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.     

On the optimization of the shell thickness of superficially porous particles

The thickness of the porous shells of superficially porous particles influences the separation power of columns packed with these packing materials. Models of the mass transfer kinetics across porous adsorbents permit the prediction of the HETP curves of columns packed with particles having shells of different thicknesses, for molecules of different sizes. Decreasing the thickness of the porous layer potentially results in lower values of the “C-term” of the HETP curve and of the minimum of these curves. The Poppe plots calculated under isocratic and gradient conditions show that the separation power of columns packed with superficially porous particles increases significantly with decreasing thickness of the porous layer but this increase is more important for larger than for smaller molecules. The resolution between pairs of compounds increases at constant values of their retention factors when the strength of the eluent must be reduced to compensate for the decrease of their retention that is caused by the reduction of the surface area of the stationary phase. Thus, the separation power of columns packed with superficially porous particles increases with decreasing shell thickness. In contrast, if analysts do not compensate for the retention decrease, the resolution between small molecular weight compounds becomes worse with thin than with thick superficially porous particles. Finally, the importance of using instruments providing low extra-column band broadening contributions is stressed.     

Fast supercritical fluid chromatography of polymers using packed capillary columns

Summary Fast separations of perfluorinated polyethers and polymethylsiloxanes that are composed of 50–80 oligomers were demonstrated in packed capillary column supercritical fluid chromatography (SFC) using a carbon dioxide mobile phase. Separations were accomplished within 10 min using a 13 cm×250 μm i.d. column packed with 2 μm porous octadecyl bonded silica (ODS) particles. Effects of particle diameter of the packing material and pressure programming on separation were investigated, and packed column SFC was compared with open tubular column SFC. Results show that as the particle diameter was decreased from 5 to 3 to 2 μm and the column length was reduced from 85 to 43 to 13 cm, the separation time could be reduced from 70 to 20 to 10 min while still maintaining similar separation (resolution). Short columns packed with small porous particles are very suitable for fast SFC separations of polymers.     

Chiral separations in capillary high-performance liquid chromatography and nonaqueous capillary electrochromatography using helically chiral poly(diphenyl-2-pyridylmethyl methacrylate) as chiral stationary phase.

Enantioseparations in nonaqueous capillary electrochromatography (CEC) are reported in this study for the first time, using wide-pore aminopropyl silica gel coated with helically chiral poly(diphenyl-2-pyridylmethyl methacrylate) (PDPM) as chiral stationary phase (CSP). The anodic electroosmotic flow (EOF) in a methanolic solution of ammonium acetate was used for the migration of neutral analytes through the packed bed in the capillaries. Four different techniques, high-performance liquid chromatography (HPLC) in common-size columns, capillary HPLC, pressure-assisted CEC and CEC were compared from the viewpoint of separation parameters. The latter three were performed with the same experimental setup, varying the relative contribution of the pressure-driven and the electrically driven flow to the overall mobility of the analyte. Capillary HPLC offers clear advantages compared to enantioseparations in common-size columns. However, for a given particle size of the packing material, CEC was not obviously advantageous compared to pressure-driven separations.     

Kinetic plot and particle size distribution analysis to discuss the performance limits of sub-2 microm and supra-2 microm particle columns

To contribute to the current debate about the "ideal" particle size range (sub-2mum vs. supra-2mum), the present study compares the kinetic performance of some commercially available sub-2mum and 3.5mum particles used under quasi-adiabatic conditions via the kinetic plot method. Under the adopted assumption that viscous heating effects can be neglected (which is uncertain in a pressure range above 400bar), the obtained kinetic plots show that, provided each particle size is used in a column with properly optimized length, the gain in separation speed that sub-2mum particle columns might have over maximally performing 2.5mum particle columns is very small. Sub-2mum particle columns can only yield a gain in separation speed in the range of high-speed/low-resolution-separations (total time based on k=10 below 5 or 10min). And even in this range, the actual gain that can be expected is only marginally small (only a few %). The present study hence suggests that the development and the use of particles in the 2-3mum range should deserve more attention than it did in the past few years. However, to be competitive, this 2-3mum material should be packed in relatively long columns, with a packing quality matching that of the current best performing 3.5mum particle columns. The supra-2mum particles should also be able to withstand the same pressures as the sub-2mum particle material one is comparing it to.     

