Particulate Stationary Phases Research Articles

Are we approaching a post‐monolithic era?

Thirty years after the introduction, monolithic stationary phases are an important member of chromatographic phases. When compared to conventional particulate materials, the continuous internal structure of both inorganic-based silica and organic polymer-based monoliths allows some hydrodynamic and analytical possibilities that are not provided by conventional particulate stationary phases. Polymer-based monolithic stationary phases offer simple preparation and straightforward surface modification which makes them very versatile materials that are applicable, for example, as chromatographic stationary phases, sample enrichment units, enzymatic reactors, and external trigger-responding materials. On the other hand, current polymer monoliths cannot compete with efficiency provided by superficially porous and sub 2 μm particles. In this highlight article, I take advantage of the thirtieth anniversary of their introduction to discuss several concerns related to polymer-based monolithic stationary phases. Particularly, I focus on preparation repeatability, porous properties, swelling of the polymers in organic solvents, column efficiency for small molecules, and heterogeneity of dominant flow-through pores. In the end, I offer three possible approaches on how to overcome drawbacks related to stationary phases heterogeneity to further increase the applicability of polymer-based monolithic stationary phases. This article is protected by copyright. All rights reserved.

Simultaneous Capillary Liquid Chromatography Determination of Drugs in Pharmaceutical Preparations Using Tunable Platforms of Polymethacrylate Monolithic Columns Modified with Octadecylamine

Nowadays, analytical techniques tend to develop cheap and green approaches to reduce the costs and minimize environmental impacts. In this study, poly(glycidyl methacrylate-co-ethylene dimethacrylate) monoliths were prepared in capillary columns (0.25 mm i.d. × 20 cm length) as reactive and tunable platforms, and then simply functionalized based on a ring-opening reaction of epoxide with octadecylamine. The columns were successfully used as reversed-phase stationary phases for determination of ascorbic acid, paracetamol, caffeine, aspirin, and ibuprofen in pharmaceutical combinations. The synthesized monoliths have been estimated by FT-IR spectroscopy, SEM and specific surface area; also, the porous and hydrodynamic properties have verified the differences before and after modification. While the columns before modification were unable to separate the drugs, the columns after amination allowed for the complete separation of the five drugs in about 10 min at 23 µL min−1 flow rate with chromatographic resolution more than 2.12. The developed assay showed excellent suitability (RSDs < 2.6%), wide linear ranges (0.07–300 µg mL−1), sensitive detection limits (0.022–0.060 µg mL−1), good repeatability (RSDs < 4.76%) and acceptable columns reproducibility (RSDs < 18.58%). The method was successfully applied for commercial pharmaceutical preparations; the average recovery values were found to be in the range of 98.03–104.54% (RSDs < 5.51%). The satisfactory recovery percentages along with the perfect correlation between the retentions of standard compounds and the active ingredients extracted from the commercial tablets indicate the accuracy and selectivity of the developed method. The prepared columns and validated method were finally compared with previous reports, particularly commercial C18 stationary phases. This is a very useful comparison due to the structural similarity between the prepared polymeric materials and C18 particulate stationary phases. The prepared capillary columns exhibited various advantages such as the fast separation time, wide linear ranges, low analysis costs, and trivial environmental wastes.

HPLC Characterization of the Association Mechanism of Organic Compounds with β Cyclodextrin Using a Novel Boron Nitride Nanotube Particulate Stationary Phase

In this paper, a simple homogeneous coating of silica spherical particles with pristine boron nitride nanotubes (BNNTs) was described. BNNTs dissolved in dimethylacetamide (DMAc) were mixed with amino-functionalized silica particles having a 5 μm diameter. Favorable interaction between the amino group and the BNNT surfaces induces the absorption of the BNNTs on the silica. The BNNT-coated silica particles were used as stationary phase for HPLC. For the first time, it was demonstrated that this new particulate BNNT stationary phase can be used for the study of the complexation of solute molecules (terpene molecules used as test drugs in this work) with β cyclodextrin (βCD). The apparent formation constants Kf of terpene derivative/βCD were in the same magnitude as those reported in the literature. The plot of Kf versus the water fraction in the methanol/water mobile phase showed that the BNNT surface played an active role in the complex formation due to terpene/BNNT-specific polar interactions. This work demonstrated that our novel particulate BNNT HPLC stationary phase was an efficient tool to study molecular recognition mechanism and more specifically the association between a drug substance and a target molecule with the aim of reaching biopharmaceutic and clinical applications.

