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.     

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.     

Effects of stationary-phase polarity on retention in reversed bonded phase HPLC columns

Summary Variations in retention and selectivity have been studied in cyano, phenyl and octyl reversed bonded phase HPLC columns. The retention of toluene, phenol, aniline and nitrobenzene in these columns has been measured using binary mixtures of water and methanol, acetonitrile or tetrahydrofuran mobile phases in order to determine the relative contributions of proton donor-proton acceptor and dipole-dipole interactions in the retention process. Retention and selectivity in these columns was correlated with polar group selectivities of mobile phase organic modifiers and the polarity of the bonded stationary phases. In spite of the prominent role of bonded phase volume and residual silanols in the retention process, each column exhibited some unique selectivities when used with different organic modifiers.     

High‐performance liquid chromatography enantioseparation of polyhalogenated 4,4′‐bipyridines on polysaccharide‐based chiral stationary phases under multimodal elution

An investigation on the high‐performance liquid chromatography enantioseparation of 12 polyhalogenated 4,4′‐bipyridines on polysaccharide‐based chiral stationary phases is described. The overall study was directed toward the generation of efficient separations in order to obtain pure atropisomers that will serve as ligands for building homochiral metal organic frameworks. Four coated columns—namely, Lux Cellulose‐1, Lux Cellulose‐2, Lux Cellulose‐4, and Lux Amylose‐2—and two immobilized columns—namely, Chiralpak IC and IA—were used under normal, polar organic, and reversed‐phase elution modes. Moreover, Chiralcel OJ was considered under normal‐phase and polar organic conditions. The effect of the chiral selector and mobile phase composition on the enantioseparation, the enantiomer elution order and the beneficial effect of nonstandard solvents were studied. The effect of water in the mobile phase on the enantioselectivity and retention was investigated and retention profiles typical of hydrophilic interaction liquid chromatography were observed. Interesting phenomena of solvent‐induced enantiomer elution order reversal occurred under normal‐phase mode. All the considered 4,4′‐bipyridines were enantioseparated at the multimilligram level.     

Comparison of common mobile‐phase volume markers with polar‐group‐containing reversed‐phase stationary phases

A systematic study of the behavior of several common mobile‐phase volume markers using traditional and polar‐group‐containing reversed‐phase stationary phases is presented. Examined mobile‐phase volume markers include two neutral molecules, uracil and thiourea, concentrated (0.10 M) and dilute (0.0001 M) KNO₃, and D₂O. Mobile‐phase volumes are examined over the entire reversed‐phase mobile‐phase range of 100% water to 100% methanol or acetonitrile. The behavior of these mobile‐phase volume markers is compared with a maximum theoretical value (i.e. the void volume), as determined by pycnometry. The data suggest that: (i) uracil begins to fail as a mobile‐phase volume marker in mobile phases below about 40% strong solvent for polar group containing phases; (ii) in nearly all cases, the mobile‐phase volume measured dynamically is smaller than the pycnometric void volume; (iii) a significant dependence of measured mobile‐phase volume on salt concentration is seen on the polar endcapped phase, which is not observed on the traditional and embedded polar group phase; and (iv) D₂O does not work well as a mobile‐phase volume marker with polar‐group‐containing phases, possibly due to interaction with the stationary phase polar group.     

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.     

