Buffer: 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.2 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 nonionic interactions with the matrix. At very low ionic strength, the presence of a small number of negatively charged groups can cause retardation of basic proteins and exclusion of acidic 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 (see Table 1.3)
Set an appropriate pressure limit on the chromatography system to avoid damage to the column packing. Use lower ﬂow rates for high viscosity solutions and low temperature
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. Prepacked columns are supplied with recommended values. See Appendix 1 on how to check column efficiency.
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.
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.
If required, Sephacryl HR may be autoclaved repeatedly at 121 °C, pH 7, for 30 min without signiﬁcantly affecting its chromatographic properties. The medium must be removed from the column as autoclaving can damage column components. Note that HiPrep columns cannot be repacked.
To remove severe contamination
Reverse the ﬂow and wash at a ﬂow rate of 10 cm/h (0.3 mL/min for column 16/60 or 0.8 mL/min for 26/60) at room temperature using the following solutions:
For extreme cases of contamination, check the instructions supplied with the product.
Reversing ﬂow through a column packed with Sephacryl® media should only be considered under cases of severe contamination. Reversing the ﬂow can cause channeling through the packed bed leading to poor resolution, reduced efﬁciency, and the need to repack the column. Professionally packed columns are less likely to be affected, but extreme care must be taken.