Polar contributions of the stationary phase to the reversed-phase ion-pair high-performance liquid chromatographic separation of quaternary ammonium drugs

The chromatographic reproducibility of a methodology, developed for the separation and determination of quaternary ammonium drugs by reversed-phase ion-pair column liquid chromatography, was studied. The results in terms of retention dependence on the residual silanol content of the octadecyl stationary phase and column aging were compared with those obtained with conventional separation techniques. By on-column silylation with N-trimethylsilylimidazole, it was demonstrated that eluents containing both amines and alkanesulphonates, beside a higher resolving power, provide reproducible separations which are far less dependent on the residual and generated silanol groups compared to those obtained with eluents containing only an organic amine.     

TLC of alkaloids on cyanopropyl bonded stationary phases. Part II. Connection with RP18 and silica plates

Some standards of the alkaloids and synthetic or natural mixtures are separated by two-dimensional thin-layer chromatography (TLC) on different adsorbent layers. Normal- and reversed-phase systems are used to obtain significant differences in the separation selectivity. Optimization of the one-dimensional TLC separation of the alkaloids' standards is performed on cyanopropyl-silica, RP18W, and silica layers in various eluents containing (besides diluent and modifier) silanol blockers, such as diethyl amine or ammonia. The most selective systems are used for the separation of the alkaloids' mixtures by two-dimensional TLC with an adsorbent gradient method. The mixtures of alkaloids or plant extracts (Chelidonium majus, Fumaria officinalis, or Glaucium flavum) are chromatographed in system I; the plates are connected with the plate pre-coated with various adsorbent, and partly separated fractions are transferred to the second layer and developed in system II. CN-silica-RP18W and CN-silica-silica are used as the connected layers. The alkaloids are identified by R(F) values of standards, and the components of plant extracts are identified in both systems, and by the comparison of UV spectra obtained in diode array detector densitometry.     

Preparation of pH-responsive stationary phase for reversed-phase liquid chromatography and hydrophilic interaction chromatography

Novel pH-responsive polymer-grafted silica was successfully synthesized through the radical "grafting from" polymerization on azo initiator-immobilized silica. The immobilization of azo initiator onto the silica surface was achieved by the reaction of surface amino groups with 4,4'-azobis(4-cyanovaleric acid chloride). The polymer-grafted silica was prepared by stirring suspension of the azo initiator-immobilized silica in anhydrous dioxane containing acrylic acid (AAc) and butyl acrylate (BA). The resulting polymer-grafted silica was demonstrated to be pH responsive to hydrophobic/hydrophilic property by reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC). In RPLC mode, the retention of aromatic compounds decreased with the increase in the pH of mobile phase. However, the opposite result was obtained in HILIC mode; the retention of soybean isoflavones was stronger with the mobile phase at higher pH. Finally, the separations of sulfonamides and soybean isoflavones were carried out in RPLC mode and the separation of some nucleotides was achieved in HILIC mode.     

Normal Phase TLC Separation of Enantiomers Using Chiral Ion Interaction Agents

Abstract

A method for the thin layer chromatographic (TLC) separation of enantiomers and diastereomers involving the use of chiral ion interaction agents is described. Several aromatic amino alcohols were resolved by TLC on diol and/or high performance silica gel plates using a mobile phase containing (1R)-(-)- ammonium-10-camphorsulfonate or N-benzoxycarbonyl-glycyl-L-proline (ZGP). Many of these chiral aromatic amino alcohols are of pharmacological importance as α- and β-adrenergic blockers, adrenergic compounds, and anti-glaucoma agents. A comparison was made between various N-CBZ-amino acid derivatives as chiral counter ions/chiral mobile phase additives (CMAs). These separations could not be achieved on other normal phase TLC stationary phases including microcrystalline cellulose, alumina and ordinary silica gel plates.     