Stationary and mobile phases in hydrophilic interaction chromatography: a review

Hydrophilic interaction chromatography (HILIC) is valuable alternative to reversed-phase liquid chromatography separations of polar, weakly acidic or basic samples. In principle, this separation mode can be characterized as normal-phase chromatography on polar columns in aqueous-organic mobile phases rich in organic solvents (usually acetonitrile). Highly organic HILIC mobile phases usually enhance ionization in the electrospray ion source of a mass spectrometer, in comparison to mobile phases with higher concentrations of water generally used in reversed-phase (RP) LC separations of polar or ionic compounds, which is another reason for increasing popularity of this technique. Various columns can be used in the HILIC mode for separations of peptides, proteins, oligosaccharides, drugs, metabolites and various natural compounds: bare silica gel, silica-based amino-, amido-, cyano-, carbamate-, diol-, polyol-, zwitterionic sulfobetaine, or poly(2-sulphoethyl aspartamide) and other polar stationary phases chemically bonded on silica gel support, but also ion exchangers or zwitterionic materials showing combined HILIC-ion interaction retention mechanism. Some stationary phases are designed to enhance the mixed-mode retention character. Many polar columns show some contributions of reversed phase (hydrophobic) separation mechanism, depending on the composition of the mobile phase, which can be tuned to suit specific separation problems. Because the separation selectivity in the HILIC mode is complementary to that in reversed-phase and other modes, combinations of the HILIC, RP and other systems are attractive for two-dimensional applications. This review deals with recent advances in the development of HILIC phase separation systems with special attention to the properties of stationary phases. The effects of the mobile phase, of sample structure and of temperature on separation are addressed, too.     

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.     

Preparation of a monolithic column with a mixed‐mode stationary phase of reversed‐phase/hydrophilic interaction for capillary liquid chromatography

A monolithic capillary column with a mixed‐mode stationary phase of reversed‐phase/hydrophilic interaction chromatography was prepared for capillary liquid chromatography. The monolith was created by an in‐situ copolymerization of a homemade monomer N,N‐dimethyl‐N‐acryloxyundecyl‐N‐(3‐sulfopropyl) ammonium betaine and a crosslinker pentaerythritol triacrylate in a binary porogen agent consisting of methanol and isopropanol. The functional monomer was designed to have a highly polar zwitterionic sulfobetaine terminal group and a hydrophobic long alkyl chain moiety. The composition of the polymerization solution was systematically optimized to permit the best column performance. The columns were evaluated by using acidic, basic, polar neutral analytes, as well as a set of alkylbenzenes and Triton X100. Very good separations were obtained on the column with the mixed‐mode stationary phase. It was demonstrated that the mixed‐mode stationary phase displayed typic dual retention mechanisms of reversed‐phase/hydrophilic interaction liquid chromatography depending on the content of acetonitrile in the mobile phase. The method for column preparation is reproducible.     

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。     

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.     

Green high‐performance liquid chromatography enantioseparation of lansoprazole using a cellulose‐based chiral stationary phase under ethanol/water mode

A simple and environmentally friendly reversed‐phase high‐performance liquid chromatography method for the separation of the enantiomers of lansoprazole has been developed. The chromatographic resolution was carried out on the cellulose‐based Chiralpak IC‐3 chiral stationary phase using a green and low‐toxicity ethanol‐aqueous mode. The effects of water content in the mobile phase and column temperature on the retention of the enantiomers of lansoprazole and its chiral and achiral related substances have been carefully investigated. A mixed‐mode hydrophilic interaction liquid chromatography and reversed‐phase retention mechanism operating on the IC‐3 chiral stationary phase allowed us to achieve simultaneous enantioselective and chemoselective separations in water‐rich conditions. The enantiomers of lansoprazole were baseline resolved with a mobile phase consisting of ethanol/water 50:50 without any interference coming from chiral and achiral impurities within 10 min.     

Combined effects of mobile phase composition and temperature on the retention of homologous and polar test compounds on polydentate C8 column

Combined effects of temperature and mobile phase on the reversed phase chromatographic behavior of alkylbenzenes and simple substituted benzenes were investigated on a Blaze C₈ polydentate silica-based column, showing improved resistance against hydrolytic breakdown at temperatures higher than 60 °C, in comparison to silica-based stationary phases with single attachment sites. For better insight into the retention mechanism on polydentate columns, we determined the enthalpy and entropy of the transfer of the test compounds from the mobile to the stationary phase. The enthalpic contribution dominated the retention at 80% or lower concentrations of methanol in the mobile phase. Entropic effects are more significant in 90% methanol and in acetonitrile–water mobile phases. Anomalies in the effects of mobile phase on the enthalpy of retention of benzene, methylbenzene and polar benzene derivatives were observed, in comparison to regular change in enthalpy and entropy of adsorption with changing concentration of organic solvent and the alkyl length for higher alkylbenzenes. The temperature and the mobile phase effects on the retention are practically independent of each other and – to first approximation – can be described by a simple model equation, which can be used for optimization of separation conditions.     

