Welcome back for the third article in our series about High Performance Liquid Chromatography (HPLC). In our first two articles we covered the principles behind HPLC, the most commonly used types of HPLC, HPLC in pharmaceutical research and HPLC in food research. If you would like to read the first or second article they are located on our Scientific Trends page. In this article we will be focusing on the biological applications of HPLC and explaining how HPLC is useful in biology, biotechnology and the life sciences.

HPLC in the Biological and Life Sciences

HPLC is widely used throughout the biological sciences. It is used by biochemists to purify peptides and proteins and used by molecular biologists to isolate nucleic acids, oligonucleotides and plasmids. It is also widely used in the biotechnology fields. For most biological samples, reverse-phase HPLC is used. Reverse-phase HPLC consists of a polar mobile phase and an apolar stationary phase.

Biological macromolecules can be either polar on non-polar dependent upon the side chain groups. For biological molecules that contain surface charges (or are surface active) or polar side chains, there are many points for intermolecular attractions to form, be it by hydrogen bonding, hydrophilic/hydrophobic interactions etc. Because of this, these biological molecules are better suited for dissolution in polar solvents and will stay longer in the mobile phase. Whereas apolar molecules will prefer to adhere to the apolar stationary phase by van der Waals and dispersion interactions. This affinity for separation is the reason why reverse-HPLC is the most common method.

If polar molecules are required to stay in the column for longer, then standard HPLC can be used as they will adhere to the polar stationary phase (and works vice versa to reverse-phase HPLC). Dependent upon the column characteristics, ion-exchange, hydrophobic interactions and size exclusion can be used to separate biological molecules. HPLC is also used for biological molecules because it is fast, accurate, allows protein to keep their biological activity and it allows enzymes to keep their enzymatic activity. These characteristics enable HPLC to effectively separate molecules with no loss of function.

Fast Protein Liquid Chromatography (FPLC)

Fast protein liquid chromatography (FPLC) is a similar analytical instrument that is specifically designed to separate, isolate and purify protein and other biomolecules from complex mixtures. The main difference between HPLC and FPLC is the internal pressure. HPLC columns can withstand up to 400 atmospheres of pressure, however FPLC can only withstand up to 50 atmospheres. The standard working pressure is between 30 and 50 atmospheres. It is possible to use FPLC columns on a HPLC machine as long as the internal pressure is limited to FPLC working pressures.

FPLC produces a high selectivity for proteins due to its stationary phase. In FPLC, the mobile phase is a solution buffer, but the stationary phase is a resin (in the form of beads) that is known to bind to the protein of interest by charge transfer processes. Similarly, as in HPLC, the proteins can bind to the column by a number of interactions including size, ion exchange, hydrophobicity and biorecognition. This allows for an efficient separation and purification of proteins. The flow rate in FPLC machines is relatively high at 1-5 mlmin-1. Unlike HPLC, which only has one external collecting container for waste; FPLC contains two- one for waste, and for the purified product.