Ultracentrifuge

ULTRACENTRIFUGE, ultracentrifugation. The U. represents a new type of instrument built by the Swedish scientist Svedberg in Uppsala. It received its name by analogy with the ultramicroscope, ultrafiltration, etc., since it is intended for studying the properties of colloidal particles or even molecules. In the U. the factor of a certain arrangement and movement of particles in a solution placed in the field of action of an extremely powerful centrifugal force is used. Three properties characterize the U. and distinguish it from ordinary centrifuges. 1. During centrifugation, convection currents in the liquid are eliminated, which could hinder the uniform settling of the smallest particles. 2. Ultracentrifuges have an attachment for the precise observation of the phenomenon of particle movement (sedimentation) during the rotation itself. For this, a beam of bright light is directed at the rotating part of the centrifuge, which passes through a cuvette with the solution being studied during its rotation, and the resulting picture is photographed; the photographs are analyzed with a microphotometer, and the degree of darkening or change in refraction is used to judge the concentrations in different parts of the liquid. 3. The U. develop centrifugal forces of extremely great intensity. Since 1924, Svedberg has built a whole series of increasingly perfected models of the U. The first U. was built with the following indicators: the centrifugal force exceeded the force of terrestrial gravity 5,000 times, and the number of revolutions equaled 10,000 per minute. The model built in 1934 develops a centrifugal force equal to 900,000 terrestrial gravities, making 145,000 revolutions per minute, at a radius of 36 mm and a height of the liquid layer of 8 mm. At present, two standard types of U. have been developed in Uppsala. The first type covers instruments with a centrifugal force equal to from 5,000 to 15,000 forces of terrestrial gravity; the second type is instruments with a centrifugal force from 15,000 to 900,000 terrestrial gravities (a model with a centrifugal force equal to 1,000,000 forces of terrestrial gravity is realized). The first type is equipped with a vertical rotor. The second has a horizontal rotor, rotating to reduce friction in a hydrogen atmosphere at reduced pressure of the latter. With the help of the U., two kinds of measurements leading to the determination of molecular weight and other molecular quantities can be performed. 1) The method of observation of sedimentation equilibrium (Sedimentations - Gleichgewichtsmethode) consists in the fact that the final distribution of concentrations at various heights of the liquid, which has occurred as a result of the action of the centrifugal force on an initially homogeneous system, is observed. This state is established when equilibrium is reached between the rate of sedimentation, on the one hand, and diffusion on the other. If now two concentrations C₁ and C₂ are measured in the layers of liquid located at a distance x₁ and x₂ from the axis of rotation, then the molecular weight M can be calculated by the following formula: M = - 2RT ln Vc where R is the gas constant, T is the absolute temperature, V is the partial specific volume of the dissolved substance, d is the density of the solvent, ω is the angular velocity. 2) The method of observation of the rate of sedimentation; in this case photographs are taken at equal intervals of time (e.g., 3 min.). From the change in concentration found in the photographs in different layers of the liquid, the rate of sedimentation of the particles is judged. This rate, referred to a unit of force of the centrifugal field, is called the sedimentation constant, the value of which at a certain temperature and in a certain solvent appears characteristic for each kind of molecules and is interesting both in itself and in that it can serve, if the diffusion constant is known, for the calculation of molecular weight and other molecular quantities. The field of application of the U. is extremely great; with these instruments it is possible to perform: 1) determination of the molecular weight of high-molecular-weight substances with low density and low-molecular-weight substances with high density. 2) Detection of homogeneity or heterogeneity (polydispersity) of particles of high-dispersion colloidal solutions and carrying out molecular weight analysis of a mixture. 3) With the determination of the sedimentation constant, it is often possible to trace the finest reactions of aggregation or dissociation in biological media. 4) By a combination of the measurement of equilibrium and the rate of sedimentation, some conclusions can be drawn regarding the external appearance of molecules, namely, to characterize to what extent the shape of the molecules differs from spherical. 5) Furthermore, the frequency distribution of particles in high-dispersion colloids can be determined. So far the following classes of substances have been studied: proteins, polysaccharides, polystyrenes, coloring matters and other synthetic high-molecular-weight organic substances, as well as some inorganic colloids and inorganic salts. Let us mention the main results obtained in this respect in view of their importance for biology and medicine. It turned out that natural proteins, in contrast to synthetic colloids, are extraordinarily homogeneous. For example, the solution of Hb of human blood (molecular weight 69,000) turns out to be highly homogeneous; furthermore, it has been established that the molecular homogeneity of a protein (especially proteins with a high molecular weight) does not remain unchanged at all pH. Each protein has its own well-defined pH range within which it remains homogeneous. When the pH passes beyond the boundaries of the stability region, a breakdown of molecules into smaller particles occurs, or, conversely, their aggregation into larger ones. This process of breakdown is reversible. In many cases, near the boundary of stability, one or several products of dissociation (whose molecular weight can also be determined) coexist next to the unchanged molecules of the substance taken. It is interesting that different proteins and their dissociation products have repeating and very few figures for molecular weights, namely multiples of the number 34,500. Thus, Svedberg was able to arrange proteins in a series according to the principle of increasing molecular weight in the following classes: 1, 2, 3, 4, 6, 12, 24, 48, 96, 192 times multiples of 34,500. The single molecular weight has egg albumin, insulin, pepsin; to the second class belong serum albumin and hemoglobin. Of carbohydrates, cellulose and starch were studied with the help of the U. For native cellulose, an approximate value of the weight of the particle was found - 300,000. Solutions of starch proved to be heterogeneous and, depending on the preparation, showed different partial weights. In conclusion, it should be mentioned that recently in Uppsala the solutions of some inorganic salts were investigated in the U. In most of the cases studied so far (CsJ, KJ0₃, T1N0₃, HgCl₂) the found molecular weights coincide well with those calculated from the chemical formula (average error 10%). The rapid improvement of the U. makes it probable that it will be possible to extend this new type of analysis also to dissolved substances with a small molecular weight.