A New Definition of the Stationary Phase Volume in Mixed-Mode Chromatographic Columns in Hydrophilic Liquid Chromatography.

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.

Dual-mode hydrophilic interaction normal phase and reversed phase liquid chromatography of polar compounds on a single column.

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.

Comprehensive two-dimensional monolithic liquid chromatography of polar compounds.

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.

Mobile phase effects in reversed-phase and hydrophilic interaction liquid chromatography revisited.

Correct adjustment of the mobile phase is equally important as the selection of the appropriate column for the separation of polar compounds in LC. Both solvophobic and selective polar interactions control the retention in the Reversed Phase and Hydrophilic Interaction modes. The retention models describing the effects of the volume fraction of the strong eluent component in binary mobile phases on the sample retention factors apply in a limited mobile phase composition range. We introduced a three-parameter retention model, which provides improved prediction of retention over a broad mobile phase range, under isocratic and gradient elution conditions. The model does not imply any assumptions concerning either adsorption or partition distribution mechanism, but allows estimating retention in pure strong and in pure weak mobile phase components. The experimental retention data for phenolic acids and flavones on several core-shell columns with different types of stationary phases agree with the theory. Many polar columns with important structural hydrophobic moieties show dual retention mechanism, (Reversed Phase in water rich mobile phases and Hydrophilic Interaction at high acetonitrile concentrations). It is possible to select the mobile phase compositions in each of the two modes for separations of samples containing compounds largely differing in polarity. The three-parameter model describes the retention in each mode, with separately determined best-fit parameters. We applied the two-mode model to the retention data of sulfonamides and benzoic acid related compounds on a new polymethacrylate zwitterionic monolithic micro-column.

Mobile phase effects on the retention on polar columns with special attention to the dual hydrophilic interaction-reversed-phase liquid chromatography mechanism, a review.

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.

Retention Models on Core-Shell Columns.

A thin, active shell layer on core-shell columns provides high efficiency in HPLC at moderately high pressures. We revisited three models of mobile phase effects on retention for core-shell columns in mixed aqueous-organic mobile phases: linear solvent strength and Snyder-Soczewinski two-parameter models and a three-parameter model. For some compounds, two-parameter models show minor deviations from linearity due to neglect of possible minor retention in pure weak solvent, which is compensated for in the three-parameter model, which does not explicitly assume either the adsorption or the partition retention mechanism in normal- or reversed-phase systems. The model retention equation can be formulated as a function of solute retention factors of nonionic compounds in pure organic solvent and in pure water (or aqueous buffer) and of the volume fraction of an either aqueous or organic solvent component in a two-component mobile phase. With core-shell columns, the impervious solid core does not participate in the retention process. Hence, the thermodynamic retention factors, defined as the ratio of the mass of the analyte mass contained in the stationary phase to its mass in the mobile phase in the column, should not include the particle core volume. The values of the thermodynamic factors are lower than the retention factors determined using a convention including the inert core in the stationary phase. However, both conventions produce correct results if consistently used to predict the effects of changing mobile phase composition on retention. We compared three types of core-shell columns with C18-, phenyl-hexyl-, and biphenyl-bonded phases. The core-shell columns with phenyl-hexyl- and biphenyl-bonded ligands provided lower errors in two-parameter model predictions for alkylbenzenes, phenolic acids, and flavonoid compounds in comparison with C18-bonded ligands.

Recent advances in stationary phases and understanding of retention in hydrophilic interaction chromatography. A review.

Hydrophilic interaction chromatography (HILIC) is a rapidly developing liquid chromatography mode suitable for separation of strongly or moderately polar samples on polar columns in highly organic mixed mobile phases. In the past five years many new types of columns appeared based on silica gel, organic polymers and other types of supports. The present work reviews the new additions to their family. Further, the progress in the understanding of theoretical principles of the HILIC mode, especially of the role of the mobile phase, and of the adsorbed water are addressed. The amount of adsorbed water strongly depends on the type of the polar column and may be used to distinguish between "conventional HILIC" and "aqueous normal phase chromatography (ANP)". Further, sample structural effects on the retention are treated in terms of the Linear Solvation Energy Relationship model adopted for partially ionized polar compounds. The impact of dual HILIC-reversed phase (RP) mechanism on a possible increase of the applications of some polar columns is discussed. Attention is paid also to the increasing role of the HILIC mode in various two-dimensional LC techniques and the related mobile phase compatibility problems.

