HILIC characterization: Estimation of phase volumes and composition for a zwitterionic column

Abstract

A methodology for the estimation of the different phase volumes in HILIC is presented. For a ZIC-HILIC column the mobile phase volume (hold-up volume) is determined in several acetonitrile-and methanol-water compositions by a Linear Free Energy Relationships (LFER) homologous series approach involving n-alkyl-benzenes, -phenones, and -ketones. We demonstrate that the column works as a HILIC column when the mobile phase contains high and medium proportions of methanol or acetonitrile. However, for acetonitrile contents below 20%, or 40% for methanol, same column works in RPLC. In between, a mixed HILIC-RPLC behavior is observed, and solutes of low molecular volume are retained as in HILIC mode, but the largest ones show RPLC retention. From the homologous series retention data and pycnometric measurements involving the pure organic solvents and their mixtures with water, the mean solvent composition of the water-rich transition layers between column functionalization and the bulk mobile phase, which act as stationary phase, is estimated. Finally, the phase ratio between stationary and mobile phases is also estimated for each eluent composition, allowing the calculation of the corresponding stationary phase volumes. All volumes are strongly dependent on the water content in the eluent, especially when acetonitrile is selected as mobile phase constituent. In HILIC mode, when the water content in the hydroorganic mobile phase increases, the volumes of mobile phase decrease, but the volumes of stationary phase (mainly the water layer adsorbed onto the bonded-phase and the water-enriched interface) increase. However, at high water concentrations, where the column works in RPLC mode, the mobile phase volume increases and the stationary phase (which is now the bonded zwitterion) decrease when increasing the water percentage in the mobile phase.

Evaluation of Hold-Up Volume Determination Methods and Markers in Hydrophilic Interaction Liquid Chromatography

“…A variation of the method derived from Linear Free Energy Relationships (LFER) models was proposed in an earlier study [ 24 ]. In the Abraham LFER model [ 25 ], the LFER variable (log k in chromatography [ 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ]), is given as a linear combination of the solute-solvent interactions modeled by the solute descriptors accounting for dispersion forces ( E ), dipolarity/polarizability ( S ), hydrogen bond acidity ( A ), hydrogen bond basicity ( B ) and molecular volume ( V ) according to Equation (4): where c is a non-solute dependent term accounting mainly for the chromatographic phase ratio, and e , s , a , b , and v the complimentary descriptors of the chromatographic system, all of them obtained by linear regression of the retention of a series of solutes against their solute descriptors. The Abraham descriptors E , S , A , and B for individual homologues in a series are almost constant and only V changes sequentially (linearly in fact) with the member number (number of -CH 2 - groups in the side alkyl chain).…”

“…Since V M and v values are expected to be independent of the specific homologous series used for the chromatographic system characterization, several homologous series can be altogether analyzed in the same fitting equation [ 35 ]: where n is the number of homologous series included in the model, and f i are binary flag descriptors (1 or 0) used as independent variables in the fitting (i.e., for homologues of i -th series, f i = 1 and f i ≠1 = 0). More precise and reliable results should be obtained from fittings to Equation (6), since homologues from different series cover a broader chemical space in terms of solute-solvent interactions.…”

“…Retention of the members of the three homologous series decreases for mobile phases with a high content of organic solvent, acetonitrile ( Figure 1 a) or methanol ( Figure 1 d), which is typical of HILIC retention. By increasing the water content in the mobile phase, 70% of acetonitrile ( Figure 1 b) and 80% of methanol ( Figure 1 e), the HILIC column starts to show a mixed retention mechanism: mainly HILIC for the smallest homologues and mainly RPLC for the largest ones [ 35 ]. When the water content in the mobile phase is high enough, for instance 50% of acetonitrile ( Figure 1 c) and 50% of methanol ( Figure 1 f), both retention trends can be clearly observed and U-shape curves are obtained.…”

The effect of root hairs on exudate composition: a comparative non-targeted metabolomics approach

“…A higher RSD might lead to an exclusion of features according to the RSD quality filter. Although HILIC and RP are offering some degree of orthogonality, not all compounds present in the sample are retained and amenable to this combination of methods, especially when the sample is a complex mixture of different compounds [ 68 , 69 ], as in the case of root exudates. Hence, some of the compounds detected with the DI-FT-ICR-MS may have eluted in the void volume of the LC-separations, which was not considered for evaluation of the TOF-MS data.…”

Automated Intact Glycopeptide Enrichment Method Facilitating Highly Reproducible Analysis of Serum Site-Specific N-Glycoproteome

“…Different methods have been developed to enrich intact glycopeptides from complex samples. , For example, lectin affinity chromatography enriches glycopeptides based on specific lectin–carbohydrate recognization, titanium dioxide (TiO 2 ) selectively enriches glycopeptides via coordination, and strong cationic exchange (SCX) via charge interaction. The TiO 2 - and SCX-based methods typically cannot enrich glycopeptides with neutral glycans. , In comparison, hydrophilic interaction chromatography (HILIC) could retain virtually all intact glycopeptides due to the high hydrophilicity of attached glycans and has become a popular method for site-specific glycoproteomic studies. − Glycoproteomic biomarker discovery usually requires the analysis of a large cohort of samples. Therefore, a highly efficient and reproducible glycopeptide enrichment method is crucial.…”