Comparison of the efficiency of microparticulate and monolithic capillary columns

A comparison is made between the efficiency of microparticulate capillary columns and silica and polymer-based monolithic capillary columns in the pressure-driven (high-performance liquid chromatography) and electro-driven (capillary electrochromatography) modes. With packed capillary columns similar plate heights are possible as with conventional packed columns. However, a large variation is observed in the plate heights for individual columns. This can only be explained by differences in the quality of the packed bed. The minimum plate height obtained with silica monolithic capillary columns in the HPLC mode is approximately 10 microm, which is comparable to that of columns packed with 5-microm particles. The permeability of wide-pore silica monoliths was found to be much higher than that of comparable microparticulate columns, which leads to much lower pressure drops for the same eluent at the same linear mobile phase velocity. For polymer-based monolithic columns (acrylamide, styrene/divinyl benzene, methacrylate, acrylate) high efficiencies have been found in the CEC mode with minimum plate heights between 2 and 10 microm. However, in the HPLC mode minimum plate heights in the range of 10 to 25 microm have been reported.     

Electrochromatography in packed tubes using 1.5 to 50 μm silica gels and ODS bonded silica gels

Summary Electrochromatography (that is HPLC where the eluent is driven along the column by electro-osmosis using fields of up to 100 kV m⁻¹) promises plate efficiencies for HPLC which are comparable to those attained in capillary gas chromatography, but this requires that narrow-bore columns can be successfully packed with submicron particles. This paper demonstrates that we have now moved a considerable distance towards this goal. We show (1) that, following theory, there is no evidence of any reduction in electroosmotic velocity in columns packed with particles down to 1.5 μm diameter, (2) that reduced plate heights as low as unity are attainable for unretained solutes using both slurrypacked and drawn-packed columns 30 to 200 μm bore and up to 1 m long when packed with conventional 3 and 5 μm silica gels or with 1.5 μm impermeable silica spheres, (3) that columns driven electrically show higher plate efficiencies than identical columns driven by pressure, and (4) that 100,000 plate HPLC separations can be achieved in relatively short times of 30 minutes using in situ derivatised drawn packed capillaries containing 3 and 5 μm ODS-silica gels.     

Silica particles encapsulated poly(styrene-divinylbenzene) monolithic stationary phases for micro-high performance liquid chromatography

In the paper we demonstrate a new approach for the preparation and application of continuous silica bed columns that involve encapsulation (entrapment) of functionalized silica microparticles, which can be used as packing material in micro high performance liquid chromatography (micro-HPLC) and capillary electrochromatography (CEC). Like traditional packed columns, these capillaries possess characterized silica particles that offer high phase ratio and narrow pore size distribution leading to high retention and separation efficiency, respectively. More importantly, immobilization of the microparticles stabilizes the separation bed and eliminates the need for retaining frits. The developed capillary columns were fabricated in exactly the same way as a packed capillary column (slurry packing) but with an additional entrapment step. This immobilization of the packed bed was achieved by in situ polymerization of styrene and divinylbenzene in presence of decanol as a porogen and azobisisobutyronitrile as thermal initiator. Silica particles with different particle sizes and pore sizes ranging from 60 to 4000 A were studied. In addition different modified silica was used, including C-18 reversed phase, anion exchange and chiral stationary phases. Efficient separation of polyphenolic compounds, peptides, proteins and even DNA mutation were achieved using the developed technique depending on the properties of the silica particles used (particles pore size). For example, using 3 microm ProntoSIL C-18 particles with 300 A pore size, separation efficiencies in the range of 120,000-200,000 plates/m were obtained for protein separation, in a 6 cm x 200 microm i.d. capillary column. Using encapsulated silica C-18 with 1000 A pore size, separation of DNA homo and hetero duplexes were achieved under denaturing HPLC conditions for mutation detection. In addition, nucleotides were separated using anion exchange material encapsulated with poly(styrene-divinylbenzene) (PS/DVB), which indicated that the chromatographic properties of the silica packing material were still active after polymerization. The prepared capillary columns were found to be stable and could easily be operated continuously up to a pressure of 350 bar without column damage and capillary can be cut to any desired length.     

