Buffer Considerations for LC and LC–MS - - Chromatography Online
Buffer Considerations for LC and LC–MS

LCGC North America
pp. 1000-1005

What Happens to an Aqueous Buffer When Organic Solvent Is Added?

Figure 2
When an organic solvent is added to an aqueous buffer to prepare a particular mobile phase, two different phenomena take place: the buffer capacity of the mixture is reduced due to the dilution effect, because β is proportional to the concentration of the buffer; and the maximum of buffer capacity is shifted according to the pK'a shift of the buffer species, including the contribution of the activity coefficients. There are data available about the pK a variation of commonly used HPLC buffers in acetonitrile–aqueous (8) and methanol–aqueous (9) solution up to 60% and 80% in volume, respectively. In this studied range of solvent compositions, the pK A of neutral and anionic acids is shifted to higher values, whereas the pK A of cationic acids shows the reverse trend. Both phenomena are depicted in Figure 2 for phosphoric and citric acids buffering systems. It must be mentioned that in the present article, the pH in the hydroorganic mixture is expressed in the s w pH scale — that is, the pH quantity is referred to water as the standard state. In other words, the pH is measured in the mixture, but the glass electrode is calibrated using standard aqueous buffers. After this clarification and coming back to Figure 2, the citric acid system shows a good buffer capacity in an outstanding wide range of pH, up to pH 6.5–7 in aqueous solution and up to pH 8 in 60% of acetonitrile, whereas in the case of the phosphoric acid system, there is a poor buffered zone around pH 5 because of the large difference between the first and the second pK a values.

MS-Friendly Buffer Mixtures: Ammonium Carbonate, Acetate, and Formate

Figure 3
Buffers composed of two buffering species are used commonly in HPLC systems. In particular, ammonium formate, ammonium acetate, and ammonium carbonate are used widely when HPLC is coupled to mass spectrometry (MS). Ammonium acetate and formate are employed in separations performed at low pH, in which the buffering species are formate or acetate, and ammonium plays only the role of a volatile MS-friendly co-ion, instead of sodium or potassium ions. Figure 3 shows buffer capacity profiles of ammonium acetate and formate in aqueous solution, and how they vary when acetonitrile is added to the aqueous buffers to prepare particular mobile phases. In these plots, the individual buffer capacities corresponding to ammonium–ammonia and acetic acid–acetate or formic acid–formate species are described, together with the contribution of a hydrogen ion (H3O+ ) at very acidic pH and hydroxyl ion (OH- ) at very alkaline pH. In the case of ammonium acetate, there is a difference of 4.6 units between the pK a values of both buffering species (ammonium–ammonia and acetic acid–acetate) in aqueous solution, leaving a broad nonbuffered zone between them. In the case of ammonium formate, this nonbuffered zone is even wider (5.5 units) because formic acid is more acidic than acetic acid. These pK a differences are reduced progressively with the addition of the organic solvent (ammonium is a cationic acid and formate and acetate are conjugate bases of neutral acids), but in spite of this fact, the difference between pK a values is still substantial at high organic solvent concentrations. Therefore, when these buffers are used for low pH separations, there is no contribution from ammonium in the improvement of acetate buffer capacity, and their buffer capacity at intermediate pH values is really poor.

Figure 4
Ammonium hydrogen carbonate has been described as an excellent buffer for the analysis of basic drugs by HPLC–MS (10). In fact, this mixed buffer presents a good buffer capacity in a relatively wide pH range, because the buffer capacity of ammonium–ammonia species is added up to the one corresponding to hydrogen carbonate–carbonate (Figure 4). In this case, in contrast to ammonium formate or acetate, the difference on aqueous pK a values is only about 1 unit. Therefore, there is a wide pH range of excellent buffer capacity at least from pH 8 to 11 in aqueous solution, and if the concentration of the buffer is high enough, it might be wider. When acetonitrile is added, ammonium becomes more acidic and hydrogen carbonate more basic, so differences in pK a values are increased. But the pK a values of the buffering species are still close enough to provide a wide range of good buffer capacity, and it widens with the addition of acetonitrile. McCalley (11) reported the instability of ammonium hydrogen carbonate solutions at pH around 7, probably because of the poor buffering capacity of this buffer in the valley between the first and the second pK a values.


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