As many we know the buffers are commonly used in mobile phases of HPLC to keep the pH stable. They are composed of a weak base or acid, combined with its conjugate acid or base. Because the retention, peak shape, column stability, reproducibility, and selectivity of ionizable analytes is very sensitive to the pH of the mobile phase, hence it is required to control the pH by adding a buffer in the mobile phase.
Maintaining the pH of the mobile phase is a significant tool for achieving proper separation of analytes in high-performance liquid chromatography (HPLC), but if the pH is poorly maintained results in several problems with separations. When an acid exceeds 2 pH units or under its pKa, it will be greater than 99% ionized or non-ionized, correspondingly. Base compounds are ionized below their pKa and non-ionized above their pKa. The non-ionized compounds form will be less polar and therefore they will more strongly retain in reversed-phase chromatography. Consequently, at low pH, the acidic molecules will be retained more, while the basic molecules will be retained more at higher pH. If the pH of the mobile phase is near to pKa value, then that small changes in pH lead to large changes in the retention time of the analytes. The resolution, peak shape, and system suitability parameters also change by changing the pH units - this is the common error in pH modification for many analyses.
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