Solvation processes on phenyl‐bonded stationary phases—The influence of polar functional groups

A series of phenyl‐bonded stationary phases with incorporated polar functional groups was subjected to an adsorption investigation. Measurement of acetonitrile and methanol adsorption was obtained using the minor disturbance method. It was observed that adsorption of organic solvent strongly depends on the presence of polar functional groups in the bonded phases that influence the hydrophobicity and polarity of the stationary phase surface. Additionally, relative adsorption of acetonitrile and methanol confirms earlier observations, that the presence of amine and amide groups in the stationary phase changes the relative elution strength of organic solvents. The heterogeneous surface of the stationary phase makes it possible to observe the competitiveness of the water and organic solvent adsorption.     

Excess isotherms as a new way for characterization of the columns for reversed-phase liquid chromatography

In this work, we present the excess isotherm of acetonitrile for stationary phases with different coverage density. Data obtained with the minor disturbance method were compared with (29)Si cross-polarization/magic-angle spinning NMR spectra to find dependence between acetonitrile adsorption on C18 chemically bonded stationary phases and coverage density of stationary phase. The preferential adsorption of acetonitrile on the bonded phase and the adsorption of water on the silica surface can be well correlated with the coverage density.     

Phenyl‐bonded stationary phases—The influence of polar functional groups on retention and selectivity in reversed‐phase liquid chromatography

The chromatographic properties of four phenyl‐bonded phases with different structures were studied. The columns used were packed with a stationary phase containing a phenyl ring attached to the silica surface using different types of linkage molecules. As a basic characteristic of the bonded phases, the hydrophobicity and silanol activity (polarity) were investigated. The presence of the polar amino and amide groups in the structure of the bonded ligand strongly influences the polarity of the bonded phase. Columns were compared according to methylene selectivity using a series of benzene homologues and according to their shape and size selectivity using polycyclic aromatic hydrocarbons. The measurements were done using methanol/water and acetonitrile/water mobile phases. The presented results show that the presence of polar functional groups in the ligand structure strongly influences the chromatographic properties of the bonded phase.     

Zeta potential determination as a new way of stationary phases characterization for liquid chromatography

A set of seven homemade octadecyl silica‐based bonded phases was investigated. Their zeta potential data in methanol and ACN as well as in methanol–water and ACN–water solution were obtained using Zetasizer. The influence of both the coverage density of bonded ligands and the end‐capping of the modified surface on these data was investigated. Presented results may give useful information about the accessibility of the residual silanols in different mobile phases during the chromatographic analysis. Those measurements may be useful to choose chemically bonded stationary phases for CEC. The results also confirm the phenomena of anion exclusion from the pores of stationary‐bonded phase.     

Comparison of Solvent Adsorption on Chemically Bonded Stationary Phases in RP-LC

The excess adsorption isotherms of organic modifiers (methanol and acetonitrile) from water on chemically bonded stationary phases were studied, using seven different packed columns with different organic ligand lengths from C4 to C30. Isotherms were measured using the minor disturbance method. Excess isotherms can give useful information about the structure of the stationary phase. The amount of “adsorbed” solvent can indicate the type of distribution mechanism.     

Study of solvation processes on cholesterol bonded phases

Four cholesterol bonded phases with different structures were investigated. The columns studied were packed with stationary phase containing cholesterol attached to the silica surface using different types of linkage molecules. The presence of the polar amino and carboxyl groups in the structure of the bonded ligand strongly influence on the solvation process. The possibility of hydrogen bonding, dipole-dipole and π-π electron interactions lead to preferential solvation of bonded ligands. The coverage density of bonded ligands and length of the linkage strongly influence the adsorption of solvent from the mobile phase. The removal of residual silanols during the hydrosilation procedure significantly influences solvation of the bonded phase. Excess isotherms of the commonly used solvents in RP HPLC (methanol and acetonitrile) were obtained using the minor disturbance method. For comparison of the stationary phases prepared on different silica gels the excess adsorbed amounts were calculated per volume of the stationary phase in the column. The hydrosilated UDC Cholesterol bonded phase is preferentially solvated by methanol whereas the highest coverage Cosmosil Cholester phase exhibit high adsorption of acetonitrile. Polar groups in the Amino-cholesterol type bonded phase are solvated with both solvent but the mechanisms of these processes are different.     

