Introduction

In reversed-phase HPLC, the pH of the eluent can significantly influence the separation of components. Buffers are required when the sample contains ionic or ionisable analytes. Without a buffer, poor peak shape and variable retention may result.
In general, for acids, increasing eluent pH leads to increased ionisation and a decrease in retention (see Figure 1). For bases, decreasing pH results in greater ionisation and decreased retention. For robust methods, separations should be developed at a pH where retention is least affected by pH change (see robust pH zones in Figure 1). 

Choice of Buffer pH

Optimum buffering capacity occurs at a pH equal to the pKa of the buffer. In general, the effective pH range for a buffer is within ± 1 pH unit of it’s pKa. Table 1 indicates the pH range of some common buffers. The buffer pH should be selected so that it is at least ± 1 pH units from the pKa of the analyte. This will ensure that the analytes are 100% ionised or 100% non-ionized, for reproducible retention. 

Further Considerations

At pH >7, phosphate buffers accelerate the dissolution of silica. For optimum column lifetime, organic buffers (eg pyrrolidine) should be used for pH above 8. Buffer concentrations in the 10 - 25mM range are recommended (maximum 50mM). Fresh buffer solutions should be prepared regularly and pre-column filters should be used to protect columns. Buffers should be flushed from the column prior to storage.
 

Buffers for LC-MS

For LC-MS analyses, volatile buffers or additives are preferred, in order to minimise MS ion suppression and maintain sensitivity. In addition, non-volatile buffers such as phosphate may lead to contamination of the ion source. Buffer concentrations should be as low as possible eg 10 to 20mM. Ammonium salts are more volatile than those of Na+ or K+. TFA should be avoided with electrospray LC-MS as it reduces sensitivity. Formic acid or acetic acid (0.01 to 1% v/v) are preferred. For higher pH applications, ammonium hydroxide is recommended.

Table 1. Common Buffers and Additives for HPLC