Large Through-pores Research Articles

On-line enrichment of N-glycans by immobilized metal-affinity monolith for capillary electrophoresis analysis

A novel N-glycan enrichment strategy is presented using unexpected but strong interactions between the sulfonate groups brought by the fluorescent dye of glycans and the Zr4+ modified poly(ethylene glycol methacrylate phosphate (EGMP)-co-acrylamide (AM)-co-bis-acrylamide (BAA)) monolith. The poly (EGMP-co-AM-co-BAA) monolith was synthesized via ultraviolet (UV) irradiation and then functionalized with Zr4+. The obtained monolith was characterized with scanning electron microscopy and mercury intrusion porosimetry. Large through-pores and a continuous skeleton with high permeability were observed. The N-glycans were labeled with the 1-aminopyrene-3, 6, 8-trisulfonic acid (APTS) and enriched by the Zr4+ modified monolith through IMAC interaction. This enrichment step was then coupled off-line to capillary electrophoresis (CE) separation with laser induced fluorescence (LIF) detection. Successful preconcentration of the APTS labeled maltooligosaccharide ladder was achieved under optimized conditions. Enrichment factors obtained for the maltooligosaccharides ranged from 9 to 24 with RSDs from 2.0% to 9.2% (n = 3). Moreover, very good repeatabilities (<6.7%) were obtained for glucose oligomers (4–15 glucose units) corresponding to sizes expected for N-glycans, demonstrating the great potential of this Zr4+ modified monolith to enrich APTS labeled glycans from N-glycoproteins. The proposed method was then successfully applied for the enrichment of N-glycans released from Ribonuclease B, in which case all five expected oligomannose glycans (Man 5 to Man 9) were successfully enriched. Thanks to the advantage of the method to enrich selectively APTS-glycans compared to the commercial SPE columns composed of HILIC or PGC materials, the first proof of concept of on-line enrichment coupled to CE-LIF separation was demonstrated for maltooligosaccharides as well.

One-pot preparation of zwitterionic sulfoalkylbetaine monolith for rapid and efficient separation of lysozyme from egg white

A porous zwitterionic monolithic column was prepared to rapidly and efficiently separate lysozyme from egg white. The monolith was synthesized in a stainless steel HPLC column (5 cm × 4.6 mm i.d.) by in-situ thermal initiated co-polymerization of N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl) ammonium betaine (MSA) and ethylene dimethacrylate (EDMA). Due to the combination of quaternary ammonium and sulfonic groups on the monolithic matrix in one-pot process, the hydrophobic carbon chain and hydrophilic radical were obtained, which provided multiple driving forces for neutral, basic and acidic analytes, thus mix-mode chromatography mechanism contributed to the retention of different charged proteins. Properties such as composition, morphology and stability of the MSA-co-EDMA monolithic column were characterized by various analytical methods and the results showed that the monolith has large through-pores, good hydrophilicity and permeability. The effects of mobile phase pH and ionic strength on proteins were investigated, drawing the conclusion that the main adsorption and elution mechanism of lysozyme on MSA-co-EDMA monolith was electrostatic interaction, while those of other proteins included hydrophobic, hydrophilic and electrostatic interactions. Therefore, efficient separation of lysozyme and other proteins could be successfully achieved by switching the pH of mobile phase. Lysozyme can be adsorbed using 20 mmol/L phosphate buffer (pH 7.0) and eluted with 20 mmol/L phosphate buffer (pH 2.0). To prove the practicality of the monolithic column, it was also applied in the separation of lysozyme in egg white, which means the work has the potential for further development in proteome analysis of real biological samples.

Preparation of molecularly imprinted hybrid monoliths for the selective detection of fluoroquinolones in infant formula powders

A novel molecularly imprinted inorganic-organic hybrid monolith (MIP hybrid monolith) was fabricated through a facile single-step polymerization strategy with levofloxacin (LEV) as the template, 3-aminopropyltriethoxysilane-methacrylic acid as the hybrid functional monomer and ethylene glycol dimethacrylate as the crosslinker in a mixed porogen of methanol, toluene and dodecanol. The optimized LEV-MIP hybrid monolith was characterized using scanning electron microscopy and fourier transform-infrared spectroscopy. Uniform monolithic matrix with large through-pores in the network skeleton of LEV-MIP hybrid monolith was observed. The influence of polymerization conditions on the specific recognition behavior of the resulting monolith was systematically investigated. The LEV-MIP hybrid monolith exhibited much better adsorption (3.62 times) and selectivity towards LEV in comparison with non-imprinted hybrid monolith. Furthermore, the LEV-MIP hybrid monolith based solid-phase extraction combining with liquid chromatography-mass spectrometry was applied for the selective determination of fluoroquinolones (FQs) in infant formula powder. The average recoveries of six FQs in milk powders spiked at 20, 50 and 100 μg kg−1 were in the range of 82.91–102.00% with the precision of 1.04–7.39%. The limit of detection and limit of quantitation of the proposed method were in a range of 0.19–1.24 μg kg−1 and 0.63–4.13 μg kg−1, respectively.

