Liquid chromatography (LC) is a separation technique used to separate, identify, and quantify each component in a mixture. It relies on the differences in chemical interactions between the analytes in a mixture and the mobile and stationary phases used for the separation. With LC, individual analytes are separated as they flow through a chromatography column under the influence of a liquid mobile phase.
High performance liquid chromatography (HPLC) was established as a powerful separation technique during this time period. Continuous developments since then have focused on improving column technology, mobile phase composition, detection methods, and software for data analysis. Today, LC is used extensively across many industries and fields of research.
The development of Liquid chromatography started in the early 1900s with the work of Russian scientist Mikhail Tswett. He used calcium carbonate to separate plant pigments and dyes through adsorption on a column. This work led to the discovery of chromatography as an analytical technique. In the following decades, researchers worked to develop new stationary and mobile phases to improve separations. By the 1950s and 1960s, reversed-phase chromatography had been introduced, using non-polar stationary phases. This allowed for the separation of more polar compounds that did not interact strongly with early normal phase systems. In the 1970s and 1980s, columns packed with porous silica particles significantly improved separation efficiency and speed due to reduced diffusion distances within the particles.
Instrumentation of a Basic Liquid Chromatography System
A basic LC system consists of five main components: the mobile phase reservoir, pump, injector, chromatography column, and detector. The mobile phase reservoir contains the liquid that will carry the sample through the system. HPLC pumps precisely control the flow rate and pressure of the mobile phase. Common pumps operate at flow rates ranging from nanoliters to milliliters per minute at pressures up to 400 bar. The injector introduces a small, reproducible volume of the sample solution into the column. Modern instruments use autosamplers for automated injection of multiple samples. The column is where separation of the sample components occurs. It contains the stationary phase, usually silica based, that interacts differently with each analyte. Detectors, such as UV-Vis or mass spectrometers, are used at the end of the column to identify and quantify the separated analytes as they elute. Chromatography software integrates the detector signal, performs calculations, and generates a chromatogram for analysis.
Separation Mechanisms in Liquid Chromatography
The separation of analytes in LC occurs primarily based on differences in their distribution between the mobile and stationary phases. This distribution depends on a complex balance of interactions like polarity, size, shape, hydrophobicity, and charge. There are two main modes of separation: normal phase LC and reverse phase LC. Normal phase uses a polar stationary phase like silica or alumina and a nonpolar mobile phase like hexane or dichloromethane. This separates compounds based mostly on differences in polarity. Reverse phase LC, the more commonly used mode, operates with a nonpolar stationary phase like C18 bonded silica and a polar mobile phase like water/acetonitrile. Here, separation is driven largely by differences in hydrophobicity. Ion-exchange chromatography also separates ions based on charge interactions with charged functional groups on the stationary phase. Other modes like size-exclusion use differences in molecular size for separation. Proper selection of the stationary and mobile phases is crucial for achieving good separation of the analytes of interest.
Applications and Development of Liquid Chromatography
LC finds widespread use across many fields and industries due to its versatility and high separation power. In pharmaceutical analysis, it is commonly employed for impurity testing, quantitative analysis of active ingredients and degradation products, dissolution testing of dosage forms, extractables/leachables analysis, and more. The food industry relies on LC for testing pesticide residues, food additives, flavors, vitamins, and more. Environmental applications include analysis of pollutants, wastewater effluents, and environmental toxins. LC-MS has become a ubiquitous tool for biomedical research in analyzing drugs and metabolites, proteins, peptides, and other biomolecules. It also enables the comprehensive analysis of complex natural products. Continuous developments are improving LC techniques to address new analytical challenges. Some promising areas include ultrahigh pressure LC and sub-2 μm particles for fast, high resolution separations, multidimensional LC for complex mixtures, and multi-mode stationary phases enabling multiple mechanisms in one column. Coupling to new detection techniques like mass spectrometry also expands the scope of analytes that can be studied by LC. With its robust performance and flexibility, liquid chromatography will certainly remain a core analytical technique across sciences for years to come.
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