Separation ultracentrifugation

Lipoproteins (from human plasma). Individual human plasma lipid peaks were removed from plasma by ultracentrifugation, then separated and purified by agarose-column chromatography. Fractions were characterised immunologically, chemically, electrophoretically and by electron microscopy. [Rudel et al. Biochem J 13 89 1974.]... 

Toyopearl HW-75 resin, with pores larger than 1000 A, have been used in place of ultracentrifugation steps for the purification of plasmid DNA. Ultracentrifugation is a time-consuming process and requires expensive chemicals, such as cesium chloride. Toyopearl HW-75 resin provides superior separation performance for plasmid DNA and also provides high yields (54). 

Separation methods based on size include size exclusion chromatography, ultra-filtration, and ultracentrifugation (see Chapter Appendix). The ionic properties of peptides and proteins are determined principally by their complement of amino acid side chains. Furthermore, the ionization of these groups is pH-dependent. 

Techniques which seem less suitable for routine size analysis are (1) analytical ultracentrifugation combined with a Schlieren optical system (Mason and Huang, 1978 Weder and Zumbuehl, 1984) (2) the sedimentation field flow fractionation (SFFF) technique to separate heterogeneous dispersions (e.g., Kirkland et al., 1982). 

Physical separation of granulometric fractions by sedimentation and ultracentrifugation could also help in constraining weathering rates at local scale. The fine fractions are useful when they exclusively contain secondary minerals, that is, when they are not polluted by... 

The components appearing on ultracentrifuging have been separated by fractional precipitation with potassium chloride,248 and the physical proper-... 

Most organelle membranes, such as the tonoplast (6) and the Golgi apparatus (7), can be separated by density gradient ultracentrifugation of plant cell homogenates. However, other effective methods for the isolation of the plasma membrane (8,9) have been described. Moreover, another method that uses an aqueous two-phase system for the isolation of ER is also described (10). Those interested in these details for these methods should consult the original articles. 

Physical separation methods can be based on equilibrium considerations, but the majority are not. Ordinary filtration is an example of a non-equilibrium, physical method and so is ordinary centrifugation— e.g.—the separation of a precipitate from the suspending liquid using an artificial gravity field. There are separation methods, which are called filtration which are not such as gel filtration. Ultracentrifugation in a salt gradient is a physical equilibrium method. 

Size analyses are commonly carried out by mixing the pigment powder with an organic solvent or with water and adding appropriate surfactants to enhance the dispersibility of the powder. Aqueous dispersions frequently undergo size separation in ultracentrifuges, while organic solvents are more appropriate for electron microscopic techniques. 

Discrete particle size separation or fractionation is carried out in the strong gravitational field of rapidly rotating ultracentrifuges. Theoretical background and experimental technique are described in depth in the literature [17-19], While there... 

Molecules that vary significantly in their size can be separated by ultrafiltration or dialysis, while molecules that are only slightly different in size can often be separated by gel permeation chromatography. Ultracentrifugal techniques, while apparently separating on the basis of size, are strictly speaking more influenced by the mass and density of the molecule and to a lesser extent by its shape. 

The movement of the analyte is an essential feature of separation techniques and it is possible to define in general terms the forces that cause such movement (Figure 3.1). If a force is applied to a molecule, its movement will be impeded by a retarding force of some sort. This may be as simple as the frictional effect of moving past the solvent molecules or it may be the effect of adsorption to a solid phase. In many methods the strength of the force used is not important but the variations in the resulting net force for different molecules provide the basis for the separation. In some cases, however, the intensity of the force applied is important and in ultracentrifugal techniques not only can separation be achieved but various physical constants for the molecule can also be determined, e.g. relative molecular mass or diffusion coefficient. 

There are basically three ways in which separation methods can be performed. They are most easily described in relation to chromatography but they are also relevant to other methods such as electrophoresis and ultracentrifugation. 

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