Buffer: 10 to 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.0 to 7.4, or select the buffer in which the sample should be stored or solubilized for the next step.
Use 150 mM sodium chloride, or a buffer with equivalent ionic strength, to avoid pH-dependent ionic interactions with the matrix. At very low ionic strength, the presence of a small number of negatively charged groups on the medium can cause retardation of basic proteins.
The sample should be fully dissolved. Centrifuge or filter to remove particulate material (Appendix 3). Always use degassed buffers and maintain a constant temperature during the run to avoid introducing air into the column.
Set an appropriate pressure limit on the chromatography system to avoid damage to the column packing.
First-time use or after long-term storage
Column performance should be checked at regular intervals by determining the theoretical plate number per meter and peak symmetry.
Make sure to not exceed the pressure limits of the column and consider flow limitations (Table 1.3). This is particularly important when working at low temperatures, such as in a cold room, or when the column is used with 20% ethanol or other viscous solutions. Exposure to temperatures outside the range 4 °C to 40 °C will negatively affect the efficiency of a packed bed and the column will need to be repacked.
Table 2.3. Recommended flow rates at different stages using prepacked columns containing Superdex Increase, Superdex, or HiLoad columns containing Superdex prep grade media
† 0.13 mL/min for Superdex 200 Increase 5/150 GL.
Further equilibration might be necessary if the buffer contains detergent.
Routine cleaning after every 10 to 20 separations is recommended, but the frequency of cleaning will also depend on the nature of the samples being applied.
Removing severe contamination
For extreme cases of contamination, check the instructions supplied with the product.