As a chromatographer not many of us are aware of how critical pH is in your method development, sometimes it is even considered as an inconsequential factor, which could have a negligible effect on our analysis.
However, in truth pH can make or break your analysis, choosing the correct or more importantly optimized pH in a method can be a single most significant step when developing a method.
In reverse-phase chromatography, most analytes are at least slightly ionized, classifying them as either weak acids or weak bases. Knowing the pKa or pKb of the compound is crucial for selecting the correct mobile phase pH.
To ensure proper resolution and peak shape, chemists aim to keep analytes in a single ionization state—either deprotonated or unionized. The ionization state affects interactions with the stationary phase, as ionized molecules are more polar, leading to less hydrophobic interaction and lower retention in an aqueous mobile phase.
When both ionized and neutral forms are present, ionized molecules show weaker hydrophobic interactions, leading to broader, asymmetrical peaks. Silanol groups or metal contaminants may also cause poor peak shape due to ionic interactions with ionized analytes.
Adjusting the mobile phase pH based on an analyte’s pKa allows control over its ionization state. For best results, the pH should be at least 2 units above or below the analyte’s pKa. Proper pH control can significantly improve the retention and peak shape of both acidic and basic compounds.
Thus, when you will be next developing or optimizing a method consider pH as one of the most important factor and you will have positive circumstances for accomplishing your desired retention, resolution and peak shape even for your most challenging analytes.
Read also:
Resource Person: Ahmad Hassan