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Some sources state that "HPLC analytical samples should be
dissolved in the mobile phase," but this is not always correct, as
the sample may not dissolve well in the mobile phase.
Dissolving the sample in the mobile phase can be effective for
avoiding ghost peaks near the t0 region.
However, in isocratic analysis, if the sample is dissolved in a
solvent similar to the mobile phase, the solute may preferentially
partition into the mobile phase rather than the stationary phase,
leading to a lack of retention. Therefore, the solubility of the
solute and the polarity of the mobile phase need to be considered
separately.
When injecting a sample into an HPLC column, one essential
requirement must be met: the solute must contact the surface of the
stationary phase on a molecular level. If a solvent that promotes
the formation of aggregates is used, the stationary phase will
attempt to interact with the solute as an aggregate. When both
monomers and aggregates are present, the resulting peak shape after
injection will deteriorate.
In the case of real samples, the
solubility of other components (matrix components) besides the
analyte must also be considered. If the matrix components are not
fully dissolved, they can impose a significant burden on the column,
leading to increased column pressure. In such cases, sample
pretreatment becomes essential.
The miscibility between the
sample solvent and the mobile phase solvent is also crucial. If the
sample solvent and the mobile phase are immiscible, the solute may
not be able to contact the stationary phase on a molecular level,
potentially compromising the analysis.
Here are examples
where the sample solvent differs from the mobile phase solvent.
Melatonin-Related Compounds
https://www.imtakt.com/TecInfo/TIE72E.pdf
In
gradient analysis, it is often advisable to match the sample solvent
to the initial pH of the mobile phase.
Bezafibrate
(Lipid-Lowering Agent)
https://www.imtakt.com/TecInfo/TIE77E.pdf
Cephalosporin Antibiotics
https://www.imtakt.com/TecInfo/TIE76E.pdf
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