Introduction -- HPLC high pressure liquid chromatography and liquid chromatography (LC) are analytical chromatographic technique useful for separating ions or molecules dissolved in a solvent. If the sample solution is in contact with a second solid or liquid phase, the different solutes will interact with the other phase to differing degrees due to differences in adsorption, ion-exchange, partitioning, or size. These differences allow the mixture components to be separated from each other by using these differences to determine the transit time of the solutes through a column.
Liquid chromatography (LC) is an analytical chromatographic technique that is useful for separating ions or molecules that are dissolved in a solvent. If the sample solution is in contact with a second solid or liquid phase, the different solutes will interact with the other phase to differing degrees due to differences in adsorption, ion-exchange, partitioning, or size. These differences allow the mixture components to be separated from each other by using these differences to determine the transit time of the solutes through a column.
Simple liquid chromatography consists of a column with a fritted bottom that holds a stationary phase in equilibrium with a solvent. Typical stationary phases are: solids (adsorption), ionic groups on a resin (ion-exchange), liquids on an inert solid support (partitioning), and porous inert particles (size-exclusion). The mixture to be separated is loaded onto the top of the column followed by more solvent. The different components in the sample mixture pass through the column at different rates due to differences in their partioning behavior between the mobile liquid phase and the stationary phase. The compounds are separated by collecting aliquots of the column effluent as a function of time.
Conventional LC is most commonly used in preparative scale work to purify and isolate some components of a mixture. It is also used in ultra trace separations where small disposable columns are used once and then discarded. Analytical separations of solutions for detection or quantification typically use more sophisticated high-performance liquid hromatography instruments. HPLC instruments use a pump to force the mobile phase through and provide higher resolution and faster analysis time.
HPLC Instrumentation -- Solvents must be degassed to eliminate formation of bubbles. The pumps provide a steady high pressure with no pulsating, and can be programmed to vary the composition of the solvent during the course of the separation. The liquid sample is introduced into a sample loop of an injector with a syringe. When the loop is filled, the injector can be inject the sample into the stream by placing the sample loop in line with the mobile phase tubing. The different types of HPLC columns are described below. The presence of analytes in the column effluent is recorded by detecting a change in refractive index, UV-VIS absorption at a set wavelength, fluorescence after excitation with a suitable wavelength, or electrochemical response. Mass spectrometers can also be interfaced with liquid chromatography to provide structural information and help identify the separated analytes.
Columns -- Conventional liquid chromatography uses plastic or glass columns that can range from a few centimeters to several meters. The most common lengths are 10-100 cm, with the longer columns finding use for preparative-scale separations. High-performance liquid chromatography (HPLC) columns are stainless steel tubes, typically of 10-30 cm in length and 3-5 mm inner diameter. Short, fast analytical columns, and guard columns, which are placed before an analytical column to trap junk and extend the lifetime of the analytical column, are 3-10 cm long.
Picture of an HPLC column
Stationary Phases
Partition Chromatography -- In partition chromatography the stationary phase is bonded to inert particles of 3-10 µm of diameter, with the smaller sizes, 3-5 µm, being used in analytical columns, and the larger particles being used in preparative-scale HPLC. Analytes separate as they travel through the column due to the differences in their partitioning between the mobile phase and the stationary phase.
Reverse-phase partition chromatography uses a relatively nonpolar stationary phase and a polar mobile phase, such as methanol, acetonitrile, water, or mixtures of these solvents. The most common bonded phases are n-octyldecyl (C18) and n-decyl (C8) chains, and phenyl groups. Reverse-phase chromatography is the most common form of liquid chromatography, primarily due to the wide range on analytes that can dissolve in the mobile phase.
Normal-phase partition chromatography uses a polar stationary phase and a nonpolar organic solvent, such as n-hexane, methylene chloride, or chloroform, as the mobile phase. The stationary phase is a bonded siloxane with a polar functional group.
Chiral stationary phases -- Separating enantiomeric mixtures is very challenging because the optical isomers are identical chemically. Chiral analytes can be separated by using a chiral stationary phase. One optical isomer of a chiral molecule is usually bonded to a polymer, which is then coated onto silica packing material. Separations occur because the chiral analytes will interact with the one isomer on the stationary phase differently.
Ion Chromatography -- Cation and anion-exchange resins are described in the ion chromatography document. A link for a web text book by Y.V. Kazakevich and H.M. McNair gives and excellent background material for high performance liquid chromatography.
