High-Performance Liquid Chromatography (HPLC) is a convective analytical or preparative technique which is used to separate the components in a mixture of chemical compounds based on differences in their physical and/or chemical properties. These properties can include size, shape, electrical charge, hydrophobicity, hydrophilicity, complementarity, or any of a large number of other features. When it’s applied to small samples for the purpose of detecting, quantifying, and characterizing the constituent components, this technique is generally referred to as ‘analytical HPLC.’ When it’s performed on a larger scale for the purpose of isolating and collecting samples of the constituents, the method is generally referred to as ‘preparative HPLC.’
The rise in the popularity and availability of HPLC methods and instrumentation over the last thirty years has coincided with the development of the biotechnology industry. Unlike small-molecule therapeutics, which usually have molecular masses of only a few hundred daltons, therapeutic proteins are typically very large molecules with masses in the thousands of daltons. Thus, gas chromatography, which works well for many small molecules, is unsuited to biotech drugs, which don’t evaporate readily and must therefore generally be handled in the liquid phase.
In HPLC, the sample is dissolved in a suitable liquid and a small aliquot is injected into the flowing mobile phase and pumped through an HPLC column packed with small particles of a stationary phase. These stationary-phase particles have specific properties which depend on the type of HPLC which is being performed – reversed-phase (RP), normal-phase (NP), ion-exchange (IEX), size-exclusion (SEC), and hydrophobic (HIC) and hydrophilic (HILIC) interaction chromatography, among others. As the mixture of analytes passes over the stationary-phase particles, each of the components interacts with the particles in a slightly different way, depending on its specific physical and chemical properties, causing each component to separate into a discrete band which elutes from the column at a different time. These discrete bands are visualized by the detector as separate ‘peaks’ which elute at characteristic ‘retention times.’ Using software, these peaks are recorded and their properties can then be measured and related back to the amounts of the various components which were present in the original sample.
A major reason that HPLC has enjoyed such enormous success is that an amazing variety of stationary phases (column packing materials) have been developed developed, so that separations based on a wide variety of physical or chemical properties can be accomplished using the same basic HPLC instrumentation. That, in turn, allows researchers to learn just one technique – HPLC – in order to be able to detect, isolate, quantify, and characterize variants of almost any analyte using an amazing variety of physical, chemical, and biological properties. This success has led to HPLC now being the dominant analytical method not just in biotechnology but also in fields ranging from small-molecule therapeutics to polymers, nanotechnology, virology, and green technology.
At Analytical Ventura, we have many years of experience with the various forms of HPLC, and we have described many of them in pages on this website. Our scientist are happy to work with you to determine what type of HPLC will work best for you and to develop a protocol using individual or combined separation modes to solve your analytical challenges.