Automated dual two-dimensional liquid chromatography approach for fast acquisition of three-dimensional data using combinations of zwitterionic polymethacrylate and silica-based monolithic columns.

A monolithic sulfobetaine polymethacrylate micro-column BIGDMA-MEDSA designed in our laboratory, shows dual retention mechanism: In acetonitrile-rich mobile phase, hydrophilic interactions control the retention (HILIC system), whereas in more aqueous mobile phases the column shows essentially reversed-phase behavior with major role of hydrophobic interactions. The zwitterionic polymethacrylate micro-column can be used in the first dimension of two-dimensional LC in alternating reversed-phase (RP) and HILIC modes, coupled with an alkyl-bonded core-shell or silica-based monolithic column in the second dimension, for HILIC×RP and RP×RP comprehensive two-dimensional separations. During the HILIC×RP period, a gradient of decreasing acetonitrile gradient is used for separation in the first dimension, so that at the end of the gradient the polymeric monolithic micro-column is equilibrated with a highly aqueous mobile phase and is ready for repeated sample injection, this time for separation under reversed-phase gradient conditions with increasing concentration of acetonitrile in the first dimension. The fast repeating reversed-phase gradients on a short silica-monolithic or core-shell column in the second dimension can be optimized independently of the actual running first-dimension gradient program. As the alternating HILIC and RP separations on the first-dimension zwitterionic methacrylate column are based on complementary retention mechanisms, the instrumental setup essentially represents two coupled two-dimensional systems. It is first time that such an automated dual LCxLC approach is reported. The novel system allows obtaining three-dimensional data in a relatively short time and can be applied not only to multidimensional gradient separations of flavones and related polyphenolic compounds.

Improvement of the sensitivity of 2D LC-MEKC separation of phenolic acids and flavonoids natural antioxidants using the on-line preconcentration step.

A 2D method was developed for separation of phenolic acids and flavonoids natural antioxidants combining LC with MEKC. The in-capillary preconcentration step was applied for the improvement of the sensitivity of 2D method before the second dimension MEKC analysis. The influence of first dimension LC mobile phase composition on migration times in the second MEKC dimension was evaluated. When gradient elution is applied in the first dimension of 2D LC-MEKC system, increasing concentration of organic solvent in the mobile phase and in fractions transferred from LC influences the electroosmotic flow, partitioning equilibria of samples in micelles and properties of the micelles, which results in shifts of migration times during the consecutive runs in the second MEKC separation dimension. The shifts of migration times caused by the influence of increasing concentration of ACN on MEKC separation in second dimension of 2D LC-MEKC system were compensated by aligning the time axis using electroosmotic flow and micellar marker migration times. The optimized LC-MEKC method was applied on the separation of natural antioxidants in the plant extracts samples.

Orthogonality of silver-ion and non-aqueous reversed-phase HPLC/MS in the analysis of complex natural mixtures of triacylglycerols.

The goal of this work is the study of possibilities of two basic separation modes used in the analysis of complex triacylglycerol (TG) samples of plant oils and animal fats, i.e. non-aqueous reversed-phase (NARP) and silver-ion HPLC coupled with atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The orthogonality of both separation modes is tested for complex TG mixtures containing fatty acids (FAs) with different acyl chain lengths, different number, positions and geometry of double bonds (DBs) and different regioisomeric positions of FAs on the glycerol skeleton. The retention in NARP mode is governed by the equivalent carbon number, while the retention in silver-ion chromatography increases with the increasing number of DBs with a clear differentiation between cis- and trans-FAs. Moreover, silver-ion mode enables at least the partial resolution of regioisomeric TG mixtures including cis-/trans-regioisomers, as illustrated on two examples of randomization mixtures. Off-line 2D coupling of both complementary modes (NARP in the first dimension and silver-ion in the second dimension) yields the superior chromatographic selectivity resulting in the highest number of identified TGs ever reported for studied samples. Off-line 2D chromatograms are processed with the home-made software providing various ways of data visualization.