The role of chromatography

Chromatographic techniques, whether liquid phase or gas phase, are a key element of most major organic testing laboratories. They are, however, only one part of a much bigger picture.

In the scientific press there has been significant coverage on the different types of detector currently available to the modern analytically laboratory. Universal detectors such as flame ionisation for total petroleum hydrocarbon (TPH) analysis, selective detectors such as electron capture for the analysis of chlorinated compounds like polychlorinated biphenyls (PCBs) or confirmatory detectors like mass spectrometry (for pretty much anything!) are all getting smaller, more sensitive and more robust. Mass spectrometry, whether it be single quad or triple quad (MS/MS), still sets the gold standard when it comes to trace level analysis but one important fact remains and that is that all of these techniques, and many more besides, require the use of chromatography in order for them to function.

Chromatography is a complex science in its own right with sixty eight million pages alone available on Google (Google, April 2021). However complex the science has become though, the purpose, and the fundamental principles that drive the technique, are still the same.

Chromatography is basically the separation of a complex mixture of components into separate entities. The complex mixture could be a blend of hydrocarbons in a fuel sample or a combination of pharmaceuticals in a waste water effluent – the main aim is to separate out these components before they arrive at the detector.

In the beginning


The beginnings of chromatography can be traced back to the beginning of the 20th century when the botanist Mikhail Tsvet created sa new technique to separate the coloured pigments within plants. Using basic chemical apparatus, Tsvet was able to separate the coloured pigments (chlorophyll – green; carotene – orange; and xanthophyll – yellow) in to three separate bands. It was this separation of the three colours that gave name to the technique – ‘Chroma’ meaning colour and ‘graphia’ meaning to write.

The basic principles of this experiment are still used today, often as introductory experiment in science classes whereby coloured sweets or ink dots can be placed on to thin filter paper, moistened, and the separation of the colours undertaken.

“there are several forms of chromatography but the two most common are liquid chromatography and gas chromatography”

A selection of samples are ‘dotted’ on to a predetermined start line on a sheet of modified paper which is then placed vertically in to a beaker containing a small volume of solvent. The solvent is slowly absorbed by the paper and starts rising up the paper. In this example the paper is referred to as a stationary phase and the solvent the mobile phase. The ink samples dissolve in the rising solvent and their component parts are carried by the solvent up the paper – the separation of the different components is dependent on the interaction of the component and the modified paper.

Components with little interaction travel faster and further, whilst components with more interactions travel slower and travel less far.

The simplified example below shows a spot of ink (Test 1) being compared against three Samples (1-3). Superficially at least the ink dots look the same, however by using chromatography the components parts are resolved and a match can be seen.


Example of an ink spot chromatogram prior to separation.


After separation a match between the test ink and sample 2 becomes evident.

Evolution of chromatography

Whilst the previous example is a useful tool in demonstrating the principles of separation, it doesn’t have the speed, sensitivity or robustness required for commercial analysis. Over the past 100 plus years, the science of chromatography has improved to enable fast reliable separations to be undertaken.

There are several forms of chromatography but the two most common, especially within environmental laboratories, are liquid chromatography and gas chromatography.

Liquid chromatography, often referred to LC or HPLC (high performance liquid chromatography), is a direct evolution of the ink spot test shown above.

Samples are introduced in to the system via high precision injectors capable of injection sub microlitre to several millilitre volumes.

Instead of a modified paper, the analytical chemist has access to a wide range of packed columns containing ultra-purified silica or polymers which have had their surfaces modified to alter their interaction characteristics. The columns can vary dramatically in size depending on the application in hand but typically in analytical applications 1-5mm in diameter and 100mm to 250mm is commonplace.

Although solvent is still used to enable the separation of the components, the flow is now under direct control by high pressure pumps capable of running at hundreds if not thousands of bar pressure.

The entire system is computer controlled with additional variables such as flow rate, temperature and solvent composition all under the analytical chemist’s control. With this the ability to separate an almost limitless range of compounds is possible (if however somewhat impractical in cases).

“gas chromatography operates in the gas phase, as a technique the components it can separate must be volatile and thermally stable and unlikely to break down”

Examples of applications that use LC as a separation technique can include; organic acids, phenolic compounds, vitamins, fluorinated surfactants such as PFOS and drugs of abuse. The choice of application is often more tailored to the detection system and the required sensitivity of the technique rather than how best to separate the analytes themselves.

The Role of Chromatography

The output of the LC system is dependant of the detector used but regardless of the specifics, a graph, or more accurately a chromatogram, is produced that shows the separation and response of the components as shown below;

Those compounds that have more affinity or interactions with the stationary phase (column) come out or elute later, whilst those that have minimal interactions will come out earlier. In this example component one has no interactions whereas component six has a much greater degree of interaction.

Generally, for any similar group of components, the greater the size or area of the peak the higher the concentration of the component present.

