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There are several possible causes for
changes in retention time and peak shape when switching HPLC
columns.
1. Instrumentation environment
If both the
column and the instrument were changed simultaneously, it is
important to suspect changes in the instrument environment. In the
case of a gradient system, differences in mixer capacity can lead to
delays in the system, which then manifest as differences in
retention time. To achieve consistent retention times in gradient
analysis, such as for inter-room reproducibility, it is essential to
use instruments from the exact same manufacturer, model, and even
the same model version.
In
low-pressure gradient systems, the mechanism for mixing the mobile
phase differs between manufacturers. Therefore, when comparing
instruments other than the same model, achieving consistent gradient
slopes and corresponding retention reproducibility becomes quite
challenging.
(Reference)
Differences in
gradient systems
2. Column-to-column variation
Even when
using the same instrument, sample, and mobile phase, replacing the
column can result in different retention times. This is due to
variations in the packing batch. Differences in ligand quantity and
residual silanol content manifest as changes in retention and peak
shape.
It is extremely risky to create a
method based on a single column and then continue using that same
column for actual analysis. Many issues arise when a column degrades
after several years, and upon replacing it with a new column,
reproducibility is lost.
It is impossible to manufacture columns with identical ligand
molecule counts on the stationary phase surface. Once a method is
created, it is essential to immediately verify the robustness of the
method using a different column (preferably from a different packing
batch) to ensure method validation.
3. Analytical conditions
When the
analytical method is not robust, even between new columns,
differences in retention, separation, and peak shape may occur. The
primary cause of this is "insufficient ion strength of the mobile
phase."
When
analyzing ionic compounds with only water/organic solvents, there is
a concern that the dissociation (high polarity) and non-dissociation
(low polarity) states become incomplete, leading to instability in
retention and peak shape. Even if pH adjusters are added to the
mobile phase, insufficient concentration may prevent proper
dissociation control of the solute, resulting in irreproducible
results. For example, a 1mM salt concentration.
Silica-based stationary phases have
acidic silanol groups on their surface, which tend to interact
ionically with basic compounds, leading to retention. Additionally,
the uneven distribution of silanol groups can cause peak shape
distortion. When changing columns, the amount of silanol also
changes, so in methods with insufficient ion strength, there is a
risk of variation in peak retention and shape.
To ensure the same results when changing columns, it is necessary to
increase the ion strength in the mobile phase. For example, using
50–100mM salt concentrations.
Some column manufacturers use
pyridine/phenol as an example to demonstrate the benefits of
endcapping, but even weak ions like pyridine can cause gradual peak
changes during repeated analyses if the pH and ion strength in the
mobile phase are not properly controlled. Moreover, if ionic
impurities in the sample adsorb onto the stationary phase, they can
have an ionic effect on the solute’s retention and peak shape.
Analyzing strong ions, such as tertiary amines, with only
water/organic solvents is even more risky.
If the solute structure contains ionic characteristics, it is
essential to design the mobile phase with not only the appropriate
pH but also adequate ion strength.
Even if the solute is not ionic, solute structures with polar
groups, such as amides, can experience strong electrostatic
interactions with silanol or siloxane groups. As a result, when
changing columns, retention and peak shape may fluctuate. Increasing
the ion strength of the mobile phase, as well as adjusting pH or
temperature, can improve reproducibility.
It is crucial not to rely solely on a pH meter when adjusting the pH
and ion strength of the mobile phase.
(Reference)
Method for
preparing mobile phase buffer
To improve inter-room
reproducibility of the method, it is crucial to have a robust mobile
phase design that ensures the same results even when changing
columns.
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