The Fundamentals of Liquid Chromatography

Chromatography is the collective term for a set of techniques used to separate mixtures. These techniques include gas chromatography (GC), thin layer chromatography (TLC), Size exclusion Chromatography (SEC), and high performance liquid chromatography (HPLC).

The Two Phases

Chromatography involves passing a mixture dissolved in a “mobile phase” through a “stationary phase”. The mobile phase is usually a liquid or a gas which transports the mixture to be separated through a column or flat sheet which has a solid stationary phase.

Liquid Chromatography

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. It can be carried out in either a column or a plane. LC is particularly useful for the separation of ions or molecules that are dissolved in a solvent.

Simple liquid chromatography consists of a column with a fritted bottom that holds a stationary phase in equilibrium with a solvent. Commonly used stationary phases include solids, ionic groups on a resin, liquids on an inert solid support and porous inert particles. The mixture to be separated is loaded onto the top of the column followed by more solvent. The different components in the mixture pass through the column at different rates because of the variations in the partitioning behaviour between the mobile liquid and stationary phases.

Liquid chromatography is more widely used than other methods such as gas chromatography because the samples analysed do not need to be vaporised. Also, the variations in temperature have a negligible effect in liquid chromatography, unlike in other types of chromatography.

High Performance Liquid Chromatography (HPLC)

Present day liquid chromatography that generally utilises tiny packing particles and a fairly high pressure is known as HPLC. It is basically a highly improved form of column chromatography often used by biochemists to separate amino acids and proteins due to their different behaviour in solvents related to the amount of electronic charge of each one.

Instead being allowed to drip through a column under gravity, the solvent is forced through under high pressures of up to 400 atmospheres, making the process much faster. Because smaller particles are used, with their sizes being determined by a particle size analyser, there is greater surface area for interactions between the stationary phase and the molecules flowing past it. This in turn allows for much better separation of the components in the mixture.

There are many advantages of HPLC. For one, it is an automated process that only takes a few minutes to produce results. This is a vast step up from liquid chromatography, which uses gravity instead of a high-speed pump to force components through the densely packed tubing. HPLC produces results that are of a high resolution and are easy to read. Moreover, the tests are easily reproduced via the automated process.

Unfortunately, there are also disadvantages of this technique. It is difficult to detect coelution with HPLC and this may result in inaccurate compound categorisation. The equipment needed to conduct HPLC is also costlier and its operation can be complex.

Thanks to rapid advances in technology, analytical instrumentation such as HPLC are increasing in popularity. For the most part, the efficiency of these techniques outweighs their disadvantages making them a popular choice particularly in the pharmaceutical and medicinal industries.