Review on the chemical and thermal stability of stationary phases for reversed-phase liquid chromatography

At present, in high-performance liquid chromatography (HPLC) for the majority of analyses, reversed-phase liquid chromatography (RPLC) is the separation mode of choice. Faster method development procedures using aggressive eluents under elevated temperature conditions, the need for improved selectivities, efficiencies and resolution, the reduction of solvent consumption and also the decrease of analysis times require reversed-phase (RP) columns of high chemical and thermal stability. Until now, the majority of columns for RPLC separations are manufactured from silica substrates. Silica has many favorable properties making this material nearly ideal as a support for RP columns. However, its solubility, that increases considerably in eluents of pH above +/-7, is a drawback preventing its widespread use over the entire pH range. In addition, also the thermal stability of silica is limited. Recently, however, substantial progress has been made in the synthesis of RPLC silica-based stationary phases showing satisfactory thermal and chemical stability under many different experimental conditions. Also, new substrates mainly based on other inorganic substrates like, e.g. alumina and zirconia have been developed now as a starting material for the preparation of RPLC stationary phases of improved chemical and thermal stability. In addition, for the same reasons, many efforts have also been made to synthesize polymer and also polymer-coated phases. These latter phases, more particularly those based on zirconia, but also polymer phases show a high degree of chemical and thermal stability compared to silica counterparts. In this paper, an overview will be given of the state-of-the-art of the thermal and chemical stability of the different available stationary phases for RPLC.     

Reversed-phase liquid chromatographic separation and simultaneous profiling of steroidal glycoalkaloids and their aglycones

Improved and simplified reversed-phase liquid chromatographic conditions for the separation and simultaneous profiling of both steroidal glycoalkaloids and their aglycones, having solanidane- or spirosolane-type structures, are described. The most reproducible retention behavior for these ionizable compounds on C18 columns was achieved under isocratic and gradient elution conditions using acetonitrile in combination with triethylammonium phosphate buffer at pH 3.0, when basic functional groups of solutes and silanol groups on the silica are fully protonated minimizing ionic interactions. Gradient elution was the only feasible approach for the simultaneous separation of steroidal glycoalkaloids and their aglycones. A Zorbax SB C18 column, specially designed for low-pH separations, showed good performance in critical separations. The impurities of the commercial tomatine and tomatidine standards were studied and confirmed using mass spectrometric, liquid chromatographic and thin-layer chromatographic methods.     

Preparation of spherical silica hydroxyl-functionalized covalent organic polymer composites for mixed-mode liquid chromatography

Covalent organic polymers are an emerging class of amorphous microporous materials that have raised increasing concerns in analytical chemistry due to their unique structural and surface chemical properties. However, the application of covalent organic polymers as mixed-mode stationary phases in chromatographic separations has rarely been reported. Herein, novel spherical silica hydroxyl-functionalized covalent organic polymer composites were successfully prepared via a layer-by-layer approach. The structure and morphology of the materials were carefully characterized by elemental analysis, Fourier-transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, X-ray diffraction, thermogravimetric analysis, Brunauer-Emmett-Teller, and contact angle measurements. Baseline separations of various alkylbenzenes, polycyclic aromatic hydrocarbons, and nucleosides and bases were achieved on the prepared stationary phase under reversed-phase/hydrophilic interaction mode. The column efficiencies of 23 853 and 36 580 plates/m were obtained for butylbenzene and uracil, respectively, and the relative standard deviation of the retention time for continuous injections was less than 1.38% (n = 10), suggesting satisfactory column efficiency and repeatability. Additionally, this novel stationary phase realized the complete separation of the endocrine-disrupting chemicals in river water. This work affords a new route for synthesizing covalent organic polymers-based mixed-mode stationary phase and further reveals their great potential in chromatographic separation.     

