Column chromatography liquid phase

Chromatographic and electrophoretic separations are truly orthogonal, which makes them excellent techniques to couple in a multidimensional system. Capillary electrophoresis separates analytes based on differences in the electrophoretic mobilities of analytes, while chromatographic separations discriminate based on differences in partition function, adsorption, or other properties unrelated to charge (with some clear exceptions). Typically in multidimensional techniques, the more orthogonal two methods are, then the more difficult it is to interface them. Microscale liquid chromatography (p.LC) has been comparatively easy to couple to capillary electrophoresis due to the fact that both techniques involve narrow-bore columns and liquid-phase eluents. 

The distinguishing features of gas chromatography are a gaseous mobile phase and a solid or immobilized liquid stationary phase. Liquid stationary phases are available in packed or capillary columns. In the packed columns, the liquid phase is deposited on a finely divided, inert solid support, such as diatomaceous earth or porous polymer, which is packed into a column that typically has a 2- to 4-mm id and is 1 to 3 m long. In capillary columns, which contain no particles, the liquid phase is deposited on the inner surface of the fused silica column and may be chemically bonded to it. In gas-solid chromatography, the solid phase is an active adsorbent, such as alumina, silica, or carbon, packed into a column. Polyaromatic porous resins, which are sometimes used in packed columns, are not coated with a liquid phase. 

Typical thin-layer separations are performed on a glass plate that is coated with a thin and adherent layer of finely divided particles this layer constitutes the stationary phase. The particles are similar to those described in the discussion of adsorption, normal- and reversed-phase partition, ion-exchange, and size-exclusion column chromatography. Mobile phases are also similar to those employed in high-performance liquid chromatography. 

Although liquid chromatography techniques have become quite popular in the separation of peptides in complex protein digests, they are yet to make an impact for the separation of protein samples for proteome-wide applications. It is envisioned that in the future their application for protein separation will increase. Various combinations of reversed-phase (RP)-HPLC with ion-exchange, size-exclusion, chromato-focusing (CF), IEF, and capillary electrophoresis (CE) have emerged for 2D separation of complex mixtures of proteins and peptides. A recent addition in this field is the use of CF as the first dimension and RP-FIPLC as the second-dimension separation device.14 CF is a column-based liquid-phase separation technique, in which proteins are fractionated on the basis of differences in their p/values in a weak ion-exchange column. 

Column chromatography Liquid chromatography performed by moving the mobile phase through a packed column using gravity. 

The aqueous phase may be injected if the sample is sufficiently concentrated. A number of problems may be encountered under these circumstances. When water is converted to steam, the volume increases dramatically 1 il of water becomes more than 1000 jtl of steam. This is larger than the injector volume of many current gas chromatographs, and the steam may degrade the performance of the system. Polar gas chromatography liquid phases such as Carbowax and PEG will degrade in the presence of steam unless they are bonded to the column. 

Liquid phase chromatography can use a supercritical fluid as an eluent. The solvent evaporates on leaving the column and allows detection by FID. At present, there are few instances in the petroleum industry using the supercritical fluid technique. 

Mixtures passed through special columns (chromatography) in the gas phase (GC) or liquid phase (LC) can be separated into their individual components and analyzed qualitatively and/or quantitatively. Both GC and LC analyzers can be directly coupled to mass spectrometers, a powerful combination that can simultaneously separate and identify components of mixtures. 

The application of pressure to the liquid phase in liquid chromatography generally increases the separation (see HPLC). Also in PIC improved efficiency of the column is observed if pressure is applied to the mobile phase (Wittmer, Nuessle and Haney Anal Chem 47 1422 1975). 

Katz et fl/.[l] searched the literature for data that could be used to identify the pertinent dispersion equation for a packed column in liquid chromatography. As a result of the search, no data was found that had been measured with the necessary accuracy and precision and under the sufficiently diverse solute/mobile phase conditions required to meet the second criteria given above. It became obvious that a... 

Figure 4-1. Components of a simple liquid chromatography apparatus. R Reservoir of mobile phase liquid, delivered either by gravity or using a pump. C Glass or plastic column containing stationary phase. F Fraction collector for collecting portions, called fractions, of the eluant liquid in separate test tubes.

The stationary phase matrices used in classic column chromatography are spongy materials whose compress-ibihty hmits flow of the mobile phase. High-pressure liquid chromatography (HPLC) employs incompressible silica or alumina microbeads as the stationary phase and pressures of up to a few thousand psi. Incompressible matrices permit both high flow rates and enhanced resolution. HPLC can resolve complex mixtures of Upids or peptides whose properties differ only slightly. Reversed-phase HPLC exploits a hydrophobic stationary phase of... 

Other modes of LC operation include liquid-liquid partition chromatography (LLC) and bonded phase chromatography. In the former, a stationary liquid phase which is immiscible with the mobile phase is coated on a porous support, with separation based on partition equilibrium differences of components between the two liquid phases. This mode offers an alternative to ion exchange in the fractionation of polar, water soluble substances. While quite useful, the danger exists in LLC that the stationary phase can be stripped from the column, if proper precautions are not taken. Hence, it is typical to pre-equil-ibrate carefully the mobile and stationary phases and to use a forecolimn, heavily loaded with stationary phase 9). 

High performance liquid chromatography (HPLC) has been by far the most important method for separating chlorophylls. Open column chromatography and thin layer chromatography are still used for clean-up procedures to isolate and separate carotenoids and other lipids from chlorophylls and for preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography. The whole analysis should be as brief as possible, since each additional step is a potential source of epimers and allomers. 

Emenhiser, C. et al.. Separation of geometrical carotenoid isomers in biological extracts using a polymeric Cjq column in reversed-phase liquid chromatography, J. Agric. Food Chem., 44, 3887, 1996. 

Tswett s initial column liquid chromatography method was developed, tested, and applied in two parallel modes, liquid-solid adsorption and liquid-liquid partition. Adsorption ehromatography, based on a purely physical principle of adsorption, eonsiderably outperformed its partition counterpart with mechanically coated stationary phases to become the most important liquid chromatographic method. This remains true today in thin-layer chromatography (TLC), for which silica gel is by far the major stationary phase. In column chromatography, however, reversed-phase liquid ehromatography using chemically bonded stationary phases is the most popular method. 

Compared with liquid column chromatography, in PLC there is a certain limitation with respect to the composition of the mobile phase in the case of reversed-phase chromatography. In planar chromatography the flow of the mobile phase is normally induced by capillary forces. A prerequisite for this mechanism is that the surface of the stationary phase be wetted by the mobile phase. This, however, results in a Umitation in the maximum possible amount of water applicable in the mobile phase, is dependent on the hydrophobic character of the stationary RP phase. To... 

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