Hydrophilic interactions change along with volume fractions

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  • Published: Aug 1, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: Laboratory Informatics / Chemometrics & Informatics
thumbnail image: Hydrophilic interactions change along with volume fractions

HILIC increasingly used for analysis

Lipophilicity is an important variable affecting drug compounds’ activities

In recent years, hydrophilic interaction liquid chromatography (HILIC) has become a more common analytical method. It can be considered a form of normal phase HPLC since it uses a polar stationary phase, such as silica or modified silica. The solvent consists of a water-miscible organic solvent, typically acetonitrile, mixed with a smaller quantity of aqueous phase. The proportion of the aqueous phase is usually increased in a gradient as the column is run, while still retaining the minor non-aqueous component.

The theory of HILIC has not yet been as exhaustively investigated as that of reverse phase HPLC. The Belgrade researchers examined the relationship between retention factors, k, and the fraction of the aqueous phase in the mobile phase, φ. The compounds initially examined were the antipsychotic pharmaceutical olanzapine and eight of its impurities.

Various models used to give HILIC retention factors

The HILIC was carried out with UV detection using a Waters Breeze HPLC system. Four different columns were examined: unmodified silica (BetaSil, Thermo Fisher scientific), diol (BetaSil Diol), cyano (BetaSil CN) and zwitterionic. (SeQuant Zic-HILIC, Merck Millipore). The organic phase was acetonitrile and the aqueous phase was 20 mM ammonium acetate buffer at pH 3.0. For the majority of runs, the aqueous phase was increased in a gradient from 5 to 20%, increasing in 3% steps. For some impurities this gave too long a retention time, so gradients from 8 to 23% aqueous and 11 to 26% aqueous were used for some ‘long runners’.

A number of different mathematical models were applied to the experimental data. A non-localised partition model, often applied to reversed phase systems, was first used, with the equation ln k = ln kw – Sφ, where k was the retention factor, kw the retention factor in neat acetonitrile, S a constant and φ the volume fraction of the aqueous phase. A localised absorption model, often used for normal phase HPLC with wholly organic solvents, was also used, with the equation ln k = ln kw – S ln φ. Neither model proved a particularly good fit to the experimental data. A so-called mixed model, with the equation ln k = ln kw + S1φ + S2 ln φ, gave a somewhat better fit.

A quadratic equation, ln k = ln kw + S1φ + S2φ2, proved to give a good fit to the experimental data, with high coefficients of determination (R2) and coefficients of prediction (Q2). However, this complex equation can be difficult to apply and does not readily lend itself to extrapolation.

Finally, a so-called spline interpolation technique was used, in which the graph of ln k vs. φ for each substance was split into segments between interpolation nodes. A different polynomial was created for each segment using Matlab 7.10.0 software. The polynomials were of the form an0 + an1φ + an2φ2 + an3φ3, for n = 1 to 5 (the number of interpolation nodes used). The spline technique gave high coefficients of prediction (Q2). It was tested for a particular solvent composition, φ = 16%, giving retention factors, k, within 10% of experimental values, which was a superior performance to that of the other models. The spline model was also applied to the anti-nausea drug granisetron and two of its impurities, again giving good coefficients of prediction.

Spline model applied successfully to HILIC

The paper showed that the mechanism of HILIC is complicated and sometimes cannot be represented by a simple partition or absorption model. A quadratic model could be successfully applied to HILIC on olanzapine. An empirical spline model was also successfully applied to HILIC, both for olanzapine and granisetron. These results suggest that the predominant mechanism in HILIC changed as the proportion of water in the eluting solvent increased. Further work is needed to clearly elucidate the mechanisms involved in HILIC.

Related Links

Journal of Chemometrics, 2017, Early View paper. Tumpa et al. Modeling of HILIC retention behavior with theoretical models and new spline interpolation technique.

Wikipedia, Hydrophilic Interaction Chromatography

Wikipedia, Spline (Mathematics)

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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