Physical properties and structure of fine core–shell particles used as packing materials for chromatography: Relationships between particle characteristics and column performance

The recent development of new brands of packing materials made of fine porous-shell particles, e.g., Halo and Kinetex, has brought great improvements in potential column efficiency, demanding considerable progress in the design of chromatographic instruments. Columns packed with Halo and Kinetex particles provide minimum values of their reduced plate heights of nearly 1.5 and 1.2, respectively. These packing materials have physical properties that set them apart from conventional porous particles. The kinetic performance of 4.6 mm I.D. columns packed with these two new materials is analyzed based on the results of a series of nine independent and complementary experiments: low-temperature nitrogen adsorption (LTNA), scanning electron microscopy (SEM), inverse size-exclusion chromatography (ISEC), Coulter counter particle size distributions, pycnometry, height equivalent to a theoretical plate (HETP), peak parking method (PP), total pore blocking method (TPB), and local electrochemical detection across the column exit section (LED). The results of this work establish links between the physical properties of these superficially porous particles and the excellent kinetic performance of columns packed with them. It clarifies the fundamental origin of the difference in the chromatographic performances of the Halo and the Kinetex columns.     

Evaluation of ultra performance liquid chromatography. Part I. Possibilities and limitations

A practical evaluation of the possibilities and limitations of ultra performance liquid chromatography (UPLC) is presented. Acquity BEH columns packed with 1.7 microm particles are evaluated by means of van Deemter and Knox plots. The columns are characterised by high optimal velocities (3.7 mm/s) and low plate heights (4.4 microm). Minimum plate heights of 2d(p) were, however, not reached and reasons are presented and discussed. Furthermore, the use of 1.7 microm particles at 1000 bar is compared, from a theoretical viewpoint, to conventional LC (3.5 and 5 microm particles at 400 bar) in terms of analysis speed and practical maximum efficiency. Experimental data are used to construct kinetic- or "Poppe-plots", which facilitate investigation of the effect of pressure and particle size on speed and efficiency. It is found that UPLC conditions hold advantages in terms of speed of analysis, for required theoretical plate counts up to approximately 80,000.     

Enantiomer separations by capillary electrochromatography using chiral stationary phases

The applicability of capillary electrochromatography (CEC) using packed capillary column to enantiomer separations was investigated. As chiral stationary phases, OD type packing materials of 5 and 3 microm particle diameters, originally designed for conventional high-performance liquid chromatography (HPLC) were employed. The chiral packing materials were packed by a pressurized method into a 100 microm I.D. fused-silica capillary. Several racemic enantiomers, such as acidic, neutral and basic drug components, were successfully resolved, typically by using acidic or basic solutions containing acetonitrile as mobile phases. The separation efficiencies for some enantiomers in the chiral CEC system using the 5 microm OD type packing were superior to those obtained in HPLC using chiral packings. The plate heights obtained for several enantiomers were 8-13 microm or the reduced plate height of 1.6-2.6, which indicates the high efficiency of this chiral CEC system.     

Review. Use of laser detectors in capillary liquid chromatography

The main relationship of high-performance liquid chromatography (HPLC) are considered. It is shown that the optimum conditions of ultrasensitive trace analysis should be achieved by using packed capillary columns manufactured from flexible quartz capillaries with dc approximately less than 0.2 mm. The main features of these columns (v opt = 0.6 v opt of that for conventional HPLC columns with double the hydraulic permeability) make it possible to obtain two or three times higher plate numbers for the same analysis time and column pressure characteristic of conventional HPLC, as a result of using a submicrometre sorbent. The main features of laser detection in capillary liquid chromatography (laser-induced fluorescence and cross-beam thermal lens absorption detectors) are considered. The requirements that should be met by a modern capillary liquid chromatograph based on using flexible quartz capillary columns with a submicrometre sorbent and laser detectors are formulated. Examples of using these systems for femtomole and attomole analyses of biological samples (amino acids and prostaglandins) are given.     

