ChromFAQ:Buffers
From ChromFAQ
Why do I have to use a buffer in reversed-phase chromatography?
If a sample contains only neutral (non-ionizable) compounds, then a buffer is usually not necessary in reversed-phase. Otherwise, use of a buffer is highly recommended.
Retention in reversed-phase chromatography is based primarily on hydrophobicity. Other things being equal, hydrophilic molecules will be weakly retained (and elute early in the chromatogram) while hydrophobic molecules are more strongly retained (and so elute later). The hydrophobicity of a weak acid or weak base is strongly affected by its degree of ionization, with the ionized form (anion or cation) being more hydrophilic and the neutral form (free acid or free base) being more hydrophobic. The degree of ionization is pH dependent, and changes most rapidly when the pH equals the pKa of the molecule. Because pH is a logarithmic function, ± 1 pH unit on either side of the pKa changes the %ionization from 10% to 90%; ± 2 pH units changes the %ionization from 1% to 99%. Therefore, to minimize the potential for problems resulting from small changes in pH, it is a good idea to stay at least 1.5 pH units away from the pKa of your analyte(s) if possible.
A buffer is used to control pH. A buffer must be able to both donate and accept protons (hydronium ions). Because buffers are themselves weak acids or bases; they are most effective at their own pKa, and become more ineffective as the pH moves more than 1 unit or so away from their pKa.
Buffer selection is determined primarily by the desired pH and secondarily by detector compatibility. Many organic buffers, for example, have fairly high UV cutoffs , while non-volatile buffers such as phosphate are unsuitable for use with mass spectrometry.
Buffer concentration is usually expressed as "millimolar" (mM). Be careful, however, because there is a bit of ambiguity here. The author has always interpreted the buffer concentration and pH as being defined with respect to the aqueous part of the mobile phase (i.e., before any organic solvent is added). A substantial number of people, however, interpret concentration and pH as being defined with respect to the final mobile phase (i.e., after addition of organic). Hopefully, the interpretation will be clear from context (e.g. "50% acetonitrile + 50% of a 25 mM, pH 2.5 potassium phosphate buffer" is different from "25mM, pH 2.5 potassium phosphate buffer in 50% acetonitrile/water").
The buffer concentration needs to be high enough to prevent pH changes caused by the presence of the sample. This is usually in the range of 10 to 50 mM (in the aqueous part of the mobile phase!). The most common values are 25 mM for UV detection and 10 mM for mass spec. You could often get by with less, and you may occasionally need more, but most people don't attempt any optimization of the buffer concentration unless they have problems.
One good rule for HPLC is "to get the best results, disturb the equilibrium of your system as little as possible". This suggests that the sample should ideally be dissolved in the mobile phase. Even if the sample must be dissolved in something other than the mobile phase (e.g., a buffer at different pH), problems are usually minimal so long as: - the injection volume is low and/or - the pH difference is not too great and/or - the mobile phase buffer capacity is sufficient to deal with the upset.
