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There is a technique known as ion-pair chromatography (IPC). This
method is employed when highly polar ionic compounds cannot be
retained hydrophobically on a reverse-phase column such as ODS. By
adding an alkyl compound (ion-pair reagent) with a counterion to the
mobile phase, these ionic compounds can interact with the stationary
phase and be retained effectively.
Although this method may seem convenient at first glance, it has
a "significant drawback." Frequent fluctuations in retention can
occur with the re-preparation of the mobile phase or when switching
columns. This variability can make it challenging to achieve
consistent results, undermining the reliability of ion-pair
chromatography.
The fundamental cause of this issue lies in the fact that there
can be two possible states of interaction between the solute and the
ion-pair reagent within the column. These two states can lead to
variability in retention, making it difficult to achieve consistent
and reproducible results in ion-pair chromatography.
1. When the Ionic Bond Between the Solute and the
Ion-Pair Reagent is Strong:
As envisioned in ion-pair chromatography, highly polar solutes
form a salt with the ion-pair reagent, transforming into hydrophobic
compounds. This enables them to adsorb and desorb hydrophobically
onto the reverse-phase stationary phase, such as ODS (as
illustrated: reversed-phase mode). This interaction allows for
retention and separation of polar ionic compounds that otherwise
would not be retained on the hydrophobic stationary phase.
2. When the Hydrophobicity of the Ion-Pair Reagent is
High (The Pitfall):
In this case, the ion-pair reagent interacts strongly with the
hydrophobic groups of the ODS or similar stationary phases through
hydrophobic interactions. As a result, the stationary phase
transitions from "reverse-phase mode" to "ion-exchange mode."
Consequently, the column begins to function as an "ion-exchange
column" with respect to the solute, breaking the original
hydrophobic interaction expected in reverse-phase mode (as
illustrated: ion-exchange mode).
When the hydrophobic interaction, as described in case (1), is
weak, reproducibility is easier to achieve. However, in case (2),
where strong ionic interactions dominate, even a slight change in
the concentration of the ion-pair reagent can significantly affect
the interaction and, consequently, retention. Retention variability
can also occur based on the order in which the mobile phase is
prepared. Ion-pair reagents are typically added in millimolar
concentrations, so even minor fluctuations in concentration can have
a pronounced impact on the retention of solutes that form ion-pairs.
When replacing a column, the primary reason for variations in
retention is that the ligand amount on the stationary phase is not
exactly the same across different batches of packing material. It is
impossible to precisely match the pore size and surface area of
silica, a polymeric material. Molecular interactions in substance
separation are the result of the interactions between individual
solute molecules and individual stationary phase ligands. Achieving
a uniform number of ligands at the molecular level on the irregular
surface area of a silica-based stationary phase is virtually
impossible. This is akin to the fact that it's nearly impossible to
ensure that the amount of active ingredient in a pharmaceutical
formulation is identical down to the Avogadro's number level.
In ion-pair chromatography, it is believed that both interactions
described in (1) and (2)—hydrophobic interaction and ionic
interaction—are simultaneously at play. Moreover, because this
technique essentially functions as ion-exchange chromatography under
isocratic conditions, it is inherently prone to reproducibility
issues and can be considered a risky method. The reproducibility
depends not only on the ion-pair reagent's ionic properties and
alkyl chain length but also on the strength of the solute’s ionic
characteristics. Therefore, it is not appropriate to discuss
reproducibility in terms of the column alone, as multiple factors
are involved in achieving consistent results.
In ion-exchange chromatography, ionic interactions are much
stronger than the hydrophobic interactions in reverse-phase
chromatography. Therefore, gradient elution is strongly recommended
over isocratic elution for better control of retention and
reproducibility.
At Imtakt Corporation, we offer the following reversed-phase +
ion-exchange "mixed-mode" columns:
Scherzo SS-C18, SM-C18, SW-C18 Columns
By using these columns appropriately, you can achieve a more
reproducible reversed-phase + ion-exchange mode compared to ion-pair
chromatography. Most importantly, the use of volatile salts makes
this method ideal for LC-MS applications.
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