The reason overloading is more problematic in formic acid than trifluoroacetic acid appears to be related to ion pairing and ionic strength effects. Mobile phases of higher ionic strength can physically screen protonated peptide cations from mutual electrostatic repulsion. A more important consideration, however, might be the formation of ion pairs between mobile phase anions and peptide cations. Those analyte ions that form "neutral" ion pairs should not undergo these mutual repulsion effects. The ion-pair properties of trifluoroacetic acid are well known and can be seen in increased peptide retention in this mobile phase (Figure 1, Table II). Note that an exactly analogous argument would hold if "dynamic ion exchange," occurred, with initial adsorption of the mobile phase anion on the stationary phase followed by interaction with the peptide cation, rather than a classic ion-pair mechanism (8). It appears also that ion pairs might form between analyte cations and inorganic anions such as chloride and perhaps even phosphate (7,13-16). Addition of potassium chloride to formic acid mobile phases was indeed shown to give improvements in column loading properties (7,13-14) for basic drugs and for peptides. Nevertheless, the addition of involatile salts to the mobile phase is hardly of practical use in applications involving MS. The adjustment of the pH of the formic acid mobile phase to 3.3 with ammonia produced a marked improvement in the column loading properties. Because formic acid is a weak acid, raising the pH in this way increases the ionic strength of the mobile phase (see Table II). Increased ionic strength gives increased opportunity for ion pairing; this effect is in addition to the physical screening of ions of the same charge. Both effects can reduce the effects of mutual repulsion and thus increase the loadability of the column. An indicator of this increased ion pairing is the increase in retention of the Alberta peptides in ammonium formate-formic acid compared with that in formic acid alone (Figure 1, Table II). Note that no exponential tailing was observed at this higher pH, which might instead have been indicative of the onset of silanol ionization. It is possible however, that competitive interactions of ammonium ions with ionized silanols could contribute to this result. The peak capacity even for dilute P3 improved from 223 to 238 and the gradient asymmetry factor from 1.74 to 1.25 using ammonium formate at higher pH indicating that silanol interactions are not a problematic factor in this particular case. Thus, use of ammonium formate buffers rather than formic acid alone can be useful in improving column loadability if scientists wish to avoid the use of trifluoroacetic acid. However, this is not likely to be true for older, more active "Type A" phases, or even all varieties of inert "Type B" phases, where silanol ionization could occur at higher pH. Clearly, some phases of even the latter group are more active than others (12).

Figure 3: (a) Overlaid chromatograms of 2.5 mg and 0.1 mg each bradykinins on PLRP-S; flow rate: 1 mL/min; mobile phase additive: formic acid (0.9 g/L). (b) As (a), but mobile phase additive: trifluoroacetic acid (0.9 g/L); acetonitrile gradient: 0.625%/min.