Influence of different polymerisation parameters on the separation efficiency of monolithic poly(phenyl acrylate-co-1,4-phenylene diacrylate) capillary columns

Monolithic poly(phenyl acrylate-co-1,4-phenylene diacrylate) (PA/PDA) capillary columns were prepared in the confines of 200 microm I.D. fused silica capillaries by thermally initiated free radical copolymerisation of phenyl acrylate (PA) and 1,4-phenylene diacrylate (PDA) in the presence of alpha,alpha'-azoisobutyronitrile (AIBN). Variation of polymerisation parameters in terms of total monomer to porogen ratio, nature of the pore-forming agent and polymerisation temperature is shown to have a significant impact on the porous properties of the supports, which was proven by inverse size-exclusion chromatography (ISEC). Monoliths of significantly different porosity (total porosity accessible to the mobile phase (epsilonT)=0.66-0.71, volume fraction of pores (epsilonP)=0.15-0.24) and hence permeability could easily be prepared. The chromatographic efficiency of the PA/PDA monoliths regarding protein and oligonucleotide separation was studied. A correlation between porosity, retention behaviour and efficiency was derived from the obtained separations. In addition to chromatographic evaluation, pressure drop versus flow rate measurements confirmed mechanical stability. Swelling propensity (SP) factors of 0.47-0.87, moreover, indicated a high degree of crosslinking.     

Monolithic poly(p-methylstyrene-co-1,2-bis(p-vinylphenyl)ethane) capillary columns as novel styrene stationary phases for biopolymer separation

Novel monolithic capillary supports (200 microm I.D.) were prepared by polymerisation of methylstyrene (MS) and 1,2-bis(p-vinylphenyl)ethane (BVPE) as a crosslinker in the presence of inert diluents (porogens). These polymeric reversed-phases (MS/BVPE) showed excellent mechanical stability and minimised swelling in organic solvents. The chromatographic potential of monolithic MS/BVPE as a stationary phase for micro-high-performance liquid chromatography (mu-HPLC) was investigated by the separation of proteins and peptides applying reversed-phase (RP) and nucleic acids applying ion-pair reversed-phase (IP-RP) conditions. The permeability and chromatographic efficiency of the capillary columns were found to be highly influenced by the total monomer to porogen content as well as by the microporogen nature and its ratio. In the course of these optimisation studies, monoliths covering a broad permeability range were fabricated. The application of volumetric flow rates up to 200 microl/min allowed swift resolution of proteins, while smaller biomolecules were successfully separated at a higher overall porosity. A protein test mixture containing ribonuclease A, cytochrome c, alpha-lactalbumin, beta-lactoglobulin B and ovalbumin was thus baseline separated in 35s, a homologous series of phosphorylated oligothymidylic acids [d(pT)12-18] in less than 2 min.     

Amino-Functionalized Monolithic Poly(glycidyl methacrylate-<Emphasis Type="Italic">co</Emphasis>-divinylbenzene) Ion-Exchange Stationary Phases for the Separation of Oligonucleotides

Monolithic capillary columns were prepared by thermal initiated copolymerization of glycidyl methacrylate (GMA) and divinylbenzene (DVB) inside silanized 200 µm i.d. fused silica capillaries. Polymerization mixtures containing different amounts of porogen (1-decanol and tetrahydrofuran (THF)) and different ratios of monomer and crosslinker were used for synthesis. For characterization the pore size distribution profiles of the resulting monoliths were determined by mercury intrusion porosimetry. The morphology of the copolymer was investigated by scanning electron micrographs (SEM). A high linear dependence between flow rate and pressure drop was achieved which indicates that the polymer is pressure-stable even at high flow rates. After characterization the produced GMA-DVB monoliths, which contain reactive epoxide groups, were modified by reaction with diethylamine to obtain a poly(3-diethylamino-2-hydroxypropyl methacrylate-co-divinylbenzene) ion-exchange monolithic stationary phase. The synthesized monoliths contain ionizable amino groups that are useful for anion-exchange chromatography (AEC). Poly(3-diethylamino-2-hydroxypropyl methacrylate-co-divinylbenzene) monolithic columns allowed a fast and highly efficient separation of a homologous series of phosphorylated oligothymidylic acids [d(pT)_12-18]. Since durability is an important parameter of chromatographic column characterization, the separation performance for d(pT)_12-18 in a freshly produced capillary column and on the same column after 100 chromatographic runs was compared.     

