Methods to determine the kinetic performance limit of contemporary chromatographic techniques

By combining separation efficiency data as a function of flow rate with the column permeability, the kinetic plot method allows to determine the limits of separation power (time vs. efficiency) of different chromatographic techniques and methods. The technique can be applied for all different types of chromatography (liquid, gas, or supercritical fluid), for different types of column morphologies (packed beds, monoliths, open tubular, micromachined columns), for pressure and electro‐driven separations and in both isocratic and gradient elution mode. The present contribution gives an overview of the methods and calculations required to correctly determine these kinetic performance limits and their underlying limitations.     

Non-particulate (continuous bed or monolithic) restricted-access reversed-phase media for sample clean-up and separation by capillary-format liquid chromatography

Restricted-access reversed-phase non-particulate (continuous bed or monolithic) stationary phases of different hydrophobicity synthesized in 100 m i.d. fused silica capillaries have been evaluated. A specific property of restricted-access media (RAM) is that they interact with small analytes and exclude big molecules, e.g. proteins, from access to the active sites and adsorption on the surface. This dual property facilitates direct injection of biological fluids for drug or drug-metabolite analysis. Different RAM and RAM-precursor capillary columns were tested to assess the influence of chromatographic bed morphology on loadability. Inverse size-exclusion chromatography was used for investigation of pore structural properties of the capillary-format continuous beds. The data obtained were used to discuss the mechanism of separation of the biological samples using capillary columns and to propose a model for the topochemical architecture of the RAM investigated. Different morphology of the non-particulate reversed-phase precursors resulted in two types of RAM material shielded with hydrophilic polymer, classified as homogeneous or heterogeneous topochemistry stationary phases. Capillary columns were applied for chromatography of biological fluids. High resolution was obtained, without the need for column switching, when capillary columns operated in gradient conditions. Extensive evaluation of the chromatographic properties (hydrophobicity, efficiency, separation impedance, and loadability) of the non-particulate reversed-phase materials was performed before and after shielding with hydrophilic polymer to generate restricted-access properties. Minor changes of hydrophobicity, efficiency, or separation impedance were observed after the shielding.     

Monolithic Stationary Phases for Capillary Electrochromatography Based on Synthetic Polymers: Designs and Applications

Monolithic materials have quickly become a well‐established stationary phase format in the field of capillary electrochromatography (CEC). Both the simplicity of their in situ preparation method and the large variety of readily available chemistries make the monolithic separation media an attractive alternative to capillary columns packed with particulate materials. This review summarizes the contributions of numerous groups working in this rapidly growing area, with a focus on monolithic capillary columns prepared from synthetic polymers. Various approaches employed for the preparation of the monoliths are detailed, and where available, the material properties of the resulting monolithic capillary columns are shown. Their chromatographic performance is demonstrated by numerous separations of different analyte mixtures in variety of modes. Although detailed studies of the effect of polymer properties on the analytical performance of monolithic capillaries remain scarce at this early stage of their development, this review also discusses some important relationships such as the effect of pore size on the separation performance in more detail.     

Fluid dynamics in capillary and chip electrochromatography

This review is concerned with the phenomenological fluid dynamics in capillary and chip electrochromatography (EC) using high-surface-area random porous media as stationary phases. Specifically, the pore space morphology of packed beds and monoliths is analyzed with respect to the nonuniformity of local and macroscopic EOF, as well as the achievable separation efficiency. It is first pointed out that the pore-level velocity profile of EOF through packed beds and monoliths is generally nonuniform. This contrasts with the plug-like EOF profile in a single homogeneous channel and is caused by a nonuniform distribution of the local electrical field strength in porous media due to the continuously converging and diverging pores. Wall effects of geometrical and electrokinetic nature form another origin for EOF nonuniformities in packed beds which are caused by packing hard particles against a hard wall with different zeta potential. The influence of the resulting, systematic porosity fluctuations close to the confining wall over a distance of a few particle diameters becomes aggravated at low column-to-particle diameter ratio. Due to the hierarchical structure of the pore space in packed beds and silica-based monoliths which are characterized by discrete intraparticle (intraskeleton) mesoporous and interparticle (interskeleton) macroporous spatial domains, charge-selective transport prevails within the porous particles and the monolith skeleton under most general conditions. It forms the basis for electrical field-induced concentration polarization (CP). Simultaneously, a finite and -- depending on morphology -- often significant perfusive EOF is realized in these hierarchically structured materials. The data collected in this review show that the existence of CP and its relative intensity compared to perfusive EOF form fundamental ingredients which tune the fluid dynamics in EC employing monoliths and packed beds as stationary phases. This addresses the (electro)hydrodynamics, associated hydrodynamic dispersion, as well as the migration and retention of charged analytes.     

