Highly efficient monolithic silica capillary columns modified with poly(acrylic acid) for hydrophilic interaction chromatography

Monolithic silica capillary columns for hydrophilic interaction liquid chromatography (HILIC) were prepared by on-column polymerization of acrylic acid on monolithic silica in a fused silica capillary modified with anchor groups. The products maintained the high permeability (K=5 x 10(-14)m(2)) and provided a plate height (H) of less than 10 microm at optimum linear velocity (u) and H below 20 microm at u=6mm/s for polar solutes including nucleosides and carbohydrates. The HILIC mode monolithic silica capillary column was able to produce 10000 theoretical plates (N) with column dead time (t(0)) of 20s at a pressure drop of 20 MPa or lower. The total performance was much higher than conventional particle-packed HILIC columns currently available. The gradient separations of peptides by a capillary LC-electrospray mass spectrometry system resulted in very different retention selectivity between reversed-phase mode separations and the HILIC mode separations with a peak capacity of ca. 100 in a 10 min gradient time in either mode. The high performance observed with the monolithic silica capillary column modified with poly(acrylic acid) suggests that the HILIC mode can be an alternative to the reversed-phase mode for a wide range of compounds, especially for those of high polarity in isocratic as well as gradient elution.     

Polymethacrylate monolithic and hybrid particle-monolithic columns for reversed-phase and hydrophilic interaction capillary liquid chromatography

We prepared hybrid particle-monolithic polymethacrylate columns for micro-HPLC by in situ polymerization in fused silica capillaries pre-packed with 3–5 μm C₁₈ and aminopropyl silica bonded particles, using polymerization mixtures based on laurylmethacrylate–ethylene dimethacrylate (co)polymers for the reversed-phase (RP) mode and [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl) zwitterionic (co)polymers for the hydrophilic interaction (HILIC) mode. The hybrid particle-monolithic columns showed reduced porosity and hold-up volumes, approximately 2–2.5 times lower in comparison to the pure monolithic columns prepared in the whole volume of empty capillaries. The elution volumes of sample compounds are also generally lower in comparison to packed or pure monolithic columns. The efficiency and permeability of the hybrid columns are intermediate in between the properties of the reference pure monolithic and particle-packed columns. The chemistries of the embedded solid particles and of the interparticle monolithic moiety in the hybrid capillary columns contribute to the retention to various degrees, affecting the selectivity of separation. Some hybrid columns provided improved separations of proteins in comparison to the reference particle-packed columns in the reversed-phase mode. Zwitterionic hybrid particle-monolithic columns show dual mode retention HILIC/RP behaviour depending on the composition of the mobile phase and allow separations of polar compounds such as phenolic acids in the HILIC mode at lower concentrations of acetonitrile and, often in shorter analysis time in comparison to particle-packed and full-volume monolithic columns.     

Retention behaviour of imidazolium ionic liquid cations on 1.7 μm ethylene bridged hybrid silica column using acetonitrile-rich and water-rich mobile phases

The potential of 1.7 μm ethylene bridged hybrid silica phase was investigated for the separation of twelve imidazolium-based ionic liquid cations. U-shaped retention profile was observed for all solutes with an increase in retention at both low and high acetonitrile content. Chromatographic behaviour of imidazolium cations in both hydrophilic interaction chromatography (HILIC) and per aqueous liquid chromatography (PALC) modes was studied by varying key parameters such as buffer concentration and pH, acid additive, organic modifier and column temperature. Experimental data provided some evidences that under PALC conditions cationic solutes are retained predominantly by mixed hydrophobic/ion-exchange interactions. In the HILIC mode, both partitioning and ion-exchange interactions are responsible for the retention of solutes. Compared to PALC, HILIC provided significantly higher efficiencies with less or even no peak tailing, better separation selectivity and greater resistance to overload. In PALC mode gradient elution was required to achieve adequate retentivity of all solutes but selectivity was not sufficient to distinguish between solutes with very similar hydrophobicity. In contrast, under HILIC conditions twelve solutes were almost completely resolved in less than 4 min by using isocratic elution. Summarizing, it could be concluded that ethylene bridged hybrid silica column providing a dual retention mechanism offers the possibility of selecting between the two retention modes with opposite separation selectivity, just by changing the composition of the mobile phase.     