Investigation of the temperature dependence of water adsorption on silica-based stationary phases in hydrophilic interaction liquid chromatography.

In the present work, the adsorption of water was investigated in aqueous normal-phase liquid chromatography on Cogent Silica C and Cogent Phenyl hydride stationary phases at different temperatures by frontal analysis - using coulometric Karl Fischer titration - to compare the temperature dependence of adsorption of water from aqueous acetonitrile. The Cogent Silica-C and Cogent Phenyl Hydride columns have a silicon hydride surface (silica hydride) with less than 2% free silanol group; therefore, they do not have a strong association with water. The adsorption behavior of water on the mentioned stationary phases was modeled by Langmuir isotherm. The preferentially adsorbed water was expressed in terms of a hypothetical monomolecular water layer equivalent in the inner pores. The uptake of water slightly depends on the temperature. The adsorbed water may fill four to eight percent of the pore volume over the studied temperature range, which approximately corresponds to the equivalent of 0.24-0.68 water layer coverage of the adsorbent surface. The phenyl hydride stationary phase shows decreased water uptake in comparison to the Silica C stationary phase.

Effect of water on the retention on diol and amide columns in hydrophilic interaction liquid chromatography.

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.

The effects of temperature and mobile phase on the retention of aliphatic carboxylic acids in hydrophilic interaction chromatography on zwitterionic stationary phases.

The influence of the mobile phase and temperature, on the retention behavior of seven aliphatic acids (pyruvic, gluconic, 2-oxoglutaric, tartaric, malic, oxalic, and citric acid) in hydrophilic interaction liquid chromatography on zwitterionic stationary phases with sulfobetaine and phosphorylcholine ligands is investigated. In agreement with the van't Hoff model, most acids show linear ln k versus 1/T plots. However, the retention of structurally symmetrical oxalic and tartaric dicarboxylic acids is almost independent of temperature, or slightly increases at rising temperature. The experimental parameters of the van't Hoff plots suggest positive entropic contributions to the retention of these symmetrical acids, possibly connected with changes in molecular symmetry on their adsorption. The type of the zwitterionic stationary phase and the mobile phase composition (the molar concentration of acetate buffer and the volume fraction of acetonitrile) affect the retention and the selectivity of the separation of the acids.

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.

Separation of flavonoids on different phenyl-bonded stationary phases-the influence of polar groups in stationary phase structure.

Four phenyl-bonded stationary phases, differing in polar embedded group between spacer and phenyl ring, were used for the separation of flavonoids in reversed-phase conditions. In addition, the work was focused on the comparison of these stationary phases in terms of retention and nature of interactions between flavonoid solutes and both, mobile and stationary phases. The differences and similarities between the columns and between individual flavonoids were evaluated by a statistical analysis. The retention over the wider range of mobile phase composition was described using well known model suggested for partition chromatographic systems. Due to differences in polarity of flavonoids, gradient elution had to be applied to achieve appropriate conditions for the successful separation. A chromatographic optimization software was employed for establish the appropriate profiles of gradient separations using UV detection at 275 nm. The most appropriate conditions for the separation of flavonoids were apparent on the phenyl and phenoxy columns.

Possibilities of retention prediction in fast gradient liquid chromatography. Part 3: Short silica monolithic columns.

