Reversed-Phase HPLC Mobile-Phase Chemistry — Explained

In reversed-phase separations, the eluent tends to be more polar (more hydrophilic) than the stationary-phase surface and is typically a mixture of water and an organic solvent, usually methanol or acetonitrile. The choice of organic modifier will affect the selectivity of the separation because methanol and acetonitrile have different solvochromatic properties, the most important of which are dipole moment, acidity, and basicity. Acetonitrile has a higher dipole moment and is more predominantly acidic (hydrogen bond donor) whereas methanol has a lower dipole moment, is more predominantly basic (hydrogen bond acceptor), and has a slightly lower elution strength. Different binary and ternary mixtures of these solvents with water can produce a wide range of selectivity options during method development. Acetonitrile is often used because of its low UV cutoff, lower viscosity (methanol forms highly viscous mixtures with water at certain concentrations), and higher boiling point. Solvent mixtures may be isocratic (fixed ratio of organic to aqueous components), or the elutropic strength can be continuously increased during the experiment by increasing the amount of organic modifier in a gradient elution experiment.

For ionizable analytes, the extent of ionization will alter the polarity of the analyte molecule as well as its ability to interact with solvent and charged species within the bonded phase or stationary-phase surface. The eluent pH can be used to influence the degree of analyte ionization and as such its polarity, which alters analyte retention based on the proximity of eluent pH to the pK_a (partial acid dissociation constant) of the analyte functional groups. If the analyte contains an acidic functional group with pK_a equal to 4.0, retention at pH 2.0 will be significantly greater because the analyte will be almost wholly nonionized (less polar) and at pH 6.0 retention will be significantly lower because the functional group will be almost wholly ionized (more polar). The opposite would be true if the analyte were basic. In this case, the relative retention times of analytes (selectivity) can be altered by changing the eluent pH until a satisfactory separation can be obtained. Eluent pH values at or near to the analyte pK_a will risk robustness issues as small changes in pH will result in relatively large changes in analyte retention.

Table I: Solvochromatic and physicochemical properties of common solvents used in reversed-phase HPLC

Modern approaches to working with ionizable analytes often involve working at pH extremes to avoid variations in selectivity because of changes in the mobile-phase pH. The pH is typically altered using trifluoroacetic acid, formic acid, ammonia, or ammonium hydroxide. This often improves method robustness, but requires selectivity to be optimized by other means such as stationary phase, organic modifier type, and eluotropic strength, which limits the extent to which separations can be optimized. For complex separations (acids, bases, or mixtures of both) more careful pH optimization may be required and the use of a buffered mobile phase will be required.

Table II: Properties of various common HPLC buffers

A particular buffer is only reliable within 1 pH unit on either side of its pK_a and, therefore, the choice of buffer will be heavily influenced by the required eluent pH. The buffer concentration must be adequate, but not excessive. Below 10 mM, buffers have very little buffering capacity and will not be able to resist changes in pH. At concentrations greater than 50 mM there is a risk of the salt being precipitated in the presence of high organic concentrations (that is, >60% acetonitrile). Buffer concentrations will normally be in the range 25–100 mM, and the effect of the buffer concentration should be investigated as part of the method development process because both retention and selectivity of the separation can be affected by changes in the type and concentration of the buffer. Remember, if a UV-based detector is being used, take note of the UV cutoff of the buffer. If mass spectrometric detection is being used, then the use of a volatile buffer is essential.

In cases where strong acids or bases are being analyzed, or when analytes are amphoteric, it may be necessary to use an ion-pairing reagent, which is an acid or base with highly hydrophobic groups that will pair with the conjugate group on the analyte molecule to neutralize charge and add hydrophobic character, which can be used to improve retention of the neutral ion pair. When an ion pair is used, the eluent pH is adjusted to ensure complete ionization of the analyte.