Determination of selectivity of HPLC systems by correspondence factor analysis

Correspondence factor analysis （CFA） was employed to study the selectivity of 14 HPLC systems. The tested LC systems were classified as reversed-phase （RP）, ion-exchange （IE） and hydrophilic interaction chromatography （HILIC） modes. It was found that the retentions of the hydrophilic solutes on HILIC column were significantly influenced by the second-order effects besides their hydrophilic properties. Organic modifiers and residue silanol groups on silica surface both participated in retention. HypersilTM amino column performed separation in the HILIC mode at appropriate conditions, and its retention mechanism was more similar to that of HILIC silica column than that of HILIC column coating poly（aspartamide） groups.     

Preparation and application of novel zwitterionic monolithic column for hydrophilic interaction chromatography

A novel zwitterionic hydrophilic porous monolithic stationary phase was prepared based on the thermal‐initiated copolymerization of N,N‐dimethyl‐N‐(3‐methacryl‐amidopropyl)‐N‐(3‐(sulfopropyl)ammonium betaine and ethylene glycol dimethacrylate. A typical hydrophilic separation mechanism was observed at a highly organic mobile phase (ACN >60%) on this optimized zwitterionic hydrophilic interaction chromatography (HILIC) monolithic stationary phase. Good permeability, stability, and column efficiency were observed on the final monolithic column. Additionally, a weak electrostatic interaction for charged analytes was confirmed in analysis of six benzoic acids by studying the influence of mobile phase pH and salt concentration on their retention behaviors on the obtained zwitterionic HILIC monolithic column. The optimized zwitterionic HILIC monolith exhibited good selectivity for a range of polar test analytes.     

Hydrophilic interaction chromatography using amino and silica columns for the determination of polar pharmaceuticals and impurities

Hydrophilic interaction chromatography (HILIC) is described as a useful alternative to reversed-phase chromatography for applications involving polar compounds. In the HILIC mode, an aqueous-organic mobile phase is used with a polar stationary phase to provide normal-phase retention behavior. Silica and amino columns with aqueous-acetonitrile mobile phases offer potential for use in the HILIC mode. An examination of the retention and separation of several pyrimidines, purines, and amides on silica and amino columns from three manufacturers revealed that mobile phases should contain a buffer or acid for pH control to achieve similar and reproducible results among columns from different sources. Amino columns may also be used in an anion-exchange mode, which provides an advantage for some applications. In some cases, silica can provide different selectivity and better separation than an amino column. Example applications include: low-molecular-mass organic acids and amides as impurities in non-polar drug substances, 5-fluorouracil in 5-fluorocytosine, guanine in acyclovir, and different selectivity for polar basic compounds compared to an ion-pairing system.     

Effects of the operation parameters on Hydrophilic Interaction Liquid Chromatography separation of phenolic acids on zwitterionic monolithic capillary columns

Strongly polar phenolic acids are weakly retained and often poorly separated in reversed-phase (RP) liquid chromatography. We prepared zwitterionic polymethacrylate monolithic columns for micro-HPLC by in situ co-polymerization in fused-silica capillaries. The capillary monolithic columns prepared under optimized polymerization conditions show some similarities with the conventional particulate commercial ZIC-HILIC silica-based columns, however have higher retention and better separation selectivity under reversed-phase conditions, so that they can be employed for dual-mode HILIC-RP separations of phenolic acids on a single column. The capillary polymethacrylate monolithic sulfobetaine columns show excellent thermal stability and improved performance at temperatures 60–80 °C. The effects of the operation conditions on separation were investigated, including the type and the concentration of the organic solvent in the aqueous-organic mobile phase (acetonitrile and methanol), the ionic strength of the acetate buffer and temperature. While the retention in the RP mode decreases at higher temperatures in mobile phases with relatively low concentrations of acetonitrile, it is almost independent of temperature at HILIC conditions in highly organic mobile phases. The best separation efficiency can be achieved using relatively high acetate buffer ionic strength (20–30 mmol L⁻¹) and gradient elution with alternately increasing (HILIC mode) and decreasing (RP mode) concentration of aqueous buffer in aqueous acetonitrile. Applications of the monolithic sulfobetaine capillary columns in alternating HILIC-RP modes are demonstrated on the analysis of phenolic acids in a beer sample.     

丙基脲硅胶毛细管整体柱的制备及性能研究

采用溶胶-凝胶技术在毛细管中原位合成硅胶整体柱,通过表面化学修饰技术制备了极性的丙基脲硅胶整体柱.对所制备的整体柱柱性能进行了评价,考察了极性物质在该整体柱上的保留行为,并对其可能的保留机理进行了探讨.研究表明,该柱在亲水作用电色谱模式下能有效分离苯酚类极性小分子化合物.     