Chiral separation of three agrochemical toxins enantiomers by high-performance liquid chromatography on a vancomycin crystalline degradation products-chiral stationary phase

This work aims to evaluate for the enantiomeric separations of three agrochemical toxins: haloxyfop-methyl, fenoxaprop-p-ethyl and indoxacarb on crystalline degradation products-chiral stationary phase (CDP-CSP) of high-performance liquid chromatography (HPLC) under normal and polar organic phases. In the normal phase, the mobile phase was n-hexane with alcohols including methanol and isopropanol as polar modifiers. In the polar organic phase mode, the mobile phase was methanol with different percentages of triethylammunium acetate. The influence of flow rate (0.3-0.9 mL/min), analyte concentration and silica gel particle sizes (10, 15 and 30 microm) was investigated. This new chiral stationary phase showed excellent stereoselectivity for the two enantiomers of haloxyfop-methyl and fenoxaprop-p-ethyl and chiral recognition for indoxacarb under normal-phase mode. However, under polar organic phase, only indoxacarb was separated (alpha < 1.5). The chromatographic results were compared with commercial chiral columns.     

Synthesis and evaluation of silica hydride‐based fluorinated stationary phases

Two novel silica hydride‐based fluorinated bonded phases have been synthesized using a hydrosilation procedure to test combined fluorine and hydride selectivity. The bonded moieties were characterized by elemental and spectral analysis. Chromatographic testing was done using hydrophilic analytes in the aqueous normal phase mode. At higher amounts of the nonpolar solvent in the mobile phase, there should be increased retention for solutes such as acids, bases and other polar compounds, whereas nonpolar solutes can be retained when water is increased as in RP chromatography. The synergistic effects of the fluorinated phase selectivity and aqueous normal phase retention on a hydride surface have been explored for small polar molecules. The stability and repeatability of the hydride‐based fluorinated stationary phases were evaluated. The use of acetone as the organic component in the mobile phase was also tested.     

Analysis of polar peptides using a silica hydride column and high aqueous content mobile phases

The retention behavior of a set of polar peptides separated on a silica hydride stationary phase was examined with a capillary HPLC system coupled to ESI‐MS detection. The mobile phases consisted of formic acid or acetic acid/acetonitrile/water mixtures with the acetonitrile content ranging from 5 to 80% v/v. The effects on peptide retention of these two acidic buffer additives and their concentrations in the mobile phase were systematically investigated. Strong retention of the peptides on the silica hydride phase was observed with relatively high‐organic low‐aqueous mobile phases (i.e. under aqueous normal‐phase conditions). However, when low concentrations of acetic acid were employed as the buffer additive, strong retention of the peptides was also observed even when high aqueous content mobile phases were employed. This unique feature of the stationary phase therefore provides an opportunity for chromatographic analysis of polar peptides with water‐rich eluents, a feature usually not feasible with traditional RP sorbents, and thus under conditions more compatible with analytical green chemistry criteria. In addition, both isocratic and gradient elution procedures can be employed to optimize peptide separations with excellent reproducibility and resolution under these high aqueous mobile phase conditions with this silica hydride stationary phase.     

Histidine‐modified organic‐silica hybrid monolithic column for mixed‐mode per aqueous and ion‐exchange capillary electrochromatography

A novel organic‐silica hybrid monolith was prepared through the binding of histidine onto the surface of monolithic matrix for mixed‐mode per aqueous and ion‐exchange capillary electrochromatography. The imidazolium and amino groups on the surface of the monolithic stationary phase were used to generate an anodic electro‐osmotic flow as well as to provide electrostatic interaction sites for the charged compounds at low pH. Typical per aqueous chromatographic behavior was observed in water‐rich mobile phases. Various polar and hydrophilic analytes were selected to evaluate the characteristics and chromatographic performance of the obtained monolith. Under per aqueous conditions, the mixed‐mode mechanism of hydrophobic and ion‐exchange interactions was observed and the resultant monolithic column proved to be very versatile for the efficient separations of these polar and hydrophilic compounds (including amides, nucleosides and nucleotide bases, benzoic acid derivatives, and amino acids) in highly aqueous mobile phases. The successful applications suggested that the histidine‐modified organic‐silica hybrid monolithic column could offer a wide range of retention behaviors and flexible selectivities toward polar and hydrophilic compounds.     

