Is 0.1% TFA (aq)/0.1% TFA in acetonitrile the ultimate robust high performance liquid chromatography (HPLC) mobile phase?
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.
(PHOTO CREDIT: WLADIMIR BULGAR/GETTY IMAGES)
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.
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 1: Selectivity and retention differences observed in the separation of 10 steroid analytes with a range of hydrophobicity.
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 2: Viscosity of various aqueous binary mixtures of solvents used for reversed-phase HPLC.
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.
Figure 3: Separations showing the various approaches to HPLC eluent optimization for ionizable compounds.
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.
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 4: Separations showing the change in retention and selectivity on changing the ionic strength of the buffer.
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.
Figure 5: Comparison of void marker and analyte retention drift to identify potential changes caused by eluent composition
in reversed-phase HPLC separations.