Fast HPLC with a silica-based monolithic ODS column

Fast high-performance liquid chromatography is becoming routine in laboratories that require high throughput or for combinatorial libraries. Reduced analysis time is commonly achieved by using shorter columns and higher flow rates. Shorter columns require smaller particles in order to maintain efficiency. However, smaller particles increase backpressure, which limits both column length and higher flow rates for typical LC pumps. This disadvantage has been addressed by the emergence of monolithic liquid chromatographic columns (1). Unlike particle-base columns, monolithic columns consist of a continuous rod-shaped porous network with a bimodal pore distribution. In this study, a commercially available 50- x 4.6-mm silica-based octadecyl silane monolithic column (Chromolith SpeedROD RP18e, EM Science, Gibbstown, NJ) was used to separate a seven-component test mixture with a wide range of polarity. The primary goals of this investigation were: (a) to study the chemistry (selectivity) of the new silica-based monolithic columns and (b) to study their run-to-run and column-to-column performance (retention times and peak areas). The selectivity (alpha factor) is a ratio of partition coefficients and, if comparable for a variety of solutes, would mean that methods could be readily transferred between particulate and monolithic columns.     

New zwitterionic polymethacrylate monolithic columns for one‐ and two‐dimensional microliquid chromatography

We prepared 0.53 and 0.32 mm id monolithic microcolumns by in situ copolymerization of a zwitterionic sulfobetaine functional monomer with bisphenol A glycerolate dimethacrylate (BIGDMA) and dioxyethylene dimetacrylate crosslinkers. The columns show a dual retention mechanism (hydrophilic‐interaction mode) in acetonitrile‐rich mobile phases and RP in highly aqueous mobile phases. The new 0.53 mm id columns provided excellent reproducibility, retention, and separation selectivity for phenolic acids and flavonoids. The new zwitterionic monolithic columns are highly orthogonal, with respect to alkyl silica stationary phases, not only in the hydrophilic‐interaction mode but also in the RP mode. The optimized monolithic zwitterionic microcolumn of 0.53 mm id was employed in the first dimension, either in the aqueous normal‐phase or in the RP mode, coupled with a short nonpolar core‐shell column in the second dimension, for comprehensive 2D LC separations of phenolic and flavonoid compounds. When the 2D setup with the sulfobetaine–BIGDMA column was used for repeated sample analysis, with alternating gradients of decreasing (hydrophilic‐interaction mode), and increasing (RP mode) concentration of acetonitrile on the sulfobetaine–BIGDMA column in the first dimension, useful complementary information on the sample could be obtained.     

Short communication performance of octadecylsilylated monolithic silica capillary columns of 530 microm inner diameter in HPLC

Monolithic silica capillary columns were successfully prepared in a fused silica capillary of 530 microm inner diameter and evaluated in HPLC after octadecylsilylation (ODS). Their efficiency and permeability were compared with those of columns pakked with 5-microm and 3-microm ODS-silica particles. The monolithic silica columns having different domain sizes (combined size of through-pore and skeleton) showed 2.5-4.0-times higher permeability (K= 5.2-8.4 x 10(-14) m2) than capillary columns packed with 3-mm particles, while giving similar column efficiency. The monolithic silica capillary columns gave a plate height of about 11-13 microm, or 11 200-13 400 theoretical plates/150 mm column length, in 80% methanol at a linear mobile phase velocity of 1.0 mm/s. The monolithic column having a smaller domain size showed higher column efficiency and higher pressure drop, although the monolithic column with a larger domain size showed better overall column performance, or smaller separation impedance (E value). The larger-diameter (530 microm id) monolithic silica capillary column afforded a good peak shape in gradient elution of proteins at a flow rate of up to 100 microL/min and an injection volume of up to 10 microL.     

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.     

