High-performance Liquid Chromatography

High-performance liquid chromatography (sometimes referred to as high-pressure liquid chromatography), HPLC, is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying and purifying the individual components of the mixture. HPLC is also considered an instrumentation technique of analytical chemistry, instead of a gravitimetric technique. HPLC has many uses including medical (e.g. detecting vitamin D concentrations in blood serum), legal (e.g.detecting performance enhancement drugs in urine), research (e.g. purifying substances from a complex biological sample, or separating similar synthetic chemicals from each other), and manufacturing (e.g. during the production process of pharmaceutical and biologic products). HPLC can alternatively be described as a mass transfer involving adsorption.

HPLC relies on the pressure of mechanical pumps on a liquid solvent to load a sample mixture onto a chemistry column, in which the separation occurs. A HPLC separation column is filled with solid particles (e.g. silica, polymers, or sorbents), and the sample mixture is separated into compounds as it interacts with the column particles. HPLC separation is influenced by the liquid solvent’s condition (e.g. pressure, temperature), chemical interactions between the sample mixture and the liquid solvent (e.g. hydrophobicity, protonation, etc…), and chemical interactions between the sample mixture and the solid particles packed inside of the separation column (e.g. ligand affinity, ion exchange, etc...).

HPLC is distinguished from ordinary liquid chromatography because the pressure of HPLC is relatively high (~150 bar, ~2000 PSI), while ordinary liquid chromatography typically relies on the force of gravity to provide pressure. Due to the higher pressure separation conditions of HPLC, HPLC columns have relatively small internal diameter (e.g. 4.6 mm), are short (e.g. 250 mm), and packed more densely with smaller particles, which helps achieve finer separations of a sample mixture than ordinary liquid chromatography can. This gives HPLC superior resolving power when separating mixtures, which is why it is a popular chromatographic technique.

The schematic of an HPLC instrument typically includes a sampler by which the sample mixture is injected into the HPLC, one or more mechanical pumps for pushing liquid through a tubing system, a separation column, a digital analyte detector (e.g. a UV/Vis, or a photodiode array (PDA)) for qualitative or quantitative analysis of the separation, and a digital microprocessor for controlling the HPLC components (and user software). Many different types of columns are available, varying in size, and in the type (i.e. chemistry) of solid packed particle types available. Some models of mechanical pumps in a HPLC instrument can also mix multiple liquids together, and the recipe or gradient of those liquids can modify the chemical interactions that occur in HPLC’s column, and thereby modify the chemical separation of the mixture.