The ultracentrifuge

Development of the ultracentrifuge with its ability to attain centrifugal forces in excess of 500,000 x g (75,000 rpm, r = 8 cm) opened entirely new fields of investigation. It permitted the fractionation of subcellular 

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The ultracentrifuge has been used extensively, especially for the study of biopolymers, and can be used in several different experimental modes to yield information about polymeric solutes. Of the possible procedures, we shall consider only sedimentation velocity and sedimentation equilibrium. We shall discuss these in turn, beginning with an examination of the forces which operate on a particle setting under stationary-state conditions. 

We have emphasized biopolymers in this discussion of the ultracentrifuge and in the discussion of diffusion in the preceding sections, because these two complementary experimental approaches have been most widely applied to this type of polymer. Remember that from the combination of the two phenomena, it is possible to evaluate M, f, and the ratio f/fo. From the latter, various possible combinations of ellipticity and solvation can be deduced. Although these methods can also be applied to synthetic polymers to determine M, they are less widely used, because the following complications are more severe with the synthetic polymers ... 

Sedimentation coefficients are a measure of the velocity witli which a particle sediments in a centrifugal force field. Sedimentation coefficients are typically expressed in Svedbergs (.symbolized S), named to honor The Svedberg, developer of the ultracentrifuge. One S equals 10 . sec. 

The second virial coefficient B in Eq. 17 refers to the static case. In the ultracentrifuge the measured value can show a speed dependence [39], an effect which can be minimized by using low speeds and short solution columns. If present it will not affect the value of after extrapolation to zero concentration. 

Svedberg T, Pedersen KO (1940) The Ultracentrifuge. Oxford University Press, Oxford, UK... 

The quantity on the left, which is determined by measuring the rate of movement of the sedimentation boundary in the ultracentrifuge operated at the constant angular velocity co, is called the sedimentation constant s. Thus... 

T. Svedberg and K. O. Pedersen, The Ultracentrifuge (Clarendon Press, Oxford, 1940). 

Add 30 mL of 15-50% (w/w) gradient solution to the ultracentrifuge tubes of the swinging bucket rotor (max vol 40 mL). Sucrose concentrations increase from the top to the bottom of the tube. 

Alternatively, in sedimentation equilibrium experiments, the ultracentrifuge is operated at slow speeds for longer time to permit the solute molecules to attain equilibrium between sedimentation and diffusion. If the sample is monodisperse, its concentrations cb c2 can be measured at 2 positions xh x2 in the cell. 

The ultracentrifuge is made up of an aluminium rotor several inches in diameter and it is rotated at high speed in an evacuated chamber. The solution to be centrifuged is kept in a small cell within the rotor near its periphery. The rotor can be driven electrically or by an oil or air turbine. 

The usefulness of the ultracentrifuge in the preparation of samples rather than in the production of analytic data should not be overlooked. Preparative ultracentrifuges have utility in fractionating polymer samples and in freeing them from easily sedimented impurities. 

Equations suitable for simulation of molecular weight distributions for any initial distribution and chosen values of G(scission) and G(crosslinking) have been developed and demonstrated. The molecular weight distributions may be obtained by GPC (with the limitation of changes in relative hydrodynamic volumes) and by sedimentation velocity in the ultracentrifuge. 

Sedimentation coefficients are expressed in Svedbergs (S), after the Swedish biochemist The Svedberg who developed the ultracentrifuge in the 1920s. While S values are indicative of molecular weight, they are not addi-tive-the 70s ribosome is made up of one 50S and one 30S subunit. 

Molecular weights of both synthetic and naturally occurring polymers have been obtained by LS almost as a routine measurement. For reasons which are not wholly evident but which probably have historical and medical connotations, sedimentation in the ultracentrifuge seems to be somewhat preferred by workers in the field of biopolymers, although this technique offers no advantages over LS. Indeed LS can frequently provide additional information and is less time consuming. 

In plasma, Lp(a) moves with a pre-beta-1 mobility (B8, B9) and has been referred to as the sinking pre-beta fraction, as it shares the mobility of VLDL yet does not float at the same density in the ultracentrifuge. Serum electrophoresis followed by a lipid staining with Oil Red O or Fat Red should be a convenient method for detection of elevated Lp(a) levels. Sample collection and preservation are, however, critical parameters in this assay (B26, K9). 

Svedberg, T. Pederson, K.O. (1940). The Ultracentrifuge. Oxford University Press. Tiselius, A. (1947). Adsorption analysis of amino-acids. Adv. Protein Chem. 3, 67-93. Young, E.G. (1963). Occurrence, Classification, Preparation and Analysis of Proteins. In Comprehensive Biochemistry (Florkin, M. Stotz, E.H., Eds.), Vol. 7, pp. 1-55. Elsevier, Amsterdam. 

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