Preparation and evaluation of open tubular C18-silica monolithic microcartridges for preconcentration of peptides by on-line solid phase extraction capillary electrophoresis

In this study, C18-silica monoliths were synthesized as a porous layer in open tubular capillary columns, to be cut later into microcartridges for the analysis of neuropeptides by on-line solid-phase extraction capillary electrophoresis with UV and MS detection (SPE-CE-UV and SPE-CE-MS). First, several types of C18-silica monolithic (MtC18) microcartridges were used to analyse standard solutions of five neuropeptides (i.e. dynorphin A (1–7), substance P (7–11), endomorphin 1, methionine enkephalin and [Ala]-methionine enkephalin). The MtC18 sorbents were especially selective against endomorphin 1 and substance P (7–11)). The best results in terms of sensitivity and inter-microcartridge reproducibility were achieved with the microcartridges obtained from a 10-cm open tubular capillary column with a thin monolithic coating with large through-pores (1–5μm). Run-to-run repeatability, microcartridge durability, linearity ranges and LODs were studied by MtC18-SPE-CE-MS. As expected due to their greater selectivity, the best LOD enhancement was obtained for End1 and SP (7–11) (50 times with regard to CE-MS). Finally, the suitability of the methodology for analysing biological fluids was tested with plasma samples spiked with End1 and SP (7–11). Results obtained were promising because both neuropeptides could be detected at 0.05μgmL−1, which was almost the same concentration level as for the standard solutions (0.01μgmL−1).

One-pot preparation of a molecularly imprinted hybrid monolithic capillary column for selective recognition and capture of lysozyme

A molecularly imprinted inorganic–organic hybrid monolithic capillary column (MIP hybrid monolith) was synthesized by one-pot process and its application in selective recognition and capture of lysozyme (Lyz) from complex biological samples was described for the first time. Due to a combination of rigid silica matrices and flexible organic hydrogels in one-pot process, stable and accessible recognition sites in the as-prepared MIP hybrid monolith could be obtained after the removal of template protein, which facilitated the rebinding of template and provided good reproducibility and lifetime of use. The morphology, permeability, and pore properties of the as-prepared MIP hybrid monolith were characterized and a uniform monolithic matrix with high surface area and large through-pores was observed. The recognition behavior of MIP and non-imprinted (NIP) hybrid monolith was evaluated by separating template protein from unfractionated protein mixture and the result indicated that the MIP hybrid monolith has much higher affinity toward the template protein than NIP hybrid monolith. High imprinted factor (IF) and separation efficiency could be obtained. In addition, the practicality of the Lyz-MIP hybrid monolith was further evaluated by selective separation of Lyz from egg white and capture of Lyz from human serum by adopting it as an in-tube solid phase microextraction (in-tube SPME), and the good results demonstrated its potential in proteome analysis.

One‐pot synthesis of (3‐sulfopropyl methacrylate potassium)‐silica hybrid monolith via thiol‐ene click chemistry for CEC

A novel (3-sulfopropyl methacrylate potassium)-silica hybrid monolithic column for CEC has been prepared by a simple one-pot approach based on efficient thiol-ene click chemistry. In this process, the polycondensation of hydrolyzed alkoxysilanes and in situ click reaction of vinyl groups on 3-sulfopropyl methacrylate potassium and thiol groups on the precondensed siloxanes simultaneously occurred in a pretreated capillary. Homogeneous monolithic matrix with large through-pores tightly bonded to the inner wall of the capillary was shown by optical microscope and SEM. The minimum plate height of this hybrid monolithic column was determined as 3.9 μm for thiourea. Anilines, alkylbenzenes, and phenols were well separated on this hybrid monolithic column by CEC, which indicated typical reversed-phase and cation-exchange chromatographic retention mechanisms of the column.