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.     

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.     

Polydopamine‐assisted immobilization of zeolitic imidazolate framework‐8 for open‐tubular capillary electrochromatography

In this work, we developed a capillary column modified with zeolitic imidazolate framework‐8 as a novel stationary phase for open‐tubular capillary electrochromatography. To immobilize zeolitic imidazolate framework‐8 onto the inner surface of silica capillary, a bio‐inspired polydopamine functionalization was used to functionalize the capillary surface with polydopamine. First, a polydopamine layer was assembled inside the capillary. Second, due to noncovalent adsorption and covalent reaction ability, polydopamine could attract and anchor zeolitic imidazolate framework‐8 onto the inner surface of capillary. It has been demonstrated that zeolitic imidazolate framework‐8 was successfully grafted on the inner wall of the capillary by scanning electron microscopy, and Fourier transform infrared spectroscopy. The electro‐osmotic flow characteristics of capillaries were also investigated by varying the pH value and acetonitrile content of mobile phase. The zeolitic imidazolate framework‐8 coating not only increased the phase ratio of open‐tubular column, but also improved the interactions between tested analytes and the stationary phase. Three groups of isomers including acidic, basic, and neutral compounds were well separated on the zeolitic imidazolate framework‐8 bonded column, with theoretic plate numbers up to 1.9 × 10⁵ N for catechol. The repeatability of the prepared columns was also studied, and the relative standard deviations for intra‐ and interday runs were less than 5%.     

Enhanced‐fluidity liquid chromatography using mixed‐mode hydrophilic interaction liquid chromatography/strong cation‐exchange retention mechanisms

The potential of enhanced‐fluidity liquid chromatography, a subcritical chromatography technique, in mixed‐mode hydrophilic interaction/strong cation‐exchange separations is explored, using amino acids as analytes. The enhanced‐fluidity liquid mobile phases were prepared by adding liquefied CO₂ to methanol/water mixtures, which increases the diffusivity and decreases the viscosity of the mixture. The addition of CO₂ to methanol/water mixtures resulted in increased retention of the more polar amino acids. The “optimized” chromatographic performance (achieving baseline resolution of all amino acids in the shortest amount of time) of these methanol/water/CO₂ mixtures was compared to traditional acetonitrile/water and methanol/water liquid chromatography mobile phases. Methanol/water/CO₂ mixtures offered higher efficiencies and resolution of the ten amino acids relative to the methanol/water mobile phase, and decreased the required isocratic separation time by a factor of two relative to the acetonitrile/water mobile phase. Large differences in selectivity were also observed between the enhanced‐fluidity and traditional liquid mobile phases. A retention mechanism study was completed, that revealed the enhanced‐fluidity mobile phase separation was governed by a mixed‐mode retention mechanism of hydrophilic interaction/strong cation‐exchange. On the other hand, separations with acetonitrile/water and methanol/water mobile phases were strongly governed by only one retention mechanism, either hydrophilic interaction or strong cation exchange, respectively.     

Characterization of calixarene‐bonded stationary phases

Calixarene‐bonded stationary phases received growing interest in HPLC as stationary phases with special retention characteristics and selectivity. The commercially available unsubstituted and p‐tert‐butyl‐substituted Caltrex^® columns have been intensively studied and characterized in our workgroup. They can be used as reversed phases, yet they support additional interactions. Especially, their steric, polar and ionic properties differ from conventional alkyl‐bonded phases. However, also the hydrophobic interaction shows differences since adsorption and partition interactions on or in a bonded layer of calixarenes are not similar to those of alkyl‐bonded layers. The relative strength of the hydrophobic properties of the stationary phases has been found depending on the methanol concentration of the mobile phase. Generally, the dependencies of their interaction strengths on mobile‐phase conditions, e.g. the change of the intensity of the hydrogen‐bonding abilities with decreasing methanol content, are not similar from phase to phase either. This probably gives calixarene‐bonded stationary phases enhanced suitability for analyses at extreme compositions of the mobile phase. An overview about the synthesis, retention and selectivity properties of Caltrex^® columns is given here.     

