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?     

Polyethyleneimine Immobilized on Silica Endcapped with Octadecyl Groups as a Stationary Phase for RP-LC

A new reversed stationary phase was prepared, based on thermal immobilization of trimethoxysilylpropyl modified polyethyleneimine onto silica particles endcapped with octadecyl molecules. The physicochemical and morphological properties of the stationary phase were characterized by solid state cross-polarization and magic angle spinning ²⁹Si nuclear magnetic resonance, infrared spectroscopy, porosimetry, and elemental analysis. For the studies on reversed phase high-performance liquid chromatography (HPLC) retention, separation of the established Tanaka and Engelhardt test mixtures was performed. The stationary phase showed a typical partition mechanism for the reversed phase; however, the low hydrophobicity required a low organic content solvent in the mobile phase for chromatographic separation of more hydrophobic compounds. The stationary phase also showed low residual silanol activity for the elution of basic compounds due to the protection offered by octadecyl endcapped molecules and the competition provided by the imine groups of the polymeric layer. The proposed stationary phase possesses interesting selectivity and is convenient for applications requiring the separation of more retentive compounds in conventional HPLC columns using more aqueous mobile phases.     

Fabrication of ZIF‐8@SiO2 Core–Shell Microspheres as the Stationary Phase for High‐Performance Liquid Chromatography

The unique features of high porosity, shape selectivity, and multiple active sites make metal–organic frameworks (MOFs) promising as novel stationary phases for high‐performance liquid chromatography (HPLC). However, the wide particle size distribution and irregular shape of conventional MOFs lead to lower column efficiency of such MOF‐packed columns. Herein, the fabrication of monodisperse MOF@SiO₂ core–shell microspheres as the stationary phase for HPLC to overcome the above‐mentioned problems is reported. Zeolitic imidazolate framework 8 (ZIF‐8) was used as an example of MOFs due to its permanent porosity, uniform pore size, and exceptional chemical stability. Unique carboxyl‐modified silica spheres were used as the support to grow the ZIF‐8 shell. The fabricated monodisperse ZIF‐8@SiO₂ packed columns (5 cm long × 4.6 mm i.d.) show high column efficiency (23 000 plates m^?1 for bisphenol A) for the HPLC separation of endocrine‐disrupting chemicals (bisphenol A, β‐estradiol, and p‐(tert‐octyl)phenol) and pesticides (thiamethoxam, hexaflumuron, chlorantraniliprole, and pymetrozine) within 7 min with good relative standard deviations for 11 replicate separations of the analytes (0.01–0.39, 0.65–1.7, 0.70–1.3, and 0.17–0.91 % for retention time, peak area, peak height, and half peak width, respectively). The ZIF‐8@SiO₂ microspheres combine the advantages of the good column packing properties of the uniform monodisperse silica microspheres and the separation ability of the ZIF‐8 crystals.     

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.     

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.     

Synthesis and characterization of a novel resorcinarene-based stationary phase bearing polar headgroups for use in reversed-phase high-performance liquid chromatography

A novel silica-bonded stationary phase containing a functionalized resorcinarene selector was prepared by a straightforward synthesis. The complete modification of all resorcinic hydroxyl groups was achieved by reaction with isopropyl isocyanate. The derivatized resorcinarene selector was subsequently immobilized via the four alkenyl chains containing a terminal double bond by a free radical-induced reaction on mercaptopropyl-functionalized silica. A comprehensive characterization of the resulting bonded stationary phase was carried out by solid state NMR, IR and elemental analysis. The resulting selector is defined as a "polar headed" reversed phase since the highly ordered polar carbamate groups of the new stationary phase are located, compared to conventional polar embedded stationary phases, at a greater distance from the silica surface. Thus a new concept is introduced in the field of polar modified reversed-phase HPLC. The properties of the novel stationary phase are demonstrated by comparison with commercially available reversed phases.     

