Buffer Selection
The typical pH range for reversed-phase on silica-based packing is pH 2 to 8. Choice of buffer is typically governed by the desired pH. It is important that the bu!er has a pKa close to the desired pH since buffers control pH best at their pKa. A rule of thumb is to choose a buffer with a pKa value <2 units of the desired mobile phase pH (see Table 1).

Buffer Concentration: Generally, a buffer concentration of 10-50 mM is adequate for small molecules.

Buffer Solubility: A general rule is no more than 50% organic should be used with a buffer. This will depend on the specific buffer as well as its concentration.

Buffer’s Effect on Detection: The choice of buffer is also dependent upon means of detection. For traditional UV detection, the buffer needs to be effectively transparent in this region, especially, critical for gradient separations. Buffers listed in

Table 1 have low enough absorption below 220 nm. Phosphoric acid and its sodium or potassium salts are the most common bu!er systems for reversed-phase HPLC. Phosphonate buffers can be replaced with sulfonate buffers when analyzing organophosphate compounds. With the growth in popularity of LC-MS, volatile bu!er systems, such as TFA, acetate, formate, and ammonia, are frequently used due to compatibility with mass spectral (MS) detection. In regard to the issue of suppression of ionization, formate and acetate are ideal choices for positive-ion mode detection. TFA, however, can negatively impact detector response even in positive-ion mode (4,5), while it strongly suppresses ionization with negative ion mode. Acetic acid is good for negative-ion mode. LC-MS applications further limit buffer selection and buffer concentration.