Getting Insights Into Chromatographic Properties of HILIС and Mixed-Mode Homemade Stationary Phases Using Principal Component and Cluster Analyses

In this work, we compared the chromatographic properties of 27 homemade monomer- and polymer-modified stationary phases synthesized via the Ugi reaction for hydrophilic interaction liquid chromatography (HILIC). These stationary phases along with the unmodified substrate were characterized by retention factors of 33 polar biologically active compounds belonging to various classes (nucleobases/nucleosides, sugars, carboxylic acids, and water-soluble vitamins). Additionally, the widely used Tanaka HILIC test was performed. The experimental data from both characterization approaches were processed using several chemometric techniques, including principal component analysis (PCA), hierarchical cluster analysis (HCA), and K-means algorithm. It was initially expected that polymer-modified phases would differ significantly from monomer-modified ones due to their mixed-mode properties. It was confirmed by the clear separation of these two types of stationary phases on the PCA score plot obtained for binary logarithms of selectivities (calculated from all 33 retention factors). Dissimilarities observed among some monomer-modified stationary phases resulted in insights into Ugi reaction conditions suitable for obtaining adsorbents with distinct chromatographic properties. Each class of test compounds required specific mobile phase composition to achieve reasonable chromatographic characteristics, such as retention times and peak shapes. To exclude the long-lasting re-equilibration stage associated with mobile phase changes, a smaller set of only three test compounds was proposed, yielding nearly the same clustering results as the complete dataset. This simplified procedure can facilitate the rapid characterization of newly synthesized stationary phases and allow for comparison with previously studied phases.