| Size exclusion chromatography (SEC) is widely used in the area of biochromatography.
The separation mechanism is based on the different size and shape of proteins. |
Small protein molecules are retarded by the column while large molecules pass through more
rapidly.
SEC is useful for the separation of proteins in the range between
5kDa and 1.000kDa, but also larger proteins or other compounds can be separated. The mechanical
stability of Fractogel® EMD BioSEC media allow higher flow rates compared to known soft gels.
This feature is very helpful during regeneration and equilibration of the columns. The pressure
stability also facilitates the packing of larger columns that might sometimes be necessary during
the polishing steps for the production of pharmaceutical proteins. The high stability against
alkali treatment enables users to set up production scale separations on Fractogel® EMD BioSEC.
SEC is a very mild separation method because any desirable buffer system can be used. Thus,
optimal conditions with respect to the protein stability can be selected.
Getting Started Now (Practical Hints)
Column dimensions and sample volumes.
Since no gradients are used for elution, the equipment necessary for SEC can be rather simple. For
SEC, packing of the column must be carried out very carefully to obtain optimal results. For
laboratory purposes, the column dimentions should range from 600mm x 16mm to 1000mm x 50mm. The volume
of the loaded sample should not exceed 5% of the column volume for preparative runs and up to 1%
for analytical applications. For production scale separation, column diameters up to 30cm and gel
bed lengths up 120cm are recommended. The resolution is not correlated to the total amount of
protein loaded on the column. Thus, highly concentrated protein samples will give the best
separations in the case of SEC.
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