Polar columns used in the HILIC (Hydrophilic Interaction Liquid Chromatography) systems take up water from the mixed aqueous-organic mobile phases in excess of the water concentration in the bulk mobile phase. The adsorbed water forms a diffuse layer, which becomes a part of the HILIC stationary phase and plays dominant role in the retention of polar compounds. It is difficult to fix the exact boundary between the diffuse stationary and the bulk mobile phase, hence determining the column hold-up volume is subject to errors. Adopting a convention that presumes that the volume of the adsorbed water can be understood as the column stationary phase volume enables unambiguous determination of the volumes of the stationary and of the mobile phases in the column, which is necessary for obtaining thermodynamically correct chromatographic data in HILIC systems. The volume of the aqueous stationary phase, Vex, can be determined experimentally by frontal analysis combined with Karl Fischer titration method, yielding isotherms of water adsorbed on polar columns, which allow direct prediction of the effects of the composition of aqueous-organic mobile phase on the retention in HILIC systems, and more accurate determination of phase volumes in columns and consistent retention data for any mobile phase composition. The n phase volume ratios of 18 columns calculated according to the new phase convention strongly depend on the type of the polar column. Zwitterionic and TSK gel amide and amine columns show especially strong water adsorption.
- Keywords
- hydrophilic interaction, liquid chromatography, polar columns, stationary and mobile phase,
- Publication type
- Journal Article MeSH
Two-dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On-line coupling of a reversed-phase column with an aqueous normal-phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on-line two-dimensional liquid chromatography needs a capillary or micro-bore column providing low-volume effluent fractions transferred to a short efficient second-dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro-columns in fused silica capillaries with structurally different dimethacrylate cross-linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed-phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on-line an organic polymer monolithic capillary column in the first dimension with a short silica-based monolithic column in the second dimension provides two-dimensional liquid chromatography systems with high peak capacities. The silica monolithic C18 columns provide higher separation efficiency than the particle-packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed-phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.
- Keywords
- comprehensive two-dimensional chromatography, monolithic columns, polymethacrylate monoliths, silica-based monolithic columns,
- Publication type
- Journal Article MeSH
We investigated the retention of phenolic acid and flavone antioxidants on five polar columns in buffered aqueous ACN mobile phases. All columns show mixed retention mechanism: RP in highly aqueous mobile phases and normal phase (hydrophilic interaction LC, HILIC) in mobile phases with high concentration of ACN. The Silica Hydride and the ZIC HILIC sulfobetaine zwitterionic columns show rather limited retention in the RP mode. The Luna HILIC column shows higher retention in both the HILIC and the RP modes in comparison to the PEG and DIOL columns. We characterized the selectivity of various HILIC systems using linear solvation energy relationship model with molecular structure descriptors characterizing selective molecular size, dipole-dipole and proton-donor/proton-acceptor interactions and we investigated the effects of the mobile phase composition on the linear solvation energy relationship characteristics of the separation phase systems to select suitable conditions for orthogonal HILIC separations in combination with RP systems. Dual retention mechanism offers possibilities for using complementary selectivity in the HILIC and the RP modes for sequential 2-D separations of natural antioxidants on a single Luna HILIC column. Column equilibration time of 15 min between alternating RP and HILIC gradient runs is sufficient for reproducible results.
- Publication type
- Journal Article MeSH
Monolithic columns for capillary hydrophilic interaction liquid chromatography (HILIC) were prepared in fused-silica capillaries by radical co-polymerization of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide and ethylene dimethacrylate in various binary and ternary porogen solvent mixtures with azobisisobutyronitrile as the initiator of the polymerization reaction. Columns showed mixed separation modes: reversed-phase (RP) in water-rich mobile phases and HILIC at high concentrations of acetonitrile (>60-80%) in aqueous-organic mobile phases. A continuous change in retention was observed at increasing concentration of water in acetonitrile, giving rise to characteristic U-turn plots of retention factors versus the concentration of water in the mobile phase, with minima corresponding to the transition between the mechanisms controlling the retention. The selectivity of organic polymer monolithic columns for HILIC separations can be varied by adjusting the concentration of sulfobetaine monomer and the composition of the porogen solvent in the polymerization mixture. Under HILIC conditions, the monolithic capillary sulfobetaine columns show separation selectivities for polar phenolic acids similar to those of a commercial silica-based sulfobetaine ZIC-HILIC column, which, however, has limited selectivity in the RP mode due to lower retention.