Development of a new extraction technique and HPLC method for the analysis of non-psychoactive cannabinoids in fibre-type Cannabis sativa L. (hemp)

The present work was aimed at the development and validation of a new, efficient and reliable technique for the analysis of the main non-psychoactive cannabinoids in fibre-type Cannabis sativa L. (hemp) inflorescences belonging to different varieties. This study was designed to identify samples with a high content of bioactive compounds, with a view to underscoring the importance of quality control in derived products as well. Different extraction methods, including dynamic maceration (DM), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) and supercritical-fluid extraction (SFE) were applied and compared in order to obtain a high yield of the target analytes from hemp. Dynamic maceration for 45min with ethanol (EtOH) at room temperature proved to be the most suitable technique for the extraction of cannabinoids in hemp samples.The analysis of the target analytes in hemp extracts was carried out by developing a new reversed-phase high-performance liquid chromatography (HPLC) method coupled with diode array (UV/DAD) and electrospray ionization-mass spectrometry (ESI–MS) detection, by using an ion trap mass analyser. An Ascentis Express C18 column (150mm×3.0mm I.D., 2.7μm) was selected for the HPLC analysis, with a mobile phase composed of 0.1% formic acid in both water and acetonitrile, under gradient elution. The application of the fused-core technology allowed us to obtain a significant improvement of the HPLC performance compared with that of conventional particulate stationary phases, with a shorter analysis time and a remarkable reduction of solvent usage.The analytical method optimized in this study was fully validated to show compliance with international requirements. Furthermore, it was applied to the characterization of nine hemp samples and six hemp-based pharmaceutical products. As such, it was demonstrated to be a very useful tool for the analysis of cannabinoids in both the plant material and its derivatives for pharmaceutical and nutraceutical applications.

Metabolite fingerprinting of Punica granatum L. (pomegranate) polyphenols by means of high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometry detection

The present study was aimed at the development of a new analytical method for the comprehensive multi-component analysis of polyphenols in Punica granatum L. (pomegranate) juice and peel. While pomegranate juice was directly analysed after simple centrifugation, different extraction techniques, including maceration, heat reflux extraction, ultrasound-assisted extraction and microwave-assisted extraction, were compared in order to obtain a high yield of the target analytes from pomegranate peel. Dynamic maceration with a mixture of water and ethanol 80:20 (v/v) with 0.1% of hydrochloric acid as the extraction solvent provided the best result in terms of recovery of pomegranate secondary metabolites.The quali- and quantitative analysis of pomegranate polyphenols was performed by high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometry detection. The application of fused-core column technology allowed us to obtain an improvement of the chromatographic performance in comparison with that of conventional particulate stationary phases, thus enabling a good separation of all constituents in a shorter time and with low solvent usage.The analytical method was completely validated to show compliance with the International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use guidelines and successfully applied to the characterisation of commercial and experimental pomegranate samples, thus demonstrating its efficiency as a tool for the fingerprinting of this plant material. The quantitative data collected were submitted to principal component analysis, in order to highlight the possible presence of pomegranate samples with high content of secondary metabolites. From the statistical analysis, four experimental samples showed a notable content of bioactive compounds in the peels, while commercial ones still represent the best source of healthy juice.

Polystyrene bound stationary phase of excellent separation efficiency based on partially sub-2 μm silica monolith particles

Partially sub-2μm porous silica monolith particles have been synthesized by a renovated procedure and modified to polystyrene coated silica particles with excellent separation efficiency when used as chromatographic media. In the procedure of preparing silica monolith particles in this study, subtle control of formulation of the reaction mixture and multi-step heating followed by calcination, without any washing and sieving process, enabled formation of silica particles characterized by proper particle and pore size distribution for high separation efficiency. 3-Chloropropyl trimethoxysilane was used as the halogen terminal spacer to combine the initiator to silica particles. Uniform and thin coating of polystyrene layer on initiator attached silica particles was formed via reversible addition-fragmentation chain transfer (RAFT) polymerization. Micro-columns (1.0mm ID and 300mm length) were packed with the resultant phase and their chromatographic performance was elucidated by HPLC. A mobile phase of 60/40 (v/v) acetonitrile/water containing 0.1% TFA and a flow rate of 15μL/min were found to be the optimized conditions leading to number of theoretical plates close to 50,000 (165,000m–1). This is the very first study to get such highly efficient HPLC columns using a silica monolith particulate stationary phase.

