Centrifugation Application Notes

the sample and gradient in the direction of the cen- trifugal field. If the rate of this increase in density is relatively rapid, a gradient is described as steep, as opposed to shallow. Steep gradients have a greater sample capacity than shallow gradients. In Figure 1 the sample zone represented by the lightly shaded region meets that criterion, while the sample zone represented by the darker area does not. In Figure 2, because the gradient is steeper, both sample zones meet that criterion even though they represent the same amount as in Figure 1. Thus, the most commonly used way to increase sample capacity is to use a steeper gradient. A variation on this technique is the use of a convex rather than a linear gradient (Figure 3). This

Bottom of Gradient

Top of Gradient

Figure 3. Plot of convex gradient where ρ is density and r is the radial distance from the center of rotation.

Once the capacity of a given gradient for a sam- ple has been estimated or determined empirically, it is important that the sample is loaded onto the gra- dient so that (1) the sample zone is as thin as possi- ble and (2) the sample zone is stable with respect to the gradient. The total density of the sample zone, including macromolecules, buffer and other salts, must be less than the density of the top of the gradi- ent. If the sample has been concentrated from a large volume (several fractions from a column sepa- ration, for example), the concentration of salts may be so high that it will be necessary to dialyze it before layering on the gradient. The previous discussion has focused primarily on rate-zonal separations where the goals of resolu- tion and detection are most likely to be in conflict. Isopycnic banding in equilibrium density gradients can also pose the problem of loss of resolution with increasing sample load, but the problem is less seri- ous for two reasons. The sample is usually incorpo- rated homogeneously within the gradient column, thus eliminating the need to layer it as a concentrat- ed solution. Secondly, gradients formed in this methodology are usually quite steep. One factor which should be taken into account when estimating sample load for this type of separa- tion is the density difference between the sample components. It has been observed (3) that for a small density difference such as 0.005 g/mL, the amount of sample which gives a good separation is as little as 5 µ g per mL of gradient when a swinging bucket rotor is used. Twice that amount is well sep- arated, however, when a fixed angle rotor is used.

Figure 2. Plot of a steeper gradient. Since the slope of the gradient is steep, both ρ 1 at r 1 < ρ 2 at r 2 and ρ 3 at r 1 < ρ 2 at r 2 . Thus, both sample loads are stable.

gradient shape can be particularly valuable when complex samples are being separated. The upper steep portion of the gradient, where most of the sample components are still present, has a high sample capacity. The bottom of the gradient, where sample load is smaller, is less steep, facilitating sep- aration of components with small differences in sed- imentation velocity. A second approach for increasing sample load on rate-zonal gradients is to use a rotor with large tubes. From the standpoint of absolute sample capacity it provides a larger volume of gradient but, more importantly, it provides a larger cross-section- al area over which the sample can be layered.

2