Structure and performance of silica-based monolithic HPLC columns

Silica-based monolithic columns were prepared for HPLC with systematic variations of the tetramethoxysilane (TMOS) and polyethylene oxide (PEO) content as reactants in a sol-gel process accompanied by phase separation. The resulting monoliths showed differences in the macropore and silica skeleton diameter as well as in the corresponding domain sizes (the sum of macropore and skeleton diameter). All monoliths were synthesized with a diameter of 4.6 mm and cladded with a suitable polyaryletheretherketone (PEEK) polymer in a standardized and optimized manner for the subsequent chromatographic evaluation of the resulting monolithic HPLC columns. The columns were tested under normal phase conditions using n-heptane/dioxane (95:5 v/v) as a mobile phase and 2-nitroanisole as a test compound for the determination of separation efficiency and permeability. Two different sets of columns were prepared: the first one in which the amount of PEO was stepwise decreased to yield monoliths with identical macropore volumes and variations in the domain sizes. The second group of materials was synthesized adjusting both TMOS and PEO quantities to yield monolithic columns with identical macropore diameters of about 1.80 microm but different skeleton diameters and macropore volumes. The chromatographic results suggest that an increase in the column performance cannot be achieved by just arbitrarily decreasing the domain size of a given column. From a certain point of "downsizing" the monolithic structure a loss of structural homogeneity can be observed, which is apparently responsible for a lower chromatographic performance.     

Voltage-assisted capillary LC of peptides using monolithic capillary columns prepared by ring-opening metathesis polymerization

We examined the use of monolithic capillary columns prepared via ring-opening metathesis polymerization (ROMP) for peptide separation in voltage-assisted capillary LC (voltage-assisted CLC). In order to demonstrate their potential for peptide separation, ROMP-derived monoliths with RP properties were prepared. The preparation procedure of monoliths was transferred from ROMP monoliths optimized for CLC. ROMP monoliths were synthesized within the confines of 200 microm id fused-silica capillaries with a length of 37 cm. After optimization of the chromatographic conditions, the separation performance was tested using a well-defined set of artificial peptides as well as two peptidic mixtures resulting from a tryptic digest of BSA as well as a collagenase digest of collagen. ROMP monoliths showed comparable performance to other monolithic separation media in voltage-assisted CLC published so far. Therefore, we conclude that by optimizing the composition of the ROMP monoliths as well as by using the controlled manner of their functionalization, ROMP monoliths bear a great potential in CLC and CEC.     

Preparation and characterization of hexyl methacrylate monolithic columns for CEC

The preparation of hexyl methacrylate (HMA) monolithic columns for CEC separations has been investigated with two initiation systems: (i) ammonium peroxodisulphate and TEMED to activate the polymerization reaction, and (ii) by thermal initiation with AIBN. For both initiators, the influence of composition of porogenic solvent on morphological and chromatographic properties of monoliths was investigated. Two porogenic solvent systems, aqueous and non-aqueous media, were also studied for monolithic beds polymerized with AIBN. Under optimal conditions, low minimum plate heights (9.6 mum for peroxodisulphate, 8.4 and 10.0 mum for AIBN in aqueous and non-aqueous porogenic solvents, respectively) were obtained. A comparison in terms of chromatographic performance of HMA monoliths with butyl methacrylate columns polymerized with both initiators was also performed. The resulting HMA-based stationary phases also exhibited a good repeatability and column-to-column reproducibility, with RSD values below 5.6% in the studied electrochromatographic parameters. The potential of HMA-based columns was demonstrated by the analysis of complex mixtures of polyaromatic hydrocarbons and anabolic steroids.     