Improvement of chromatographic performances of in-situ synthesized hybrid C 8 silica monoliths by reduction of structural radial heterogeneities

Several modifications of a previously described protocol are proposed to improve the performances of in-situ synthesized C 8 hybrid silica monoliths. Our attention was focused on reducing the sources of radial heterogeneity that may be responsible for the poor efficiencies observed in the hydrodynamic elution mode. It was demonstrated that a decrease in the temperature of the capillary during the filling step equally to that of the polymerization mixture (0 °C), associated with a decrease of the gelation temperature to 20 °C along with a new pre-treatment of the capillary's internal walls [with a mixture of tetraethoxysilane (TEOS)/EtOH (1/3, v/v)] allows (i) increasing the radial homogeneity of the monolith, thus further enhancing the performances in the nano-liquid chromatography (nano-LC) mode, (ii) improving the capillary to capillary reproducibility in terms of permeability and efficiencies. In fact, the average minimum plate height H min was lowered from 24 to 14 μm and the capillary-to-capillary reproducibility of the synthesis was widely improved by factors two and three of reduction on the calculated standard deviation, respectively for both the efficiency in the nano-LC mode and the permeability. At last, the improved radial homogeneity and anchoring of the synthesized monoliths allowed increasing the inner diameter of the capillary (up to 150 μm) without any significant loss in efficiency. Finally, long term stability of the as-obtained monolithic stationary phases in terms of retention and efficiency was studied. In addition, the evaluation of their chromatographic behaviour was also achieved with the Tanaka test and the results were compared to those already published for commercial monoliths (Chromolith) as well as for particulate stationary phases.

Chromatographic Properties of Ethanol/Water Mobile Phases on Silica Based Monolithic C18

For almost 10 years, silica-based monolithic stationary phases have been used in numerous fields of analytical chemistry, especially when high flow rate is required or for the separation of high molecular weight compounds such as peptides. In comparison with particulate stationary phases, the macroporous structure of the silica rod induces low pressure drops, which allows the use of high flow rates, leading to a dramatic reduction of the analysis duration. We suggest another use of this type of phases with mobile phases having a high viscosity, such as ethanol–water mixtures. These mobile phases are environmentally friendly, avoiding the use of toxic solvents such as methanol and acetonitrile, and consequently the necessary cleaning of waste is reduced. In this paper, the eluting strength of different hydro-organic mobile phases and their efficiencies are compared on Chromolith RP-18e. Due to higher eluting strength, the use of ethanol–water mixtures also allows decreasing the amount of organic solvent required to achieve a separation, whereas, despite a higher viscosity, the chromatographic efficiencies are as good as those obtained with classical mobile phases. Finally, separation of pesticides (triazines) and sunscreen molecules are presented.

Porous polymer monoliths for extraction: Diverse applications and platforms

Polymer monoliths are becoming increasingly popular as sorbent materials, and, along with silica monoliths, they are sometimes touted as replacements for the particulate stationary phases used in HPLC. This critical and prospective review shows how polymer monoliths are in fact finding numerous extraction roles that do not resemble HPLC. They are showing great promise as extractors in a remarkable range of platforms, formats and hyphenated systems with functions ranging from chromatographic preconcentration to large-scale preparative extraction. Monolith surface chemistry, morphology and the approaches to monolith synthesis are discussed with regards to these emerging roles.

A kinetic parameter concerning mass transfer in silica monolithic and particulate stationary phases measured by the peak‐parking and slow‐elution methods

Mass transfer in monolithic C18-silica stationary phases and C18-silica gel particles was studied. A traditional kinetic parameter, gamma(s)D(s), which is a diffusion coefficient of solute molecules in the stationary phase, was measured by two unusual approaches, i.e., peak-parking and slow-elution methods. The correlation between the ratio of gamma(s)D(s) to molecular diffusivity (Dm) and the retention factor (k) was represented by one common curve, irrespective of the RPLC conditions. A similar curved profile was also observed between another kinetic parameter (D(Ls)), which is related to the axial diffusive molecular migration in the stationary phase, and the retention equilibrium constant (Ka). The values of D(Ls) and Ka were calculated from those of gamma(s)D(s) and k, respectively. The ratio of D(Ls)/Dm increases with decreasing Ka and seems to approach around unity when Ka is infinitely small. The dependence of D(Ls) on Ka was also studied from extra-thermodynamic points of view. The linear correlation between In D(Ls) and In Ka suggests the existence of a kind of linear free energy relationship between the mass transfer in the stationary phase and the retention equilibrium. Because these characteristics of D(Ls) are similar to those of the surface diffusion coefficient (D(sur)), D(Ls) seems to correspond to D(sur).