Characterization of stationary phases by a linear solvation energy relationship utilizing supercritical fluid chromatography

Supercritical fluid chromatography was utilized in combination with the Abraham model of linear solvation energy relationship to characterize 11 different HPLC stationary phases. System constants were determined at one supercritical fluid chromatography condition for each stationary phase. The results indicate that several types of silica columns, including type B silica, type C silica, and fused core silica, are very similar in their retention behavior. Several aromatic stationary phases were characterized and it was found that, in contrast to the other phases studied, all of the aromatic stationary phases had positive contributions from the dispersion/cavity (v) term of the linear solvation energy relationship. Several aliphatic phases were characterized and there were several linear solvation energy relationship constants that differentiated the phases from each other, mainly the polar terms (dipolarity and hydrogen bonding). One stationary phase, a fused core pentafluorophenyl (PFP) phase, had very poor regression quality. The column volume of this phase was lower than the others in the study, which may have had some impact on the results of the regression.     

Preparation and evaluation of a hydrolytically stable amide-embedded stationary phase

We have developed a new hydrolytically stable amide-embedded stationary phase via a simple and effective synthetic method. The preparation of the new phase involves the synthesis of multifunctional silane ligands and the surface modification of porous silica particles via multiple attachments of these ligands to the silica surface. A hydrolytically stable coating was produced as a result of multiple covalent linkages formed between silane ligands and the silica surface, and cross-linking between adjacent ligands. The resulting amide-embedded stationary phase showed excellent hydrolytic stability over a wide pH range. Like other existing amide-embedded columns, this new stationary phase exhibits higher retention for polar compounds and different selectivity as compared to conventional C18 columns. The new phase is compatible with 100% aqueous mobile phases, and also provides high column efficiency and good peak shapes for both acidic and basic compounds.     

Rapid, high-resolution high-performance liquid chromatographic analysis of antibiotics

A HPLC column devised for high separation speed combined with highly practical operating features has been found useful for separating antibiotics. Important characteristics involve compromises in packing particle size, column configuration and support-stationary phase combinations. We determined that these columns are useful for rapid, high-resolution separations with unmodified state-of-the-art HPLC equipment without the extra-column band-broadening effects typical of so-called “fast” HPLC columns. The proposed columns feature efficient sterically-protected monofunctional silane stationary phases that provide good separation reproducibility and high column stability. The combination of these unique bonded silanes and a highly purified, less-acidic silica support give superior peak shapes for antibiotic compounds. The proposed column configuration can halve separation times and double peak heights without loss in resolution, compared to widely used analytical columns. Increased mobile phase flow-rates permit even faster separations of antibiotics with only modest loss in resolution and peak heights for trace analyses in biological systems.     

Reversal of the enantiomeric elution order of some aromatic amino acids using reversed-phase chromatographic supports coated with the teicoplanin chiral selector

In this paper, two chiral stationary phases were prepared by coating the surface of both C8 and C18 high-performance liquid chromatography (HPLC) supports with the teicoplanin chiral selector. The hydrophobic C11 acyl side chain, attached to the d-glucosamine group of teicoplanin, served as anchor moiety for the immobilization of the chiral selector on the apolar support material. The retention and enantioselectivity of these coated stationary phases were studied using some aromatic amino acids as probe solutes and an aqueous solution as mobile phase. It was found that the enantiomer elution order on the modified C8 and C18 stationary phases was reversed (l > d) relatively to that classically observed with a teicoplanin covalently immobilized on a silica support (d > l). Such a dynamic coating on the reversed-phase supports was found to be of interest since the apparent enantioselectivity was not significantly changed by the use during an extended period of time or following a long-term storage of the columns.     

Separation behavior of basic compounds on unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases with reversed‐phase eluents

Unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases have been evaluated and compared for the separation of basic compounds of differing molecular weight, pK_a, and log D using aqueous/organic mobile phases. The influences of percentage of organic modifier, buffer pH, and concentration in the mobile phase on base retention were investigated on unbonded silicon oxynitride and silica phases. The results confirmed that unbonded silicon oxynitride and silica phases demonstrated excellent separation performance for model basic compounds and both the unbonded phases examined possessed a hydrophobic/adsorption and ion‐exchange character. The silicon oxynitride stationary phase exhibited high hydrophilicity compared with silica with a reversed‐phase mobile phase. An ion‐exclusion‐type mechanism becomes predominant for the separation of three aimed bases on the silicon oxynitride column at pH 2.8. Different from silicon oxynitride stationary phase, no obvious change for the retention time of three model bases on silica stationary phase at pH 2.8 can be observed.     

