Centrifugation Application Notes

A-2037A

A PPLICATION I NFORMATION

U l t r a c e n t r i f u g a t i o n

O PTIMIZING C ENTRIFUGAL S EPARATIONS : S AMPLE L OADING

One of the critical factors in obtaining satisfactory separations by density gradient centrifugation is the relationship between sample size and gradient capacity. What is the sample capacity of a given gradient? How best can the sample be applied to the gradient? These concerns are particularly important in rate-zonal separations where the sample is applied as a narrow zone and gradient capacities are generally more limited than those of isopycnic gra- dients. A number of investigators have explored this question (1,2), especially in zonal rotors where maximizing sample load is important. There has, however, been a general failure to develop a model that would predict with any degree of accuracy the sample capacity of a given gradient in a way that today’s investigators would find useful. Most equa- tions require knowledge of the molecular weight and/or the partial specific volume of the macromol- ecules of interest. However, even when those prop- erties are known, the sample amounts which prove to be optimum experimentally are about 60% of the predicted values. Many factors play a role in determining how much sample a gradient will hold, but observation and experience have shown that 10 µ g-0.5 mg of sample for each mL of gradient volume is a reason- able first approach. Within that range, the type of gradient used, the number of components in the sample and their sedimentation coefficients, and the resolution desired will all affect the optimum sam- ple load. From the standpoint of higher quality sep- arations, the lower end of the range is preferred; less is definitely best. However, since the majority of gradients are fractionated and assayed, the actual

determinant of how small a sample can be put on a gradient is often the sensitivity of the detection method. Given these dual but opposing goals, optimum resolution and enough sample material to detect, what factors might be manipulated to maximize sample load while retaining adequate resolution? An overriding rule which cannot be violated whether the sample is a zone on top of a sucrose density gradient or a band in an equilibrium gradi- ent, is that the density should increase throughout Figure 1. Plot of a relatively shallow gradient. For the lighter area, a sample of density 1 ( ρ 1 ) loaded at the radial position 1 (r 1 ) is stable since ρ 1 at r 1 < ρ 2 at r 2 . However, a sample of density ρ 3 (darker area) produces a density inversion since ρ 3 at r 1 > ρ 2 at r 2 .