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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".
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