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In HPLC, there are two primary separation modes: "normal-phase
mode" and "reverse-phase mode." Commonly used stationary phases like
C18 (ODS) and C8, which have alkyl groups as ligands, operate
primarily in "reverse-phase mode."
The majority of HPLC applications use reverse-phase mode because
the target substances in HPLC are mainly organic compounds that
contain carbon. Carbon atoms exhibit hydrophobic (water-repelling)
properties, and the carbon chains of alkyl stationary phases, like
ODS, tend to attract carbon-containing compounds (retaining them on
the column) through what is known as "hydrophobic interaction." As
the polarity of the mobile phase decreases, for example, by
increasing the concentration of acetonitrile, retention becomes
shorter, and compounds elute more easily from the column.
In contrast, "normal-phase mode" operates based on "electrostatic
interactions" between a highly polar stationary phase, such as
silica, and polar molecules containing oxygen, nitrogen, or other
electronegative atoms. As the polarity of the mobile phase
increases, such as by decreasing the concentration of acetonitrile,
retention decreases, and compounds elute more easily.
In terms of defining normal-phase and reverse-phase modes, it is
purely the relationship between the polarities of the stationary
phase and the mobile phase that matters, and it is not dependent on
the specific names or structures of these phases. The presence or
absence of water is irrelevant, meaning that normal-phase mode can
occur in both non-aqueous and aqueous systems. Therefore, even HILIC
(Hydrophilic Interaction Liquid Chromatography) is considered a type
of normal-phase mode. The separation of sugars using amino columns,
which has long been performed in normal-phase mode, often uses
"water/acetonitrile" as the mobile phase, leading to confusion and
the misconception that it operates in reversed-phase mode. The
emergence of terms like HILIC also reflects a lack of proper
understanding of normal-phase interactions.
Because the polarity of the stationary and mobile phases is
reversed in normal-phase and reversed-phase modes, the elution order
of compounds tends to "reverse," though it does not always do so.
The selectivity of the stationary phase varies depending on the
structure of the compounds.
Generally, for highly polar substances that cannot be retained on
an ODS column unless the organic solvent concentration is below 10%,
it is recommended to explore using a normal-phase column.
Examples of IMTAKT Normal-Phase Columns:
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