Chrominfo: Selection of Mobile Phase in HPLC

The mobile phase is the most significant parameter in HPLC and all types of chromatography analysis. The type of mobile phase used can have a huge impact on separation as well as the retention time. This may suppress or promote the ionization of the molecules that are analyzed. The selection of an appropriate mobile phase is the very key step in the chromatographic method development.

The essential requirement for the mobile phase is to dissolve the components to the appropriate concentrations to detect it. There are different types of high-performance liquid chromatography and they need different types of mobile phase requirements. In which the stationary phase is fixed, while the sample mixture carries by the mobile phase composition, which can be varied as per the requirement. The speed of the molecules in the mobile phase is managed by the importance of their interactions with the stationary phase / mobile phase. Due to differences in factors, like the strength of components richness for the stationary phase and the solubility in the mobile phase, several compounds will travel quicker than others, therefore make it easy to separate the compounds within that sample mixture.

The selection of the mobile phase in HPLC separation modes are as follows.

Reversed-phase chromatography (RP-HPLC) separates the analytes based on nonpolar interactions and involves a polar mobile phase and a nonpolar stationary phase. In which the more polar compound is eluted first from the HPLC column. Usually, the mobile phase in RP-HPLC consists of a mixture of methanol, acetonitrile, tetrahydrofuran (THF), propanol, and water sometimes buffer (e.g. acetate buffer, phosphate buffer).
NP-HPLC uses a nonpolar mobile phase and a polar stationary phase for the separation of polar components of the sample. In normal phase chromatography, the mobile phase is usually used with non-polar solvents, for example, hexane, iso-octane, and heptane, with slightly more polar solvents like isopropanol, chloroform or ethyl-acetate. If the nonpolar solvent quantity increases in the mobile phase, the retention time of the analytes will increase.

The separation in ion-exchange chromatography is depending on ionic interactions between ionic and polar molecules; hence, the aqueous salt solution is commonly used as mobile phases. The medium amounts of water-miscible polar organic solvents like HPLC grade methanol can be added to the buffer. The strength and selectivity of the solvent can be adjusted by the managing of the buffer, pH, and salt concentrations. In IEC, the retention time of a molecule depends on the affinity of various molecule ions and counter ions for the charged sites on the surface of the stationary phase and several other parameters of a solution containing concentration and strength.
Size-exclusion chromatography (SEC) is used to separate the analytes according to their molecular size it is also recognized as gel permeation chromatography when the mobile phase is an organic solvent. The choice of the mobile phase in the SEC is based on its ability to dissolve and maintain a constant viscosity at operating temperature. The choice of the mobile phase is dependent on its ability to dissolve samples and maintain consistent viscosity at working temperature. Mobile Phases for size exclusion chromatography contain the high polar solvents like alcohol, DMSO, acetone, and water are used, and also of salts the ionic strength is maintained.