Correct preparation of samples and buffers and application of a high salt wash (1 M NaCl) at the end of each separation should keep most columns in good condition. However, reduced performance, a slow flow rate, increasing back pressure or complete blockage are all indications that the medium needs to be cleaned using more stringent procedures in order to remove contaminants.
It is recommended to reverse the direction of flow during column cleaning so that contaminants do not need to pass through the entire length of the column. The number of column volumes and time required for each cleaning step may vary according to the degree of contamination. If the cleaning procedure to remove common contaminants does not restore column performance, change the top filter (when possible) before trying alternative cleaning methods. Care should be taken when changing a filter as this may affect the column packing and interfere with performance.
The following procedure should be satisfactory to remove common contaminants:
To remove precipitated proteins:
* If contamination is thought to be significant, use a lower flow rate to increase the contact time when using 1 M NaOH.
** When cleaning larger columns, allow a contact time of 1–2 hours for any solution that is used as an initial cleaning step.
Organic solvents or detergents may be required to completely remove contaminants of this type.
Before using organic solvents, wash the medium with at least 4 column volumes of distilled water to avoid any salts precipitating on the column.
When applying organic solvents or solutions it may be necessary to reduce the flow rate to avoid over-pressuring the column.
Use cleaning solutions such as up to 100% isopropanol, up to 100% methanol, up to 100% acetonitrile, up to 2 M NaOH, up to 75% acetic acid, up to 100% ethanol, ionic or non-ionic detergents.
Always check for solvent compatibility in the instructions supplied with the medium or column.
Avoid anionic detergents with Q, DEAE and ANX charged groups. Avoid cationic detergents with S, SP and CM charged groups.
Examples of cleaning procedures:
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