- Publication type
- Journal Article MeSH
Adopting a stationary phase convention circumvents problematic definition of the boundary between the stationary and the mobile phase in the liquid chromatography, resulting in thermodynamically consistent and reproducible chromatographic data. Three stationary phase definition conventions provide different retention data, but equal selectivity: (i) the complete solid phase moiety; (ii) the solid porous part carrying the active interaction centers; (iii) the volume of the inner column pores. The selective uptake of water from the bulk aqueous-organic mobile phase significantly affects the volume and the properties of polar stationary phases. Some polar stationary phases provide dual-mode retention mechanism in aqueous-organic mobile phases, reversed-phase in the water-rich range, and normal-phase at high concentrations of the organic solvent in water. The linear solvation energy relationship model characterizes the structural contributions of the non-selective and selective polar interactions both in the water-rich and organic solvent-rich mobile phases. The inner-pore convention provides a single hold-up volume value for the retention prediction on the dual-mode columns over the full mobile phase range. Using the dual-mode monolithic polymethacrylate zwitterionic micro-columns alternatively in each mode in the first dimension of two-dimensional liquid chromatography, in combination with a short reversed-phase column in the second dimension, provides enhanced sample information.
Hydrophilic interaction liquid chromatography is a separation technique suitable for the separation of moderately and highly polar compounds. Various stationary phases (SPs) for hydrophilic interaction liquid chromatography are commercially available. While the SPs based on the same type of ligand are available from different providers, they can display a distinct retention characteristics and separation selectivity. The current work is focused on characterization and comparison of the separation systems of two amide-based HPLC columns from two producers, i.e. XBridge Amide column and TSK gel Amide-80 column. Several characterization procedures (tests) were used to investigate the differences between these columns. The chromatographic behavior of selected analytes indicates that multimodal interactions are responsible for retention and separation on these columns. Multiple testing approaches were used in order to reveal subtle differences between the SPs. Both amide-based columns showed certain differences in retention, selectivity, and plate counts. Based on the tests used in this study, we conclude that the investigated columns provide a different degree of H-bonding interactions.
- Keywords
- Amide-based columns, Hydrophilic interaction liquid chromato-graphy, Linear free energy relationship, Peptides, Selectivity tests,
- MeSH
- Amides isolation & purification MeSH
- Chromatography, Liquid instrumentation MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Amides MeSH
Hydrophilic interaction liquid chromatography on polar columns in aqueous-organic mobile phases has become increasingly popular for the separation of many biologically important compounds in chemical, environmental, food, toxicological, and other samples. In spite of many new applications appearing in literature, the retention mechanism is still controversial. This review addresses recent progress in understanding of the retention models in hydrophilic interaction liquid chromatography. The main attention is focused on the role of water, both adsorbed by the column and contained in the bulk mobile phase. Further, the theoretical retention models in the isocratic and gradient elution modes are discussed. The dual hydrophilic interaction liquid chromatography reversed-phase retention mechanism on polar columns is treated in detail, especially with respect to the practical use in one- and two-dimensional liquid chromatography separations.
- Keywords
- dual retention mechanism, gradient elution, hydrophilic interaction liquid chromatography, two-dimensional separation, water adsorption,
- MeSH
- Chromatography, Liquid methods MeSH
- Chromatography, Reverse-Phase methods MeSH
- Diffusion MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Indicators and Reagents MeSH
- Ions MeSH
- Linear Models MeSH
- Organic Chemicals MeSH
- Silicon Dioxide MeSH
- Polymers chemistry MeSH
- Solvents MeSH
- Static Electricity MeSH
- Water chemistry MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Names of Substances
- Indicators and Reagents MeSH
- Ions MeSH
- Organic Chemicals MeSH
- Silicon Dioxide MeSH
- Polymers MeSH
- Solvents MeSH
- Water MeSH
The amount of water adsorbed on polar columns plays important role in hydrophilic interaction liquid chromatography. It may strongly differ for the individual types of polar columns used in this separation mode. We measured adsorption isotherms of water on an amide and three diol-bonded stationary phases that differ in the chemistry of the bonded ligands and properties of the silica gel support. We studied the effects of the adsorbed water on the retention of aromatic carboxylic acids, flavonoids, benzoic acid derivatives, nucleic bases, and nucleosides in aqueous-acetonitrile mobile phases over the full composition range. The graphs of the retention factors versus the volume fraction of water in mobile phase show "U-profile" characteristic of a dual hydrophilic interaction-reversed phase retention mechanism. The minimum on the graph that marks the changing retention mechanism depends on the amount of adsorbed water. The linear solvation energy relationship model suggests that the retention in the hydrophilic interaction liquid chromatography mode is controlled mainly by proton-donor interactions in the stationary phase, depending on the column type. Finally, the accuracy of hydrophilic interaction liquid chromatography gradient prediction improves for columns that show a high water adsorption.
