Sephadex® LH-20 should be packed in a solvent-resistant (SR) column selected from Table 6.3 according to the column volume required for the separation.
Simple steps to clarify a sample before applying it to a column will avoid the risk of blockage, reduce the need for stringent washing procedures and extend the life of the chromatography medium. Filter or centrifuge all solvents and samples before use.
In solvents such as chloroform, Sephadex® LH-20 is less dense than the solvent and the medium will ﬂoat. Pour the medium into the column and drain until the second adapter can be inserted. Lock the adapter in position at the surface of the medium and direct the ﬂow of chloroform upwards. The bed will be packed against the top adapter and the lower adapter can be pushed slowly upwards towards the lower surface of the medium.
Close the column outlet when moving the adapter to avoid compressing the bed.
Start at a linear ﬂow of 1 cm/h to check resolution. Low ﬂow rate improves the resolution.
Wash the column with 2 to 3 column volumes of the solvent, followed by re-equilibration in a new solvent if changing the separation conditions.
Sephadex® LH-20 is stable in most aqueous and organic solvent systems. The medium is not stable below pH 2.0 or in strong oxidizing agents.
Store dry at 4 °C to 30 °C. Store packed columns and used medium at 4 °C to 8 °C in the presence of a bacteriostatic agent.
Transfer Sephadex® LH-20 from an aqueous solution to the organic solvent by moving through a graded series of solvent mixtures. This will ensure efﬁcient replacement of the water by the required solvent.
To transfer from aqueous solution or organic solvent (100% A) to a new organic solvent (100% B), proceed as follows: transfer to 70% A:30% B then to 30% A:70% B and ﬁnally to 100% B. If A and B are not mutually miscible, make the transfer via an intermediate solvent, for example from water to chloroform via acetone, as shown in Figure 6.3.
Figure 6.3.Suggested routes for changing to organic solvents.