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Good reproducibility may be obtained when the same column is used
repeatedly, but when it is replaced with another column of the same
brand and type, the retention times may change and reproducibility
may be lost.
There are two major causes: differences in the packing
material and the robustness of the analytical
method.
■ Different Packing Material Batches Can Give Different
Retention
In an HPLC column, retention occurs through interactions between
the solute and the stationary phase, such as ODS groups bonded to
the silica surface. Therefore, if the amount of stationary phase
differs, the magnitude of these interactions also changes, resulting
in changes in retention time.
For example, if the specific surface area of the base silica
changes, the amount of stationary phase that can be bonded to its
surface also changes. Different production batches of packing
material may have slight differences in such physical properties.
Therefore, some change in retention between different packing
material batches is not unusual.
This effect is particularly noticeable in isocratic
elution.
In isocratic analysis, the solute travels through the column
while repeatedly adsorbing to and desorbing from the stationary
phase under a mobile phase of constant composition. Therefore, even
a small change in the interaction with the stationary phase can
affect the retention time.
In gradient elution, on the other hand, the elution strength of
the mobile phase is continuously increased to elute the solute.
Therefore, the effect of small differences in the stationary phase
on retention time tends to be smaller.
■ Another Cause Is “Method Robustness”
Changes in retention time after replacing a column cannot always
be attributed to variation in the packing material. The analytical
conditions themselves may also be insufficiently robust.
If functional groups of the solute interact nonspecifically not
only with the stationary phase but also with polar sites such as
silanols or siloxanes near the stationary phase, even small
differences in the surface properties of the packing material may
affect retention.
A typical example is the analysis of compounds containing ionic
functional groups using only water/acetonitrile as the mobile phase.
Under these conditions, neither pH nor ionic strength is
sufficiently controlled.
As a result, not only the intended hydrophobic
interactions, but also secondary ionic interactions
and electrostatic interactions may contribute to
retention.
Under such conditions, even a slight difference in the column
surface can change the balance of these interactions, resulting in a
change in retention time simply by replacing the column.
■ pH and Ionic Strength Are Important Even in Reversed-Phase
Analysis
Just because an analysis is performed in reversed-phase mode does
not necessarily mean that water and acetonitrile alone are
sufficient.
If the solute contains ionic or polar functional groups and
secondary interactions such as ionic or electrostatic interactions
may occur, it is important to properly control the pH and
ionic strength of the mobile phase.
In other words, improving column-to-column reproducibility
requires not only consistent columns but also a robust analytical
method.
When a method shows poor reproducibility, or when
solute–stationary phase interactions are strong, it is also
recommended to consider gradient analysis as the standard
approach rather than isocratic analysis.
Why Is Gradient Elution the Standard Approach in HPLC?
In general, columns manufactured from the same batch of packing
material should provide nearly identical chromatograms when one
column is replaced with another.
However, this alone does not demonstrate that the analytical
method itself is reproducible.
■ A Practical Approach to Method Development
There is no problem with using an old column while developing
analytical conditions. Once the analytical method has been
established, replace it with a new column and check the
reproducibility.
If the old and new columns provide the same results, the method
can be considered robust.
For this reproducibility check, or robustness evaluation, it is
even more important, whenever possible, to confirm that the same
separation can be obtained using columns manufactured from
different batches of packing material.
If columns from different packing material batches produce
essentially the same analytical results, the method can be
considered robust and relatively insensitive to small
column-to-column differences.
■ Summary
“Obtaining reproducible results from repeated analyses using the
same column” and “obtaining reproducible results after replacing the
column” are not the same kind of reproducibility.
Checking the method by replacing the column — robustness
evaluation — is an important part of developing a reliable
HPLC method.
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