Methods and Techniques: Silica Gel C18 Cleaning Procedure
Reversed Phase Silica Maintenance Procedures 
In liquid chromatography pharmaceutical processes, there are 4 procedures, which are operated for GMP to ensure a maximum of reliability in the final product obtained.
1. Regeneration:
Normally operated with a more polar solvent, 2 column volumes are enough to remove low migrating and/or strongly bounded impurities from the surface of the RP silica.
This procedure intends to retrieve the original capacity of the RP silica.
2. Re-equilibration:
After the regeneration, the RP silica should be returned to the starting conditions of the chromatography run before the next injection, 2 to 3 column volumes with a short gradient are recommended and often necessary to get and warranty base line UV detection returned to the starting conditions. This procedure ensures the efficiency for the next run.
3. Cleaning:
After a certain number of cycles, dependent of the raw material injected (feed stream), it is necessary to regularly proceed with a “cleaning step”. Most of the time reversed flow in the column is chosen with an adapted protocol.
4. Sanitization:
Between batches, it is recommended to ensure column integrity to perform sanitization cycle to prevent microbial growth and cross-contamination from batch-to-batch.
It is recognized that sodium hydroxide (Na OH) solution 0.1 to 0.5 M is one of the most popular sanitizing agent, its effectiveness, low cost and low toxicity are especially attractive for process chromatography.
Despite extensive bonding chemistry with end-capping protecting the silica, the silica is dissolved at high pH, thus decreasing the lifetime of the RP silica. One method, developed some years ago by Grace-Amicon, (Journal of Chromatography, 503, (1990) p.385-401) utilized the addition of aluminium salt (ion) to mediate the action of the sodium hydroxide during the sanitization cycle.
The experiment in this publication shows that after more than 100 wash cycles with 0.1 to 0.5 M Na OH containing 0.005 M Al NO3, there is no “detectable” deterioration of the silica bed.
Recommendation:
In any case the contact time with NaOH solution should be maximum 30 minutes, and by reversed flow (after the Cleaning cycle) to ensure a maximum of efficient sanitation. It is preferable to decrease the flow rate and to elute half of the column volume with NaOH solution, followed by a strong polar solvent such as methanol or acetonitrile.
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