Wednesday, October 2, 2019

What is the Role of the Mobile Phase in HPLC?



Chromatography in chemistry is the science of separating mixtures. To do this, the sample goes through two distinct phases, named the mobile phase and the stationary phase. In which, the stationary phase remains fixed, while the mobile phase flows from the system. The mobile phase is the liquid or gas that runs through the system separates the molecules in the stationary phase at different rates. The stationary phase is either the solid or liquid (e.g. Column, silica, paper, TLC plate) at which molecules are to be separated.
The mobile phase is an important parameter in the chromatographic separation process, and many of its other properties are significant in HPLC analysis. The mobile phase is a developing liquid or solution combination that travels up to the stationary phase, moving samples with it. The analytes of the sample solution separate, based on how much they adsorb onto the stationary phase versus how easily they dissolve in the system of the mobile phase. This is an important role of the mobile phase in HPLC and other chromatography. The retention time of each molecule will contrast, it depends on the interaction between the stationary phase, molecules, and solvents are being used in the analysis. As the sample travels through the HPLC column, it interacts at different rates between the two phases, mainly due to different polarities in the molecules. Generally in RP-HPLC consists of a mixture of methanol, acetonitrile, tetrahydrofuran (THF), propanol, and water sometimes buffer (e.g. Acetate buffer, phosphate buffer) used as a mobile phase.
The mobile phase of HPLC should have the following important features to meet the essential objectives:
  • The sample to be analyzed has to be soluble in the mobile phase.
  • The components of the solvent system have to be non-toxic and non-hazardous.
  • The mobile phase must be inert; they should not react with the sample components and the stationary phase.
  • When it passes through the detector it should not give the response.
  • The mobile phase should be inexpensive and make the proportion of solvents used cost-effectively viable.


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