Reversed-phase chromatography (RP-HPLC) contrasts from other chromatographic techniques where the attractive forces among the mobile phase and the stationary phase are prominent. In RP-HPLC, the stationary phase is usually an inert hydrocarbon that is interacting just with hydrophobic solute or molecule, in which selectivity is dominated by solvent effects. Generally, reversed-phase chromatography consists of only water in the mobile phases, or water plus is an organic solvent e.g. Methanol, acetonitrile, etc. Despite recent interest has been expressed in non-aqueous mobile phases. However, very recent interest has been expressed in non-aqueous mobile phases.
Here are mentioned some factors affecting reversed-phase chromatography.
The flow rate of the system:Flow rate is relied upon to be a significant factor in the resolution of small analytes, including protein digests and, small peptides in reversed-phase separations.
The length of the column used:The column length affects the retention time. If you reduce the length of the column the RT of the molecule also reduce proportionally.
The temperature of the system:Temperature can have an intense effect on RP-HPLC, specifically for the low atomic weight molecules. The mobile phase viscosity used in the reverse phase chromatography reduces with the rising temperature of the HPLC column. As the mass transport of molecules is a diffusion-controlled process between the stationary phase and the mobile phase, low solvent viscosity usually leads to high efficient mass transfer, consequently, high resolution.
The gradient elution method:In some biomolecules and some analytes, gradient elution methods have to be used because they are difficult to separate or the number of components is more in the sample. The gradient elution methods can affect some separations of reversed-phase chromatography.
The mobile phase of the system:The mobile phase of rp hplc chromatography consists of organic and aqueous phases such as methanol, acetonitrile, water, and buffer. Though, mobile phase solutions have slightly buffering capacities because they generally have a strong acid in low pH with high amounts of organic solvents. While working close to physiological conditions, adequate buffering capacity should be maintained.
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