A unified classification of stationary phases for packed column supercritical fluid chromatography

The use of supercritical fluids as chromatographic mobile phases allows to obtain rapid separations with high efficiency on packed columns, which could favour the replacement of numerous HPLC methods by supercritical fluid chromatography (SFC) ones. Moreover, despite some unexpected chromatographic behaviours, general retention rules are now well understood, and mainly depend on the nature of the stationary phase. The use of polar stationary phases improves the retention of polar compounds, when C18-bonded silica favours the retention of hydrocarbonaceous compounds. In this sense, reversed-phase and normal-phase chromatography can be achieved in SFC, as in HPLC. However, these two domains are clearly separated in HPLC due to the opposite polarity of the mobile phases used for each method. In SFC, the same mobile phase can be used with both polar and non-polar stationary phases. Consequently, the need for a novel classification of stationary phases in SFC appears, allowing a unification of the classical reversed- and normal-phase domains. In this objective, the paper presents the development of a five-dimensional classification based on retention data for 94-111 solutes, using 28 commercially available columns representative of three major types of stationary phases. This classification diagram is based on a linear solvation energy relationship, on the use of solvation vectors and the calculation of similarity factors between the different chromatographic systems. This classification will be of great help in the choice of the well-suited stationary phase, either in regards of a particular separation or to improve the coupling of columns with complementary properties.     

Evaluation of silica from different vendors as the solid support of anion‐exchange chiral stationary phases by means of preferential sorption and liquid chromatography

Chromatographic performance of a chiral stationary phase is significantly influenced by the employed solid support. Properties of the most commonly used support, silica particles, such as size and size distribution, and pore size are of utmost importance for both superficially porous particles and fully porous particles. In this work, we have focused on evaluation of fully porous particles from three different vendors as solid supports for a brush‐type chiral stationary phase based on 9‐O‐tert‐butylcarbamoyl quinidine. We have prepared corresponding stationary phases under identical experimental conditions and determined the parameters of the modified silica by physisorption measurements and scanning electron microscopy. Enantiorecognition properties of the chiral stationary phases have been studied using preferential sorption experiments. The same material was slurry‐packed into chromatographic columns and the chromatographic properties have been evaluated in liquid chromatography. We show that preferential sorption can provide valuable information about the influence of the pore size and total pore volume on the interaction of analytes of different size with the chirally‐modified silica surface. The data can be used to understand differences observed in chromatographic evaluation of the chiral stationary phases. The combination of preferential sorption and liquid chromatography separation can provide detailed information on new chiral stationary phases.     

Epoxide‐derived mixed‐mode chromatographic stationary phases for separation of active substances in fixed‐dose combination drugs

A method for the preparation of novel mixed‐mode reversed‐phase/strong cation exchange stationary phase for the separation of fixed‐dose combination drugs has been developed. An epoxysilane bonded silica prepared by vapor phase deposition was used as a starting material to produce diol, octadecyl, sulfonate, and mixed octadecyl/sulfonate groups bonded silica phases. The chemical structure and surface coverage of the functional groups on these synthesized phases were confirmed by fourier‐transform infrared and solid‐state ¹³C NMR spectroscopy and elemental analysis. Alkylbenzene homologs, basic drugs, nucleobases and alkylaniline homologs were used as probes to demonstrate the reversed‐phase, ion exchange, hydrophilic interaction and mixed‐mode retention behaviors of these stationary phases. The octadecyl/sulfonate bonded silica exhibits pronounced mixed‐mode retention behavior and superior retentivity and selectivity for alkylaniline homologs. The mixed‐mode retention is affected by either ionic or solvent strength in the mobile phase, permiting optimization of a separation by fine tuning these parameters. The mixed‐mode stationary phase was applied to separate two fixed‐dose combination drugs: compound reserpine tablets and compound methoxyphenamine capsules. The results show that simultaneous separation of multiple substances in the compound dosage can be achieved on the mixed‐mode phase, which makes multi‐cycles of analysis for multiple components obsolete.     

