When an acid is more than 2 pH units above or below its pKa, it will be >99% ionised or non ionised, respectively. Bases are ionised below their pKa and non ionised above. The non ionised form will be less polar (more hydrophobic), and so more strongly retained in a reversed-phase system. As a result, at low pH, acids will be more retained, whereas bases will be more retained at high pH.
If the mobile phase pH is near pKa, you can see that slight changes in pH can make substantial changes in retention – not what is desired for a robust separation. Some compounds are very sensitive to very small changes in pH. Here the resolution changes by a factor of two for a change of only 0,1 pH units – this is the amount of error in pH adjustment common to many laboratories. Besides the instability of retention times when the pH is near pKa, relative peak spacing (selectivity) can change if compounds of similar structure are present.
Another factor that should be considered when choosing the mobile phase pH is the stability of the column. As a rule, silica-based columns should be operated at 2
Since the retention of ionisable compounds are very sensitive to the mobile phase pH, it is necessary to control the pH of the mobile phase by the addition of a buffer. A buffer maintains the pH when a small amount of acid or base is added.
Many different substances have been used for buffering in HPLC.
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