Short Column Sedimentation Equilibrium Analysis for Rapid Characterization of Macromolecules in Solution


Short column sedimentation equilibrium analysis offers many advantages for researchers wanting quick, sure characterization of the solution properties of macromolecules. Presented here is a list of when short column methods are an appropriate choice, a brief theoretical treatment and an overview of short column sedimentation methods, including ones diagnostic for self-association and nonideality. Short column methods were first described by Van Holde and Baldwin in 1958, and centerpieces specifically designed for these methods were described by Yphantis in 1960. Their distinguishing characteristics are:

  1. The parallel rows of four, small, sample viewing (1.2 mm diameter) holes 4 mm apart.
  2. The two rows of larger (2.4 mm) holes used for filling the cell.
  3. The connecting groove between the fill hole and sample viewing holes.

Despite their early development, short column sedimentation equilibrium methods did not enjoy extensive use with the early users of sedimentation equilibrium for several reasons:

  1. Manual data acquisition from short columns required nearly the same amount of time as from longer columns and did not yield as much information per data set
  2. They required higher concentrations of material
  3. They could not be used with the photoelectric scanner of the Model E
  4. They often required that a separate experiment be conducted to measure the sample concentration. However, a great deal has changed in the intervening years, and all of these limitations have been overcome.

Data acquisition takes seconds now, whereas it could require days in the early ’60s. Thus, there is a distinct advantage to the rapid equilibration available with short columns in that they provide at least a 20-fold increase in the number of samples that can be examined in a given period of time. Thus, with a few exceptions, the quantity of information available from short columns exceeds that from the longer columns. Short column centerpieces can be used with the photoelectric scanner of the Proteomelab XL-A analytical ultracentrifuge, hence lower concentrations may be used and a separate concentration determination is not necessary. Finally, improvements in data analysis have made it possible to combine the data from several short column experiments in order to evaluate the molecular parameters of interest. These same programs eliminate the need for separate concentration determinations for interference optics, saving material and time. With these advances, the advantages of short columns make them the method of first-choice for many routine analyses.

The advantages of short column centerpieces are:

  1. Only a small volume (15 μL vs. 100 μL) of material is required at moderate concentrations (0.1 OD)
  2. Short equilibration times are needed (60–90 min vs. 16–18 h for most molecules)
  3. A large number of samples can be examined simultaneously
  4. There is minimal radial redistribution of solutes

The first advantage is a clear benefit when only small quantities of a material are available for analysis. Even more attractive is the possibility of recovering much of the sample volume (75–90%) after analysis. When it is desirable to examine a sample over a wide range of buffer conditions, the first three advantages are of interest. The final advantage is of particular interest when it is necessary to perform titrations or when characterizing an association between dissimilar macromolecules (i.e., heteroassociation).

These advantages are so compelling, it is useful to outline when it is better to use the longer, 3-mm column centerpieces. The longer columns are better to use when:

  1. Examining heterogeneous samples
  2. Examining low molecular weight materials
  3. When only very dilute samples are available
  4. When the sticking of a sample to centerpiece walls makes it desirable to exploit the lower surface area-to-volume ratio of the 3-mm column centerpieces

Below are described some typical applications for short column centerpieces. These uses have been arranged in sequence ranging from the most simple to the more complicated. It is important to note, however, that the basic methodology is the same and that it is the data analysis and interpretation that vary in complexity. This has the fortunate consequence that even if diagnostic analysis indicates that a chemical system is exhibiting complicated behavior, it is often only the method of analysis that needs to be modified, and there is no need to repeat experiments.

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