We studied possibilities of prediction of the gradient elution data for alkylbenzenes, flavones and phenolic acids on two short octadecyl silica gel monolithic columns, namely a Chromolith Flash C18, 25×4.6mm, and a "new generation" Chromolith High Resolution C18, 50×4.6mm, in fast 1-2min gradients. With fixed short gradient times and varying gradient ranges of acetonitrile concentration in water, high flow rates of the mobile phase (3-5mL/min) could be used. The gradient elution data were predicted from four gradient models based on two-parameter and three-parameter isocratic retention equations. Various gradient retention models can be used for prediction of chromatograms and optimization of separation within a fixed gradient time. A two-parameter log-log model introduced in 1974 and a three-parameter model introduced in 1980 provided slightly more accurate prediction than the Linear Solvent Strength (LSS) semi-logarithmic two-parameter model, most frequently used in reversed-phase LC. A three-parameter model introduced in 1978 provided slightly improved accuracy of prediction of gradient data with respect to two-parameter models, in contrast to another, more recent three-parameter empirical model introduced in 2010 (which failed for gradients starting at a non-zero concentration of acetonitrile). Both a longer (5cm) and more efficient Chromolith HR column and a shorter (2.5cm) slightly less efficient Chromolith Flash column provide useful separations in fast gradients (1-2min) at high flow rates (3.5-5mL/min), especially in second dimension of two-dimensional LC×LC, in combination with HILIC separation on monolithic microcolumn in D1.

Polymethacrylate monolithic columns for hydrophilic interaction liquid chromatography prepared using a secondary surface polymerization.

Zwitterionic methacrylate based polymeric monolithic columns were prepared in two-step polymerizations, with reduced polymerization times. Characteristic properties such as hydrodynamic permeability, porosity, retention factors, and pore size distribution charts were used for column evaluation. A scaffold column was fabricated by polymerization of poly(lauryl methacrylate-co-tetraethyleneglycol dimethacrylate) and was used without further modification as a support for a poly(N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine-co-bisphenol A glycerolate dimethacrylate) second monolith layer with zwitterionic functionality, for HILIC separations. An additional internal structure was formed by the second monolithic layer. The fabrication procedure was reproducible with RSD<5%. Field emission scanning electron microscopy has also been used to investigate column pore morphology, using a novel technique where the polymeric material is imaged directly, without coverage with a conducting film or particles. The new polar monolithic columns were used for HILIC separations of phenolic acids, flavones, nucleosides, and bases of nucleic acids, with similar efficiencies but different selectivities for zwitterionic methacrylate monolithic columns recently prepared by single step polymerization.

Thermodynamics Study of Solvent Adsorption on Octadecyl-Modified Silica.

Elution and solvation processes in liquid chromatography may be controlled by temperature changes. In the case of solvent adsorption, the temperature influences the amount of adsorbed solvent as well as the enthalpy and entropy of the solvation process. In this work, the thermodynamic parameters of organic solvents used as organic modifiers in the reversed-phase high-performance liquid chromatography elution process were determined. The changes of enthalpy and entropy in a series of chemically bonded stationary phases were measured to determine the effects of the temperature and surface coverage density of octadecyl ligands on the thermodynamic parameters of the solvation. For both the enthalpy and entropy a parabolic trend was observed with the minimum for medium surface coverage. The correlation of solvent adsorption values with the enthalpy of solvation was also investigated. The highest influence of the temperature on solvation process was observed for stationary phases with high surface coverage.

Monolithic and core-shell columns in comprehensive two-dimensional HPLC: a review.

The crucial point affecting the separation time in comprehensive two-dimensional liquid chromatography is the performance of the column used in the second dimension, which should allow highly efficient fast chromatographic separations in the short time available for the analysis of fractions transferred from the first to the second dimension (often 1 min or less). This can be accomplished on short columns packed with sub-2-µm particles, at the cost of very high operation pressure. Core-shell or silica monolithic columns have better permeability, and their use in the second dimension of comprehensive two-dimensional liquid chromatography with conventional liquid chromatography instrumentation is continuously increasing. Monolithic columns based on organic polymer matrices offer a wide selection of stationary phase chemistries, including new hydrophilic interaction liquid chromatography materials, which can be used in the design of novel two-dimensional separations. Some organic polymer monolithic materials offer a dual retention mechanism (reversed-phase hydrophilic interaction liquid chromatography), so a single column can be used in alternating runs for highly orthogonal off-line two-dimensional and even three-dimensional separations. In the present work, the properties of core-shell and silica gel monolithic columns are briefly summarized and their applications in two-dimensional separations of peptides, proteins, oligomer surfactants, fats and oils, carotenoids, phenolic and flavone compounds in plant extracts, food, and beverages are reviewed.

Determination of the polyphenolic content of a Capsicum annuum L. extract by liquid chromatography coupled to photodiode array and mass spectrometry detection and evaluation of its biological activity.

