Technically the ammonium acetate and formic acid are not acting as a buffer but will be affecting the pH of the mobile phase. A buffer is used to resist small changes in pH and must be made from the acid and corresponding salt, i.e. ammonium acetate and acetic acid or ammonium formate and formic acid.
Therefore, in this case it may have been that the initial user of this method wanted to buffer the system and, hence, chose ammonium acetate and formic acid; however, as mentioned previously this is not a buffer as the correct choice of salt and acid would be ammonium acetate and acetic acid or ammonium formate and formic acid. This may have just been a mistake during development but the method worked, or perhaps the pH was lowered using ammonium acetate and formic acid was added to aid ionization of the analytes, however, formic acid alone would have done this.
Another point to note is the concentration (1 M) of the “buffering” component in the mobile phase. There are cases where analyte retention in reversed phase HPLC is affected by buffer concentration. These cases are usually confined to situations where there are ion exchange interactions taking place between basic solutes and acidic silanols on the surface of the silica stationary phase support (separation of basic compounds) using reversed phase stationary phases that have significant silanol activity with mobile phase pH > 3. Increasing the concentration of the mobile phase buffer, and thereby increasing the ionic strength of the mobile phase, will sometimes suppress this ion exchange interaction and reduce this ‘secondary retention’ effect.
It should be noted that as the buffer concentration is increased the mobile phase is made more polar (ionic). This can affect analytes in differing ways depending on their chemistry; some analytes may experience reduced retention while some will exhibit slightly increased retention. The main objective is to use an appropriate buffer (i.e. buffer pKa vs. mobile phase pH) in the correct concentration to have sufficient buffering capacity to overcome peak shape and retention time irreproducibility.
Buffer capacity is a measure of the efficiency of a buffer to resist small changes in pH. Buffer capacity (β) can be expressed as the amount of strong acid or base (in gram equivalents) that is required to change 1 liter of a buffer solution by one pH unit.