Optimization of chromatographic systems for the high-performance thin-layer chromatography of flavonoids

Summary To characterize the retention and selectivity of separations of 23 flavonoids (aglycones and glycosides) relationships betweenR _F and modifier concentration were determined for silica and diol adsorbents (with mixtures of ethyl acetate and methanol as mobile phases), for cyanopropyl silica (with mixtures of ethyl acetate and dichloromethane as mobile phases), for aminopropyl silica (with mixtures of ethyl acetate, methanol and water as mobile phases) and for octadecyl silica (with mixtures of methanol and water as mobile phases). Owing to large polarity differences between aglycones and glycosides, these groups of compounds cannot be separated other than by use of reversed-phase systems, for which the selectivity is lower. It follows from correlation plots ofR _F1 againstR _F2 that for some pairs of adsorbents (e. g. silica and diol) selectivity differences are small; for others the points in the plot are widely dispersed, indicating selectivity differences. The chemometric database obtained can be used to choose optimum chromatographic systems for the separation of given sets of flavonoids and for planning gradient elution programs for separation of flavonoid aglycones and glycosides in a single TLC experiment.     

Imidazoline type stationary phase for hydrophilic interaction chromatography and reversed-phase liquid chromatography

An imidazoline was prepared by solvent-free microwave-assisted organic synthesis and immobilized on porous silica particles by polymerization. The resulting material was composed of both hydrophobic alkyl ester chains and hydrophilic imidazoline rings, which gave it both hydrophilic interaction and reversed-phase characteristics. The titration curve suggests that the new material has buffering capacity and acquires increasing positive charge over the pH range 9-4, and is "zwitterionic" in the upper part of this pH range. Through investigating the effect of column temperature, the water content, pH and ion strength of mobile phase on the retention time of polar compounds in highly organic eluents, it was found that the new material could be used as a hydrophilic interaction liquid chromatography (HILIC) stationary phase which involved a complex retention process consisting of partitioning, surface adsorption and electrostatic interactions. In addition, the retention behavior of aromatic compounds in different mobile phase conditions was also studied, which showed the new material mainly exhibited a partitioning mechanism in the reversed-phase liquid chromatography (RPLC) mode. The separation of six water-soluble vitamins and five aromatic compounds were achieved by using the new material in the HILIC and RPLC modes, respectively.     

Surface modification for PDMS-based microfluidic devices

This review focuses on advances reported from April 2009 to May 2011 in PDMS surface modifications for the application in microfluidic devices. PDMS surface modification techniques presented here include improved plasma and graft polymer coating, dynamic surfactant treatment, hydrosilylation-based surface modification and surface modification with nanomaterials such as carbon nanotubes and metal nanoparticles. Recent efforts to generate topographical and chemical patterns on PDMS are also discussed. The described surface modifications not only increase PDMS wettability, inhibit or reduce non-specific adsorption of hydrophobic species onto the surfaces in the act, but also result in the display of desired functional groups useful for molecular separations, biomolecular detection via immunoassays, cell culture and emulsion formation.     

Impact of molecular surface characteristics on the reversed-phase retention behaviour of synthetic dyes

The lipophilicity and specific hydrophobic surface area of 42 synthetic dyes were determined on reversed-phase alumina layers using water-methanol mixtures as eluents, and their relationship with the molecular surface characteristics was elucidated by principal component analysis followed by a two-dimensional nonlinear map and cluster analysis. Four dyes remained on the origin in each eluent system. Except for two dyes, the majority showed regular retention behaviour with their retention decreasing steadily with increasing concentration of methanol in the mobile phase. Calculations indicated that both hydrophobicity parameters decrease with increasing polar surface area of the molecules.     

Modification of porous silica particles with poly(acrylic acid)

The surface of porous silica particles was modified with poly(acrylic acid) by reacting the carboxyl groups on poly(acrylic acid) with the amino groups of pregrafted aminopropyltriethoxysilane (APS). The chemical modifications by APS and polymer were characterized by infrared spectroscopy and the amount of APS and poly(acrylic acid) grafted to the surface were determined by thermal gravimetric analyses. The wettability of the modified silica particles, based on the rate of water penetration, was pH‐dependent with PAA; at pH 1.5 the wettability increased but at pH 5.5 it decreased dramatically. The pore size and size distribution of the silica particles decreased with APS and polymer grafting. Copyright © 2000 John Wiley & Sons, Ltd.     

