There is no standardized method for cleaning HPLC columns, as the approach varies depending on the type of stationary phase used.

Below is a general cleaning procedure for "silica-based columns". However, it is important to note that manufacturers may have different recommendations, so caution is advised.
Furthermore, the method outlined below does not guarantee successful cleaning in every case. It is a general approach, and adjustments to the cleaning process may be necessary depending on the specific method being used.

0) Common Procedures and Limitations of Cleaning
The elution time for cleaning is typically about 20 minutes at the analysis flow rate. Overly extensive cleaning can damage the column.
Column cleaning involves harsh conditions for the column itself, and therefore the cleaning process has the potential to cause column degradation. It is important to understand that in some cases, avoiding column cleaning can lead to a longer column lifespan.

If the purpose of column cleaning is due to "increased pressure resulting from column clogging", in most cases, the pressure does not decrease after cleaning. This is because column cleaning is designed to clean the "surface of the stationary phase", not to remove debris that is trapped between the packing particles. Generally, debris lodged between the particles cannot be removed by cleaning. It is necessary to pre-treat the sample in advance to ensure that no colloids or fine particles are present in the injected sample.

Biological samples that tend to clog columns include "proteins", "nucleic acids (DNA, RNA)", "polysaccharides", or complex high-molecular-weight compounds of these. These materials cannot be removed by the following cleaning methods. Therefore, it is necessary to perform pre-treatment steps that reliably remove these substances from the sample before injection.

1) Reverse Phase Columns (Cadenza CD-C18, Unison UK-C18, C8, C1, Phenyl)
Generally, increasing the concentration of organic solvent can elute hydrophobic compounds. However, for basic compounds that ionically bind to residual silanol groups, we recommend cleaning with the following solvent mixture containing acetic acid:
Acetonitrile / Water / Acetic Acid = 90 / 10 / 1

For cleaning on the neutral side, the following solvent is recommended:

Acetonitrile / 100mM Ammonium Acetate = 80 / 20

2) Silica Columns (Unison UK-Silica)
Silica columns are sensitive to conditions above weak acidic pH, so it is necessary to clean them at acidic pH. For example, the following methods are available:

[For non-aqueous mobile phase:]
Methanol / Acetic Acid = 100 / 1

[For aqueous mobile phase:]
Water / Acetic Acid = 100 / 1

3) Amino Columns (Unison UK-Amino)
Amino columns are normal phase columns, so they are cleaned by increasing the polarity of the mobile phase. However, general amino columns are weak to aqueous mobile phases, and cleaning with water may result in the loss of ligands.
Unison UK-Amino is highly durable against aqueous mobile phases, so the following cleaning example can be applied:

100mM Ammonium Acetate

4) Ion Exchange Columns (Scherzo SS-C18, SM-C18, SW-C18)
For the cleaning of mixed-mode or multi-mode columns like "reverse phase + ion exchange", cleaning is required by organic solvents and ionic strength.

[Cleaning Example]
Acetonitrile / 100mM Ammonium Acetate = 50 / 50

Even for the same column, the cleaning method varies depending on the method. There is no "standard procedure" for cleaning. Column cleaning must be designed as part of "method development".

WH18 / YAZAWA Itaru,