Wednesday, October 2, 2019

What is the Mobile Phase in HPLC?

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.



You may also like this

No comments:

Post a Comment