gel filtration 2

In order to minimize sample dilution use a maximum sample volume that gives the resolution required between the peaks of interest.
The ratio of sample volume to column volume influences resolution, where higher sample volume to column volume ratios give lower resolution.
Resolution in gel filtration increases as the square root of bed height. Doubling the bed height gives an increase in resolution equivalent to = 1.4 (40%). For high resolution fractionation long columns will give the best results and a bed height between 30–60 cm should be satisfactory. Sufficient bed height together with a low flow rate allows time for all intermediate molecules to diffuse in and out of the matrix pores and give sufficient resolution.
Media Selection
Chromatography media for gel filtration are made from porous matrices chosen for their inertness and chemical and physical stability. The size of the pores within a particle and the
particle size distribution are carefully controlled to produce a variety of media with different selectivities. Today s gel filtration media cover a molecular weight range from 100 to 80 000 000, from peptides to very large proteins and protein complexes.
Superdex is the first choice for high resolution, short run times and high recovery.
Sephacryl is suitable for fast, high recovery separations at laboratory and industrial scale.
Superose offers a broad fractionation range, but is not suitable for large scale or industrialscale separations.
After deciding upon Superdex, Sephacryl or Superose, select the medium with the fractionation range that covers the molecular weight values of interest in your sample. In cases where two media have a similar fractionation range: select the medium with the steepest selectivity curve for best resolution of all components in the sample. When you are interested in a specific component, select the medium where the log molecular weight of the target component falls in the middle of the selectivity curve.
Sephadex is ideal for rapid group separations such as desalting and buffer exchange. Sephadex is used at laboratory and production scale, before, between or after other chromatography purification steps.
Sephadex G-25 is recommended for the majority of group separations involving globular proteins. This medium is excellent for removing salt and other small contaminants away
from molecules that are greater than Mr 5 000.