Applications of monolithic columns in gas chromatography and supercritical fluid chromatography

Porous monoliths are well‐known stationary phases in high‐performance liquid chromatography and capillary electrochromatography. Contrastingly, their use in other types of separation methods such as gas or supercritical fluid chromatography is limited and scarce. In particular, very few studies address the use of monolithic columns in supercritical fluid chromatography. These are limited to silica‐based monoliths and will be covered in this review together with an underlying reason for this trend. The application of monoliths in gas chromatography has received much more attention and is well documented in two reviews by Svec and Kurganov published in 2008 and 2013, respectively. The most recent studies, covered in this review, build on the previous findings and on further understanding of the influence of preparation conditions on porous properties and chromatographic performance of poly(styrene‐co‐divinylbenzene), polymethacrylate, and silica‐based monolithic columns while expanding to polymer‐based monoliths with incorporated metal organic frameworks and to vinylized hybrid silica monoliths. In addition, the potential application of porous layer open tubular monolithic columns in low‐pressure gas chromatography will be addressed.     

A new easy-to-prepare homogeneous continuous electrochromatographic bed for enantiomer recognition

Completely homogeneous polyacrylamide-based gels were used for capillary electrochromatography (CEC) of drug enantiomers. Like continuous beds (also called continuous polymer rods, silica rods, monoliths) they do not require frits to support the bed because it is covalently linked to the capillary wall. A long lifetime is an important feature of the beds. The gel matrices can be prepared in any laboratory and for specific interactions they can be derivatized with appropriate ligands. The application range is, therefore, broad. For chiral electrochromatography, negatively and positively charged polyacrylamide gels copolymerized with 2-hydroxy-3-allyloxy-propyl-beta-cyclodextrin (allyl-beta-CD) were prepared. The latter monomer was synthesized from beta-CD and allylglycidyl ether by a very simple one-step procedure. Eight acidic, neutral and basic drug compounds were resolved into their enantiomers, most of them with baseline separation. Interestingly, the resolution is independent of the electroendosmotic velocity, i.e., rapid analyses will not give low resolution. Upon increasing this velocity, the plate height for the fast enantiomer did not change (or decreased slightly), whereas that for the slow enantiomer increased. Only the last term in the van Deemter equation contributed significantly to the total plate height. The composition of the gel was chosen such that the "pores" became large enough to guarantee a satisfactory electroendosmotic flow (EOF). This open gel structure explains why acetone diffused as in free solution, i.e., independently of the presence of the gel matrix. This finding also indicates that the separation of small molecules in polyacrylamide gels cannot be explained by "molecular-sieving", but rather by some type of adsorption ("aromatic adsorption"?).     

Monolithic capillary columns based on pentaerythritol acrylates for molecular‐size‐based separations of synthetic polymers

Monolithic capillary columns based on pentaerythritol triacrylate and pentaerythritol tetraacrylate were synthesized using different compositions of polymerization mixtures and different polymerization conditions. The impact of porogen type and porogen/monomer ratio on the porosity of synthesized monoliths was investigated. Porogen type appears to be the main factor influencing the separating properties of the monolithic sorbent. Using optimal polymerization conditions (porogen type, porogen/monomer ratio, reaction temperature, time etc.) monoliths with a porous structure optimized for polymer separations can be obtained. The monolithic capillary columns containing porous sorbents with optimized porosity are capable of separating 10 to 12 polystyrene standards in one chromatographic run utilizing both size exclusion chromatography and hydrodynamic chromatography separation mechanisms.     

Development of fluorinated,monolithic columns for improved chromatographic separations of fluorous-tagged analytes

With applications that take advantage of highly selective fluorine–fluorine interactions appearing with greater frequency in the literature, the development of porous polymer monoliths from fluorous components is reported here for the purpose of chromatography of tagged analytes. With potential uses in fields as diverse as separation science and proteomics, facile fabrication of materials with fluorous specificity that can be applied in a high-throughput manner is greatly desirable. To this end, we have developed porous polymer capillary columns with varied fluorous content using a simple UV-initiated radical polymerization process and characterized them using flow-induced backpressure and scanning electron microscopy (SEM). With structural similarities assured (visually, and by backpressure variations of less than 42%), the monoliths were tested as chromatographic columns for the separation of a series of fluorous-tagged analytes under gradient conditions. It was found that columns made with fluorinated components exhibited greater selectivity for fluorous analytes than did equivalent, non-fluorinated monoliths, retaining analytes with either one or two fluorous tags for approximately 6% and 13% longer, respectively. This supports the idea of differences existing between fluorous and reverse-phase separation mechanisms, and encourages a broader range of potential applications for fluorous monoliths of this type.     

