Chromatography, Liquid
Authors: Lunn George 
Published: July 2004

This article describes the modern practice of analytical high performance liquid chromatography (HPLC). Liquid chromatography involves the separation of compounds by differential migration as a liquid mobile phase flows over a solid stationary phase. The mode of separation varies depending on the mobile and stationary phases. In HPLC small stationary‐phase particle sizes and highly controlled conditions are used to achieve high resolutions.

A representative HPLC instrument consists of a mobile phase reservoir, a high pressure pump, an injection device, a separation column, a detector, and a data system.

In some cases, sample preparation is as simple as filtering the sample before injection. However, in many cases, particularly drugs in biological fluids, complex sample preparation procedures are required to obtain reproducible results. Methods such as liquid–liquid extraction, solid‐phase extraction, and column‐switching are frequently used.

Derivatization may be used to improve the detectability of the compounds of interest. Chiral separations can be achieved by using chiral derivatization reagents. Postcolumn reactions can also be used to derivatize molecules after they have been chromatographed.

The most commonly used detectors detect eluting compounds by their ultraviolet (uv) absorbance, fluorescence, mass spectrum, or electroactivity (electrochemical detection).

As originally developed, liquid chromatography (LC) involved an unmodified polar solid stationary phase, such as alumina or silica, and a nonpolar liquid mobile phase, such as octane. Separation is achieved by the adsorption of analyte molecules on the surface of the stationary phase. Less polar (or hydrophobic) molecules are more weakly adsorbed, and hence elute quicker, than more polar (or hydrophilic) molecules. Normal phase chromatography may also take place using some kinds of modified silica stationary phases, eg, cyano, diol, or amino.

By far, the most common HPLC technique is reversed‐phase HPLC. In reversed‐phase HPLC stationary phases consisting of chemically modified silica are used with polar mobile phases, eg, methanol:water 50:50. Typically, these chemically modified stationary phases have nonpolar long‐chain hydrocarbon groups (eg, an aliphatic chain containing 18 carbons, designated as C18) bonded to the surface but many variations have been developed, particularly in recent years. The long‐chain organic groups behave like an organic liquid and molecules of the analytes partition between this nonpolar stationary phase and the polar mobile phase. Less polar (or hydrophobic) molecules spend more time in the stationary phase, and hence elute slower, than more polar (or hydrophilic) molecules. This is the reverse of the situation with normal phase chromatography.

Other common techniques are ion‐exchange chromatography, size‐exclusion chromatography (also known at gel‐permeation chromatography), and chiral chromatography.

HPLC remains the most important chromatographic method for pharmaceutical applications. Pharmaceutical applications can be divided into two broad categories: analysis of the drug substance or dosage form and the analysis of drugs in bodily fluids (eg, blood or urine). The drug substance is a chemical substance that contains the active ingredient (eg, aspirin or ibuprofen). The drug substance is often formulated with excipients and made into a dosage form (eg, tablets, capsules) that can be consumed by the patient.

HPLC is also used extensively in environmental applications and is used as an analytical technique in many other fields.