Ultracentrifuge. Early High-Tech Machine. Vacuum Ultracentrifuge
As a scientific instrument, the ultracentrifuge is used primarily in biochemical research to sediment substances from a solution. A rotor with containers holding the solution is spun at high speeds, causing the solute to move towards the periphery of the container under influence of the generated gravitational fields. Historically, however, ultracentrifuges were first developed for analytical purposes to determine the size of colloidal particles.
Early High-Tech Machine. Beginning about 1910 and through the 1920s, Swedish chemist Theodor Svedberg analyzed the particle sizes of colloids. Under the influence of gravity, these particles gradually separated from a solvent at a speed depending upon their size. To improve the accuracy of his method, he began to use centrifuges to generate forces many times that of the earth’s gravitational field.
He developed this idea into a practical method and could subject the colloid particles to forces up to 5000 g (5000 times the force of gravity). In analogy with the common methods of ultrafiltration and the ultramicroscope, he called the apparatus an ultracentrifuge.
Svedberg used the new apparatus to determine particle sizes of both inorganic and organic colloids including proteins, expecting that these, like inorganic colloids, would show a wide distribution of particle sizes. However, when he analyzed hemoglobin, all particles seemed to have the same size. This suggested that this protein could be a well-defined molecule, which was a revolutionary thought at the time. To be sure, however, further analyses would be needed using fields of the order of 100,000g.
This 20-fold improvement was difficult to achieve, but he succeeded after investing huge sums of money and conquering a variety of problems. The rotor, driven by oil turbines to facilitate lubrication of the bearings, spun in a hydrogen atmosphere to reduce heat production. The substance to be analyzed was placed in a special container and the sedimentation of the particles caused by the gravitational forces was recorded by illuminating the process with a special light source and taking photos at regular intervals. Some hours of calculation were needed to deduce the particle sizes from these photographs.
After experiments with this new ultracentrifuge Svedberg concluded that hemoglobin was indeed a monodisperse protein. After this surprising result, research at his laboratory became almost exclusively focused on proteins. In the following decade he tried to improve his ultracentrifuges, especially to increase the forces that could be generated. Almost all parts of the apparatus were optimized, including the shape of the turbines and turbine chambers, oil inlets, bearings, type of oil used, rotor balancing method, rotor size, and so on.
By 1937 Svedberg considered that he had reached the limit of modifications. He used his latest ultracentrifuge, which generated 400,000 g, until his retirement in 1949, after which the protein research was continued by his former colleagues. It was not until the mid-1970s, half a century after the first apparatus was developed, that the oil-turbine ultracentrifuge was taken out of use.
Vacuum Ultracentrifuge. American physicist Jesse Wakefield Beams studied optical phenomena in the 1920s. In his research he used rapidly rotating mirrors mounted on small conically shaped spinning tops (of the order of 1-2 cm in diameter) that were driven by compressed air. After 1930 he started to make these tops hollow, which allowed him to use them as small centrifuges. Because of the high speeds that could be achieved, Beams called them ultracentrifuges. He identified a wide variety of applications for the apparatus, including Svedberg’s method of determining molecular weights.
Edward Greydon Pickels, one of Beams’ students, developed the apparatus further for this application. This proved difficult because the high speeds heated the rotor and caused convection currents in the sedimentary solution. Pickels tried various solutions until in 1935 he produced a design in which the rotor spun in vacuum. A small wire, passing through a vacuum-tight gland, connected the rotor to the driving air turbine. This design solved all convection problems, allowing forces up to 1,000,000 g.
This design attracted the attention of scientists at the Rockefeller Institute for Medical Research in New York, and Pickels developed two types of ultracentrifuge further for them. The first type, the analytical ultracentrifuge, was used to determine particle sizes and was analogous to Svedberg’s method. The second type, the preparative ultracentrifuge, could separate a substance from a solvent and was primarily used for concentrating viruses.
Beams attempted to make his apparatuses as simple as possible to allow many scientists to use them. His instrument makers made some ultracentrifuges for others, which led to a limited distribution in the scientific world. Around 1937 his vacuum ultracentrifuge was marketed by an American company, but this resulted in commercial failure.
Svedberg was also prepared to sell his ultracentrifuges to others, but the apparatus was extremely expensive—in the order of $20 000—which was an enormous amount of money for scientists in those days. In the early 1940s the apparatus was marketed by a Stockholm company, but once again it was a commercial failure.
In 1946 Pickels was approached by a salesman who wanted to market an analytical ultracentrifuge based on Pickels’ design. Together they formed Spinco, or Specialized Instruments Corporation. Pickels considered his design at the time too complicated and developed a more easily operated, foolproof version. Sales, however, remained low, and Spinco nearly went bankrupt. Subsequently, Pickels concentrated on developing the preparative ultracentrifuge, which seemed to sell reasonably well. This gave Spinco sufficient financial power to continue the production of the analytical ultracentrifuge as well, although in small numbers. Over the years, that number gradually rose.
Increasing numbers of scientists used ultracentrifuges for biochemical research, forming a research community that met at symposia and conferences at regular intervals. The ultracentrifuge was still not fully developed. As more scientists used them, each with their own approach and interest, new desires and problems in connection with the apparatus were articulated. Over the years a variety of ultracentrifuges have been developed to the point that it became a very common instrument in laboratories for many types of biochemical research.
Date added: 2023-11-02; views: 254;