Liquid Chromatography A Method of Chemical Separation

Liquid chromatography A method of chemical separation that involves passage of a liquid phase through a solid phase and relies
on subtle chemical interactions to resolve complex mixtures into pure compounds. A small amount of the sample to be separated is injected onto the top of a column that is densely packed with spherical particles of small diameter, that is, the stationary phase. A liquid solvent, the mobile phase, flows through the column continuously to carry the sample from the top to the bottom of the column. During passage through the column, the components of the sample are transferred back and forth continuously between the two phases, and small thermodynamic differences in the chemical interactions of the various sample components with the mobile and stationary phases slow the passage of some solutes more than others and lead to their separation. The technique can be performed on very small scales for chemical analysis, dealing with micrograms or even nanograms of sample, or it can be performed on an industrial scale for purification of commercial products. The technique has great resolving power. In the late 1960s, workers realized that to achieve maximum
performance they needed to make the stationary-phase particles very small. As the stationary-phase particles became smaller, they packed too densely to permit gravity-driven flow of the mobile phase. It was necessary to force the mobile phase through the column under high pressure, and the technique was named high-pressure liquid chromatography (HPLC). The meaning of the acronym has been changed to high-performance liquid chromatography, for the elegant separations that are possible. Typical stationary-phase particles are monodisperse, macroporous silica particles either 3 or 5 micrometers in diameter, and the column lengths for analytical scale separations are on the order of 2 10 in. (525 cm), with inside diameters of about 0.16 in. (4 mm). The columns are made of stainless steel, which is relatively inert chemically and able to withstand the high pressures applied to the top of the column. Since these columns require pressures of a few hundred to a few thousand pounds per square inch, depending on the mobile-phase flow velocity desired, a high-pressure metering pump is an integral part of a modern liquid chromatograph. Most instruments include a means of performing gradient elution, that is, making a continuous change in
the composition of the liquid mobile phase during the separation process. Gradient elution can be performed by using a separate pump for each solvent and changing the relative proportions during the separation, or by using a proportioning valve between the solvent reservoirs and the pump. An injector valve is used to introduce a small volume of sample, typically 5100 microliters, onto the top of the column without interrupting the mobilephase flow. These valves can be operated manually, or they can be programmed to perform injections from a tray of samples for routine analyses. After the sample traverses the column, a flowthrough detector is employed to generate the chromatogram, which is the visual representation of the separation. Detectors can provide both quantitative and qualitative information about the separated components. Temperature control of the column and detector is important; they are generally operated at or near room temperature, and temperature fluctuations can adversely affect the reproducibility of the separation and detection steps. Liquid chromatography very much depends on the highly selective chemical interactions that occur in both the mobile and stationary phases. Rapid separations have become possible for
compounds whose difference in free energy of transfer between the two phases is only a few calories per mole. Columns exhibiting virtually every type of possible selectivity exist, including selectivity by shape, charge, size, and optical activity. Additional selectivity can be generated through manipulations of the mobile phase; additives that incan create unique selectivities in columns that do not normally show that type of selectivity. In addition to facilitating chemical analysis, liquid chromatography can be used to obtain physicochemical information. Diffusion coefficients, kinetic parameters, critical micelle concentrations of surfactants, and other information have been estimated from chromatographic data. The most common application is the estimation of hydrophobic parameters, especially as models of biological or environmental partitioning processes (most frequently, of octanol-water partitioning). Bioavailability, bioaccumulation, soil sorption, and various other factors are estimated based on linear free-energy relationships.teract with the solute in the mobile phase

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Liquid chromatography A method of chemical separation that involves passage of a liquid phase through a solid phase and relies

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