High-performance liquid chromatography, or HPLC, is a method used in chemistry to separate compounds for analysis. As with other chromatographic methods, a sample of the compound is carried by a liquid solvent (called the mobile phase) through an adsorbent material (called the stationary phase). The components of the compound travel through the stationary phase at different rates, causing them to separate. HPLC uses pressure to force the mobile phase and the sample through an HPLC column that contains the stationary phase. Some applications for HPLC include tests to detect vitamin D levels in blood, tests for performance enhancing drugs, scientific research, and pharmaceutical manufacturing.
Different types of HPLC methods are used depending on the type of compound being tested. Normal-phase chromatography separates the components of the compound (called analytes) based on their polar interaction with the sorbent. Molecules that have a strong polarity for the stationary phase have a long retention time. Reversed-phase chromatography employs a polar mobile phase to elute polar molecules early in the process. Ion-exchange chromatography works by causing an analyte to separate based on its ionic interactions with the mobile or stationary phase, in which analyte molecules bond with molecules of an opposite charge.
HPLC columns contain the stationary phase. They are commonly made of stainless steel, though some may be made of polymers. Considerations for choosing columns include the dimensions, the type of adsorbent and its characteristics. Columns range from 5cm to 25cm in length. Shorter columns are used for faster methods, while longer columns provide increased resolution of separations. The internal diameter of a column can range from narrow bore (1mm to 2mm) to larger columns (over 10mm), though the most common size is 4mm to 4.6mm. Narrow bore columns reduce solvent usage and can improve sensitivity of the separation. Larger columns can handle larger sample sizes for purification applications. The adsorbent in a column is typically silica gel for its porosity, and ranges in size from 3μm to 5μm.