Reversed-Phase HPLC Buffers

By: Shyam Verma, Reporter US Volume 27.4

High quality buffers (solutions, solids or concentrates)

Preparation of aqueous mobile phase is the most critical step in reversed-phase chromatography (RPC) method development for ionic analytes. This includes consideration of the affects of pH on analyte retention, type of buffer to use, and its concentration, solubility in the organic modifier and its affect on detection, among other considerations. The improper choice of buffer, in terms of buffering species, ionic strength and pH, can result in poor or irreproducible retention and tailing in reversedphase separation of polar and ionizable compounds.

Problems like partial ionization of the analyte and strong interaction between analytes and residual silanoles or other active sites on the stationary phases can be overcome by proper mobile phase buffering (maintaining the pH within a narrow range) and choosing the right ionic species and its concentration (ionic strength) in the mobile phase (1-2). In sensitive LC-MS separations that depend heavily on the correct choice of acid, base, buffering species and other additives (3), a buffer must be chosen based on its ability to maintain, and not suppress analyte ionization in the MS interface.

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Mobile Phase pH and Reversed-Phase Retention

Retention of ionic analytes in RPC is fundamentally affected by mobile phase pH. The dissociation properties of the ionic functional groups also impact analyte retention. Retention of non-ionic analytes is minimally affected by mobile phase pH.

For acidic moieties (usually carboxylates), a pH below the pKa (within limits) and for basic moieties (usually amines), a pH above the pKa (within limits) of the compound enhances retention. Dramatic effects are observed on retention of these analytes in pH range near the pKa of a given functional group. This becomes apparent in consideration of chemical dissociation as illustrated in Figure 1.

Figure 1. Chemical Dissociation of an Ionic Analyte

The last two equations are commonly known as the Henderson-Hasselbach equation. These equations suggest that for pH far removed from the pKa, a small change in the pH has a minimal affect on the ratio of unprotonated-toprotonated species. Therefore, moderate change in pH will not affect retention significantly. However, at pH near the pKa, a small change in pH will produce a significant change in the ratio of the two species. Therefore, changing the pH within a range of values sufficiently close to the pKa will dramatically affect retention. When pH is used to increase reversed-phase retention, the pH should be changed in the direction that decreases analyte ionization.

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Buffer Selection

Buffers are solutions of a weak acid and its conjugate base, or a weak base and its conjugate acid. They mitigate the infl uence of hydrogen/hydronium and hydroxide ions and subsequently reduce the pH fluctuations, even upon dilution. The typical pH range for reversed-phase on a silica-based packing is pH 2 to 8. Choice of buffer is typically governed by the desired pH. It is important that the buffer 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).

Table 1. HPLC Buffers, pKa Values and Useful pH Range

Phosphoric acid and its sodium or potassium salts are the most common buffer systems for reversed-phase HPLC. Phosphate’s two pKa values, 2.1 and 7.1, and UV transparency make it ideal for most HPLC separations. Its pKa of 12.3 is suitable for buffering in 11.3-13.3 pH range. Phosphonate buffers can be replaced with sulfonate buffers when analyzing organophosphate compounds. With the growth in popularity of LC-MS, volatile buffer systems, such as TFA, acetate, formate, and ammonia, are frequently used.

Buffer Concentration: A higher buffer concentration that enhance buffer capacity will give more reproducible separation of compounds partially ionized at the pH of the mobile phase, by reducing local perturbations of the pH of the migrating analyte peak. Generally, a buffer concentration of 10-50 mM is adequate for small molecules.

Buffer Solubility: It is especially important when performing gradient separations. Solubility can be empirically determined by mixing given volume fractions of buffer and the organic solvent. Appearance of precipitates or opaque solution indicates solubility issues. 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.

Effects 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.

More common issues today are related to compatibility with mass spectral (MS) detection. Preferred buffers addressing the issue of volatility are formate, acetate, and ammonia. 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.

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  1. McMaster, M.C. HPLC A Practical User’s Guide, VCH Publishers, Inc.: New York, NY, 1994; 85.
  2. Poole, C.F. and Poole, S.K. Chromatography Today, Elsevier Science: Amsterdam, The Netherlands, 1991; 431.
  3. Analytix, Five-part series on Mobile Phase Additives for LC-MS, Issue 3, 2006 (
  4. Temesi, D., Law, B., The Effect of LC Eluent Composition on MS Response Using Electrospray Ionization, LC-GC, 17:626. 1999.
  5. Apffel, A. et. al. Enhanced Sensitivity for Peptide Mapping with Electrospray Liquid Chromatography-Mass Spectrometry in the Presence of Signal Suppression Due to Trifl uoroacetic Acid-Containing Mobile Phases, J. Chrom. A. 712:177. 1995.

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