High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC)

A bit of history

Chromatography, from the Greek chroma (colour) and grapho (writing), comprises a set of techniques aimed at separating mixtures based on the different interaction capacity of each component on/with other substances in solid or liquid state. In other words, it consists of passing a mixture of substances, or mobile phase, over a stationary phase which will retard the flow of the substances in the mixture so that as the mixture moves over it, at different speeds(depending on the different interactions produced between the solutes in the mobile phase as they move around or over the stationary solid phase), the different components of the mixture are separated.

1850 (Philipp Otto Runge)

This German painter observed that by depositing drops of colouring substances on blotting paper, differentiated coloured areas were created

1906(Mikhail Semyonovich Tsvet)

The Russian botanist, known as Tsvet, coined the term “chromatography” through his initial experiments on the separation of chlorophylls from plant tissues(mobile phase) in calcium carbonate columns(stationary phase).

1931(Richard Kuhn, Edgar Lederer, Alfred Winterstein) These researchers employed the

chromatography to study the carotenoids present in various plants.

At the same time, Paul Karrer in Zurich (Switzerland) and LászlóZechmeister in Hungary were also developing advances in the study of plant pigments. According to some articles, it was a scientific contest where gentleness and chivalry prevailed between the various scientists.

Moving on to chromatography

Think of chromatography as a race and you will see that it is much simpler than it looks. Waiting at the starting line, we have a mixture of compounds, either in liquid or gas, as if they were a group of runners together and grouped together. When the race starts, the runners soon separate from each other because they have different abilities. In the same way, chemical compounds separate at different speeds on a stationary solid. The key is to remember that chromatography is a surface effect.

As the liquid passes through the solid (stationary phase) the molecules are adsorbed on the surface and temporarily retained. This effect is known as adsorption. Each compound undergoes adsorption in a different way and will therefore be retained in the solid phase for more or less time.

These retained molecules are then “eluted” or entrained by a liquid (mobile phase) and carried to a detector where they give a signal known as a chromatogram (Figure 1).