Ion‐exchanger synthesis using reversible addition‐fragmentation chain transfer polymerization

An ion‐exchanger with polyanionic molecular brushes was synthesized by a “grafting from” route based on “surface‐controlled reversible addition‐fragmentation chain transfer polymerization” (RAFT). The RAFT agent, PhC(S)SMgBr was covalently attached to monodisperse‐porous poly(dihydroxypropyl methacrylate‐co‐ethylene dimethacrylate), poly(DHPM‐co‐EDM) particles 5.8 μm in size. The monomer, 3‐sulfopropyl methacrylate (SPM), was grafted from the surface of poly(DHPM‐co‐EDM) particles with an immobilized chain transfer agent by the proposed RAFT protocol. The degree of polymerization of SPM (i. e. the molecular length of the polyanionic ligand) on the particles was controlled by varying the molar ratio of monomer/RAFT agent. The particles carrying polyanionic molecular brushes with different lengths were tested as packing material in the separation of proteins by ion exchange chromatography. The columns packed with the particles carrying relatively longer polyanionic ligands exhibited higher separation efficiency in the separation of four proteins. Plate heights between 130–200 μm were obtained. The ion‐exchanger having poly‐(SPM) ligand with lower degree of polymerization provided better peak‐resolutions on applying a salt gradient with higher slope. The molecular length and the ion‐exchanger group content of polyionic ligand were adjusted by controlling the degree of polymerization and the grafting density, respectively. This property allowed control of the separation performance of the ion‐exchanger packing.     

A drying step in the protocol to pack capillary columns by centripetal forces for capillary electrochromatography.

Capillary columns have been packed for capillary electrochromatography (CEC) using centripetal forces. The packed columns were maintained under wet conditions or they were dried with nitrogen gas prior to forming the retaining frits. Upon fabrication of the retaining frits, the dried columns were resolvated with the mobile phase. The performance of the columns was evaluated to determine the effect of the drying step during the packing procedure. The columns submitted to the drying step showed improved separation efficiencies and stronger retention characteristics than those kept under wet conditions. The drying step allows the silica-based packing material to be better accommodated inside the capillary column. Upon solvation, the packing material "swells," resulting in a greater packing density, which allows for a stronger retention and improved separation efficiencies. The drying step led to a 13% increase in retention on columns packed with isopropanol. An increase of 15-20% in theoretical plates for the most retained compounds was also observed in such columns.     

Packing capilllary electrochromatography columns using vacuum--a preliminary study

This paper introducesa novel method for packing Capillary Electrochromatography Columns (CEC). Using vacuum packing methodology, silica particles as small as 1 microm were successfully packed into the capillary columns with 75 microm inner diameter. The columns are verystable and show no noticeable loss in efficiency after 200 sample injections. The performance of these vacuum packed capillary columns was evaluated with a mixture of aromatic and non-aromatic compounds. A 24 cm long capillary column can produce peak efficiencies of around 45,000 plates for benzene.     

Crystalline degradation products of vancomycin as chiral stationary phase in microcolumn liquid chromatography

The ability of crystalline degradation products (CDPs) of vancomycin as a chiral stationary phase was reported in a previous study for enantioselective separation of drugs, amino acids and agrochemical toxins by conventional LC column (250 x 4.6 mm). In this work, the potential of CDP of vancomycin for the enantiomeric separation in micro-LC (200 x 1 mm) has been studied. The obtained separation results are better than in our previous study with conventional LC columns. The enantiomers of D,L-phenylalanine, D,L-alanine, methyldopa, atropine and propranolol were used for this evaluation. Experiments have been carried out in a stainless steel tube that was packed with chiral silica particles of 3 and 12 microm diameters. Also, three different ratios of 3 and 12 microm silica particles were used for packing material of chiral columns and the effect on aspect ratio and resolving powers was compared.     

