24 February / March 2020
The use of Mobile Phase pH as a Method Development Tool
In LC separations, the mobile phase pH determines the ionisation state of ionisable analytes. The mobile phase pH can therefore be varied and used as a powerful tool to control analyte retention, peak shape and selectivity. This short article explains how retention of acidic, basic and neutral analytes is affected by mobile phase pH, as well as the requirements for carrying out separations at high and low pH. It also discusses how the chromatographer can utilise pH during method development.
Introduction
The fundamental goal of any LC separation is to obtain suitable resolution of the key analytes of interest. The fundamental resolution equation states that the resolution between two analyte peaks is governed by the separation effi ciency (N), analyte retention (k) and separation selectivity (α) [1]. Of these three parameters, α has the biggest impact on resolution and therefore, it pays to invest method development time in optimising the separation selectivity. Many analytical parameters can be used to affect the selectivity, in particular, optimisation of the column stationary phase, the organic modifi er, percentage organic and temperature etc. For separations involving ionisable analytes, mobile phase pH can have a profound effect on analyte retention and selectivity. It is, therefore, an important parameter to investigate during method development.
Analyte retention at different mobile phase pH
To a large degree, analyte retention in reversed-phase is dictated by analyte hydrophobicity. For ionisable analytes, as
the degree of ionisation increases, retention typically decreases (providing that no alternative modes of interaction such as ion exchange are present). Figure 1 summarises how the mobile phase pH affects the degree of ionisation for simple acidic and basic compounds. For basic analytes, at mobile phase pH’s below their pKa
, the analyte will
primarily be positively charged. At high pH (above their pKa
), they will be in their neutral
form and will be better retained by reversed- phase. Conversely, acidic species show their strongest retention with a mobile phase below their pKa
and are more weakly retained at high pH, in their deprotonated form.
To demonstrate the effect of mobile phase pH on analyte retention, a set of basic, acidic and neutral analytes were chromatographed at different pH’s (Figure 2) on a high pH stable reversed-phase C18 column. All other separation parameters such as gradient profi le, buffer concentration and temperature were kept constant and changes in analyte retention are therefore determined by the mobile phase pH.
Toluene contains no ionisable functionality and is therefore neutral over the entire pH range. This means that mobile phase pH
has no signifi cant effect on the retention of toluene. At low pH, the acidic analytes (3,4-dichlorobenzoic acid and mefenamic acid) are present in their non-ionised, neutral form and therefore show their strongest retention. As the mobile phase pH is increased to the analytes pKa
and beyond,
the degree of ionisation increases and a gradual decrease in retention is observed. In contrast, the basic analytes (nortriptyline and carvedilol) are positively charged at low pH and consequently show shorter retention. As the pH increases, the ionisation is suppressed and analyte retention increases. Protonated basic analytes may exhibit low retention and/ or poor peak shape when analysed at low pH. Performing the analysis at high pH (neutral form), is an approach which can dramatically improve both peak shape and retention and also provide gains in sensitivity (Figure 3).
It is important to note that, for maximum column lifetime, many silica based reversed- phase columns should be used within a limited pH range of approximately 2-8 and are therefore not suited to high pH work. To perform a high pH separation, it is therefore essential to use a stationary phase that is compatible with high pH mobile phases. A number of columns that can
Figure 1: Percentage ionisation of acidic (red) and basic (blue) analytes at various pH values.
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