“Click Chemistry” in the Preparation of Porous Polymer-Based Particulate Stationary Phases for μ-HPLC Separation of Peptides and Proteins

With the use of the copper(I)-catalyzed (3 + 2) azide-alkyne cycloaddition, an element of "click chemistry," stationary phases carrying long alkyl chains or soybean trypsin inhibitor have been prepared for use in HPLC separations in the reversed-phase and affinity modes, respectively. The ligands were attached via a triazole ring to size monodisperse porous beads containing either alkyne or azide pendant functionalities. Alkyne-containing beads prepared by direct copolymerization of propargyl acrylate with ethylene dimethacrylate were allowed to react with azidooctadecane to give a reversed-phase sorbent. Azide-functionalized beads were prepared by chemical modification of glycidyl methacrylate particles. Subsequent reaction with a terminal aliphatic alkyne produced a reversed-phase sorbent similar to that obtained from the alkyne beads. Soybean trypsin inhibitor was functionalized with N-(4-pentynoyloxy)succinimide to carry alkyne groups and then allowed to react with the azide-containing beads to produce an affinity sorbent for trypsin. The performance of these stationary phases was demonstrated with the HPLC separations of a variety of peptides and proteins.

Monolithic Materials: Promises, Challenges, Achievements

Nowadays, there is a lot of buzz in separations science about the porous materials known as monoliths. Monoliths are used mostly as separation media and supports, and each one can be roughly compared to a single porous ''particle''. Monoliths are an alternative to columns packed with particulate stationary phases, which chromatographers have used for >100 years. Although that technology is well developed, it is not flawless. Packed columns have rather large void volumes; this is an inevitable result of the particulate character of the packing. Even in a perfectly organized array of monodisperse spheres, {approx}30% of the volume is interstitial voids. In reality, the percentage is even larger. As a consequence, a significant part of the column volume is not used for separation. However, this is not the only problem. Consider what happens when a mobile phase is pumped through a column packed with a standard porous packing. The liquid flows readily through the interstitial voids between the particles, where resistance to its flow is the smallest (Figure 1a). In contrast, the liquid in the pores remains stagnant. If a sample is injected into the stream of the mobile phase, the compounds in the sample will also be carried throughmore » the voids. However, diffusion occurs because of the concentration gradient between the compounds in solution flowing through the interstices and the stagnant liquid within the pores of the packing, and transport of these compounds into the pores results. Once the concentration ''pulse'' has passed by the bead, the compounds diffuse back from the pores into the surrounding liquid; eventually, only the original stagnant phase remains within the pores.« less

The Use of 3 and 12 Micron Particulate Stationary Phases in Voltage-Assisted Micro-LC for the Separation of Mixtures Containing Neutral, Basic and Acidic Analytes

The application of voltage-assisted micro-LC using CEC instrumentation (capable of applying 1.2 MPa of pressure to supplement the electroosomotic flow) has been demonstrated. This has been achieved by the use of 100 μm × 24 cm capillaries packed with 12 μm stationary phase material; alternatively 7.5 cm capillaries packed with 3 μm material using the short-end injection approach with reversed polarity can be used. The paper highlights how both approaches can be successfully employed to control the separation selectivity of a range of mixtures containing acidic, basic and neutral components by the concomitant addition of voltage (either positive or negative) to the applied micro-LC flow.

Development and assessment of a miniaturised centrifugal chromatograph for reversed-phase separations in micro-channels.

This paper describes the micro-fabrication and preliminary assessment of a miniature polydimethylsiloxane (PDMS) device for performing rapid, parallel liquid phase chromatographic separations driven by centrifugal force in microchannels. Device components include a main separating channel, into which a high performance liquid chromatography (HPLC) particulate stationary phase was packed under pressure by application of centrifugal force, in addition to solvent and sample reservoirs. Also described are methods for sealing such devices based upon partial polymerisation of PDMS. The mobile phase flow rate through a typical device was measured and several important chromatographic parameters determined from a test separation. An expression describing mobile phase flow through packed channels was also developed, based upon work on liquid flow in open micro-channels. Good agreement between predicted and measured flow rates were observed. Some predictions for potential uses of such devices and possibilities for further miniaturisation are discussed.

