A detailed evaluation of the advantages and limitations of online RP-LC xHILIC compared to HILIC xRP-LC for phenolic analysis

The combination of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC) has proved effective in the LC x LC analysis of polyphenols due to the high degree of orthogonality associated with these separation modes for various classes of phenolic compounds. However, despite the growing number of such applications, HILIC is almost exclusively used as the first dimension ( 1 D) separation mode, and RP-LC in the second dimension ( 2 D). This is somewhat surprising in light of the potential advantages of swapping these separation modes. In this contribution, we present a detailed evaluation of the potential of online RP-LC x HILIC-MS for the analysis of phenolic compounds, comparing the performance of this system to the more established HILIC x RP-LC-MS configuration. Method development was performed using a predictive optimisation program, and fixed solvent modulation was employed to combat the solvent incompatibility between HILIC and RP-LC mobile phases. Red wine, rooibos tea, Protea and chestnut phenolic extracts containing a large diversity of phenolic compound classes were analysed by both HILIC x RP-LC- and RP-LC x HILIC-MS in order to compare the separation performance. Overall, the kinetic performance of HILIC x RP-LC was found to be clearly superior, with higher peak capacities and better resolution obtained for the majority of samples compared to RP-LC x HILIC analyses using similar column dimensions. Dilution of the 1 D solvent combined with large volume injections proved insufficient to focus especially phenolic acids in the 2 D HILIC separation, which resulted in severe 2 D peak distortion for these compounds, and negatively impacted on method performance. On the other hand, a noteworthy improvement in the sensitivity of RP-LC x HILIC-MS analyses was observed due to higher ESI-MS response for the 2 D HILIC mobile phase and greater sample loading capacity of the 1 D RP-LC column, brought on by the high solubility of phenolic samples in aqueous solutions. As a result, a significantly higher number of compounds were detected in the RP-LC x HILIC-MS separations. These findings point to the potential advantage of RP-LC x HILIC as a complementary configuration to HILIC x RP-LC for phenolic analysis.

Automated summary

The combination of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC) in LC x LC analysis of polyphenols is highly orthogonal for various classes of phenolic compounds. However, the use of HILIC as the first dimension (1D) separation mode and RP-LC as the second dimension (2D) is surprising considering the potential advantages of swapping these separation modes. In this study, the potential of online RP-LC x HILIC-MS for phenolic compound analysis was evaluated and compared to the established HILIC x RP-LC-MS configuration. The kinetic performance of HILIC x RP-LC was found to be superior, but RP-LC x HILIC-MS showed improved sensitivity and detected a higher number of compounds. These findings suggest that RP-LC x HILIC could be a complementary configuration for phenolic analysis.