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Of pH and adsorption isotherms

In the last issue of KromaNews we looked at the importance of performing overloading studies during preparative HPLC method development. It was shown that switching the stationary phase, while keeping all other chromatographic parameters constant, altered the non-linear adsorption behavior.
     In this issue, the adsorption behavior of a peptide on the stationary phase at different buffer pH will be shown.

The general method used for the experiments is the following:

  • Column:Kromasil 100-10-C18 4.6 × 250 mm
  • Mobile phase: 0.2 M buffer/EtOH gradient
  • Load: 1.2 g/kgmedia
  • Flow rate: 1 ml/min
  • Temperature: ambient
  • Detection: 280 nm

326 – 3598.097.3Langmuir
426 – 3598.197.8Langmuir
531 – 4098.076.5Anti-Langmuir
631.5 – 40.598.072.3S-shaped
732 – 4198.078.9S-shaped
832 – 4198.078.9Langmuir

The reconstructed elution profiles clearly show that altering pH greatly affects the adsorption behavior of the product. At pH 3-4, a typical langmurian-shaped elution pattern is seen. However, when further increasing pH, the elution pattern changes to anti-langmurian, and finally, at pH 8, the langmurian peak shape is restored. It can also be seen that this greatly affects the chromatographic results. At every pH, yield at 98 % purity differs substancially: from more than 97 % yield at pH 3 and pH 4, down to 72 % yield at pH 5 - 6. These results show that buffer pH is one of the most important factors in preparative RP-HPLC. A minor change can significantly alter the separation behavior and product purity.

The results presented herein can only be seen when performing overloading studies. Therefore, overloading studies should always be performed for preparative HPLC method development. Apart from the stationary phase and pH, other parameters that may have a similar effect are buffer type, additives, temperature, and loading amount.