A computational study of the porosity effects in silica monolithic columns

We report on a theoretical study of the influence of the through-pore porosity on the main chromatographic performance parameters (reduced theoretical plate height, flow resistance, and separation impedance) of silica monoliths. To investigate this problem devoid of any structural uncertainties, computer-generated structural mimics of the pore geometry of silica monolithic columns have been studied. The band broadening in these synthetic monoliths was determined using a commercial Computational Fluid Dynamics (CFD) software package. Three widely differing external porosities (epsilon = 0.38, epsilon = 0.60, and epsilon = 0.86) are considered and are compared on the basis of an identical intra-skeleton diffusivity (Ds = 5 x 10(-10)m2/s), internal porosity (epsilon(int) = 0.5), and for the same phase retention factor (k' = 1.25). Since the data are obtained for perfectly ordered structures, the calculated plate heights and separation impedances constitute the ultimate performance ever to be expected from a monolithic column. It is found that, if silica monoliths could be made perfectly homogeneous, domain size-based reduced plate heights as small as h(min) approximately 0.8 (roughly independent of the porosity) and separation impedances as small as Emin approximately 130 (epsilon = 0.60) and Emin approximately 40 (epsilon = 0.86) should be achievable with pure water as the working fluid. The data also show that, although the domain size is a much better reduction basis than the skeleton size, the former is still not capable of bringing the van Deemter curves of different porosity columns into perfect agreement in the C term dominated velocity range. It is found that, in this range, large porosity monoliths can be expected to yield smaller domain size-based reduced plate heights than small porosity monoliths.     

Review on recent and advanced applications of monoliths and related porous polymer gels in micro-fluidic devices

This review critically summarises recent novel and advanced achievements in the application of monolithic materials and related porous polymer gels in micro-fluidic devices appearing within the literature over the period of the last 5 years (2005-2010). The range of monolithic materials has developed rapidly over the past decade, with a diverse and highly versatile class of materials now available, with each exhibiting distinct porosities, pore sizes, and a wide variety of surface functionalities. A major advantage of these materials is their ease of preparation in micro-fluidic channels by in situ polymerisation, leading to monolithic materials being increasingly utilised for a larger variety of purposes in micro-fluidic platforms. Applications of porous polymer monoliths, silica-based monoliths and related homogeneous porous polymer gels in the preparation of separation columns, ion-permeable membranes, preconcentrators, extractors, electrospray emitters, micro-valves, electrokinetic pumps, micro-reactors and micro-mixers in micro-fluidic devices are discussed herein. Procedures used in the preparation of monolithic materials in micro-channels, as well as some practical aspects of the micro-fluidic chip fabrication are addressed. Recent analytical/bioanalytical and catalytic applications of the final micro-fluidic devices incorporating monolithic materials are also reviewed.     

Rapid reversed-phase separation of proteins and peptides using optimized 'moulded' monolithic poly(styrene-co-divinylbenzene) columns

Monolithic macroporous poly(styrene-co-divinylbenzene) stationary phases have been prepared by free radical polymerization within the confines of 4.6-mm I.D. chromatographic columns. The optimized porous properties allow the mobile phase to flow through these columns at flow-rates of up to 10 ml/min. As opposed to the simultaneously tested columns packed with either silica or synthetic polymer beads, the monoliths exhibit only modest back pressure. The monolithic columns were able to separate mixtures of peptides and proteins in a very short time. Under the optimized conditions, the separation of five proteins can be easily achieved in less than 20 s.     

毛细管电色谱中整体式高聚物毛细管柱技术的进展

对近期毛细管电色谱（CEC）中利用高分子聚合反应制备整体式（monolithic)毛细管柱的技术，特别对各种不同类型的整体式毛细管的制备方法进行了详细介绍；对该类毛细管用于不同样品的分离情况进行了总结。     

Non-particulate (continuous bed or monolithic) acrylate-based capillary columns for reversed-phase liquid chromatography and electrochromatography