Comments on the paper “Characterization of stationary phases by a linear solvation energy relationship utilizing supercritical fluid chromatography” by C. R. Mitchell,N. J. Benz,S. Zhang

In a recent paper published by Mitchell et al. in this journal, some results obtained in supercritical fluid chromatography and interpreted with the solvation parameter model to characterize interactions for “novel stationary phases” were surprising to us. Indeed, we had already published results for most of the stationary phases reported, but, except for polar phases, our results were not in agreement with those, despite the use of identical mobile phases in both studies. These data were disturbing because they suggest that supercritical fluid chromatography is always a normal‐phase mode, while we have shown that it is a reversed‐phase mode when working with non‐polar stationary phases. In the process of establishing the reason for the differences between our works, we examined several different factors. This paper deals with practice of linear solvation energy relationships: choice of dead‐volume marker, choice of test‐solutes to adequately probe the possible interactions and appropriate column length for characterization of chromatographic systems with highly eluting mobile phases are discussed. The importance of control experiments to validate retention models and confirm their accordance with the chromatographer's experience is evidenced. Recommendations for good linear solvation energy relationship practice are suggested in order to avoid the publication of results leading to erroneous conclusions.     

A simple approach to prepare a sulfone‐embedded stationary phase for HPLC

In the present study, a polar‐embedded reversed‐phase liquid chromatographic stationary phase that contained internal sulfone groups was prepared. The synthesis involved the “thiol‐ene” click chemistry between the vinyl functionalized silica and 1‐octadecanethiol, followed by the oxidization of sulfide to sulfone groups. The resulting material simultaneously possessed the alkyl chain, i.e. C18, and the internal sulfone groups. Elemental analysis demonstrates that the element contents of the C18/sulfone silica were C 8.94%, H 1.87% and S 0.66%. Chromatographic evaluations indicate that the C18/sulfone stationary phase exhibited a little less retention than the C18/sulfide one. A comparable chromatographic performance of neutral analytes was obtained on these two columns, but much better chromatographic performance in the case of basic and acid analytes was obtained on C18/sulfone stationary phase with additional features such as lower silanol activity, better stability (stable working conditions of pH 1.0–10.0), and better compatibility with 100% aqueous mobile phases. The batch‐to‐batch reproducibility was acceptable (the RSDs of retention times for the probes were no higher than 1.73%), demonstrating the suitability of the applied synthetic strategy for the new stationary phase. The C18/sulfone is a promising polar‐embedded RPLC stationary phase.     

Capillary liquid chromatography as a tool for separation of hydrophobic basic drugs. Relation between tests for column characterization and real analysis

Two capillary columns for reversed phase (RP) capillary liquid chromatography (CLC), viz. Nucleosil 100‐5 C18 and LiChrosorb RP‐select B, were characterized by the Walters test, i.e. the chromatographic test proposed for RP stationary phases. Hydrophobicity indices were determined not only in acetonitrile/water mobile phase, as proposed in the test, but they were also measured in buffered systems. This approach was used to quantify the influence of mobile phase composition on the modification of the surface of the stationary phases. In the next step, small basic compounds differing in their hydrophobicity and basicity were selected and their retention on the stationary phases in mobile phases of the same composition as used for column testing was examined. Furthermore, the retention of newly synthesized drugs, chemotherapeutics derived from thioacridine and pyridoquinoline, differing in their structures, basicity, and hydrophobicity, was also studied. The composition of the mobile phases had to be shifted to higher contents of organic modifiers – acetonitrile or methanol – in order to elute these hydrophobic compounds from the columns. The question we wanted to answer was: How is the method for testing of reversed phases related to retention, separation efficiency, and peak symmetry of various analytes?