The mobile phase or solvent system used in
high-performance liquid chromatography is selected based on the type of HPLC.
The reversed-phase HPLC typically consists of an organic solvent and a
water/aqueous solution/buffer in the mobile phase. To control retention and
peak size, buffers and other additives may be present in the aqueous phase
while our analysis of ionizable molecules. In RP-HPLC is the weakest solvent is
water. Since water is the most polar, this repels hydrophobic components in the
stationary phase compared to any other solvent and therefore retention times of
components are longer. Organic modifiers are added, and since these are less
polar, the molecules are no longer powerfully repelled in the stationary phase,
it will use less time, and consequently elute earlier. This is the reason it is
chromatographically stronger as it reduces retention time by accelerating
elution. If the increasingly more organic solvent is used in the mobile phase,
the retention time of the compound will reduce. The choice solvent might be one
of the important parameters in an HPLC, because of the impact it can have on
the selectivity. Actually, for optimizing resolution the selectivity can be
the most effective tool.
In RP-HPLC, the selected organic solvent must be miscible
with water and prioritize the low viscosity mobile phase to minimize dispersion
and keep low backpressure. Water is usually a base solvent and other organic
solvents such as acetonitrile, methanol, etc. are added in as per the required
proportions. The pH adjusted buffers (e.g. Phosphate, acetate) is also used in
the HPLC mobile phase to adjust the separations of ionizable compounds.
Ion-pairing reagents also improve the separation selectivity of charged
molecules by enhancing the RT on hydrophobic bonding phases.
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