Ultracentrifuge

[uhl-truh-sen-truh-fyooj]

The ultracentrifuge is a centrifuge optimized for spinning a rotor at very high speeds, capable of generating acceleration as high as 1,000,000 g (9,800 km/s²). There are two kinds of ultracentrifuges, the preparative and the analytical ultracentrifuge. Both classes of instruments find important uses in molecular biology, biochemistry and polymer science. Theodor Svedberg invented the analytical ultracentrifuge in 1923, and won the Nobel Prize in Chemistry in 1926 for his research on colloids and proteins using the ultracentrifuge.

The vacuum ultracentrifuge was invented by Edward Greydon Pickels. It was his contribution of the vacuum which allowed a reduction in friction generated at high speeds. Vacuum systems also enabled the maintenance of constant temperature.

In 1946, Pickels cofounded Spinco (Specialized Instruments Corp.) and marketed an ultracentrifuge based on his design. Pickels, however, considered his design to be complicated and developed a more “foolproof” version. But even with the enhanced design, sales of the technology remained low, and Spinco almost went bankrupt. The company survived and was the first to commercially manufacture ultracentrifuges, in 1947. In 1949, Spinco introduced the Model L, the first preparative ultracentrifuge to reach a maximum speed of 40,000 rpm. In 1954, Beckman Instruments (now Beckman Coulter) purchased the company, forming the basis of its Spinco centrifuge division.

Analytical ultracentrifuge

In an analytical ultracentrifuge, a sample being spun can be monitored in real time through an optical detection system, using ultraviolet light absorption and/or interference optical refractive index sensitive system. This allows the operator to observe the evolution of the sample concentration versus the axis of rotation profile as a result of the applied centrifugal field. With modern instrumentation, these observations are electronically digitized and stored for further mathematical analysis. Two kinds of experiments are commonly performed on these instruments: sedimentation velocity experiments and sedimentation equilibrium experiments.

Sedimentation velocity experiments aim to interpret the entire time-course of sedimentation, and report on the shape and molar mass of the dissolved macromolecules, as well as their size-distribution. The size resolution of this method scales approximately with the square of the particle radii, and by adjusting the rotor speed of the experiment size-ranges from 100 Da to 10 GDa can be covered. Sedimentation velocity experiments can also be used to study reversible chemical equilibria between macromolecular species, by either monitoring the number and molar mass of macromolecular complexes, by gaining information about the complex composition from multi-signal analysis exploiting differences in each components spectroscopic signal, or by following the composition dependence of the sedimentation rates of the macromolecular system, as described in Gilbert-Jenkins theory.

Sedimentation equilibrium experiments are concerned only with the final steady-state of the experiment, where sedimentation is balanced by diffusion opposing the concentration gradients, resulting in a time-independent concentration profile. Sedimentation equilibrium distributions in the centrifugal field are characterized by Boltzmann distributions. This experiment is insensitive to the shape of the macromolecule, and directly reports on the molar mass of the macromolecules and, for chemically reacting mixtures, on chemical equilibrium constants.

The kinds of information that can be obtained from an analytical ultracentrifuge include the gross shape of macromolecules, the conformational changes in macromolecules, and size distributions of macromolecular samples. For macromolecules, such as proteins, that exist in chemical equilibrium with different non-covalent complexes, the number and subunit stoichiometry of the complexes and equilibrium constant constants can be studied.

Preparative ultracentrifuge

Preparative ultracentrifuges are available with a wide variety of rotors suitable for a great range of experiments. Most rotors are designed to hold tubes that contain the samples. Swinging bucket rotors allow the tubes to hang on hinges so the tubes reorient to the horizontal as the rotor initially accelerates. Fixed angle rotors are made of a single block of metal and hold the tubes in cavities bored at a predetermined angle. Zonal rotors are designed to contain a large volume of sample in a single central cavity rather than in tubes. Some zonal rotors are capable of dynamic loading and unloading of samples while the rotor is spinning at high speed.

Preparative rotors are used in biology for pelleting of fine particulate fractions, such as cellular organelles (mitochondria, microsomes, ribosomes) and viruses. They can also be used for gradient separations, in which the tubes are filled from top to bottom with an increasing concentration of a dense substance in solution. Sucrose gradients are typically used for separation of cellular organelles. Gradients of caesium salts are used for separation of nucleic acids. After the sample has spun at high speed for sufficient time to produce the separation, the rotor is allowed to come to a smooth stop and the gradient is gently pumped out of each tube to isolate the separated components.

Hazards

The tremendous rotational kinetic energy of the rotor in an operating ultracentrifuge makes the catastrophic failure of a spinning rotor a serious concern. The stresses of routine use and harsh chemical solutions eventually cause rotors to deteriorate. Proper use of the instrument and rotors within recommended limits and careful maintenance of rotors to prevent corrosion and to detect deterioration are necessary to avoid this hazard.