The Analytical Ultracentrifuge

The Business Centre has a Beckman XL-A analytical ultracentrifuge (AUC), equipped with scanning absorption optics. We also have access via the main NCMH Laboratory to another XL-A, an XL-I (with Rayleigh interference optics) and two Model E centrifuges. One Model E has been adapted with on-line data capture and analysis software for schlieren and interference optics, the other has been adapted for high temperature (up to 70? C) experiments.

Sedimentation Equilibrium

All samples sent to NCMH should preferably be thoroughly dialysed against the selected buffer which should, when absorption optics are being used, be free of components (e.g. partially oxidised thiols) which may absorb in the chosen wavelength range. If this is not possible, then one of NMCH staff may advise of an alternative procedure. Interference or Schlieren optics (Beckman XL-I or Model E) have no problems in this area, but careful dialysis against solvent remains essential in most cases.

Molecular mass is most often determined by sedimentation equilibrium on an analytical ultracentrifuge. For this experiment approximately 100 microlitres of sample are needed, extensively dialysed against the chosen buffer. This will provide the mass at one solute concentration. Often it is useful to perform a dilution series to screen for self-interaction or thermodynamic non-ideality. Up to 9 samples can be run at one time for this purpose.

The concentration of sample required depends on the chosen optical system for observation of the equilibrium solute distribution. The Beckman Optima XL-A analytical ultracentrifuge (AUC) follows the absorption of the system and can be set to wavelengths of between 190 - 400nm or 401 - 800nm. If this system is used the OD of the sample in a 12mm path-length cell should lie in the range 0.1 < OD12mm < 0.5 at the chosen wavelength. If the absorption exceed this limit there are two options: the use of a 3mm path-length cell or the use of schlieren optics (on-line Model E) which result from the refractive index of the solute. For these optics there is no upper limit to solute concentration but the lower limit is typically 1 mg/ml for proteins and polysaccharides. For more dilute samples which have no chromophore highly precise Rayleigh interference optics (Beckman XL-I or on-line Model E) may be the best option. Here the concentrations must lie in the range 0.2 - 2.0 mg/ml for proteins and polysaccharides. Only 3 samples can be run at one time with both these types of optics. An equilibrium study can last from hours to days, dependent on the molecular weight and the sample volume.