Ultracentrifugation, problems

The reports constitute a complete set of recommendations for the ultracentrifugation problem posed to SpinPro. Thus, SpinPro performs the advisory role of an ultracentrifugation expert interviewing the investigator for the problem description, offering expert advice on the most appropriate centrifugation procedure, and finally, comparing alternative procedures. 

Reliance on procedures reported in the literature has compounded the problem. Often these procedures, perhaps initiated by investigators with a limited set of rotors, are inefficient by today s standards the rotor is inappropriate, the run speed is too slow, or the run time is too long. A new investigator applying this procedure does not take full advantage of the potential of ultracentrifugation. 

The Optimal Plan Report describes an optimal ultracentrifugation procedure designed to solve the problem described in the dialogue. It uses the most appropriate rotor from the... 

A summary of the report follows The problem is to separate proteins. Furthermore, SpinPro should pay particular attention to the purity of the separation. The sample is not negatively affected by sucrose, has a sedimentation coefficient of 16 Svedbergs, and is in liquid form of 3 mL and a concentration of 1% w/w. The protein of interest should be placed 45% from the top of the gradient at the end of the run. Of the gradient concentrations 10-40% and 5-20%, the 10-40% is preferred by the investigator. There are no solvents in the sample that are harmful to the tubes. Finally, from the lab, SpinPro should use the L2-75B ultracentrifuge and the SW 41 Ti rotor, which does not require a speed derating due to its age. 

Updates to SpinPro continue as new rotors and new techniques are developed or as inadequacies are found. New expert systems techniques, such as the ability to incorporate the principles of a problem domain, rather than just the experience of the expert, should give SpinPro the ability to design procedures for novel problems and to explain its reasoning. The updates insure that SpinPro will be a repository of knowledge about the current state of ultracentrifugation SpinPro s expertise should continue to improve. Furthermore, the expert remains gainfully employed as a final arbitrator on the inclusion or exclusion of any new knowledge. 

It would be of considerable interest to extend the technique just presented to problems involving nonlinear equations because there are many situations in ultracentrifugation where nonideality is a dominant feature. Furthermore, it is known (4, 14) that even for two-component systems with nonideality the theory for estimating the sedimentation constant based on a diffusion-free (c = 0) approximation can lead to systematic error. Therefore, the development of an approximate procedure for nonlinear equations would be useful for further progress in analytical separation methods. 

The sedimentation equilibrium experiment requires much smaller volumes of solution, about 0.15 ml. With six-hole rotors and multichannel centerpieces (41) it is potentially possible to do fifteen experiments at the same time. For situations where the photoelectric scanner can be used one might (depending on the extinct coefficients) be able to go to much lower concentrations. Dust is no problem since the centrifugal field causes it to go to the cell bottom. For conventional sedimentation equilibrium experiments, the analysis of mixed associations under nonideal conditions may be virtually impossible. Also, sedimentation equilibrium experiments take time, although methods are available to reduce this somewhat (42, 43). For certain situations the combination of optical systems available to the ultracentrifuge may allow for the most precise analysis of a mixed association. The Archibald experiment may suffer some loss in precision since one must extrapolate the data to the cell extremes (rm and r6) to obtain MW(M, which must then be extrapolated to zero time. Nevertheless, all three methods indicate that it is quite possible to study mixed associations. We have indicated some approaches that could be used to overcome problems of nonideality, unequal refractive index increments, and unequal partial specific volumes. 

In general, we are satisfied with the performance of our OMA-based light detector system for the absorption optical system in the ultracentrifuge. Many of the problems that we had to solve were inherent in the optical system and the cells, but were hidden to users with photomultiplier detector systems. Other problems associated with the SIT vidicon have been satisfactorily solved. 

A number of methods have been developed for studying self-assembly processes including light scattering, ultracentrifugation, and electron microscopy. All of these methods have some associated problems when applied to studying self-assembly however, applying several of these... 

We did not succeed in isolating the presumed colloidal carbon from a degraded PVC solution either by electrolysis or by means of an ultracentrifuge. With highly viscous solutions and maximum carbon activity this task would have been a great problem. 

With respect to the virtually very low sample loads, one could argue that the determined diffusion coefficient is at infinite dilution. However, there is no possibility in FFF techniques to perform a safe extrapolation to infinite dilution as in analytical ultracentrifugation or dynamic light scattering. In addition there is a severe problem for all particles deviating from the spherical shape (see Sect. 4.2.3 for a detailed discussion), as the evaluation of D from the FFF experiments using the conventional theory is inappropriate. 

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