Bile acids as stationary phase in liquid chromatography

Summary Bile acids chemically bonded onto aminopropylsilica have been employed as stationary phases in liquid chromatography. Bile acid aggregates were dynamically formed around molecules chemically anchored on the supports when the eluent contained bile salts. The bile salt aggregates achieved the separation of 1,1′-binaphthyl-2,2′-diyl-hydrogenphosphate enantiomers and dansyl amino acids.     

Preparation and evaluation of a chiral HPLC stationary phase based on cone calix[4]arene functionalized at the upper rim with l‐alanine units

Here we report a new chiral stationary phase (CSP) immobilized on silica gel based on cone calix[4]arene functionalized at the upper rim with two l ‐alanine units as new chiral selector that has been used in high‐performance liquid chromatography. The CSP was prepared by covalently bonding the allyl groups at the lower rim of calix[4]arene to silica gel by thiol‐ene click chemistry reaction. Elemental analysis of the CSP showed that 120 μmol of chiral selector bonded per gram of silica gel. 1‐Hexene was used for end‐capping of unreacted mercapto groups on silica gel. Since the CSP is chemically bonded to the silica, it can be used in the normal‐phase and reversed‐phase mode and with halogenated solvents as mobile phases, if desired. The chromatographic performance of the CSP was evaluated in the enantioseparation of the 3,5‐dinitrobenzoyl derivatives of some amino acids, diclofop‐methyl and dl ‐mandelic acid.     

Influence of preferential adsorption of mobile phase on retention behavior of amino acids on the teicoplanin chiral selector

The adsorption behavior of two amino acids, i.e., l,d-threonine and l,d-methionine has been investigated on the chiral stationary phase (CSP)column packed with teicoplanin bonded to a silica support. The study has been performed under non-linear conditions of adsorption isotherm for various types of organic modifiers (methanol, ethanol, propan-2-ol and acetonitrile) in the reversed-phase mode. A heterogeneous adsorption mechanism of amino acids has been identified that was strongly affected by the nature of organic modifier. Generally, isotherm non-linearity and retention decreased with decrease of the modifier content in the mobile phase exhibiting a minimum at water-rich mobile phases. These trends were suggested to result from a combined effect of the mobile as well as the adsorbed phase composition. To determine the composition of the adsorbed phase the excess adsorption of modifiers in aqueous solutions has been measured and their binary adsorption equilibria have been quantified and compared. Strongly non-ideal behavior of solvents in the mobile phase and the adsorbed phase has been accounted for by activity coefficients. The fraction of the modifiers in the adsorbed phase decreased in the sequence: methanol, ethanol, propan-2-ol and acetonitrile.     

Preparation and evaluation of a deoxycholic‐calix[4]arene hybrid‐type receptor as a chiral stationary phase for HPLC

We report the synthesis and enantioseparation characteristics of two novel covalently immobilized deoxycholic acid derivatives as chiral stationary phases for high‐performance liquid chromatography. In the structure of the first stationary phase, the 3‐position of deoxycholic acid is substituted with a 3,5‐dinitrophenylcarbamoyl group and the second one has an additional calix[4]arene attached to the carboxylic group of the deoxycholic acid. The chromatographic performance of the stationary phases was evaluated with enantioseparation of N‐(3,5‐dinitrobenzoyl)‐dl ‐leucine, N‐(3,5‐dinitrobenzoyl)‐dl ‐valine, omeprazole, diclofop‐methyl, dl ‐mandelic acid and (RS)‐pregabalin. Comparison of the performance characteristics of the prepared chiral stationary phases provided evidence for the active involvement of the calix[4]arene unit in the chiral recognition process. Both stationary phases are chemically bonded to the silica and can be used in both normal‐phase and reversed‐phase modes.     

