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The sedimentation velocity method can be employed to estimate sample purity.

Sedimentation patterns can be obtained using the Schlieren optical system. This

method measures the refractive index gradient at each point in the ultracentrifugation

cell at varying time intervals. During the entire duration of the sedimentation velocity

analysis, a homogeneous preparation forms a single sharp symmetrical sedimenting

boundary. Such a result demonstrates that the biological macromolecules analysed

exhibit the same molecular mass, shape and size. However, one can not assume that

the analysed particles exhibit an identical electrical charge or biological activity.

Only additional biochemical studies using electrophoretic techniques and enzyme/

bioassays can differentiate between these minor subtypes of macromolecules with

similar molecular mass. The great advantage of the sedimentation velocity method is

Toroidal

diffraction

grating

Incident

light

detector

Reflector

Sample/reference

cell assembly

Rotor

Imaging system for

radial scanning

Slit (2 nm)

Photomultiplier tube

Aperture

Xenon

flash lamp

Reference

Top

view

Sample

Fig. 3.8Schematic diagram of the optical system of an analytical ultracentrifuge. The high-intensity xenon

flash lamp of the Beckman Optima XL-A analytical ultracentrifuge shown here allows the use of wavelengths

between 190 nm and 800 nm. The high sensitivity of the absorbance optics allows the measurement of

highly dilute protein samples below 230 nm. (Courtesy of Beckman-Coulter.)

96 Centrifugation