The principles of modern chromatography

Whether looking in water, soil or in the air there are an enormous number of chemical substances surrounding us that have the potential to affect the health and wellbeing of both people and the environment. Analytical laboratories are tasked with the challenge of monitoring the presence and quantities of these compounds in the environment. In order to do so, a vast selection of analytical techniques have been developed and adopted.

There are a number of different subsections within analytical chemistry, but the two most commonly encountered are organic and inorganic chemistry. As a general rule organic chemistry deals with compounds that primarily rely on carbon molecules to form the basis of their structure and inorganic chemistry covers those that do not. This rule is not 100% definitive but can be assumed as a good place to start when thinking about how the analysis of a specific substance is to be carried out. For example, if the analysis of petroleum type compounds were required an organic chemistry lab would be needed (most likely using GC). If on the other hand testing for mercury was needed, the techniques used would be found in an inorganic lab.

The analytical techniques that fall within the category of organic chemistry are varied but two of the most widely used for environmental samples are gas chromatography (GC) and liquid chromatography (LC). The principle of these techniques is effectively the same and involves the components of a mixture being separated through a chromatography column but, as their names suggest, one achieves this whilst operating in the gaseous state and the other with them in a liquid state.

Setting up an analytical method for environmental samples is a complex process as there are so many variables that need to be considered when doing so. Preparing the sample so that it is suitable for analysis, the way that the sample is introduced into the system, how chromatographic separation is achieved and how the detector is programmed to monitor what has been found must all be taken into consideration during method development.

All of these will need to complement each other in order to create a robust method for analysis and will be greatly influenced by the nature of the compounds in question as well as the sample matrix itself.

LC or GC?

The decision to use LC or GC to carry out analysis for any given compound is not always an easy one, especially as there are examples where both techniques produce successful results. Compounds that work well using GC tend to be smaller molecules with low boiling points, meaning that they are volatile and are happy to exist in a gas state. Although volatility is a must for compounds in GC analysis it is also important that they are thermally stable so that they do not decompose upon heating.

LC is a good complimentary technique to GC in that it is able to separate those compounds that are non-volatile or thermal labile. Rather than rely on the use of gas phase chemistry to separate the compounds of interest, LC uses liquids – often mixtures of solvent and water – to separate and resolve analytes. This liquid is called a mobile phase, whilst the material making up the column is called the stationary phase.

The diagram below shows some examples of the types of compound analysed by each technique.

Sample preparation

Before any separation can take place, a sample needs to be prepared to make it suitable for analysis. The aim of effective sample preparation is to take the sample whether it is a solid, liquid or gas through a process that will result in it being in an appropriate container and state so that it can be introduced into the analytical system for analysis. Sometimes this process may be as simple as transferring a portion of the sample into a vial that can then be tested but it can be a far more complex procedure. The choice of how a sample is prepared for analysis will be dependent on the nature of the compounds of interest, the sample matrix that is to be tested as well as the type of sample introduction technique that is to be used.

Gas chromatography

Most organic chemistry analysis is now carried out using automated equipment that combines the sample introduction, chromatography and detection into one functional instrument. The different components of these instruments are often interchangeable depending on the requirements of the lab, so it is not uncommon for a lab to have multiple GC systems with different types of sample introduction apparatus set up with them.

“the aim of effective sample preparation is to take the sample whether it is a solid, liquid or gas through a process, so that it can be introduced into the analytical system for analysis”

One example of a sample introduction technique used with GC is headspace analysis. It’s primarily used when looking for extremely volatile organic compounds normally containing fewer than 10 carbon atoms, e.g. CFCs, solvents, gases. The sample preparation for headspace analysis is often minimal as a portion of the sample is transferred to a specially designed container where it is sealed to be air tight. The sample vial is then heated to drive the analytes from the sample into the gas phase within the vial. A needle within the autosampler then samples the vial, allowing the gas and the volatile compounds that it contains to be transferred into the GC to be analysed. Such a technique will only work with volatile compounds, so in order to look at compounds containing upwards of 10 carbons a different sample introduction approach is required; liquid injection also known as split/splitless injection.

Split/splitless injection is an alternative to headspace and is a commonly used technique when dealing with heavier, semi-volatile, compounds in GC analysis. In this technique the compounds of interest need to be removed from the sample through an extraction process typically ending up in an organic solvent. An aliquot, often only 1μl or 2μl, of the organic solvent containing the compounds is removed from the sample vial using a syringe whereby it is introduced into the inlet of the GC. In most applications the inlet is heated to a high temperature exceeding the boiling point of the solvent and this heat, combined with a flow of inert gas, vaporises the sample and enables transfer from the inlet to the GC column where separation can take place.

In GC the speed at which compounds pass through the column is controlled by a combination of applied temperature and carrier gas flow. As the carrier gas passes through the column any compounds that are travelling within it will interact with the stationary phase and will consequently be slowed down as they travel through. The interactions that occur will be dependent on what stationary phase is being used as well as the chemistry of the compounds. When a mixture of compounds is passed through the analytical column the degree to which each component is slowed down by the interactions with the stationary phase will determine how quickly each one reaches the detector.