Practical comparison of LC columns packed with different superficially porous particles for the separation of small molecules and medium size natural products

Commercial C(18) columns packed with superficially porous particles of different sizes and shell thicknesses (Ascentis Express, Kinetex, and Poroshell 120) or sub-2-μm totally porous particles (Acquity BEH) were systematically compared using a small molecule mixture and a complex natural product mixture as text probes. Significant efficiency loss was observed on 2.1-mm id columns even with a low dispersion ultra-high pressure liquid chromatography system. The Kinetex 4.6-mm id column packed with 2.6-μm particles exhibited the best overall efficiency for small molecule separations and the Poroshell 120 column showed better performance for mid-size natural product analytes. The Kinetex 2.1-mm id column packed with 1.7-μm particles did not deliver the expected performance and the possible reasons besides extra column effect have been proved to be frictional heating effect and poor column packing quality. Different column retentivities and selectivities have been observed on the four C(18) columns of different brands for the natural product separation. Column batch-to-batch variability that has been previously observed on the Ascentis Express column was also observed on the Kinetex and Poroshell 120 column.     

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.     

Hydrodynamic chromatography for the size classification of micron and sub-micron sized packing materials

Hydrodynamic chromatography (HDC) was used as a size classification and purification method for porous bridged ethyl hybrid (BEH) packing materials (particles) in the micron to sub-micron range. Using packed column HDC, a batch of particles with size 0.76 ± 0.26 μm was fractionated to yield classified material of 1.05 ± 0.16 μm, reducing the relative standard deviation from 33% to 15%. Subsequent chromatographic evaluation of this packing material showed significant improvement in column performance and decrease in flow resistance over the unclassified material. Comparing a column packed with the classified versus non-classified material, the effective flow resistance of the two columns was decreased by 58% and the minimum HETP for the packing material was improved from 4 to 2.5 μm.     

Packed-column hydrodynamic chromatography using 1-μm non-porous silica particles

150×3 mm I.D. columns, packed with 1-μm non-porous spherical silica particles, were used to separate soluble synthetic polymers by hydrodynamic chromatography. The columns exhibited a plate height of about 1.4 μm allowing very fast and efficient separations of polymers in the molecular mass range 10³−2·10⁶ g/mol. The migration behaviour of polymers could be well described by a simple theoretical model. The applicability of packed bed HDC for the fast separation of polymers was illustrated with separations of polystyrene and poly(methyl methacrylate) mixtures.     

Use of high-performance size-exclusion chromatography for the separation of poliovirus and subviral particles

The size-dependent separation of viral and subviral particles in the range 10(5)-10(7) daltons was undertaken by high-performance liquid chromatography. A combination of Ultrahydrogel 2000 and 1000 size-exclusion columns, equilibrated and developed with Tris buffer (pH 7.4), was used to fractionate extracts of cells infected with radiolabelled poliovirus. Poliovirions (30 nm) and subviral particles (20 nm) were separated according to size with full retention to their biological activities. Procapsids (same size as virions, but devoid of RNA) could not be separated from virions. Sample recoveries as determined with radiolabelled material constantly exceeded 70%. The method was successfully applied to the separation of viral and subviral particles from complex mixtures.     

Packing columns for capillary electrochromatography

Considering the current interest in capillary electrochromatography (CEC), performed in packed columns, we present the different methods used to pack capillary columns for use in CEC. General considerations on column packing are given and the column fabrication process is discussed in sufficient detail to allow instruction to those who are not experienced in the field. Five different packing methods are discussed to deliver packing material into the capillary column from a practical view point: slurry pressure packing, packing with supercritical CO2, electrokinetic packing, using centripetal forces, and packing by gravity. Entrapment of particulate material by sintering and sol-gel technology is also mentioned. Although slurry pressure packing procedures are most common, higher separation efficiencies are obtained using other packing approaches. Electrokinetic packing seems to be the simplest technique to deliver the packing material into the capillary columns. Nevertheless, as with the other packing techniques, skill and experience are required to complete all the steps involved in the fabrication of packed columns for CEC.     

