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Buffer Capacity – an underestimated parameter in prep RP-HPLC

A chromatographic system is defined by the nature of the stationary and mobile phase. While there are a large, although discrete number of stationary phases to choose from, the selection of the right mobile phase composition renders almost infinite possibilities. We are elucidating one of the important parameters, namely the buffer capacity of a reversed phase eluent.

The purpose of a buffer is to counteract pH fluctuations that will occur if an acid or base is added to an aqueous system (injection of an acid or base into the mobile phase stream). Buffer capacity β is a function of pH, concentration, and pKA of the weak acid. The buffer capacity reaches its maximum at pH = pKA and is specific for different buffer systems as shown below:

Strong protolyte

e.g. TFA

Multivalent protolyte

e.g. H3PO4

Weak protolyte

e.g. HAc

example profile of strong protolyte example profile of polyvalent protolyte example profile of weak protolyte

The amount of acid or base that can be added to a volume of a buffer solution before its pH changes significantly:

equation for buffer capacity for strong protolyte equation for buffer capacity for polyvalent protolyte equation for buffer capacity for weak protolyte
Figure 1: Buffer capacity as a function of pH for strong, multivalent, and weak protolytes.

Double peaks or distorted peak profiles in preparative RPC are often a sign of insufficient buffer capacity. The solute elutes in two different forms due to non-uniform protonation. While low buffer concentrations in the range of 10-50 mM generally are adequate for analytical separations, the buffer concentration usually needs to be increased by a factor of 10-20 in order to render sufficient buffer capacity for overloaded injections. For strong protolytes, like amines, an additional increase of the buffer concentration in the feed solution is required in order to assure uniform protonation/deprotonation of the entire amount of the sample.

Figure 2 shows how the increase of the buffer concentration from 0.1M to 1.0 M TEA acetate was necessary in order to deprotonate the entire amount of the basic solute, thus achieving a proper retention (green trace). In the case of insufficient buffer capacity, roughly half of the amine is unretained and elutes almost with the void.

example profile of strong protolyte
Figure 2: Purification of a basic tricyclic API.
  • Mobile Phase:0.1M or 1M TEA acetate pH 10.0 / MeOH (20/80).
  • Column:Kromasil 100 Å, 10 µm, C18, 4.6 x 250mm.
  • Flow rate:1 ml/min.

For weak protolytes, such as peptides and proteins, the effect is less pronounced, however the importance of making sufficient buffer capacity available remains. Figure 3 depicts the improvement that was achieved by increasing the buffer concentration from 50 mM to 200 mM for the preparative separation of a 39 amino acid peptide. As can be seen, the low buffer concentration leads to a distorted peak profile. As a result of that, the targeted product purity of ≥ 98.0% was not reached, and the recovery for 97.6% pure product was as low as 57%. By increasing the buffer capacity, the peak profile changed towards an anti-langmuirian shape, and the recovery for the set purity target of ≥ 98.0% increase to 91%. Moreover, the product eluted as a more concentrated fraction, which is generally beneficial for further work-up.

example on peptide
Figure 3: Influence of buffer concentration on the preparative separation of a 39 amino acid peptide.
 Purity [%]Recovery [%]cel(Product) [mg/mL]
50 mM97.6573.3
200 mM98.0914.5