Chromatographic characterization of hydrophilic interaction liquid chromatography stationary phases: hydrophilicity, charge effects, structural selectivity, and separation efficiency

Fourteen commercially available particle-packed columns and a monolithic column for hydrophilic interaction liquid chromatography (HILIC) were characterized in terms of the degree of hydrophilicity, the selectivity for hydrophilic-hydrophobic substituents, the selectivity for the regio and configurational differences in hydrophilic substituents, the selectivity for molecular shapes, the evaluation of electrostatic interactions, and the evaluation of the acidic-basic nature of the stationary phases using nucleoside derivatives, phenyl glucoside derivatives, xanthine derivatives, sodium p-toluenesulfonate, and trimethylphenylammonium chloride as a set of samples. Principal component analysis based on the data of retention factors could separate three clusters of the HILIC phases. The column efficiency and the peak asymmetry factors were also discussed. These data on the selectivity for partial structural differences were summarized as radar-shaped diagrams. This method of column characterization is helpful to classify HILIC stationary phases on the basis of their chromatographic properties, and to choose better columns for targets to be separated. Judging from the retention factor for uridine, these HILIC columns could be separated into two groups: strongly retentive and weakly retentive stationary phases. Among the strongly retentive stationary phases, zwitterionic and amide functionalities were found to be the most selective on the basis of partial structural differences. The hydroxyethyl-type stationary phase showed the highest retention factor, but with low separation efficiency. Weakly retentive stationary phases generally showed lower selectivity for partial structural differences.     

Monolithic silica columns with various skeleton sizes and through-pore sizes for capillary liquid chromatography

Reduction of through-pore size and skeleton size of a monolithic silica column was attempted to provide high separation efficiency in a short time. Monolithic silica columns were prepared to have various sizes of skeletons (approximately 1-2 microm) and through-pores (approximately 2-8 microm) in a fused-silica capillary (50-200 microm I.D.). The columns were evaluated in HPLC after derivatization to C18 phase. It was possible to prepare monolithic silica structures in capillaries of up to 200 microm I.D. from a mixture of tetramethoxysilane and methyltrimethoxysilane. As expected, a monolithic silica column with smaller domain size showed higher column efficiency and higher pressure drop. High external porosity (> 80%) and large through-pores resulted in high permeability (K = 8 x 10(-14) -1.3 x 10(-12) m2) that was 2-30 times higher than that of a column packed with 5-mirom silica particles. The monolithic silica columns prepared in capillaries produced a plate height of about 8-12 microm with an 80% aqueous acetonitrile mobile phase at a linear velocity of 1 mm/s. Separation impedance, E, was found to be as low as 100 under optimum conditions, a value about an order of magnitude lower than reported for conventional columns packed with 5-microm particles. Although a column with smaller domain size generally resulted in higher separation impedance and the lower total performance, the monolithic silica columns showed performance beyond the limit of conventional particle-packed columns under pressure-driven conditions.     

Naphthyl methacrylate‐based monolithic column for RP‐CEC via hydrophobic and π interactions

A neutral naphthyl methacrylate‐based monolith (NMM) was introduced for RP‐CEC of various aromatic compounds via hydrophobic and π interactions. It was characterized over a wide range of elution conditions to gain insight into its RP retention mechanism toward the various solute probes under investigation. First, the NMM column exhibited cathodal EOF at various mobile phase compositions and pH suggesting that although the NMM column is void of fixed charges, it acquires a negative zeta potential. It is believed that the negative zeta potential is imparted by the adsorption of mobile phase ions to the NMM surface. The NMM column exhibited π–π interactions in addition to hydrophobic interactions due to the aromatic and nonpolar nature of its naphthyl ligands. In all cases, the retention of the various aromatic test solutes including PAHs, benzene derivatives, toluene derivatives, anilines and toluidine, tolunitrile and nitrotoluene positional isomers on the NMM column were compared to their retention on an octadecyl acrylate‐based monolithic column. Not only were the values of the retention factors of the various solutes on the NMM column higher than those obtained on the octadecyl acrylate‐based monolithic column under otherwise the same CEC conditions, but the elution orders were also different on both columns with a superior and unique selectivity exhibited by the NMM column.     

Cyclofructan 6 based stationary phases for hydrophilic interaction liquid chromatography

New stationary phases for hydrophilic interaction liquid chromatography (HILIC) were synthesized by covalently attaching native cyclofructan 6 (CF6) to silica gel. The chromatographic characteristics of the new stationary phases were evaluated and compared to three different types of commercial HILIC columns. The CF6 columns produced considerably different retention and selectivity patterns for various classes of polar analytes, including nucleic acid compounds, xanthines, β-blockers, salicylic acid and its derivatives, and maltooligosaccharides. Univariate optimization approaches were examined including organic modifier (acetonitrile) contents and buffer pH and salt concentration. The thermodynamic characteristic of the CF6 stationary phase was investigated by considering the column temperature effect on retention and utilizing van't Hoff plots. CF6 based stationary phases appear to have exceptionally broad applicability for HILIC mode separations.     