Study of the influence of the aspect ratio on efficiency, flow resistance and retention factors of packed capillary columns in pressure- and electrically-driven liquid chromatography

The influence of the aspect ratio, rho (rho = column diameter/particle diameter), on column parameters such as efficiency, retention factors and flow resistance was studied in both high-performance liquid chromatography and capillary electrochromatography with packed capillary columns. In order to compare the true efficiencies of different columns, a procedure to account for external band broadening was applied. High efficiencies (reduced plate height h approximately 2) were obtained with capillary columns with internal diameters of 150-, 100-, and 75-microm, packed with 10-microm particles. In contrast to previous reports in the literature, no significant improvements in efficiency or flow resistance were observed when the aspect ratio of such columns was decreased. Our observations suggest that the wall effect in these types of columns is not significant. When the aspect ratio was decreased by increasing the particle size, a decrease in reduced plate height was observed. However, the results of flow resistance measurements showed that the latter effect should be attributed to differences in packing and particle batch quality rather than to differences in the aspect ratio.     

Characterization of the efficiency of microbore liquid chromatography columns by van Deemter and kinetic plot analysis

The efficiency of miniaturized liquid chromatography columns with inner diameters between 200 and 300 μm has been investigated using a dedicated micro‐liquid chromatography system. Fully porous, core–shell and monolithic commercially available stationary phases were compared applying van Deemter and kinetic plot analysis. The sub‐2 μm fully porous as well as the 2.7 μm core–shell particle packed columns showed superior efficiency and similar values for the minimum reduced plate heights (2.56–2.69) before correction for extra‐column contribution compared to normal‐bore columns. Moreover, the influence of extra‐column contribution was investigated to demonstrate the difference between apparent and intrinsic efficiency by replacing the column by a zero dead volume union to determine the band spreading caused by the system. It was demonstrated that 72% of the intrinsic efficiency could be reached. The results of the kinetic plot analysis indicate the superior performance of the sub‐2 μm fully porous particle packed column for ultra‐fast liquid chromatography.     

Magnetically immobilized frits for the preparation of packed columns used in capillary electrochromatography

Fabrication of porous frits to retain stationary phases is a critical issue in column preparation for capillary electrochromatography (CEC). In this work, porous frits were prepared by applying an external magnetic field to magnetically responsive particles placed inside a fused-silica capillary. Three batches of uniform magnetite spheres with particle diameters of 0.3, 0.4, and 0.6 μm and saturation magnetization values of 73.03, 74.41, and 77.83 emu/g, respectively, were used as frit particles and octadecyl- and phenyl-bonded silica gels were packed successfully into frit-containing capillaries. The performance of the resulting magnetically immobilized frits and packed columns was evaluated. The electroosmotic mobilities in capillaries containing outlet frit only were found to be reduced by 2–4% whereas the plate heights of an unretained marker increased by 30–50% as compared to those in open capillaries. These variations are believed to be associated with the inhomogeneities of the packed structure of the frits. The magnetically immobilized frits showed adequate mechanical strength to withstand the flow drag force, allowing separation in capillaries packed with 5-μm stationary phases up to 10–15 cm, thus rendering column efficiency and reproducibility comparable with those obtained with sintered frits. Taken together, retaining frits made of uniform magnetite particles serves as a viable alternative to sintered frits for column preparation, which offers several distinct advantages such as ease of preparation, improved durability as compared to sintered frits where the removal of the polyimide coating makes the packed column susceptible to breakage, and use of large-bore capillaries for semipreparative separations.     

Entrapment of functionalized silica microspheres with photo-initiated acrylate-based polymers

The entrapment of silica-based microspheres, commonly used as stationary phases in chromatography, with an organic porous polymer based on poly(butyl acrylate-co-1,3-butanediol diacrylate) was explored. The spheres were immobilized by photopolymerization leading to entrapped beds within 75 microm i.d. fused silica capillaries, and were mechanically stable, resisting pressure drops of over 5600 psi (38.6 MPa) for only 1 cm of material. The morphology of the polymer formation around the spheres was investigated by SEM and corroborated with back pressure measurements, which indicated that the spheres were held together by encapsulating polymer. The entrapped material was extruded from the capillary in some cases to facilitate imaging. The entrapment conditions were explored, varying the polarity of the sphere surface, the solvent, and the monomers, revealing that polymer formation is based on partitioning of the monomers between the surface and solvent. The resulting polymer morphology is discussed with respect to the effects of confinement, supported by experiments with varying microsphere diameters. The columns described here have favourable properties for use in capillary chromatography and supported catalysis among other applications, and is suitable for lab-on-a-chip devices.     