Hydroxyl Functionalized Uniform‐Porous Beads,Synthesis and Chromatographic Use

Uniform‐porous poly(dihydroxypropyl methacrylate‐co‐ethylene dimethacrylate), poly(DHPM‐co‐EDM) particles were synthesized as an alternative packing material for reversed phase chromatography. In the synthesis, poly(glycidyl methacrylate‐ethylene dimethacrylate), poly(GMA‐co‐EDM) particles were obtained by a multi‐stage swelling and polymerization protocol, the so called “modified seeded polymerization”. For this purpose, 2.4 µm polystyrene seed particles were first swollen by dibutyl phthalate (DBP) and then by a monomer mixture including glycidyl methacrylate and ethylene dimethacrylate. The repolymerization of monomer phase in the swollen seed particles provided porous uniform particles approximately 7 µm in size. Poly(DHPM‐co‐EDM) particles were obtained by the acid hydrolysis of the particles synthesized with different GMA feed concentrations. These particles were used as column‐packing material in the reversed phase separation of alkylbenzenes. The retention factor‐acetonitrile concentration diagrams clearly showed that the polarity of packing material could be controlled by changing the GMA feed concentration in the “modified seeded polymerization”. The packing materials with more hydrophobic character (i.e., poly(EDM) and poly(DHPM‐co‐EDM) particles produced with the GMA feed concentrations up to 20%) exhibited better chromatographic performance in the reversed phase mode.     

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.     

Affinity chromatography of proteins on non-porous copolymerized particles of styrene, methyl methacrylate and glycidyl methacrylate.

Non-porous particles having an average diameter of 2.1 microm were prepared by co-polymerization of styrene, methyl methacrylate and glycidyl methacrylate, which was abbreviated as P(S-MMA-GMA). The particles were mechanically stable due to the presence of benzene rings in the backbone of polymer chains, and could withstand high pressures when a column packed with these particles was operated in the HPLC mode. The polymer particles were advantaged by immobilization of ligands via the epoxy groups on the particle surface that were introduced by one of the monomers, glycidyl methacrylate. As a model system, Cibacron Blue 3G-A was covalently immobilized onto the non-porous copolymer beads. The dye-immobilized P(S-MMA-GMA) particles were slurry packed into a 1.0 cm x 0.46 cm I.D. column. This affinity column was effective for the separation of turkey egg white lysozyme from a protein mixture. The bound lysozyme could be eluted to yield a sharp peak by using a phosphate buffer containing 1 M NaCl. For a sample containing up to 8 microg of lysozyme, the retained portion of proteins could be completely eluted without any slit peak. Due to the use of a shorter column, the analysis time was shorter in comparison with other affinity systems reported in the literature. The retention time could be reduced significantly by increasing the flow-rate, while the capacity factor remained at the same level.     

Packed-column hydrodynamic chromatography using 1-μm non-porous silica particles

150×3 mm I.D. columns, packed with 1-μm non-porous spherical silica particles, were used to separate soluble synthetic polymers by hydrodynamic chromatography. The columns exhibited a plate height of about 1.4 μm allowing very fast and efficient separations of polymers in the molecular mass range 10³−2·10⁶ g/mol. The migration behaviour of polymers could be well described by a simple theoretical model. The applicability of packed bed HDC for the fast separation of polymers was illustrated with separations of polystyrene and poly(methyl methacrylate) mixtures.     

Ultrahigh-pressure liquid chromatography using a 1-mm id column packed with 1.5-microm porous particles

The evolution of chromatography has led to the reduction in the size of the packing materials used to fabricate HPLC columns. The increase in the backpressure required has led to this technique being referred to as ultrahigh-pressure liquid chromatography (UHPLC) when the column backpressure exceeds 10000 psi (approximately 700 bar). Until recently, columns packed with sub-2-microm materials have generally fitted into two classes; either short (less than 5 cm) columns designed for use on traditional HPLC systems at pressures less than 5000 psi (350 bar), or capillary columns (inner diameters less than 100 microm). By using packing materials with diameters <2 microm to fabricate UHPLC columns, there is an increase in efficiency and a decrease in the analysis time that are directly proportional to the size of the packing material. In order to realize and exploit the increase in efficiency, however, the columns must maintain lengths typically associated with analytical columns (15-25 cm). We have packed 1 mm diameter, 150 mm in length columns with 1.5 microm packing material, and evaluated their performance in UHPLC. The pressure required to achieve optimum linear velocities in plots of plate height versus linear velocity was in the vicinity of 1104 bar (16000 psi). The 1.5 microm particle-packed column was compared with the more traditional 150 mm long analytical columns packed with 3 microm materials. This column showed an efficiency that was approximately twice that observed with the 3 microm packed column and a concomitant reduction in the analysis time, theoretically predicted.