The analytical column is where the components of the gaseous mixture are separated to ensure that they do not all reach the detector at the same time. An analytical column in gas chromatography is essentially a very thin glass tube, normally between 15 and 60 metres in length, with its internal surfaces coated in a specially designed stationary phase. The nature of this stationary phase is variable and many different types are available depending on the application required by the user.

“an analytical column in gas chromatography is essentially a very thin glass tube, normally between 15 and 60 metres in length”

The time taken between injection and the compound reaching the detector is known as its retention time; this will be consistent for a compound providing the setup of the GC system remains unchanged. Other factors that can help speed up or slow down the rate at which samples flow through the column include the temperature of the GC oven, the flow rate of the carrier gas, the column dimensions (e.g. length) and the type of stationary phase being used. Once the compounds have travelled through the entire length of the column they will be transferred directly into the detector where they can be detected and their response converted to an electronic signal.

Liquid chromatography

Sample introduction into an LC tends to be a more straightforward process than in GC as it is essentially just an injection of an aliquot of liquid into the system. It is possible to introduce sample pre-treatment steps that are automated processes that occur prior to injection of the sample but the final transfer into the system is a direct injection. Water samples may be injected directly into some of the LC systems with more powerful detectors, but often extraction procedures are carried out on samples to concentrate the compounds they contain and transfer them into a suitable solvent ready for analysis.

As the names suggest the major difference between LC analysis and GC is that the carrier gas used to transport the compounds through a GC system is now replaced with a liquid. The liquid that flows through an LC system via a high pressure pump is referred to as the mobile phase and normally consists of a combination of water and organic solvent(s). When a sample is injected it is combined with the mobile phase, which then flows through the system towards the analytical column where the separation of the compounds can take place.

Separation of a given mixture in LC again occurs by interactions between the compounds of interest and the stationary phase of the column but now these interactions are partially governed by the chemistry of the compound and partially by the chemistry of the mobile phase. Mobile phase composition typically starts with a high water content and uses other non-aqueous solvents such as methanol or acetonitrile to elute the compounds from the stationary phase.

Good solubility in the selected mobile phase is essential for a compound to work using LC. As volatility of compounds is not important in LC analysis larger molecules often work better using this technique instead of GC.

The columns used for LC are quite different in appearance to the ones used for GC analysis although their main function of allowing compounds in a mixture to travel to the detector at different rates remains the same. LC columns are typically small rigid metal tubes that typically range from 10 cm – 30 cm in length that are often packed with a either a modified silica or polymeric material which forms the solid phase. As was the case in a GC column it is the interactions between the compounds and the material contained within the column that regulates how quickly they are able to move on towards the detector. There are many types of LC columns available on the market, all with a variety of dimensions and packing materials to cover a wide range of applications. Careful column selection combined with finding the perfect mobile phase composition within a method will enable an analyst to separate the components from a sample effectively. This will make it easier to detect the individual components, therefore helping to improve the quality and accuracy of the overall analysis.


With the compounds in a sample separated through either gas or liquid chromatography it is then necessary to use a detector of some kind to determine what is in the mixture that has been separated. There are a wide variety of detectors available that can be coupled with either an LC or a GC system that will allow chemical analysis to be completed and the choice of detector used will very much be dependent on the nature of the analysis required.

Some detector types such as a fluorescence detector will be exclusively for LC systems, whereas flame ionisation detectors (FID) for example may only be used only for GC. Detectors are available that are capable of working with either technique and a widely used example of one of these is mass spectrometry (MS). Liquid chromatography mass spectrometry (LC-MS) and gas chromatography mass spectrometry (GC-MS) are two very powerful and widely used techniques in environmental organic analysis and consequently a great deal of research and development has been carried out on these techniques to provide greater selectivity and lower concentrations of substances to be detected.

The theory behind mass spectrometry (MS) is highly complex but in its simplest form it revolves around the creation of charge particles or ions through the addition or removal of electrons, which are recorded as a mass-to-charge ratio (m/z). The m/z values of the positive and negative ions viewed in a mass spectrum can be used to calculate the masses of the molecules that they came from and therefore compounds that were present in the sample can be identified.

“as technology evolves the instrumentation will continue to improve, meaning that compounds can be looked for at incredibly low levels with even more accuracy”

It is the use of chromatographic separation in tandem with mass spectrometry that makes the analysis of samples containing a complex mixture of compounds possible. As the compounds in a sample are separated by the analytical column they will reach the detector at different times and so a mass spectrum can be obtained each time one of the compounds registers. By plotting the response intensity observed by the MS against time a chromatogram can be generated with each peak representing a compound that has been detected. The size of the peak is proportionate to the concentration of each analyte.

The peaks seen in a chromatogram will have a unique mass spectra associated with them that can be used to confirm the identity of the compound.

MS is but one of many detectors, but for an advanced environmental analytical laboratory it is the gold standard in analysis.


Liquid and gas chromatography are both very useful techniques when it comes to analysing environmental samples containing complex mixtures of compounds. Depending on the sample type and the chemistry of the compounds of interest the most appropriate techniques can be chosen and, when combined with an efficient sample extraction process and suitable detector, accurate analysis can be achieved. As technology evolves the instrumentation required to carry out such analysis will continue to improve, meaning that compounds can be looked for at incredibly low levels with even more accuracy.