Phosphate is a particularly valuable buffer because it can be used at wavelengths shorter than 220 nm, making it the most commonly used buffer in HPLC.
The use of buffers for reversed-phase separations of polar and ionizable molecules necessitates precise pH control. This assures repeatable retention times (RT) and is free from peak tailing and fronting. Buffers are commonly used in chromatographic mobile phases to keep the pH constant. They are made up of a weak acid or weak base combined with its conjugate acid or base in a partially aqueous solution. These pair of molecules is in equilibrium, which means that when an acid or basic is added, the equilibrium moves to compensate, keeping the pH constant.
Buffer solutions are used in high-performance liquid chromatography (HPLC) to control pH and resist changes in the pH of the mobile phase. The separation of polar and ionization analytes in RP-HPLC needs specific pH control using buffers. This makes sure reproducible retention time and symmetrical peak shape it also prevents interactions between molecules and residual silanols on the stationary phase.
Reverse phase separations can be easily performed in the pH range of 2.0 to 7.5. However, the results are reproducible if the pH is maintained inside the +/- 1 pH unit of the pKa value of the component. The phosphate and acetate are the most popular and used buffers for HPLC with UV detection. Phosphate is a particularly useful buffer, as it can be used at wavelengths less than 220 nm hence it is the most used buffer in the HPLC.
Phosphate and acetate are the most commonly used buffers for HPLC with UV/VIS detectors since they can be used at wavelengths below 220 nm. However, phosphate buffer is non-volatile, so it is not used when a mass spectrometer is employed as an LC detector.
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