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Frequently Asked Questions

How should a buffered mobile phase be prepared?

Buffered mobile phases can basically be prepared in two ways:

  1. preparing the buffer, including pH adjustment prior to mixing with organic modifier.
  2. mixing buffer and organic modifier prior to pH adjustment.

While the pH is a well defined parameter in aqueous systems, it is not as straight forward to define or measure proton concentration in partially organic solvent systems.

It is therefore recommended to prepare the aqueous buffer, pH adjust it, filtering it through 0.4 ┬Ám and mixing it thereafter with the organic modifier. Attention should be given to the following points:

  • buffers are effective within +/- 1 pH unit from their pKA ⇒ choose correct buffer system
  • consider the risk of precipitation if high buffer concentrations are mixed with organic solvents
  • always filter buffers through 0.4 mm filter prior to use
  • be aware of the risk for bacterial or fungal growth in pure aqueous buffers
  • the pH generally shifts upon the addition of an organic modifier (pH increases for inorganic buffers (e.g. phosphates) and decreases for organic buffers (e.g. acetates)
    Example: phosphate buffer pH 7.6 ⇒ addition of 40% MeOH ⇒ pH 8.5