Gas chromatography still follows the same principles of LC but rather than using liquid solvent as a mobile phase to drive the separations a combination of heat and gas flows are utilised.

Gas chromatography often referred to GC as the name suggests operates in the gas phase. This means that as a technique, the components it can separate must be volatile and thermally stable and unlikely to break down.

The schematic shows this below:

In the image, 1 shows the high-pressure gas supply required for GC analysis. Depending on the application this may be nitrogen, argon or more commonly helium.

2: Shows the heated injection port.

3: Shows the analytical column inside of a temperature-controlled oven. The oven is capable of accurate and accelerated temperature control.

4: An example of a basic detector commonly used with GC. In this case it is a FID or flame ionisation detector.

5: The chromatogram produced by the combined GC-FID technique.

Like the HPLC system, liquid samples are introduced in to the GC via a high precision injector but injection volumes are typically only 1-5 microlitres. The injection port of the GC is heated (200-350°C) so that the liquid injected is quickly vaporised and becomes a gas.

The vaporised sample is swept in to the analytical column by a flow of high purity gas. Whist in LC the mobile phase is a liquid, in GC it is a gas; although it is most commonly referred to as a carrier gas rather than a mobile phase.

The columns for GC are typically much thinner and much, much, longer than for an LC application. Although the specifics of the GC column are dependent on the application and the analytical chemist’s requirements, they are often between 10 metres and 60 metres long yet only 1mm in diameter. They are made of drawn glass tubing coated with special polymers to ensure the glass does not break under the extreme temperature changes of even in general use.

The entire system is again computer controlled with additional variables such as flow rate and temperature under the analytical chemist’s control. The efficiency of a GC column is often much greater than that of an LC column meaning that peaks in GC are often much sharper and many more components can be serrated with a given time frame.

“the efficiency of a GC column is often much greater than that of an LC column meaning that peaks in GC are often much sharper”

Examples of applications that use GC as a separation technique can include; petroleum hydrocarbons, polychlorinated biphenyls (PCBs), pesticides such as DDT and Lindane and many more besides.

Multidimensional analysis

The basic examples above, whether they be LC or GC, have used only a single column to separate the components of our theoretical mixture. Technology now exists to enable multidimensional separations to take place using two columns effectively simultaneously.

Starting with GC back in the 1990s the technique of multidimensional analysis has recently become available to commercial laboratories and has recently expanded to LC too. The technique is referred to as GCxGC (or LCxLC depending on the application) and enables significantly greater separations to be undertaken when compared to the standard single column approaches given in the examples above.

The premise of multidimensional analysis is that two analytical columns of different chemistries (e.g. stationary phases) are connected together prior to the detector. Whilst they are in series, the flow of mobile phase or carrier gas does not travel linearly through them to the detector but more of a modulated approach.

The modified image above has the same gas supply, heated injection port and initial analytical column as the earlier example, but here the output of the first column (3) directed to a modulator (4) prior to being transferred to a second analytical column (5).

In GCxGC, the second analytical column is often considerably shorter than the main primary column.

Multidimensional analysis

The first column separates on boiling point providing the initial separation of the components prior to entering the modulator. After transfer to the second column which may separate, based on for example polarity, the components are detected as previously.

The combination of columns arranged in this way gives a significant increase in the chromatographic separation of components as now, rather than just having a standard x axis vs. y axis chromatogram as shown earlier, we have the ability to introduce a third axis, z.

Advanced separations

In its broadest sense, Total Petroleum Hydrocarbons (TPH) covers a massive range of organic compounds right from the most volatile of gases right through to tar like substances that can be found in crude oil. Analytically, most laboratories in the UK focus on a selected band of hydrocarbons from C6 up to C40 – a group that covers several hundred different compounds including both aliphatic (saturated hydrocarbons such as octane) as well as aromatics like benzo(a)pyrene.

As the individual components of this group are so varied, laboratories are often asked to subdivide the TPH total into different banded fractions e.g. aliphatic vs. aromatics or C8-C10, C10-C12 etc.

Using just a single column that separates based on boiling point we would not be able to distinguish the aliphatic from aromatic compounds without the use of prior sample treatment or high-end specialist detectors. With GCxGC however we can employ the second column, the column that separates based on polarity, to perform this task.

The power of GCxGC is even greater for analysis of unknown TPH sources, for example in the case of environmental spills. Here the comprehensive technique allows not only separation of the both aliphatic and aromatic hydrocarbons but also of other co-extracted material that may have otherwise adversely affected the interpretation of the results – compounds such as oxygenates, natural organics acids and phenolics.

Chromatography has advanced significantly since its inception but the fundamentals that formed the basis of modern analytical chemistry are still in use today. The development of GCxGC and other multidimensional techniques will enable greater understanding of samples and open up more opportunities to undertake analysis that has until now been impractical to undertake outside of a research facility.