Effect of pH on HPLC columns
The proper use of HPLC columns is of utmost significance for the life span of a column. Generally, columns of reversed-phase chromatography is stable within a pH range of 2 to 8. If you determine a pH value, the measurement should be performed before mixing with organic solvents in aqueous media. Nowadays HPLC columns are available to use outside that pH range. However, if the pH range of the mobile phase is outside the pH range of 2 to 8, ensure the seller's product information before using silica-based columns.
As the pH of the mobile phase/buffer/sample is considered as a parameter in RP-HPLC, not only its effects on retention time but also the variation in asymmetry and efficiency of the chromatograph need to be considered. The stationary phase (column) is affected by pH. At very basic pH i.e. below pH 02.00, the bonded stationary phase will be stripped of the silica support. Silica at high pH i.e. above pH 08.00 will be damaged by self-dissolution.
The separation of basic molecules at low pH is often recommended in RP-HPLC since symmetric peak shape and maximum column efficiency are usually the result. However, analysis at low pH (below pH-3) is not possible due to of instability of solute or band-spacing issues. In such cases, the pH between 04.00 to 08.00 or higher is used to separate. Even though working on the intermediate pH can generate useful band spacing for ionizable molecules, difficulties with retention reproducibility and band size may occur as a result of partial ionization of the basic molecules.
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