Recent Applications of Organic Monoliths in Capillary Liquid Chromatographic Separation of Biomolecules

Monolithic columns are an attractive alternative to traditional particulate solid phases for capillary liquid chromatography. A monolith is a continuous interconnected skeleton with large through-pores. This structure reduces the diffusion path and provides high permeability, resulting in excellent separation efficiency. The integral structure enhances the mechanical strength, while the large through-pores (a few microm) have very low flow impedance. This combination allows smaller diameter monolithic columns to be operated at higher flow-rates, simultaneously increasing both sensitivity and throughput. Polymeric monoliths were first described back in the 1960s, but the first successful ones designed for protein separations appeared much later, in the late 1980s. Organic monoliths are based upon copolymerization of a monofunctional and a bifunctional (uncommonly trifunctional) organic precursor in the presence of a suitable initiator and porogenic solvents. During the last 15 years, a vast number of different monomers and crosslinkers have been introduced and copolymerized using different polymerization techniques and initiators. Various mechanisms, including thermally- and UV-initiated free radical polymerization, as well as ring opening metathesis copolymerizations, have been demonstrated for the preparation of monolithic columns. In this review, we summarize the recent application of different organic monoliths, including styrene-, acrylate-, methacrylate-, and acrylamide for the liquid separation of biomolecules (e.g., proteins, peptides, and oligonucleotides).

Mass transfer kinetic mechanism in monolithic columns and application to the characterization of new research monolithic samples with different average pore sizes

A general reduced HETP (height equivalent to a theoretical plate) equation is proposed that accounts for the mass transfer of a wide range of molecular weight compounds in monolithic columns. The detailed derivatization of each one of the individual and independent mass transfer contributions (longitudinal diffusion, eddy dispersion, film mass transfer resistance, and trans-skeleton mass transfer resistance) is discussed. The reduced HETPs of a series of small molecules (phenol, toluene, acenaphthene, and amylbenzene) and of a larger molecule, insulin, were measured on three research grade monolithic columns (M150, M225, M350) having different average pore size ( ≃ 150 , 225, and 350 Å, respectively) but the same dimension ( 100 mm × 4.6 mm). The first and second central moments of 2 μ L samples were measured and corrected for the extra-column contributions. The h data were fitted to the new HETP equation in order to identify which contribution controls the band broadening in monolithic columns. The contribution of the B-term was found to be negligible compared to that of the A-term, even at very low reduced velocities ( ν < 1 ). At moderate velocities ( 1 < ν < 3 ), the contribution of the A-term decreases with increasing mesopore size and molecular diffusivity of the compound studied due to faster mass transfer across the column. Experimental chromatograms exhibited variable degrees of systematic peak fronting, depending on the column studied. The heterogeneity of the distribution of eluent velocities from the column center to its wall (average 5%) is the source of this peak fronting. At high reduced velocities ( ν > 5 ), the C-term of the monolithic columns is controlled by film mass transfer resistance between the eluent circulating in the large throughpores and the eluent stagnant inside the thin porous skeleton. The experimental Sherwood number measured on the monolith columns increases from 0.05 to 0.22 while the adsorption energy increases by nearly 6 kJ/mol. Stronger adsorption leads to an increase in the value of the estimated film mass transfer coefficient when a first order film mass transfer rate is assumed ( j ∝ k f Δ C ). The average pore size and the trans-skeleton mass transfer have no ( < 0.5 %, small molecules) or little ( < 10 %, insulin) effect on the overall C-term.

Less common applications of monoliths: I. Microscale protein mapping with proteolytic enzymes immobilized on monolithic supports

This review summarizes the recent contributions to the rapidly growing area of immobilized enzymes employing both silica and synthetic polymer-based monoliths as supports. Focus is mainly on immobilized proteolytic enzyme reactors designed for studies in proteomics. Porous monoliths emerged first as a new class of stationary phases for HPLC in the early 1990s. Soon thereafter, they were also used as supports for immobilization of proteins and preparation of both stationary phases for bioaffinity chromatography and enzymatic reactors. Organic polymer-based monoliths are typically prepared using a simple molding process carried out within the confines of a "mold" such as chromatographic column or capillary. Polymerization of a mixture comprising monomers, initiator, and porogenic solvent affords macroporous materials. In contrast, silica-based monoliths are first formed as a rigid rod from tetraalkoxysilane in the presence of PEG and subsequently encased with a plastic tube. Both types of monolith feature large through-pores that enable a rapid flow-through. Since all the solutions must flow through the monolith, the convection considerably accelerates mass transfer within the monolith. As a result, reactors including enzyme immobilized on monolithic support exhibit much higher activity compared to the reactions in solution.