硅胶色谱柱的亲水作用保留机理及其影响因素

亲水作用色谱（HILIC）是替代反相色谱（RPLC）分离强极性及亲水性化合物的另一色谱模式,其分离机理与RPLC有很大不同,具有和RPLC互补的选择性。在HILIC模式中,采用正相色谱（NPLC）中的极性固定相及含高浓度有机溶剂（通常为乙腈）的水溶液为流动相。硅胶是开发最早、研究最为深入及应用最为广泛的HILIC固定相,本文介绍了硅胶色谱柱的HILIC保留机理,详细概述了操作条件如硅胶柱类型、流动相组成及柱温对HILIC分离的影响,并对硅胶填料色谱柱的HILIC模式的发展方向与应用前景进行了展望。     

Preparation of high efficiency and highly retentive monolithic silica capillary columns for reversed-phase chromatography by chemical modification by polymerization of octadecyl methacrylate

High efficiency and highly retentive monolithic silica capillary columns were obtained by polymerization of octadecyl methacrylate using alpha,alpha'-azobis-isobutyronitrile (AIBN) as a free radical initiator. Hybrid type monolithic silica columns (25 cm total length x 200 microm I.D.) prepared from a mixture of tetramethoxysilane and methyltrimethoxysilane were used as a support. The effects of the monomer and the radical initiator concentrations in the reaction mixture were examined. The performance of the columns was tested in terms of column efficiency and retention behavior by using alkylbenzenes and a few other compounds as solutes and compared with that of hybrid monolithic silica columns modified with octadecylsilyl-(N,N-diethylamino)silane (ODS-DEA). Highly retentive monolithic silica columns were obtained by polymerization at high monomer concentrations. Although a decrease in column efficiency was observed with the increase in the monomer concentration in a feed mixture, an improvement in efficiency was achieved (a plate height value lower than 10 microm) by increasing an initiator concentration without significant variations in column retention properties. Results obtained by polymerization using other monomers are also presented to demonstrate the applicability of the preparation method.     

Probing the interaction mode in hydrophilic interaction chromatography

This work aims at characterizing interactions between a select set of probes and 22 hydrophilic and polar commercial stationary phases, to develop an understanding of the relationship between the chemical properties of those phases and their interplay with the eluent and solutes in hydrophilic interaction chromatography. "Hydrophilic interaction" is a somewhat inexact term, and an attempt was therefore made to characterize the interactions involved in HILIC as hydrophilic, hydrophobic, electrostatic, hydrogen bonding, dipole-dipole, π-π interaction, and shape-selectivity. Each specific interaction was quantified from the separation factors of a pair of similar substances of which one had properties promoting the interaction mode being probed while the other did not. The effects of particle size and pore size of the phases on retention and selectivity were also studied. The phases investigated covered a wide range of surface functional groups including zwitterionic (sulfobetaine and phosphocholine), neutral (amide and hydroxyl), cationic (amine), and anionic (sulfonic acid and silanol). Principal component analysis of the data showed that partitioning was a dominating mechanism for uncharged solutes in HILIC. However, correlations between functional groups and interactions were also observed, which confirms that the HILIC retention mechanism is partly contributed by adsorption mechanisms involving electrostatic interaction and multipoint hydrogen bonding. Phases with smaller pore diameters yielded longer retention of solutes, but did not significantly change the column selectivities. The particle diameter had no significant effect, neither on retention, nor on the selectivities. An increased water content in the eluent reduced the multipoint hydrogen bonding interactions, while an increased electrolyte concentration lowered the selectivities of the tested columns and made their interaction patterns more similar.     

Tetrazole-Functionalized Silica for Hydrophilic Interaction Chromatography of Polar Solutes

In this work, tetrazole-functionalized stationary phase was prepared with nitrile-modified silica by an ammonium-catalyzed (3 + 2) azide-nitrile cycloaddition reaction. The prepared stationary phase was used for separation of nucleobases and nucleosides by hydrophilic interaction chromatography (HILIC) mode. A typical HILIC mechanism was observed at higher content of acetonitrile (>85%, v/v) in the mobile phase. The retention mechanism of the column was investigated by the models used for describing partitioning and surface adsorption through adjustment ratio of water in the mobile phase, and by the influence of salt concentration, buffer pH, and temperature on the retention of solutes. The results illustrated that the surface adsorption through hydrogen bonding dominated the retention behavior of nucleobases/nucleosides under HILIC mode. From the separation ability, the tetrazole-functionalized stationary phase could become a valuable alternative for the separation of the compounds concerned.