The present study was aimed to investigate the polyphenolic profile of a pepper (Capsicum annuum L.) extract from Algeria and evaluate its biological activity. The total polyphenol content of the extract was determined as 1.373 mg of gallic acid equivalents (±0.0046), whereas the flavonoids were determined as 0.098 mg of quercetin (±0.0015). The determination of the complete polyphenolic profile of the extract was achieved by liquid chromatography with an RP-amide column in combination with photodiode array and mass spectrometry detection through an electrospray ionization interface. A total of 18 compounds were identified, of which five were reported for the first time in the sample tested. Quercetin rhamnoside was the most abundant compound (82.6 µg/g of fresh pepper) followed by quercetin glucoside (19.86 µg/g). The antioxidant activity and antimicrobial effects were also determined. For the antimicrobial tests assessed against Gram-positive and Gram-negative bacteria, kaempferol showed the strongest inhibitory effect followed by quercetin and caffeic acids. In the study of the cytotoxicity of the extract, the cancer cells (U937) were more affected than the normal cells (peripheral blood mononucleated cells), with more than 62% inhibition at the highest concentration.

Adsorption of water from aqueous acetonitrile on silica-based stationary phases in aqueous normal-phase liquid chromatography.

Excess adsorption of water from aqueous acetonitrile mobile phases was investigated on 16 stationary phases using the frontal analysis method and coulometric Karl-Fischer titration. The stationary phases include silica gel and silica-bonded phases with different polarities, octadecyl and cholesterol, phenyl, nitrile, pentafluorophenylpropyl, diol and zwitterionic sulfobetaine and phosphorylcholine ligands bonded on silica, hybrid organic-silica and hydrosilated matrices. Both fully porous and core-shell column types were included. Preferential uptake of water by the columns can be described by Langmuir isotherms. Even though a diffuse rather than a compact adsorbed discrete layer of water on the adsorbent surface can be formed because of the unlimited miscibility of water with acetonitrile, for convenience, the preferentially adsorbed water was expressed in terms of a hypothetical monomolecular water layer equivalent in the inner pores. The uptake of water strongly depends on the polarity and type of the column. Less than one monomolecular water layer equivalent was adsorbed on moderate polar silica hydride-based stationary phases, Ascentis Express F5 and Ascentis Express CN column at the saturation capacity, while on more polar stationary phases, several water layer equivalents were up-taken from the mobile phase. The strongest affinity to water was observed on the ZIC cHILIC stationary phases, where more than nine water layer equivalents were adsorbed onto its surface at its saturation capacity. Columns with bonded hydroxyl and diol ligands show stronger water adsorption in comparison to bare silica. Columns based on hydrosilated silica generally show significantly decreased water uptake in comparison to stationary phases bonded on ordinary silica. Significant correlations were found between the water uptake and the separation selectivity for compounds with strong polarity differences.

Retention and bandwidths prediction in fast gradient liquid chromatography. Part 2-Core-shell columns.

Recently, we confirmed that the well-established theory of gradient elution can be employed for prediction of retention in gradient elution from the isocratic data, method development and optimization in fast gradient chromatography employing short packed fully porous and monolithic columns and gradient times in between 1 and 2min, or even less. In the present work, we extended this study to short core-shell reversed-phase columns. We investigated the effects of the specification of the stationary phase in the core-shell structure on the prediction of gradient retention data. Two simple retention models describing the effects of the mobile phase on the retention by two-parameter equations yield comparable accuracy and can be used for prediction of elution times. The log-log model provides improved prediction of gradient bandwidths, especially for less retained compounds. A more sophisticated three-parameter model did not offer significant improvement of prediction. We compared the efficiency, selectivity and peak capacity of fast gradient separations of alkylbenzenes, phenolic acids and flavones on seven core shell columns with different lengths and chemistry of bonded shell stationary phase. Within the limits dictated by a fixed short separation time, appropriate adjustment of the range of the composition of mobile phase during gradient elution is the most efficient means to optimize the gradient separation. The gradient range affects sample bandwidths equally or even more significantly than the column length. Both 5-cm and 3-cm core-shell columns may provide comparable peak capacity in a fixed short gradient time.