Rigid gigaporous chromatographic media and their potential impact on downstream processing

More and more biomolecules are being produced by the biotechnology industry for applications ranging from medicine and food to engineering materials. Liquid chromatography plays a center-stage role in a typical downstream process producing biomolecules such as recombinant proteins. Rigid gigaporous media are porous particles possessing large transecting through-pores with a pore-to-particle diameter ratio of d pore/ d particle> 0.01. They allow convective flow in the large through-pores, while the smaller diffusion-pores (typically several hundred angstroms in size) supply the needed surface areas. Because of the transecting gigapores, a portion of the mobile phase flows through the pores in addition to fluid flow in the interstitial spaces between the particles in a packed-bed column. This considerably lowers the operating column pressure drop. This lower pressure drop makes axial-direction scale-up of chromatographic columns possible to avoid pancake columns that invariably degrade separation resolution. The large gigapores also make the binding sites on the diffusion pore surfaces more accessible, thus increasing the loading capacity of large protein molecules that can be hindered sterically if only diffusion pores are present. This work discusses the development of rigid gigaporous media and their potential impact on the design of multi-stage downstream process from the angle of multi-scale analysis.

Organic polymer monoliths as stationary phases for capillary HPLC

Modern rigid porous polymer monoliths were conceived as a new class of stationary phases in classical columns in the early 1990s and later extended to the capillary format. These monolithic materials are typically prepared using a simple molding process carried out within the confines of the capillary. Polymerization of a mixture comprising monomers, initiator, and porogenic solvent affords macroporous materials with large through-pores that enable applications in a rapid flow-through mode. Since all the mobile phase must flow through the monolith, convection considerably accelerates mass transport within the monolithic separation medium and improves the separations. As a result, monolithic columns perform well even at very high flow rates. Various mechanisms including thermally and UV initiated free radical polymerization as well as ring opening metathesis copolymerizations were demonstrated for the preparation of monolithic capillary columns. The versatility of these preparation techniques was demonstrated by their use with hydrophobic (styrene, divinylbenzene, butyl methacrylate, ethylene dimethacrylate), hydrophilic (2-hydroxyethyl methacrylate, methacrylamide, methylenebisacrylamide), ionizable (vinylsulfonic acid, 2-acrylamido-2-methyl-propanesulfonic acid), and tailor-made (norborn-2-ene, 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo,endo-dimethanonaphthalene) monomers. Variation of polymerization conditions enables control of the porous properties of the monolith over a broad range and mediates the hydrodynamic properties of the monolithic columns. The applications of polymer-based monolithic capillary columns are demonstrated for numerous separations in the microHPLC mode.

Preparation and HPLC applications of rigid macroporous organic polymer monoliths.

Rigid porous polymer monoliths are a new class of materials that emerged in the early 1990s. These monolithic materials are typically prepared using a simple molding process carried out within the confines of a closed mold. For example, polymerization of a mixture comprising monomers, free-radical initiator, and porogenic solvent affords macroporous materials with large through-pores that enable applications in a rapid flow-through mode. The versatility of the preparation technique is demonstrated by its use with hydrophobic, hydrophilic, ionizable, and zwitterionic monomers. Several system variables can be used to control the porous properties of the monolith over a broad range and to mediate the hydrodynamic properties of the monolithic devices. A variety of methods such as direct copolymerization of functional monomers, chemical modification of reactive groups, and grafting of pore surface with selected polymer chains is available for the control of surface chemistry. Since all the mobile phase must flow through the monolith, the convection considerably accelerates mass transport within the molded material, and the monolithic devices perform well, even at very high flow rates. The applications of polymeric monolithic materials are demonstrated mostly on the separations in the HPLC mode, although CEC, gas chromatography, enzyme immobilization, molecular recognition, advanced detection systems, and microfluidic devices are also mentioned.

Fast protein liquid chromatography.