Synthesis of zirconia monoliths for chromatographic separations

The aim of this work is to join the advantages of two different kinds of stationary phases: monolithic columns and zirconia-based supports. On the one hand, silica monolithic columns allow a higher efficiency with a lower back-pressure than traditional packed columns. On the other hand, chromatographic stationary phases based on zirconia have a higher thermal and chemical stability and specific surface properties. Combining these advantages, a zirconia monolith with a macroporous framework could be a real improvement in separation sciences. Two main strategies can be used in order to obtain a zirconia surface on a monolithic skeleton: coating or direct synthesis. The coverage by a zirconia layer of the surface of a silica-based monolith can be performed using the chemical properties of the silanol surface groups. We realized this coverage using zirconium alkoxide and we further grafted n-dodecyl groups using phosphate derivatives. Any loss of efficiency was observed and fast separations have been achieved. The main advance reported in this paper is related to the preparation of zirconia monoliths by a sol-gel process starting from zirconium alkoxide. The synthesis parameters (hydrolysis ratio, porogen type, precursor concentration, drying step, etc.) were defined in order to produce a macroporous zirconia monoliths usable in separation techniques. We produced various homogeneous structures: zirconia rod 2 cm long with a diameter of 2.3 mm, and zirconia monolith inside fused silica capillaries with a 75 microm I.D. These monoliths have a skeleton size of 2 microm and have an average through pore size of 6 microm. Several separations have been reported.     

HPLC-separation of enantiomers using quinine,covalently bonded to silica as stationary phase

Summary Stationary phases for chiral separations have been synthesized by chemical modification of porous small particle silica using new procedures of fixation of the chiral moiety. So called pre-polymers of the methylpolysiloxane polysiloxane type are immobilized on silica surfaces by different procedures. These pre-polymers are substituted by chiral groups. Their synthesis is done externally i.e. not in-situ on the support surface. The immobilization on the silica surface is achieved by crosslinking and/or by chemical bonding. Anchor groups within the pre-polymer (e.g. SiH) as well as on the silica surface (SiOH) give rise to the chemical bonding therewith. Chiral phases with quinine as the chiral moiety were obtained which show high separation efficiency as well as chemical stability, in addition to the enantiomeric selectivity required for the separation of certain types of aliphatic and aromatic alcohols. Presented at the 16th International Symposium on Chromatography, Paris, September 1986     

MBAMT,a new locating and chelating agen in TLC and ring colorimetric determination of Cu(II), Co(II), Ni(II), Pd(II), Pt(IV), and Rh(III)

Summary Synthesis of MBAMT (3-methyl-4-benzylideneamino-5-mercapto-1,2,4-triazole) and its IR and NMR spectral data are reported. The high stability of the characteristically coloured chelates with Cu(II), Co(II), Ni(II), Pd(II), Pt(IV) and Rh(III) has been made the basis for their efficient ascending TLC separations on silica gel G layers, when present together. Results of four different solvent systems are included to assess efficient resolution of the chelates along with their limits of identification and separation. TLC separations, followed by the ring colorimetric determination of the six metal ions (as ternary mixtures) are tabulated.MBAMT=3-methyl-4-benzylideneamino-5-mercapto-1,2,4-triazole.     

Characterization of covalently bonded and adsorbed polymer coatings on silica, alumina and zirconia by means of physico-chemical and chromatographic methods

Polymer coatings on porous silica, alumina and zirconia were prepared by covalent bonding of a copolymer of styrene and vinylsilane on the oxidic surface with or without subsequent cross-linking of the immobilized polymeric layer. Polybutadiene and polychloromethylstyrene were adsorbed on the surface of porous alumina without covalent bonding and were cross-linked after deposition. Analysis of the pore structure of coated oxides by means of nitrogen adsorption, mercury porosimetry and inverse size-exclusion chromatography revealed different polymer distributions across the modified surface and different changes of the porosity of the initial oxides depending on the method of polymer immobilization. By covalent bonding of the copolymer of styrene and vinylsilane to alumina and zirconia with subsequent cross-linking the modifying polymeric layer, highly hydrolytic stable packings were synthesized and their application in separations with aggressive eluents were demonstrated.     

