|Buffer choice for HPLC separations|
The mobile phase pH can change on standing, with ingress of CO2 from the atmosphere for example, and a buffer can help to combat this effect to a certain extent. Similarly, volatile reagents, such as TFA, may also selectively evaporate, thus changing the eluent pH. There is, however, no substitute to regularly replacing the buffer on our HPLC system!
Perhaps the largest potential for pH change is on mixing of the injection slug with the mobile phase within the tubing and components of the autosampler, or at the head of the HPLC column, where more extensive mixing of the sample diluent and eluent occurs. If the sample diluent pH differs greatly from the eluent, the ‘local’ pH will change as the two mix — leading to retention time variability and peak distortion as not all analyte molecules experience the same solution pH and, therefore, may exhibit different partitioning behaviour.
Why is pH Important?
The mobile phase pH value will depend upon the analytes pKa (partial acid dissociation constant) value and may be derived by experimentation or computer simulation or both.
HA A− + H+
to represent the dissociation of the acid.
The dissociation constant (Ka) for this equilibrium can be written as;
Because of the wide range of Ka values possible (several orders of magnitude), it is more convenient to talk about the partial acid dissociation constant, which can be written
pKa = −log10Ka
We can also express the pKa in terms of pH using the Henderson Hasselbalch equation;
It should be obvious that pH is equal to pKa when the associated and dissociated forms of the acid are present in equal concentrations (i.e., when [A− ] = [HA]). This represents the value at which the acid (or base) is 50% ionized [dissociated if acidic (A− ), associated if basic (BH+ )].
Regarding buffer concentration with respect to buffer capacity and whether there is an easy formula to calculate the concentration of buffer required taking into account the buffer capacity. There is no easy theoretical answer to this question, so we tend to take a very empirical approach. In most situations, 5–15 mM will be sufficient. Start with 25 mM of buffer and adjust lower if unpredictable selectivity changes are observed during method development and higher if a lack of robustness in retention time or poor peak shape is noted — otherwise it will remain unchanged! Note that at higher concentrations (especially above 60% acetonitrile), the inorganic buffers risk precipitation.
Volatile Buffers for MS Work
The volatile buffers usually associated with mass spectrometry are also highlighted in Table 1. These are typically used to avoid fouling of the atmospheric pressure ionization
It should be noted that TFA (trifluoroacetic acid) is not a buffer. It has no useful buffering capacity in the pH range usually associated with reversed-phase HPLC. Instead it is used to adjust the mobile phase pH well away from the pKa of the analytes such that small changes in pH that occur will not affect the chromatographic retention or selectivity.
However, a significant disadvantage of TFA is its ion-pairing capability and its tendency to ion pair with ionized analyte molecules in the gas phase within the API interface and potentially drastically reduce MS sensitivity for certain analytes. TFA is best avoided unless one knows something about the interaction of TFA with the analytes under investigation.
Generally Useful Buffer Facts!
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