The LCGC Blog: Robustness in HPLC Eluents - - Chromatography Online
The LCGC Blog: Robustness in HPLC Eluents

The Column
Volume 10, Issue 13, pp. 14–21

Is 0.1% TFA (aq)/0.1% TFA in acetonitrile the ultimate robust high performance liquid chromatography (HPLC) mobile phase? Maybe not...

It would be great if we had arrived at a "global" high performance liquid chromatography (HPLC) mobile phase, but we haven't. Here is the reason why along with some suggestions on how to achieve high quality separations with robust eluent systems.

When developing a separation method, some fundamental choices have to be made — primarily the mode of chromatography used and the way that the retention and separation (selectivity) of analyte components will be controlled and optimized. In reversed-phase mode, retention and separation will depend upon the physico-chemical properties of our analytes. Knowing some of this information will help us to make more informed decisions about the stationary phase bonded chemistry (C18, C8, and so on) that is needed; however, this is outside the scope of this piece and I'm going to assume that a stationary phase has been chosen or that a range of phases will be screened to assess their suitability.

Figure 1: Selectivity and retention differences observed in the separation of 10 steroid analytes with a range of hydrophobicity.
More hydrophobic analytes (Log P > 1) will require more organic modifier, whereas more polar analytes that are less hydrophobic (Log P < 1) require less modifier. Selectivity is affected by the type of modifier used, which for many modern applications is either methanol or acetonitrile, both of which have different properties and interact differently with both our analytes and the stationary phase surface. Some simple separations may result in using a simple binary mixture of water and one of these modifiers in either an isocratic or gradient programmed mode. However, even in these simple situations we should be mindful of the pros and cons of the modifier and ways that we can ensure the method is robust.

Figure 2: Viscosity of various aqueous binary mixtures of solvents used for reversed-phase HPLC.
Different organic modifiers interact differently with analytes and the important properties that govern the selectivity of the common modifiers can be classified by their solvochromatic parameters. Dipole character (π*) is a measure of the ability of the solvent to interact with a solute via dipolar and polarization forces and will promote retention of polarizable analytes. Acidity (α) is a measure of the ability of the solvent to act as a hydrogen bond donor towards basic (acceptor) solutes and so will promote retention of bases. Basicity (β) is a measure of the ability of the solvent to act as a hydrogen bond acceptor towards an acidic (donor solute), therefore it will retain acidic analytes well. These characteristics, along with knowledge of the analyte chemistry, can be used to manipulate elution. In general, in a 50:50 mixture of solvent and water, acetonitrile will have a higher elution (eluotropic) strength than methanol.

Figure 3: Separations showing the various approaches to HPLC eluent optimization for ionizable compounds.
Acetonitrile typically gives better efficiency at higher linear velocity (flow rate) as it is a lower viscosity solvent, which is good for higher throughput analysis. It also has a lower UV cut-off, which can be beneficial when working at lower UV wavelengths.

Certain combinations of methanol and water form high viscosity azeotropic mixtures that can affect the use of certain solvent combinations at higher flow rates on instruments with lower back pressure limits.

Figure 4: Separations showing the change in retention and selectivity on changing the ionic strength of the buffer.
When using premixed mobile phases (both organic and aqueous portions combined in a single reservoir), one should always measure the relative amounts of each solvent required and then combine them. This is instead of "topping off" one solvent and making to volume with the other, because exothermic events can mean that the final aqueous/organic ration is incorrect. This is especially important with methanol/water mixtures. Furthermore, once mixed, one should guard against selective evaporation of the more volatile organic components, which may lead to a gradual shift in retention time of analytes (typically to later elution times) over extended campaigns of analysis. Try not to filter premixed mobile phases under vacuum, as we again risk loss of the more volatile organic component, which is an insidious and irreproducible problem.

Figure 5: Comparison of void marker and analyte retention drift to identify potential changes caused by eluent composition in reversed-phase HPLC separations.
If the analyte contains ionizable functional groups we may need to use pH to control the retention and selectivity of analytes. For acid species, a lower pH tends to result in longer retention and the opposite is true for basic analytes. The selectivity of the separation will depend upon the eluent pH and how close this value is to the pKa of the various analytes (the pH at which the ratio of ionized to non-ionized forms of the analyte is exactly 1:1). In this situation, two approaches are common. Adjust the pH to a high or low value (2.2 or 10.0 are typical values) to ensure that all analytes are either fully ionized or fully non-ionized and the eluent pH is well away from the pKa of the various analytes; or carry out a series of experiments to find an eluent pH, which gives good selectivity and resolution for analytes that may vary in their degree of ionization.


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