Consideration of the effects of pH on analyte retention, the type of buffer to use, its concentration, solubility in the organic modifier, and its effect on detection are important in the reversed-phase chromatography (RPC) method development of ionic analytes. An improper choice of buffer, in terms of buffering species, ionic strength, and pH, can result in poor or irreproducible retention and tailing in the reverse-phase separation of polar and ionizable compounds.
Problems, such as partial ionization of the analyte and strong interaction between analytes and residual silanols 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.
Making an aqueous mobile phase is the most important stage of RP-HPLC method development for ionic molecules. This contains a consideration of the effects of pH on the retention time of a component, concentration, type of buffer, solubility, and its effect on detection.
Inappropriate selection of buffer, in terms of pH, ionic strength, and buffering species may result tailing and irreproducible retention time of polar and ionizing compounds in the reversed-phase separation.
Complexities such as interactions between molecules and partial ionization, molecules, and other active sites on stationary phases can be conquered by selecting the appropriate buffer its correct ionic species, and ionic strength in the mobile phase. In separations LC-MS that rely too much on the correct choice of acid, base, buffer species, and other additives, a buffer has to be selected based on its ability to suppress and not inhibit the ionization of the molecule at the MS interface.
The retention time of ionic components in reversed-phase chromatography is fundamentally influenced by the pH of the mobile phase. The RT of non-ionic components is minimum affected by the pH of the mobile phase. Buffer is a solution of a weak base and its conjugate acid or weak acid and its conjugate base.
They reduce the effect of hydroxide and hydrogen ions and reduce pH fluctuations upon dilution. Usually, the range of pH is 2.00 to 8.00 for RP-HPLC on silica-based packing. The selection of a buffer solution is usually governed by the preferred pH. If the pKa is close to the pH then the buffers best control the pH. The most commonly used buffer systems for reversed-phase chromatography are phosphoric acid and its potassium or sodium salts, and acetate buffers, which are also frequently used for separation.
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The phosphate buffer is ideal for most HPLC separations, as it has two pKa values, (2.1 and 7.1) and UV transparency. But the use of phosphate buffer in LC-MS is not appropriate, it prefers volatile buffer systems i.e. Acetate, TFA, ammonia, and format, etc.
Buffer Selection
- The typical pH range for reversed-phase on silica-based packing is pH 2 to 8. The choice of buffer is typically governed by the desired pH. The buffer must have 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).
Buffer Concentration:
- A higher concentration of the buffer that increases the buffer capacity will give a more reproducible separation of partially ionized analytes at the pH of the mobile phase, typically, a buffer concentration of 10 to 50 mM is sufficient for the separation of components. A highly concentrated buffer will cause a high backspace for the system.
Buffer pH:
- The retention time of ionic components in reversed-phase chromatography are basically affected by the pH of the mobile phase as compared with non-ionic components. In general, the pH range of buffer solution or mobile phase for RP-HPLC is 2.00 to 8.00. The selection of buffer solutions is usually controlled by the preferred pH.
Effects on Detection:
- The selection of buffer is also reliant on the means of detection. For conventional UV/VIS detectors, the buffer solution must be transparent, and effectively in this range, particularly important for gradient elution separation.
Buffer Solubility:
- This is particularly significant while working with the gradient elution method. The solubility can be determined empirically by mixing the specified volume fractions of the organic solvent and the buffer solution. The presence of precipitates solutions specifies the solubility issues with the molecule and mobile phase or solvent. A general rule is that the organic phase should not exceed 50%, which should be used with the buffer.
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