Efficiency of methacrylate monolithic columns in reversed-phase liquid chromatographic separations

Butyl-methacrylate-based porous monoliths were prepared inside fused-silica capillaries as reversed-phase separation media for liquid chromatography (LC) and capillary electrochromatography (CEC). During our previous research on methacrylate-based monoliths for reversed-phase separations, we noticed that a separation efficiency of up to 300,000 plates/m can easily be obtained in the CEC mode for unretained compounds. However, the efficiencies for retained compounds were much lower in reversed-phase systems, especially in pressure-driven LC. In this work methacrylate-based columns were prepared and characterized in terms of efficiency and retention in reversed-phase (pressure-driven) LC and in CEC. Much attention has been paid to the mass-transfer mechanism in the stationary phase. Factors that affect the plate heights for specific compounds have been investigated. A possible explanation for the relatively low separation efficiency of retained compounds and suggestions to improve molecular mass transfer are provided.     

Silicate entrapped columns--new columns designed for capillary electrochromatography

Designed especially for capillary electrochromatography (CEC), silicate-entrapped columns are made by trapping particles of chromatographic packing material in a network of silica. Once entrapped, the capillary no longer requires frits. This renders a more homogeneous and stable packed bed. Accidental breakage of the fragile frits is not an issue with these robust columns. Columns packed with reverse-phase material subjected to silicate entrapment demonstrated faster separations of retained analytes and increased efficiencies compared with nonentrapped columns. The method was also used to prepare chiral CEC columns by entrapping a molecular imprinted polymeric (MIP) packing having minimal surface charge density, thus being unable alone to support sufficient electroosmotic flow for CEC.     

加芯毛细管填充柱的理论研究

 从速率方程和柱压降两个方面对加芯毛细管填充柱的柱效和渗透性进行了探讨，并详细讨论了柱直径、芯直径、芯根数和柱压降的关系。综合考虑柱压降、板高和拉制柱子时颗粒在柱内的镶嵌状况可知最佳柱型是３芯的毛细管填充柱。     

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.     

High-speed gas chromatography: an overview of various concepts.

An overview is given of existing methods to minimise the analysis time in gas chromatography (GC) being the subject of many publications in the scientific literature. Packed and (multi-) capillary columns are compared with respect to their deployment in fast GC. It is assumed that the contribution of the stationary phase to peak broadening can be neglected (low liquid phase loading and thin film columns, respectively). The treatment is based on the minimisation of the analysis time required on both column types for the resolution of a critical pair of solutes (resolution normalised conditions). Theoretical relationships are given, describing analysis time and the related pressure drop. The equations are expressed in reduced parameters, making a comparison of column types considerably simpler than with the conventional equations. Reduction of the characteristic diameter, being the inside column diameter for open tubular columns and the particle size for packed columns, is the best approach to increase the separation speed in gas chromatography. Extremely fast analysis is only possible when the required number of plates to separate a critical pair of solutes is relatively low. Reducing the analysis time by reduction of the characteristic diameter is accompanied by a proportionally higher required inlet pressure. Due to the high resistance of flow of packed columns this seriously limits the use of packed columns for fast GC. For fast GC hydrogen has to be used as carrier gas and in some situations vacuum-outlet operation of capillary columns allows a further minimisation of the analysis time. For fast GC the columns should be operated near the conditions for minimum plate height. Linear temperature programmed fast GC requires high column temperature programming rates. Reduction of the characteristic diameter affects the sample capacity of the "fast columns". This effect is very pronounced for narrow-bore columns and in principle non-existing in packed columns. Multi-capillary columns (a parallel configuration of some 900 narrow-bore capillaries) take an intermediate position.     