Influence of the charge distribution on the stationary phases zeta potential

A set of seven home‐made silica based bonded phases with different functional groups was investigated. Their zeta potential data in methanol and acetonitrile as well as in methanol/water and acetonitrile/water solution were obtained by using a Zetasizer. The influence of polar functional groups on a zeta potential was investigated. The results show that the amines incorporated in the structure of chemically bonded phases of reversed‐phase materials are protonated during chromatographic analysis, resulting in changes of the zeta potential from negative to positive values. Acetonitrile causes more negative values and methanol provides positive (or less negative) values of the zeta potential.     

Insights into the retention mechanism of neutral organic compounds on polar chemically bonded stationary phases in reversed-phase liquid chromatography

The solvation parameter model is used to characterize the retention properties of a 3-aminopropylsiloxane-bonded (Alltima amino), three 3-cyanopropylsiloxane-bonded (Ultrasphere CN, Ultremex-CN and Zorbax SB-CN), a spacer bonded propanediol (LiChrospher DIOL) and a multifunctional macrocyclic glycopeptide (Chirobiotic T) silica-based stationary phases with mobile phases containing 10 and 20% (v/v) methanol-water. The low retention on the polar chemically bonded stationary phases compared with alkylsiloxane-bonded silica stationary phases arises from the higher cohesion of the polar chemically bonded phases and an unfavorable phase ratio. The solvated polar chemically bonded stationary phases are considerably more hydrogen-bond acidic and dipolar/polarizable than solvated alkylsiloxane-bonded silica stationary phases. Selectivity differences are not as great among the polar chemically bonded stationary phases as they are between the polar chemically bonded phases and alkylsiloxane-bonded silica stationary phases.     

Signal enhancement in supercritical fluid chromatography‐diode‐array detection with multiple injection

To circumvent the detrimental effects of large‐volume injection with fixed‐loop injector in modern supercritical fluid chromatography, the feasibility of performing multiple injection was investigated. By accumulating analytes from a certain number of continual small‐volume injections, compounds can be concentrated on the column head, and this leads to signal enhancement compared with a single injection. The signal to noise enhancement of different compounds appeared to be associated with their retention on different stationary phases and with type of sample diluent. The diethylamine column gave the best signal to noise enhancement when acetonitrile was used as sample diluent and the 2‐picolylamine column showed the best overall performance with water as the sample diluent. The advantage of multiple injection over one‐time large‐volume injection was proven with sulfanilamide, with both acetonitrile and water as sample diluents. The multiple injection approach exhibited comparable within‐ and between‐day precision of retention time and peak area with those of single injections. The potential of the multiple injection approach was demonstrated in the analysis of sulfanilamide‐spiked honey extract and diclofenac‐spiked ground water sample. The limitations of this approach were also discussed.     

Use of Chemically Bonded β‐Cyclodextrin Silica Stationary Phase for Liquid Chromatographic Separation of Structural Isomers

The retention behavior of five disubstituted benzene derivatives and two naphthalene derivatives is examined by using a chemically bonded β‐cyclodextrin silica stationary phase with the moiety containing the s‐triazine. The chromatographic results of five disubstituted benzene derivatives and two naphthalene derivatives show that effective separation is achieved on this stationary phase by high‐performance liquid chromatography. The results of the present investigation indicate that the formation of inclusion complexes plays a dominant role in the separation mechanism. However, the selectivity can be significantly enhanced by the n‐n interactions between the s‐triazine ring of the chemically bonded β‐cyclodextrin silica stationary phase and the aromatic ring of solutes. For example, the effective separation of the o‐, m‐, and p‐toluidine isomers on this stationary phase with the moiety containing the s‐triazine ring was better than on that of some β‐cyclodextrin bonded stationary phases without the moiety containing s‐triazine ring.