离心技术基础

The single most important advance in the use of centrifugal force to separate biologically important substances. was the coupling of mechanics, optics and mathematics by T. Svedberg and J.W. Williams in the 1920's. They initiated the mathematics and advanced the instrumentation. 1 to a point where it was possible to prove that proteins were large molecules that could be weighed in a centrifuge. 2. In honor of that work, the value for a molecule's (or organelle's) sedimentation velocity in a centrifugal field is known as its Svedberg constant or S value for short.

The instrumentation has progressed quite far since the early work of Svedberg and Williams. Today, any technique employing the quantitative application of centrifugal force is known as ultracentrifugation. The design of the basic instruments, the rotors and the optical systems for measurement are too extensive to cover in this volume. For our purposes, we will concentrate on two types of rotor, and a few selected parameters to be measured.

ROTORS

Table F.1. Rotor Characteristics

Figure F.1. Cross section of Sorvall SS-34 rotor.  

Rotors for a centrifuge are either fixed angles , swinging buckets , continuous flow, or zonal, depending upon whether the sample is held at a given angle to the rotation plane, is allowed to swing out on a pivot and into the plane of rotation, designed with inlet and outlet ports for separation of large volumes, or a combination of these. Figure F.1 demonstrates the characteristics of each of these.

Fixed angles generally work faster; substances precipitate faster in a given rotational environment, or they have an increased relative centrifugal force for a given rotor speed and radius. They also have few (or no) moving parts on the rotor itself and thus have virtually no major mechanical failures, other than potential metal stress, which all rotors undergo. These rotors are the work-horse elements of a cell laboratory, and the most common is a rotor holding 8 centrifuge tubes at an angle of 34 ° C from the vertical (such as the Sorvall SS-34 rotor or the Beckman JA-20). Figure F.1 presents a cross- sectional diagram of the Sorvall SS-34.

Swinging bucket rotors (also known as horizontal rotors) have the advantage that there is usually a clean meniscus of minimum area. In a fixed angle rotor, the materials are forced against the side of the centrifuge tube, and then slide down the wall of the tube. This action is the primary reason for their apparent faster separation, but also leads to abrasion of the particles along the wall of the centrifuge tube. For a swinging bucket, the materials must travel down the entire length of the centrifuge tube and always through the media within the tube. Since the media is usually a viscous substance, the swinging bucket appears to have a lower relative centrifugal force, that is it takes longer to precipitate anything contained within. If, however, the point of centrifugation is to separate molecules or organelles on the basis of their movements through a viscous field, then the swinging bucket is the rotor of choice. Moreover, if there is a danger or scraping off an outer shell of a particle (such as the outer membrane of a chloroplast), then the swinging bucket is the rotor of choice. Most common clinical centrifuges 1 have swinging buckets. Since the buckets are easy to interchange, this type of rotor is extremely versatile. Its major drawback is the number of moving parts which are prone to failure with extended use.

Nearly all cell biology laboratories will have several examples of fixed angle and horizontal rotors. While the sample volumes of these rotors can be significant, they are limiting. To overcome this limitation, a continuous flow centrifuge can be used. Limnologists often employ such a device to separate plankton from gallons of lake water. Cell biologists employ zonal rotors for the large scale separation of particles on density gradients. Zonal rotors can contain up to 2 liters of solution and can work with tissue samples measured in ounces (or even pounds). The rotors are brought up to about 3000 RPM while empty, and the density media and tissues are added through specialized ports. This type of rotor has a distinct preparative advantage over the gradient capacity of more typical rotors.