Intraparticle Flow and Plate Height Effects in Liquid Chromatography Stationary Phases

Abstract

Velocity independent plate heights were apparently first recognized for hydrodynamic chromatography columns, packed with nonporous, 115 micron glass beads which were run at reduced mobile phase velocities of 10 to 10,000. Hydrodynamic chromatography separates based on the tendency of small molecules (or particles) to associate with slower moving fluid streamlines near the surfaces of particles, compared to larger molecules which seek faster streamlines. Consequently, the larger molecules elute first. Velocity independent plate heights in liquid chromatography have also been observed for nonadsorbed solutes in paniculate and fibrous stationary phases. These stationary phases have pores which exceed 10^?4 to 10^?5 cm in dimension. The flat plate height is attributed to flow in the channels formed by these large intraparticle spaces. The development of plate height expressions which represent dispersion at interstitial velocities above 10 cm/min are discussed. Explanations of the uncoupling of dispersion from eluent flow rate in continuous stationary phases, membranes, and gigaporous particles is shown to have their origins in the studies of distribution of particles and molecules in hydrodynamic chromatography columns, and to be adequately described by modifications of the van Deemter equation.     

Monolithic silica columns with various skeleton sizes and through-pore sizes for capillary liquid chromatography

Reduction of through-pore size and skeleton size of a monolithic silica column was attempted to provide high separation efficiency in a short time. Monolithic silica columns were prepared to have various sizes of skeletons (approximately 1-2 microm) and through-pores (approximately 2-8 microm) in a fused-silica capillary (50-200 microm I.D.). The columns were evaluated in HPLC after derivatization to C18 phase. It was possible to prepare monolithic silica structures in capillaries of up to 200 microm I.D. from a mixture of tetramethoxysilane and methyltrimethoxysilane. As expected, a monolithic silica column with smaller domain size showed higher column efficiency and higher pressure drop. High external porosity (> 80%) and large through-pores resulted in high permeability (K = 8 x 10(-14) -1.3 x 10(-12) m2) that was 2-30 times higher than that of a column packed with 5-mirom silica particles. The monolithic silica columns prepared in capillaries produced a plate height of about 8-12 microm with an 80% aqueous acetonitrile mobile phase at a linear velocity of 1 mm/s. Separation impedance, E, was found to be as low as 100 under optimum conditions, a value about an order of magnitude lower than reported for conventional columns packed with 5-microm particles. Although a column with smaller domain size generally resulted in higher separation impedance and the lower total performance, the monolithic silica columns showed performance beyond the limit of conventional particle-packed columns under pressure-driven conditions.     

Electrically driven capillary size exclusion chromatography

Summary Electrically driven size exclusion chromatography (ED-SEC) of polystyrenes in packed capillaries using dimethylformamide as solvent is demonstrated. The efficiency and retention behaviour of polystyrenes under pressure and electro drive were investigated. Under pressure drive the plate height (H) increases steadily with increasing linear velocity (u) whereas under electro drive the H-u curves largely coincide and are very flat. At higher velocities the plate heights are about 50% smaller with electro drive than with pressure drive. Calculations show that with increasing ionic strength, the flow through the particles may increase causing a clear diminishing of the elution window.     

Stabilizing structure-switching signaling RNA aptamers by entrapment in sol-gel derived materials for solid-phase assays

Structure-switching, fluorescence-signaling DNA and RNA aptamers have been reported as highly versatile molecular recognition elements for biosensor development. While structure-switching DNA aptamers have been utilized for solid-phase sensing, equivalent RNA aptamers have yet to be successfully utilized in solid-phase sensors due to their lack of chemical stability and susceptibility to nuclease attack. In this study, we examined entrapment into sol-gel derived organic-inorganic composite materials as a platform for immobilization of structure-switching fluorescence-signaling RNA aptamer reporters, using both the synthetic theophylline- and naturally occurring thiamine pyrophosphate-binding RNA aptamers as test cases. Structure-switching versions of both aptamers were entrapped into a series of sol-gel derived composites, ranging from highly polar silica to hydrophobic methylsilsesquioxane-based materials, and the target-binding and signaling capabilities of these immobilized aptamers were assessed relative to solution. Both immobilized aptamers demonstrated sensitivity and selectivity similar to that of free aptamers when entrapped in a composite material derived from 40% (v/v) methyltrimethoxysilane/tetramethoxysilane. Importantly, this material also conferred protection from nuclease degradation and imparted long-term chemical stability to the RNA reporter systems. Given the versatility of sol-gel entrapment for development of biosensors, microarrays, bioaffinity columns, and other devices, this entrapment method should provide a useful platform for numerous solid-phase RNA aptamer-based devices.