Chromatographic performance of monolithic and particulate stationary phases: Hydrodynamics and adsorption capacity

Monolithic chromatographic support structures offer, as compared to the conventional particulate materials, a unique combination of high bed permeability, optimized solute transport to and from the active surface sites and a high loading capacity by the introduction of hierarchical order in the interconnected pore network and the possibility to independently manipulate the contributing sets of pores. While basic principles governing flow resistance, axial dispersion and adsorption capacity are remaining identical, and a similarity to particulate systems can be well recognized on that basis, a direct comparison of sphere geometry with monolithic structures is less obvious due, not least, to the complex shape of the skeleton domain. We present here a simple, widely applicable, phenomenological approach for treating single-phase incompressible flow through structures having a continuous, rigid solid phase. It relies on the determination of equivalent particle (sphere) dimensions which characterize the corresponding behaviour in a particulate, i.e. discontinuous bed. Equivalence is then obtained by dimensionless scaling of macroscopic fluid dynamical behaviour, hydraulic permeability and hydrodynamic dispersion in both types of materials, without needing a direct geometrical translation of their constituent units. Differences in adsorption capacity between particulate and monolithic stationary phases show that the silica-based monoliths with a bimodal pore size distribution provide, due to the high total porosity of the material of more than 90%, comparable maximum loading capacities with respect to random-close packings of completely porous spheres.

An Integrated Fritless Column for On-Chip Capillary Electrochromatography with Conventional Stationary Phases

A new polymer device for use with conventional particulate stationary phases for on-chip, fritless, capillary electrochromatography (CEC) has been realized. The structure includes an injector and a tapered column in which the particles of the stationary phase are retained and stabilized. The chips were easily fabricated in poly(dimethylsiloxane) using deep-reactive-ion-etched silicon masters, and tested using a capillary electrophoretic separation of FITC-labeled amino acids. To perform CEC, the separation channel was packed using a vacuum with 3-microm, octadecylsilanized silica microspheres. The packing was stabilized in the column by a thermal treatment, and its stability and quality were evaluated using in-column indirect fluorescence detection. The effects of voltage on electro-osmotic flow and on efficiency were investigated, and the separation of two neutral compounds was achieved in less than 15 s.

Sintered octadecylsilica as monolithic column packing in capillary electrochromatography and micro high-performance liquid chromatography

Fused-silica capillaries were packed with porous 6-μm octadecylated silica microspheres and subjected to thermal treatment in order to obtain a column with porous silica based monolithic packing. After sintering, the monolithic packing was reoctadecylated in situ with dimethyloctadecylchlorosilane. The mechanical strength and stability of the monolithic column were significantly greater than those of conventional columns packed with particulate stationary phase. The performance of the columns was evaluated in both μ-HPLC and capillary electrochromatography (CEC) of small aromatic compounds and polycyclic aromatic hydrocarbons with 10 m M borate, pH 8.0, in acetonitrile–water mixtures as the mobile phase. Since no untoward bubble formation was observed, CEC could be performed in a CZE unit without the need for pressurizing the monolithic column. The plate efficiency of the monolithic column was similar to that of columns freshly packed with the same particles and was always higher in the CEC than in the μ-HPLC under otherwise identical conditions. The electrosmotic flow (EOF) velocity increased with the acetonitrile content of the eluent in open fused-silica capillaries with octadecylated innerwall and in columns packed with sintered octadecylsilica. In contradistinction, the EOF velocity decreased with the organic strength of the eluent in raw fused-silica capillaries. The opposite trend is attributed to the different effect of changing organic modifier concentration on the accessibility of silanol groups at the raw and octadecylated silica surfaces.

Characterization of LC Stationary Phases by Inverse Size Exclusion Chromatography

Abstract The most efficient use of a porous particulate stationary phase can only be made when the material in the column is fully characterized. While complete characterization includes both chemical and physical aspects, the macro structure provides the framework for the microscopic chemical interactions. The physical analysis is therefore critically important for a more complete calibration of the chromatographic effects.