High-resolution protein separation techniques critically depend on the availability of column packings of small average particle size. This gives a minimum of peak broadening on the column owing to the direct relationship between the theoretical plate height parameter, H, and particle size (lowest values of H give the highest resolution). High-performance liquid chromatography (HPLC) procedures exploit column packings with average diameters of as small as 5–40 μm. However, these are used in high-pressure systems (up to 400 bar) often with organic solvents and are generally limited to rather low sample loadings (). To provide a more biocompatible high-resolution separation of biopolymers, including (although not exclusive to) proteins, Pharmacia (Uppsala, Sweden; now known as, Amersham Biosciences) developed fast protein liquid chromatography (FPLC) in 1982 (). More recently, Regnier and his colleagues developed “perfusive” packings that contain large through-pores (). These allow high-resolution chromatography with ion-exchange groups similar to those of Amersham Biosciences columns but at much faster flow rates, giving remarkably short separation times (e.g., 5–7 min). Perfusion chromatography columns are also compatible with the FPLC system.

Cyclodextrin-modified monolithic columns for resolving dansyl amino acid enantiomers and positional isomers by capillary electrochromatography.

We describe beta- and gamma-cyclodextrins (beta- and gamma-CD)-modified monolithic columns prepared by sol-gel process and chemical modifications. The monolithic silica column was fabricated inside a fused-silica capillary with 100 microm inner diameter by sol-gel process. The monolithic silica matrix was chemically modified chiral selectors of beta- or gamma-CDs with a spacer of 3-glycidoxypropyltrimethoxysilane by on-column reactions. Gamma-CD-modified monolithic column has successfully been applied for the separation of dansyl amino acid enantiomers. Beta-CD-modified monolithic column has been used for the separation of the positional isomers of o-, m-, and p-cresols and the enantioseparation of racemates of benzoin and several dansyl amino acids by capillary electrochromatography, respectively. For the separation of neutral positional isomers, a positive electric field was applied. However, for the separation of negatively charged analytes, a negative electric field was applied at the inlet of column. The separation efficiency of 5.0 x 10(4) theoretical plates/m for dansyl-L-threonine was obtained at electric field strength of -300 V/cm in the mobile phase of 50 mM 2-(N-morpholino)ethanesulfonic acid (MES)-Tris/methanol (70/30) buffer at pH 7.0. L-enantiomers were eluted as the first peak. Scanning electron micrograph showed that monolithic columns have the morphology of continuous skeleton and large through-pores.

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 (∼1–2 μm) and through-pores (∼2–8 μm) in a fused-silica capillary (50–200 μm I.D.). The columns were evaluated in HPLC after derivatization to C 18 phase. It was possible to prepare monolithic silica structures in capillaries of up to 200 μm 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=8·10 −14–1.3·10 −12 m 2) that was 2–30 times higher than that of a column packed with 5-μm silica particles. The monolithic silica columns prepared in capillaries produced a plate height of about 8–12 μm 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-μm 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.

Monolithic silica columns for high-efficiency separations by high-performance liquid chromatography

Generation of a large number of theoretical plates was attempted by capillary HPLC. Monolithic silica columns having small skeletons (ca. 2 μm) and large through-pores (ca. 8 μm) were prepared by a sol–gel method in a fused-silica capillary (50 μm I.D.), and derivatized to C 18 phase by on-column reaction. High external porosity (>80%) and large through-pores resulted in high permeability ( K=1.2×10 −12 m 2). The monolithic silica column in the capillary produced a plate height of about 12 μm in 80% acetonitrile at a linear velocity of 1 mm/s. Separation impedance, E value, was found to be as low as 200, that was about an order of magnitude lower than reported values for conventional columns packed with 5 μm particles. Reproducibility of preparation within ±15% was obtained for column efficiency and for pressure drop. It was possible to generate 100,000 plates by using a 130-cm column at very low pressure (<7 kg/cm 2). A considerable decrease in column efficiency was observed at high linear velocity, and for solutes with large retention factors due to the slow mobile-phase mass transfer in the large through-pores. The monolithic silica columns, however, showed performance beyond the limit of conventional particle-packed columns in HPLC under favorable conditions.

Porous polymer monoliths: an alternative to classical beads.

Porous polymer monoliths are a new category of materials developed during the last decade. These materials are prepared using a simple molding process carried out within the confines of a closed mold. Polymerization of a mixture that typically contains monomers, free-radical initiator, and porogenic solvent affords macroporous materials with large through-pores that enable flow-through applications. The versatility of the preparation technique is demonstrated by its use with hydrophobic, hydrophilic, ionizable, and zwitterionic monomers. The porous properties of the monolith can be controlled over a broad range. These, in turn, determine the hydrodynamic properties of the devices that contain the molded media. Since all the mobile phase must flow through the monolith, the mass transport within the molded material is dominated very much by convection, and the monolithic devices perform well even at very high flow rates. The applications of monolithic materials are demonstrated on the chromatographic separation of biological compounds and synthetic polymers, electrochromatography, gas chromatography, enzyme immobilization, molecular recognition, and in advanced detection systems. Grafting of the pore walls with selected polymers leads to materials with completely changed surface chemistries.