Poly(vinylpyrrolidone)-coated silica: A versatile,polar stationary phase for HPLC

Summary Poly(vinylpyrrolidone) (PVP) is immobilized on both, small- and large-pore silicas by thermal treatment, γ-radiation, or peroxide initiated polymerization. The hydrolytic stability of such a highly polar stationary phase significantly exceeds that of a comparable phase prepared by the chemical reaction of silica with a pyrrolidone ethyl dimethylchlorosilane silanization reagent. The properties of the different PVP-silicas are evaluated by elemental analysis, spectroscopy, and chromatography. Columns of PVP-silica packings can be used in several modes: a) under normal-phase conditions as a polar bonded stationary phase, b) under reversed-phase conditions, for the separation of organic proton-donor and hydrogen-bonding compounds, c) for the aqueous size exclusion chromatography of proteins, and d) with salt gradients for the hydrophobic interaction chromatography of proteins. The minimum observable reduced plate-height of PVP-silica columns is about 3. Double-layer polymer coating experiments using PVP-silica covered with poly(methyloctadecylsiloxane) have been performed to study diffusion and shielding effects of different polymer layers in the stationary phase. Depending on separation conditions, one or the other polymer governs the retention process. A mixed selectivity was observed in a reversed phase mode with acidic eluents.     

Chromatographic and electrophoretic behaviour of primary aromatic amines on anion-exchange thin layers

The use of polystyrene-based anion exchangers with aqueous eluents for the separation of primary aromatic amines has been investigated, and extended with satisfactory results to microcrystalline cellulose, cellulose-based anion-exchangers and sodium CM-cellulose layers.Interesting separations of the amines have also been effected electrophoretically on AG 1-X4 layers.The use of water-organic solvent mixtures on polystyrene-based cation exchangers is discussed.     

Two-dimensional thin-layer chromatography of selected coumarins

Polar-bonded stationary phases (CN-silica and Diol-silica) are used with nonaqueous eluents in adsorption mode or with aqueous eluents in partition mode. This enables the application of these systems in two-dimensional separations because of the different selectivity and application to the separation of closely related compounds of similar physicochemical properties and retention behaviour. Similarly, multiphase plates, connected with C18 strips and silica layers, are used with aqueous and nonaqueous eluents. Such layers were applied for the separation of selected coumarins. Thus, differences in separation selectivity are applied for the separation of coumarin fractions from plant extracts of the Apiaceae family by two-dimensional thin-layer chromatography.     

Comparison of methods for characterization of reversed-phase liquid chromatographic selectivity

The goal of the present work is to obtain a better understanding of the chemical factors affecting liquid chromatographic retention. One of the most commonly used formats for liquid chromatographic separations is based on a nonpolar stationary phase, typically an octadecyl-derivatized silica material. A wide variety of these reversed-phase columns are commercially available that differ significantly in their chromatographic retention and selectivity. We seek to quantitatively characterize these differences. Retention data for a range of compounds with many diverse characteristics have been measured on several different octadecyl silica columns (J. Chromatogr. A, submitted for publication). Principal components analysis is used to characterize the different properties of these stationary phases and predict retention factors. The key set factor analysis method and the typical solute method are used in conjunction with the principal components analysis to identify small subsets of solutes that can be used to quantitatively describe the retention of a broad range of compounds. In addition, a quantitative comparison to alternative data analysis methods is made, including linear solvation energy relationships and an iterative subtraction method based on linear regression techniques. Although many earlier studies have reported the application of these methods, this study is the first to make a quantitative comparison of these methods using a highly precise and structurally variable set of test compounds.