High Performance Liquid Chromatography

High performance liquid chromatography (HPLC), formerly known as high pressure liquid chromatography, is an analytical chemistry technique which separates, identifies, and quantifies components of a mixture.  HPLC can be used to identify a range of biological compounds including peptides, proteins, small molecules, RNA, DNA, and polymers.  In HPLC, pumps pass a pressurized liquid solvent containing the sample mixture through a solid adsorbent material.  Each unique component in the sample flows at a different rate through the solid material depending on its interaction with the adherent.  The difference in flow rates enables the separation of the components as they exit the column.

HPLC differs from traditional “low pressure” liquid chromatography due to the employment of operational pressures which can reach up to 400 atmospheres (atm).  Traditional liquid chromatography relies on gravity to pull the mobile phase through the chromatography column.  The increased pressure results in a faster analysis time, because there is no waiting on gravity to force the sample through.  Analytical HPLC samples are very small, typically 1-100 milliliters (mL), therefore the typical HPLC column is less than 5 milllimeters (mm) in diameter.

The HPLC column contains particles of packing material, and as the compound bands flow through the particles and exit from the pressurized column, a detector measures the desired fractions.  The detector is connected to a computer software program and produces a chromatogram.  A chromatogram is a graphical representation of the separation of chemical compounds which has occurred during the HPLC process.  Each peak on a chromatogram corresponds a different compound, and each peak has its own retention time.

HPLC systems can also be connected to and used in conjunction with other types of detectors such as an ultraviolet (UV) light detector to measure UV fluorescence, or a mass spectrometer (MS) to provide detailed spectral analysis of a chromatographic separation.  The use of HPLC in conjunction with other quantitative techniques provides comprehensive quantitative information about an analyte.

A typical HPLC system is comprised of a pump, HPLC column, and detector, which is connected to a computer.  The solvent (mobile phase) enters the pump, where the sample material is also added.  An injector introduces the sample into the continuously flowing mobile phase stream to carry the sample into the HPLC column to begin the stationary phase.

The solvent reservoir is usually glass and contains the solvent used for the mobile phase, which can vary as described below.  The pump then aspirates the mobile phase and forces it through the column.  The operating pressure of the pump depends on multiple factors including column dimensions, particle size of the stationary phase, and the flow rate and composition of the mobile phase.  Operating pressures can reach up to 42000 kPa, or 6000 psi.  Injection can be automatic or can be done singly, and should be reproducible as determined.

High Performance Liquid Chromatography columns are typically made of stainless steel and are between 50 and 300 millimeters (mm) in length.  The column is filled with the stationary phase, which typically has a particle size of 3 to 10 micrometers (um).  The temperature of the oven containing the column should remain constant throughout the analysis.  As the analytes elute off of the column, they are detected by UV, fluorescence, MS, or electrochemical (ECD) detectors.  The detector then sends electronic signals to the connected computer software program.  The computer accepts the detector signals and creates a peak chromatograph that is easy to interpret.

There are several different variations of the HPLC stationary phase.  Normal phase, reverse phase, size inclusion, and ion exchange have stationary phases that differ slightly.  Normal phase HPLC separates analytes based on their polarity.  The polar stationary phase is typically silica, and the non-polar mobile phase can be hexane, methylene chloride, chloroform, diethyl ether, or any mixture containing two or more of these solvents.  In reverse phase HPLC, the stationary phase is non-polar and hydrophobic (water resistant), while the mobile phase is polar.  Mixtures of methanol and water are commonly used for the mobile phase in reverse phase HPLC.

In size-exclusion HPLC, the column has a material that controls the pore size, so the sample is separated by particle size.  Larger particles are flushed out of the column quickly, and smaller particles remain in the column and take longer to elute.  Ion-exchange HPLC uses an ionically charged column to attract the sample, which leads to higher charged ions quickly attaching and remaining on the column for longer times, compared to minimally charged ions leaving the column at earlier times.

The versatility of High Performance Liquid Chromatography allows it to be used for a multitude of scientific analyses.  High Performance Liquid Chromatography is used in pharmaceutical and environmental labs in addition to forensics, food and flavor, and clinical applications.  The development of HPLC has allowed for higher throughput of sample analysis, thus allowing researchers and technicians to increase the amount of sample analyses that can be done in a short amount of time.