Three approaches are described to synthesize acrylic non-particulate beds (also called continuous beds or monoliths) in aqueous polymerization media for reversed-phase capillary liquid chromatography/electrochromatography. In the first, hexyl acrylate comonomer was dissolved together with water soluble polar comonomers using a non-ionic detergent. In the second, a new alkyl ammonium salt comonomer, (3-allylamino-2-hydroxypropyl)dodecyldimethylammonium chloride was used, which is water soluble and has detergent properties itself. The alkyl group of this comonomer provides hydrophobicity while the ionic groups generate electroosmosis in the non-particulate bed. In the third approach, the alkyl comonomer was used as a detergent to dissolve another hydrophobic comonomer in an aqueous polymerization medium. All three approaches were evaluated with respect to hydrophobicity, efficiency and electroosmotic properties of the beds. Hydrophobicity expressed as methylene group selectivity for the three types of the beds in 50% methanol mobile phase was 1.86, 1.16 and 1.78, electroosmotic mobility -5.14 x 10(-5), 6.89 x 10(-5) and 6.37 x 10(-5) cm2 V(-1) s(-1) and efficiency for the retained compound (methylparabene) 67,000, 93,000 and 110,000 plates m(-1) correspondingly. The columns were tested using pressure driven capillary chromatography and capillary electrochromatography. The influence of polymerization temperature on hydrodynamic permeability, separation impedance and inverse size exclusion porosimetry characteristics were used to evaluate the separation columns. The increase of the polymerization temperature resulted higher permeability of the bed, separation impedance and lower polymeric skeleton porosity. Further characterisation was provided by examining the separation efficiency observed for a series of benzoic acid esters and alkyl parabens.     

Properties and performance of novel high-resolution/high-permeability ion-exchange media for protein chromatography

There is continued interest in the development of stationary phases for protein chromatography that can provide high resolution at elevated flow rates of the mobile phase. When using porous particles, resolution and dynamic binding capacity decline rapidly as the flow rate is increased. Monolithic columns have been developed to overcome these limitations. However, there are difficulties in manufacturing homogeneous larger scale monoliths. In this paper we investigate the morphology and performance characteristics of columns based on new ion exchangers obtained by mechanically disrupting continuous beds of acrylamido-based polymeric media. Near colloidal suspensions of loose particles obtained with this procedure can be flow-packed in ordinary chromatography columns resulting in beds of unexpectedly high hydraulic permeability. Columns up to 2.2 cm in diameter were studied with both Q and S functionalized media. The hydraulic permeability and interparticle porosity of these columns were rather high. The permeabilities of the S and Q media were 1.5 x 10(-13) and 2.4 x 10(-13) m2, respectively, while the corresponding porosities were 60 and 70%. These porosity values are similar to those of monoliths, suggesting that these particles assemble under flow to give high-porosity bridged structures. The structure of these packed beds was further characterized by embedding small packed columns in resins and obtaining sections for microscopic observation. The sections reveal the presence of small aggregates of non-porous 1-3 microm particles, surrounded by flow channels several micrometers in size. The height equivalent to a theoretical plate under isocratic and gradient elution conditions and the dynamic binding capacity were determined for several proteins and were found to be virtually independent of flow.     

Towards a microchip-based chromatographic platform. Part 1: Evaluation of sol-gel phases for capillary electrochromatography

Silica monolithic columns suitable for implementation on microchips have been evaluated by ion-exchange capillary electrochromatography. Two different silica monoliths were created from the alkyl silane, tetramethyl orthosilicate (TMOS), by introducing a water-soluble organic polymer, poly(ethylene oxide) (PEO), with varying molecular weights into the prehydrolyzed sol. Silica monoliths created using 10 kDa PEO were found to have a much more closed gel structure with a smaller percentage of pores in the microm size range than gels created using 100 kDa PEO. Additionally, the size of the mesopores in the 100 kDa PEO monolith was 5 nm, while those in the 10 kDa PEO gel were only 3 nm. This resulted in a strong dependence of the electroosmotic flow (EOF) on the ionic strength of the background electrolyte, with substantial pore flow through the nm size pores observed in the 10 kDa PEO gel. The chromatographic performance of the monolithic columns was evaluated by ion-exchange electrochromatography, with ion-exchange sites introduced via dynamic coating with the cationic polymer, poly(diallyldimethylammonium chloride) (PDDAC). Separating a mixture of inorganic anions, the 10 kDa PEO monolithic columns showed a higher effective capacity than the 100 kDa PEO column.     

微波聚合快速制备分子印迹毛细管电色谱整体柱

以甲基丙烯酸为功能单体、己二醇二甲基丙烯酸酯为交联剂、 对羟基苯甲酸为模板分子, 采用微波辐射聚合的方式快速制备了分子印迹毛细管电色谱整体柱, 并取得了较好的印迹效果. 分子印迹材料的原位制备5 min即可完成, 大大快于国内外传统的方法.