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— The
Limitations and Reproducibility of Isocratic Analysis —
In HPLC, gradient elution should
be considered the fundamental approach.
Isocratic analysis should be regarded as the exception.
This becomes easier to understand
when we consider gas chromatography (GC).
In GC, temperature programming is standard practice, and no one
considers constant-temperature operation to be the default method.
This is because GC utilizes the fact that the strength of
solute–stationary phase interactions changes with temperature.
The same principle applies in
HPLC.
The strength of
solute–stationary phase interactions changes with mobile phase
composition. Therefore, performing separation while varying the
mobile phase is a natural and rational design strategy.
The Risks of Isocratic Analysis
When the interaction between
the solute and the stationary phase is strong, isocratic analysis
tends to become unstable.
Even slight changes in
retention time may lead to complaints such as:
“The column lacks
reproducibility.”
However, in many cases, the
true cause is not the column but insufficient robustness of the
method itself.
Under isocratic conditions,
even small differences in mobile phase composition directly
translate into differences in retention.
This is particularly critical when ionic interactions are involved.
Minor variations in pH or ionic strength can significantly influence
separation behavior.
In such systems, it is
difficult to expect high reproducibility from isocratic analysis.
Problems with Isocratic
Analysis Using Multi-Solvent Pump Mixing
In recent years, it has become
common to perform isocratic analysis by mixing solvents using two-
to four-solvent pump systems.
Although this approach appears
convenient, the mobile phase may not be perfectly homogeneous at the
molecular level. As a result, solute–stationary phase interactions
may become microscopically non-uniform, potentially reducing
retention and separation reproducibility.
While this method can be
effective during method development for condition screening, once
development is complete, the mobile phase should be prepared as a
single premixed solution. Final reproducibility should be confirmed
using one pump with a single, well-defined mobile phase.
Conditions Under Which
Isocratic Analysis Can Work
Isocratic analysis tends to be
relatively stable only when weak interactions, such as hydrophobic
interactions, are dominant.
In simple reversed-phase
separations using ODS columns, small fluctuations in organic solvent
ratio do not usually cause dramatic changes in retention or
separation behavior.
However, the situation differs
when ionic interactions are involved.
Consider whether the analyte
is:
In such systems, even slight
differences in mobile phase composition alter intermolecular
interaction energies, resulting in significant retention changes.
The Nature of the Stationary
Phase
HPLC packing materials are
polymer-based materials.
It is
impossible to reproduce surface-bonded ligands with exact
molecular-level uniformity. This is fundamentally similar to the
impossibility of producing polymer molecules with identical
molecular weights.
Therefore, the assumption that
“stationary phases are completely identical” is not scientifically
valid.
When strong interactions
dominate under isocratic conditions, these subtle structural
differences are more likely to appear as retention variability.
A Historical Misconception
In the early days of HPLC, when
instrumentation was extremely expensive, ODS columns were commonly
operated using a single pump.
Because this configuration
persisted for many years, a misconception spread:
“In HPLC, isocratic analysis is
the standard.”
However, a method developed
under instrumental limitations is not necessarily the theoretically
optimal one.
When Isocratic Elution Is
Unavoidable
In the separation of isomers or
closely related analogues, it may be necessary to use long columns
with shallow gradients or even isocratic elution.
Even in such cases, careful
selection of mobile phase composition is essential to achieve
reproducibility.
Particular attention must be
paid to minimizing variables such as:
-
Ionic strength
-
pH
-
Solvent purity
Advantages of Gradient Elution
Gradient methods offer clear
advantages:
Strongly retained components
can be efficiently eluted, enabling both shorter analysis times and
improved sensitivity.
The Importance of Instrument
Selection
Low-pressure gradient systems
differ among manufacturers and models in their mixing mechanisms.
As a result, the following
parameters vary between instruments:
For high-speed analysis and
high reproducibility, the use of high-pressure gradient systems with
binary pumps is strongly recommended.
(Reference)
High-Pressure vs. Low-Pressure
Gradient in HPLC
How to Find
an HPLC Analytical Method
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