Performance of a monolithic silica column in a capillary under pressure-driven and electrodriven conditions

A continuous macroporous silica gel network was prepared in a fused-silica capillary and evaluated in reversed-phase liquid chromatography. Under pressure-driven conditions, the monolithic silica column derivatized to C18 phase (100 microns in diameter, 25 cm in length, silica skeleton size of approximately 2.2 microns) produced plate heights of about 23 and 81 microns at 0.5 mm/s with a pressure drop of 0.4 kg/cm2, and at 4.0 mm/s with 3.6 kg/cm2, respectively, in 90% acetonitrile for hexylbenzene with a k value of 0.7. The separation impedance, E, calculated for the present monolithic silica column was much smaller at a low flow rate than those for particle-packed columns, although higher E values were obtained at a higher flow rate. Considerable dependence of column efficiency on the linear velocity of the mobile phase was observed despite the small size of the silica skeletons. A major source of band broadening in the HPLC mode was found in the A term of the van Deemter equation. The performance of the continuous silica capillary column in the electrodriven mode was much better than that in the pressure-driven mode. Plate heights of 7-8 microns were obtained for alkylbenzenes at 0.7-1.3 mm/s, although the electroosmotic flow was slow. In HPLC and CEC mode, the dependency of plate height on k values of the solutes was observed as seen in open tube chromatography presumably due to the contribution of the large through-pores. Since monolithic silica capillary columns can provide high permeability, the pressure-driven operation at a very low pressure can afford a separation speed similar to CEC at a high electric field.

Effect of skeleton size on the performance of octadecylsilylated continuous porous silica columns in reversed-phase liquid chromatography

We prepared continuous porous silica rods that had silica skeletons with sizes of 1.0–1.7 μm and through-pores of 1.5–1.8 μm, and evaluated their performance as a column in reversed-phase liquid chromatography. The mesoporous silica monoliths (mesopore size: 14 or 24 nm) were derivatized to C 18 phase by on-column reaction with octadecyldimethyl-N,N-diethylaminosilane. The C 18 silica rods gave minimum plate heights of 10–15 μm for aromatic hydrocarbons in 80% methanol and 20–30 μm for insulin in acetonitrile-water mixtures in the presence of trifluoroacetic acid. The performance of the silica rods at a high flow-rate was much better than that of conventional columns packed with 5 μm C 18 silica particles with pores of 12 or 30 nm, especially for high-molecular-mass species. Silica rods with the smaller sized silica skeletons resulted in Van Deemter plots showing a minimum plate height at the higher linear velocity of the mobile phase and a smaller dependence of plate height on the linear velocity. Separation impedance of less than 1000 was achieved with the continuous silica columns. The higher performance and lower pressure drop of silica rods at high flow-rates compared with particle-packed columns is provided by the small silica skeletons and large through-pores.

Solute retention of lysozyme in hydrophobic interaction perfusion chromatography

Perfusion chromatography is a rather new HPLC concept using packing materials having large through-pores, which were developed to overcome the problems of stagnant mobile mass transfer without loss of separation speed and column capacity. The perfusion media have been developed for both analytical and preparative separations in a number of distinct HPLC modes including hydrophobic interaction chromatography (HIC) where the separation principle is based on the surface hydrophobicity of the solute, which is adsorbed to the mildly hydrophobic stationary phase in an aqueous mobile phase at high salt concentration and eluted at a lower salt concentration. In this study, the retention behaviour of egg white lysozyme in semi-preparative hydrophobic interaction perfusion chromatography is examined using the BioCAD Workstation. Retention data of lysozyme are measured on four perfusive HIC media at different ammonium sulphate concentrations in the eluent in both isocratic and gradient mode at three different pH values representing more than 400 chromatographic runs. The solute retention behaviour of lysozyme on the HIC columns is described by a model of the capacity factor, k′, as a function of the mobile phase pH and the ionic strength. The capacity factor is a function of the protein activity coefficient in the mobile phase, modelled by a modified Debye-Hückel equation, and the protein activity coefficient on the stationary phase, expressed by a simple non-linear term. The model is capable of correlating ln k′ from zero to high ionic strength.