Column washing and system cleaning – regular care keeps the system clean

Analytes and impurities in the sample matrix may adsorb longer to HPLC columns and parts of the HPLC system such as tubings, valves or detector chambers than we like sometimes. Therefore, it is important to regularly wash the column and to remove undesired substances in our system. That also prevents column degradation and the need to regenerate the column, a more intensive way to clean columns.

Regular HPLC column washes

Chromatography columns are regularly washed in order to remove all sample and matrix from the system and prevent column degradation. As this happens with the column attached to an HPLC or UHPLC system, the system is consequently also affected by the column washing.

A regular column wash can be integrated either in each injection, after each sequence, daily or even a combination of those. These are established ways for regular column cleanings:

• Column wash after each injection:

This part overlaps with the overall goal of not only separating a sample by chromatography but also eluting all of the injection (sample and matrix) from the system. To this end, an injection contains elongated elution steps, extended gradients or a step gradient to finish an injection.

• Column wash after each sequence:

An established method to clean a column after a sequence of sample injections is to add a separate injection with a cleaning solution. That depends on the type of chromatography mode, sample and column that is used. This can be accompanied by an injection of a standard that serves to prove if the performance of a column is as expected.

• Column wash at the end of the day:

Comparable with wash steps in between different sequences, at the end of the day injection of one or several cleaning solutions serve to remove residual deposits from the column and HPLC system. The system is then changed to the buffer that will be needed the following day and the system is flushed overnight at a low flow rate.

Cleaning of HPLC systems – when and how

Of course, cleaning an HPLC system without a column is possible as well. It becomes necessary when the required conditions are not compatible with the column material and if the source of contamination is suspected to be the system. Especially when changing or adding detection methods, for instance from UV to the more sensitive multi-angle light scattering, contaminations become visible that were not detected before. Depending on materials used in the flow path of the system and on possible contaminations the following cleaning solutions are used:

• Combinations of organic solutions to remove hydrophobic substances and prevent microbial growth (e.g. “Magic Mix” water:acetonitrile:methanol:isopropanol 1:1:1:1 with up to 10% of formic acid)
• Antimicrobial solutions such as sodium azide (0.05 % w/v) to prevent microbial growth
• Basic or acidic solutions to remove charged molecules