Chromatography: A Concise Guide

chromatography columns resins art

Chromatography techniques are widely used, with the bulk of them being variations of liquid and gas chromatography. As a scientist, it is important to know the tools for analysis available, and how to apply them to your research. This guide touches on the principles behind chromatography and compares some of the most common techniques in use today.

Some Basic Theory

Although many forms of chromatography exist, the main goal of the technique is simply to separate a mixture of compounds. This is done by passing the mixture through reactive surfaces, such that the interactions change the time (retention time) at which each compound comes out the other side. Ideally, each compound – or analyte – will possess a good separation profile (resolution), making it easy to differentiate and identify them.

Each molecule has different properties, and hence the challenge of chromatography is to discover how to exploit them. This, in turn, means that is not possible for a single technique to be universally useful. The reactive surface (stationary phase), the medium that pushes the mixture through (mobile phase), and other equipment-specific parameters provide for optimization options.

paper chromatogram of marker ink
Yup, you were running chromatography experiments even before high school!


Liquid-Solid Phase Chromatography

This technique, also known as liquid chromatography (LC), employs the use of a liquid mobile phase and a solid stationary phase. It is highly versatile, able to separate virtually any mixture of compounds – from small molecules to entire large proteins.

The benefit of having a liquid mobile phase is that it allows for modification of the solution. It is possible to increase or decrease the flow rate of the liquid at different points of the run, optimizing the retention times of the analytes.

The more common detectors for LC include UV-Vis spectrophotometers, refractive index and fluorescence detectors, as well as mass spectrometry (MS) detectors. In general, MS is the most robust, able to determine the quantity as well as the structure of the analytes. However, it also requires the most maintenance and optimization in order to work.

High Performance Liquid Chromatography (HPLC)

HPLC is a more efficient form of LC, able to provide much quicker retention times and better resolution. It uses very thin columns for the stationary phase, creating high pressures of up to 400 atm to force the liquid through. These thin columns are full of tiny particles, increasing the surface area available for interactions with the compounds.

scientist operating high performance liquid chromatography machine
A typical HPLC setup with attached mass spectrometer detector.

Normal and Reverse-Phase LC

Normal-phase LC employs the use of a silica gel stationary phase; a cross-linked structure of silicon dioxide (SiO2). Silica is extremely polar, hence polar molecules bind (adsorb) onto its surface. The corollary is that non-polar molecules pass through the column, eluting at a faster rate.

Reverse-phase LC, as the name suggests, makes use of the opposite – a non-polar stationary phase. The most common are long-chain hydrocarbons (such as C18) attached to a bed of silica. The hydrocarbon chains on the column bind strongly to non-polar molecules in the mobile phase, therefore polar molecules elute first.

Ion Exchange Chromatography

This technique involves the separation of ions, either positively (cations) or negatively (anions) charged. The stationary phase in this case must have the opposite charge to the molecule(s) of interest, such that they attract and bind. Washing of the column removes impurities and other compounds, since they tend not to bind as strongly. The next step is the addition of a salt, in order to dissociate the molecules from the surface of the column.

A key advantage of this method is the ability to selectively bind and release molecules, making it useful in purification. This also means that regeneration is possible by washing with an acid or base, such that it regains its initial charge. Ion exchange chromatography is widely used in large scale processes, such as in water treatment and the recovery of uranium from nuclear reactors.

Related: Ion Exchange Chemistry

This technique is most common in amino acid separation, since many of them contain ionizable functional groups. Their charge can be modified as desired based on their amino acid sequence, by using a pH buffer in the liquid mobile phase.



Size Exclusion Chromatography

Using molecular sieves, it is possible to separate a mixture of compounds by their size alone. Molecular sieves contain small pores that can ‘trap’ small compounds, allowing the larger ones simply pass through. Think of the sieves as being a ‘maze’ for molecules small enough to enter, while those that don’t fit skip this maze altogether. Therefore, larger compounds have a faster retention time!

chromatogram size exclusion
A size exclusion chromatogram, with a single peak showing protein elution.

Size exclusion is ‘gentler’ than most other chromatography techniques as there is no binding reaction between the sieves and the compounds. As such, this technique is most popular for the purification of proteins. In the chromatogram above, the peak corresponds to a protein of interest. It can be seen that the protein was collected between tubes 10-12, with the help of a fraction collector.

Affinity Chromatography

This technique employs highly specific interactions, such as antigen-antibody, receptor-ligand, enzyme-substrate, etc. It is similar to ion exchange in that one half of the binding pair is immobilized, forming the stationary phase. The liquid mobile phase flows through, with the corresponding half (if present) binding specifically to form the pair. It is common to see detection compounds in use with this technique, such as in chromogenic enzyme-linked immunosorbent assays (ELISA).

Virtually any target can be purified in this manner, as long as the binding interaction is specific and high affinity. One common method is the use of protein A, an antibody binding compound. Protein A is attached to a resin, where it is stable and can immobilize antibodies passing through.

Related: Monoclonal Antibodies in Drug Development

Gas Chromatography

Gas chromatography (GC) gets its name from the inert gas that forms the mobile phase. As such, the sample – usually small hydrocarbons – is heated to the point of vaporization before entering the column. The column itself can either be a capillary or glass tube, with the inner surface consisting of a stationary phase.

gas chromatography machine agilent 6890
A typical GC machine, with capillary column attached.

The sample adsorbs onto the stationary phase and the entire system is heated, so that separation is based on the boiling points of the analytes. Typically, small organic substances tolerate this treatment well, whereas larger molecules and biologicals such as proteins tend to disintegrate.

Which to Choose? LC or GC?

Compare this to LC, where ‘gentler’ methods exist that can handle larger molecules without destroying them. However, GC remains popular as its sample profiles are usually cleaner and faster. An added benefit is the use of an inert gas for the mobile phase means there is little to no sample-mobile phase interaction, leading to a cleaner analysis.

Conclusion

The list above is not an exhaustive list of chromatography equipment, but just an introduction to the more common ones. In order to obtain a specific analyte for detection or collection, these methods usually have to undergo lots of optimization. Oftentimes, the sample must be put through multiple chromatography and filtration steps to ensure the purity of the final product.

It is important to remember that chromatography is simply a useful tool, and should be applied as such. It is up to the scientist to use critical thinking to analyze the data, and then design the most suitable methods.

Cover graphic: artist’s rendition of samples flowing through different chromatography columns by Melanie (@nanoclustering)

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