Efficiency enhancements in micellar liquid chromatography through selection of stationary phase and alcohol modifier

Micellar liquid chromatography (MLC) remains hindered by reduced chromatographic efficiency compared to reversed phase liquid chromatography (RPLC) using hydro-organic mobile phases. The reduced efficiency has been partially explained by the adsorption of surfactant monomers onto the stationary phase, resulting in a slow mass transfer of the analyte within the interfacial region of the mobile phase and stationary phase. Using an array of 12 columns, the effects of various bonded stationary phases and silica pore sizes, including large-pore short alkyl chain, non-porous, superficially porous and perfluorinated, were evaluated to determine their impact on efficiency in MLC. Additionally, each stationary phase was evaluated using 1-propanol and 1-butanol as separate micellar mobile phase alcohol additives, with several columns also evaluated using 1-pentanol. A simplified equation for calculation of A' and C' terms from reduced plate height (h) versus reduced velocity (nu) plots was used to compare the efficiency data obtained with the different columns and mobile phases. Analyte diffusion coefficients needed for the h versus nu plots were determined by the Taylor-Aris dispersion technique. The use of a short alkyl chain, wide-pore silica column, specifically, Nucleosil C4, 1000A, was shown to have the most improved efficiency when using a micellar mobile phase compared to a hydro-organic mobile phase for all columns evaluated. The use of 1-propanol was also shown to provide improved efficiency over 1-butanol or 1-pentanol in most cases. In a second series of experiments, column temperatures were varied from 40 to 70 degrees C to determine the effect of temperature on efficiency for a subset of the stationary phases. Efficiency improvements ranging from 9% for a Chromegabond C8 column to 58% for a Zorbax ODS column were observed over the temperature range. Based on these observed improvements, higher column temperatures may often yield significant gains in column efficiency, assuming the column is thermally stable.     

Chromatographic behavior of new deazapurine ribonucleosides in hydrophilic interaction liquid chromatography

The chromatographic behavior of new biogenic purine nucleosides in hydrophilic interaction liquid chromatography was examined on three different stationary phases, namely bare silica, and amide‐ and cyclofructan‐based stationary phases. The effects of buffer concentration, pH and acetonitrile‐to‐aqueous‐part ratio in the mobile phase on retention and peak shape were assessed. The retention coefficients and peak symmetry values substantially differed with respect to analytes´ structures, stationary phase properties and mobile phase composition. The bare silica column was unsuitable for these compounds under the chromatographic conditions tested due to very broad and asymmetrical peaks. Furthermore, the cyclofructan‐based stationary phase provided almost Gaussian peak shapes of all deazapurine nucleosides under most conditions tested. Therefore, the cyclofructan‐based stationary phase is the most suitable choice for the chromatographic analysis of nucleosides.     

Ultra‐high performance separation of basic compounds on reversed‐phase columns packed with fully/superficially porous silica and hybrid particles by using ultraviolet transparent hydrophobic cationic additives

The use of the tetrabutylammonium additive was investigated in the ultra‐high performance reversed‐phase liquid chromatographic elution of basic molecules of pharmaceutical interest. When added to the mobile phase at low pH, the hydrophobic tetrabutylammonium cation interacts with the octadecyl chains and with the residual silanols, thus imparting a positive charge to the stationary phase, modulating retention and improving peak shape of protonated basic solutes. Two sources of additive were tested: a mixture of tetrabutylammonium hydroxide/trifluoroacetic acid and tetrabutylammonium hydrogen sulfate. Retention and peak shape of 11 basic pharmaceutical compounds were evaluated on commercially available ultra‐fast columns packed with octadecyl stationary phases (Ascentis Express C18 2.0 µm, Acquity BEH C18 1.7 µm, Titan C18 1.9 µm). All columns benefit from the use of additive, especially tetrabutylammonium hydrogen sulfate, providing very symmetric peaks with reasonable retention times. Focusing on the probe compounds amitriptyline and sertraline, efficiency and asymmetry values were investigated at increasing retention factor. The trend is very different to that obtained in reversed‐phase conditions and the effect lies in the complex molecular interaction mechanisms based on hydrophobic and ion exchange interactions as well as electrostatic repulsion.     

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.