Mobile-Phase Optimization Strategies in Reversed-Phase HPLC

There are many ways to improve retention or selectivity in reversed-phase high performance liquid chromatography (HPLC), and knowledge of the key eluent variables to achieve this is essential reading.

Much has been written about the most effective ways to optimize retention and selectivity in reversed-phase HPLC; however, a working knowledge of the major and minor eluent variables that affect these parameters is often lacking in practical analytical science laboratories. Retention and selectivity can be affected by altering the type and amount of organic modifier used in the eluent system. In reversed-phase mode, more hydrophobic analytes will be eluted later and more hydrophilic (polar, ionizable) molecules will be eluted earlier, although much depends on the nature of the stationary phase chosen and it is important to understand the stationary phase factors that affect retention and selectivity. Increasing the percentage of organic modifier causes a reduction in retention, and a 10% change in modifier can be expected to produce a 2–3-fold increase in analyte retention. The range of retention factor should ideally lie between 2 and 10 for all analytes of interest, and analytes with k < 2 risk coelution with poorly retained matrix components and a susceptibility to small changes in eluent composition (including pH). Analytes with k > 10 risk a reduction in efficiency (and resolution) because of increased band-broadening effects.

Each of the common reversed-phase HPLC solvents possess different solvatochromatic properties: Methanol is more acidic; acetonitrile is able to enter into dipole–dipole type interactions; and tetrahydrofuran is more basic. However, all three solvents possess each of the properties described to a greater or lesser extent. Switching between these solvents is a good way to investigate the various selectivity options afforded by the eluent system. Solvent nomograms are useful to maintain the overall range of analyte retention (that is, maintaining analysis time) when switching modifiers to alter the selectivity of the separation, and an interactive example can be found at www.chromacademy.com. Solvent systems that give the same retention range but altered selectivity are known as isoeluotropic. It should be noted that differing organic modifiers and eluent compositions will give rise to differing eluent viscosity and UV cut-off, and these factors should also be considered when designing eluent systems. When "screening" samples for suitable eluent composition, it is usual to run a gradient over a wide elutropic range (5–95% B is typical) and from the resulting chromatogram, decisions can be made regarding the most suitable isocratic eluent composition or the initial and final eluent composition as well as the slope, if a gradient separation is required. We have discussed this topic in the "Essentials" column previously (LCGC North Amer. 31[7], 578 [2013]), and further information can be found at www.chromacademy.com.

When dealing with ionizable analytes, eluent pH needs to be carefully considered because it will affect the degree of ionization, and hence the relative hydrophobicity of analytes. When analytes are ionized, their retentions times in reversed-phase HPLC will decrease. A common approach is to adjust the pH well away from the pK_a of analytes (the pH at which 50% of the analyte molecules will be in the ionized form) to impart robustness to the method, which is exemplified in the proliferation of methods that use 0.1% trifluoroacetic acid or formic acid to achieve low analyte pH, while also being liquid chromatography–mass spectrometry (LC–MS) friendly. Although it is true that this approach reduces the need for care in mobile-phase preparation, it should be noted that any basic analytes are likely to be protonated and therefore care is required to maintain retention using a combination of special stationary-phase chemistry and modifier concentration. Trifluoroacetic acid is a reasonably strong ion pairing reagent, and neither of them constitute a "buffer" in the true sense. Separations involving several ionizable analytes, zwitterionic analytes, or mixtures of acidic and basic analytes require care in the optimization of the eluent pH to achieve a suitable separation, and it is typical to screen several pH values or use computer simulation or optimization to shorten the development time. In all cases, whenever an eluent pH is within 1 pH unit of the pK_a of an analyte, greater care is required in reproducibly obtaining the correct eluent pH to avoid retention and selectivity changes.

Buffers in HPLC are used to resist changes in the eluent pH that would lead to changes in retention and selectivity of the analytes. Most often these changes (potentially) occur when the sample diluent and eluent are mixed within the autosampler, tubing, and at the head of the analytical column), or as the eluent stands within the reservoir for prolonged periods. A buffer is a weak acid or base in co-solution with its conjugate acid or base — that is, a solution of ammonium formate at a specified concentration adjusted to a specified pH using formic acid. The choice of buffer will depend on the detector system used — for example, volatile buffers need to be used for MS applications — but primarily on the required eluent pH, which must be within ±1 pH unit of the buffer pK_a value to realize the full buffering potential. Below 10 mM, buffers of this type have little buffering capacity (that is, they cannot counteract anything but the smallest changes in pH) and above 50 mM the solid buffers risk precipitation in higher organic compositions.

It should also be noted that eluent temperature can be used to affect selectivity changes and the retention of ionizable analytes is usually affected most, especially relative to nonionizable species. Thus, variations of as little as 5 °C can profoundly affect selectivity in some cases.