- Keywords
- amide-bonded phases, diol-bonded phases, dual retention mechanism, hydrophilic interaction chromatography, water adsorption on columns,
- Publication type
- Journal Article MeSH
Monolithic continuous separation media are gradually finding their way to sample pre-treatment, isolation, enrichment and final analytical separations of a plethora of compounds, occurring as food components, additives or contaminants, including pharmaceuticals, pesticides and toxins, which have traditionally been the domain of particulate chromatographic materials. In the present review, recent advances in the technology of monolithic columns and the applications in food analysis are addressed. Silica-based monoliths are excellent substitutes to conventional particle-packed columns, improving the speed of analysis for low-molecular weight compounds, due to their excellent efficiency and high permeability. These properties have been recently appreciated in two-dimensional HPLC, where the performance in the second dimension is of crucial importance. Organic-polymer monoliths in various formats provide excellent separations of biopolymers. Thin monolithic disks or rod columns are widely employed in isolation, purification and pre-treatment of sample containing proteins, peptides or nucleic acid fragments. Monolithic capillaries were originally intended for use in electrochromatography, but are becoming more frequently used for capillary and micro-HPLC. Monoliths are ideal highly porous support media for immobilization or imprinting template molecules, to provide sorbents for shape-selective isolation of target molecules from various matrices occurring in food analysis. The separation efficiency of organic polymer monoliths for small molecules can be significantly improved by optimization of polymerization approach, or by post-polymerization modification. This will enable full utilization of a large variety of available monomers to prepare monoliths with chemistry matching the needs of selectivity of separations of various food samples containing even very polar or ionized compounds.
- Keywords
- Disks, Food analysis, MIPS, Organic polymer monoliths, Silica monolithic columns,
- MeSH
- Food Analysis methods MeSH
- Chromatography instrumentation MeSH
- Molecular Imprinting instrumentation MeSH
- Silicon Dioxide chemistry MeSH
- Polymers chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
- Names of Substances
- Silicon Dioxide MeSH
- Polymers MeSH
The polarity of (poly)methacrylate monolithic capillary columns was varied by using alkylmethacrylate monomers with butyl, cyclohexyl, 2-ethylhexyl, lauryl, and stearyl functional groups in the polymerization mixture. The hydrodynamic properties, as well as the retention characteristics in RP-LC of small molecules (alkylbenzenes) and of proteins under gradient elution conditions were studied. The RP selectivity depends on the type of alkyl chain in methacrylate monomer; however, there was no direct correlation between the size of the monomer molecule and methylene or aromatic selectivity of the monlithic column. The lowest selectivity was found for column based on lauryl methacrylate monomer. On the other hand, butyl methacrylate column shows high phenyl selectivity and the column with stearyl methacrylate possesses the highest methylene selectivity for small molecules. The retention increases with longer alkyl chain in methacrylate monomer, especially for high molar mass proteins on all prepared columns and showed gradient elution behavior of proteins in agreement with the linear solvent strength gradient model. The poly(laurylmethacrylate) column showed lowest hydrophobicity but best efficiency for proteins of all columns tested.
- Keywords
- Efficiency, Functional monomers, Polarity, Polymethacrylate monolithic columns, Proteins,
- MeSH
- Polymethacrylic Acids chemistry MeSH
- Organic Chemicals chemistry isolation & purification MeSH
- Proteins chemistry isolation & purification MeSH
- Chromatography, High Pressure Liquid instrumentation methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Polymethacrylic Acids MeSH
- Organic Chemicals MeSH
- polymethacrylic acid MeSH Browser
- Proteins MeSH
