsvet, who wanted a way of separating pigments from plant extracts. The word chromatography comes from the Greek words chroma, meaning 'colour', and graphein, meaning 'to write'. Interestingly enough, Tsvet's surname is Russian for 'colour' as well.
Chromatography achieved most of its major breakthroughs in the 1940s and 1950s, thanks to Archer Martin and Richard Synge. In fact, they won the 1952 Nobel Prize in Chemistry for their invention of a particular type of chromatography, known as partition 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.
The stationary phase is a static solid, liquid, or gel. The solvent carries the soluble mixture up the stationary phase.
The stationary phase is - as the name suggests - well, stationary. It doesn't move. Examples include paper and silica powder.
The mobile phase is the solvent used to carry the mixture analysed through the stationary 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.
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.
A chromatogram is a column or strip of material containing components separated from a mixture by chromatography. It is essentially the output of a chromatography experiment.
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
We'll briefly explore paper chromatography in a bit. However, if you can't wait, check out Paper Chromatography for a more detailed look.
Let's focus on two new terms: relative affinity, and retention factor.
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.
In chromatography, relative affinity describes how well a component is attracted to either the stationary or mobile phase. It determines how quickly the component moves through the stationary phase.
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.
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.
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:
Rf values have no units and are generally given to two decimal places.
Some types of chromatography use retention times instead of retention factors. These measure the time taken for each component to move through the stationary phase.
Now that you know about relative affinities, can you predict how Rf values vary between components?
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:
Here's a handy table to help you compare them.
Name | Stationary phase | Mobile phase | Notes |
TLC | Plate covered in thin layer of silica gel | Liquid solvent | Accurate.Uses small samples. |
Paper | Paper | Liquid solvent | Cheap.Uses small samples. |
Gas | Tube filled with silica powder | Gaseous solvent | Requires heating.Extremely sensitive. |
Column | Column filled with silica powder | Liquid solvent | Mostly used for separation. |
Partition | Column filled with liquid held in place on a solid support | Liquid solvent | Separation occurs due to differing solubilities in the two liquids. |
HPLC | Column filled with silica powder | Liquid solvent | Uses pressure to speed up the process. |
If you want to find out more about some of these types of chromatography, we've got you covered - check out Thin-Layer Chromatography, Column Chromatography, Gas Chromatography, Paper Chromatography and Ion Exchange 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:
Do you remember the horse meat scandal from 2013? Some meat products sold as beef were actually found to contain horse. But scientists were only able to prove the meat's origins by blending it up and analysing it using chromatography alongside samples of pure horse meat.
Chromatography is a separation and analytical technique used to split a soluble mixture into its component parts.
Chromatography has a variety of uses. These include analysing waste water, separating mixtures, purifying compounds and extracting active ingredients from drugs.
Chromatography has a few basic principles. In chromatography, a solvent, known as the mobile phase, carries a soluble mixture up a static medium, known as the stationary phase. Different components within the mixture have different relative affinities to each of the phases. This causes them to travel through the stationary phase at different speeds and separate out into distinct spots or layers.
In chromatography, different components within the mixture have different affinities to the mobile and stationary phases. This means that some components move faster through the stationary phase than others and causes the components to separate out into distinct spots or layers.
There are multiple different types of chromatography. These include TLC, paper chromatography, ion exchange chromatography and column chromatography.
What is the stationary phase in TLC?
A thin metal or plastic plate covered in a layer of silica gel or alumina.
What is the mobile phase in TLC?
A solvent.
How do you view colourless components on TLC chromatograms? Give two methods.
Give three advantages of TLC over paper chromatography.
A mixture contains an alcohol and an alkene. Predict which molecule will travel further up the plate in TLC.
The alkene
Substance A has an Rf value of 0.8. Substance B has an Rf value of 0.4. Predict which substance has a higher affinity to the mobile phase.
Substance A
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