Column Solvent Screening

It’s all about the Chemistry

The Quality by Design methodology intrinsic to Fusion QbD LC Method Development software dictates that primary effects are studied first. These include column type, aqueous pH, and organic solvent selection. In short the need is to determine which chemistry will give the best chance of a robust HPLC method. The experiments performed to determine this chemistry are known as HPLC Column Solvent Screening.

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HPLC column solvent screening presents many challenges as the experimental parameters are hard to change physical objects rather than easy to change instrument settings. Traditionally this often led to a best guess approach to chemistry selection followed up with one factor at a time “tinkering” of the easy to change instrument parameters. Only if this failed to find a “workable” method was an alternative “chemistry” considered. Advances in instrumentation has lead to the availability of column and mobile phase selection valves which have gone some way to enabling a fully automated column solvent screening platform. However the instruments controlling software has not had the inbuilt intelligence to make this a reality.

Interfaced with Fusion QbD, your HPLC becomes an automated machine for the execution of column solvent screening experiments in strict accordance with the experiment design determined by Fusion QbD. Fusion QbD will first build the necessary instrument methods automatically within the data system and then order them in a logical manner within a ready to run Sequence / SampleSet. All you need do is pop your sample in the autosampler!

Solvent screening necessitates the preparation of multiple mobile phases covering the range of aqueous pH under study. These may be prepared individually in the normal way and made available via a solvent selection valve. Experiments requiring a given pH will then switch the valve to the position plumbed to that pH. This arrangement can be used on any pump fitted with a selection valve (internal or external). If no switching valve exists, Fusion will split the experimental design into separate pH specific sequences / samplesets. This allows for the manual swapping of mobile phases between executing each sequence.

If a quaternary pump is available Fusion QbD can do away for the need for manual preparation of different pH phases altogether. Instead a buffer system may be chosen and the required pH levels selected for experimentation. Only the acid / base solutions are required to be prepared and Fusion will blend these in the required proportions to automatically attain the required pH. This removes manual workload for the analyst, enabling a greater number of levels to be studied, so ensuring the correct characterization of the pH effect. This also removes the issue of dead volume between a solvent selection valve and the pump head which can be an issue at low flow rates, necessitating a longer equilibration time.

Analogous functionality exists for the study of Buffer Strength, and additive concentration. It can also be used to hold the buffer/ionic strength constant over a gradient.

HPLC Column Solvent Screening by definition involves experimenting with different chemistry systems, where different selectivity (order of elution) is very much the aim. Therefore peak exchange and co-elution is to be expect. Traditionally this has caused a headache for the chromatographer as they struggle to correctly track and identify individual peaks. This has lead to incomplete data sets and poorly predicting models.

Fusion QbD’s patented “Trend Response” feature is a game changer. Simply put, it challenges the notion that peaks need to be identified during column solvent screening. Trend Responses are parameters that characterise the whole chromatogram rather than those of individual peaks. Examples include:

  • No. of integrated peaks
  • No. of peaks meeting a defined condition, e.g.
    •  resolution <1.5
    • As <0.9, >1.1
  • RT of last retained peak
  • K Prime of first eluting peak
  • Resolution of the “n” largest peak

Modelling and setting goals against such characteristics is a highly effective way to determine the best separation chemistry for your sample, setting you on the right path towards a robust HPLC method.