Biochemical Techniques and Instrumentation

4.2.1.2. Analytical ultracentrifuge

Analytical ultracentrifuge can generate a centrifugal field of 2,50,000g in its analytical cell to separate high molecular weight sub-cellular molecules or organelles that differ only slightly in density. It contains a solid rotor which has one analytical cell and one counterbalancing cell. The sample is placed in a simple analytical cell at a right angle to the axis of rotation. Then, it is placed in a cylindrical hole on the rotor, balanced by a counter  balancing blank cell with the same weight on the opposite side. The rotor is then placed on a spindle attached to a motor below the centrifuge.

Rotors are mostly made out of titanium alloy covered with a polyurethane layer. The rotor chamber is evacuated with a vacuum pump to reduce air friction, and cooled with a cooling jacket to maintain a constant temperature and prevent overheating. An optical system enables the sample to be observed throughout the duration of centrifugation. This light absorption system is known as Schlieren system. The beam of light is passed through a quartz window in the bottom of the chamber, through transparent glass windows on the top and bottom of the sample cell, and pass out through another window in the top of the chamber, where it is directed by mirrors to an optical detection device. The light is always ON and a signal is detected only when the sample cell passes through the detection path.

The solution of molecules is then introduced into the sample cell along with a solvent while the rotor is spinning. It requires only small sample size of 20-120 μl at low particle concentrations of 0.01-1g/l. The solvent, cesium chloride dissociates into high-density Cs⁺ ions that migrate outwards in the direction of the centrifugal force, forming a shallow density gradient. The molecules in solution diffuse centripetally. For eg., when there are two molecular species, the lighter ones migrates faster, and the denser ones lags behind. The discrete change in density at the interface between the two regions bends the light passing through the sample cell at that point, and superimposition of adjacent signals is seen as a spike at the transition point. The size and position of the spike are indications of quantity and molecular weight, respectively. This is mainly used for sedimentation equilibrium analysis. These observations are electronically digitized and stored for further mathematical analysis.