Separation of oligonucleotides on novel monolithic columns with ion-exchange functional surfaces.

Porous monolithic columns have been prepared by the direct free radical copolymerization of glycidyl methacrylate and ethylene dimethacrylate within the confines of a 50x8 mm I.D. chromatographic column in the presence of porogens. The epoxide groups of these monoliths were modified to different extents by reaction with diethylamine to afford 1-N,N-diethylamino-2-hydroxypropyl functionalities useful for ion-exchange chromatography. Following characterization of the monoliths, the columns were tested in the chromatographic separation of a homologous series of oligodeoxyadenylic [pd(A)(12-18)] and oligothymidylic acids [d(pT)(12-24)] at different flow-rates. Very good separations of the oligonucleotides were achieved even at the high flow-rate of 4 ml/min.     

Mixed-mode reversed-phase and ion-exchange monolithic columns for micro-HPLC

This paper describes the fabrication of RP/ion-exchange mixed-mode monolithic materials for capillary LC. Following deactivation of the capillary surface with 3-(trimethoxysilyl)propyl methacrylate (gamma-MAPS), monoliths were formed by copolymerisation of pentaerythritol diacrylate monostearate (PEDAS), 2-sulphoethyl methacrylate (SEMA) with/without ethylene glycol dimethacrylate (EDMA) within 100 microm id capillaries. In order to investigate the porous properties of the monoliths prepared in our laboratory, mercury intrusion porosimetry, SEM and micro-HPLC were used to measure the monolithic structures. The monolithic columns prepared without EDMA showed bad mechanical stability at high pressure, which is undesirable for micro-HPLC applications. However, it was observed that the small amount (5% w/w) of EDMA clearly improved the mechanical stability of the monoliths. In order to evaluate their application for micro-HPLC, a range of neutral, acidic and basic compounds was separated with these capillaries and satisfactory separations were obtained. In order to further investigate the separation mechanism of these monolithic columns, comparative studies were carried out on the poly(PEDAS-co-SEMA) monolithic column and two other monoliths, poly(PEDAS) and poly(PEDAS-co-2-(methacryloyloxy)ethyl-trimethylammonium methylsulphate (METAM)). As expected, different selectivities were observed for the separation of basic compounds on all three monolithic columns using the same separation conditions. The mobile phase pH also showed clear influence on the retention time of basic compounds. This could be explained by ion-exchange interaction between positively charged analytes and the negatively charged sulphate group.     

Size-exclusion separation of proteins using a biocompatible polymeric monolithic capillary column with mesoporosity

Biocompatible poly(ethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monoliths were prepared for size exclusion chromatography (SEC) of proteins in the capillary format using Brij 58P in a mixture of hexanes and dodecanol as porogens. The monolithic columns provided size separation of four proteins in 20 mM sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl, and there was a linear relationship between the retention times and the logarithmic values of the molecular weights. Compared to SEC monoliths previously synthesized using a triblock copolymer of polyethylene oxide and polypropylene oxide, an increase in mesoporosity was confirmed by inverse size exclusion chromatography. As a result, improved protein separation in the high molecular weight range and reduced column back-pressure were observed.     

Preparation of polymeric monoliths by copolymerization of acrylate monomers with amine functionalities for anion-exchange capillary liquid chromatography of proteins

Two novel polymeric monoliths for anion-exchange capillary liquid chromatography of proteins were prepared in a single step by a simple photoinitiated copolymerization of 2-(diethylamino)ethyl methacrylate and polyethylene glycol diacrylate (PEGDA), or copolymerization of 2-(acryloyloxy)ethyl trimethylammonium chloride and PEGDA, in the presence of selected porogens. The resulting monoliths contained functionalities of diethylaminoethyl (DEAE) as a weak anion-exchanger and quaternary amine as a strong anion-exchanger, respectively. An alternative weak anion-exchange monolith with DEAE functionalities was also synthesized by chemical modification after photoinitiated copolymerization of glycidyl methacrylate (GMA) and PEGDA. Important physical and chromatographic properties of the synthesized monoliths were characterized. The dynamic binding capacities of the three monoliths (24 mg/mL, 56 mg/mL and 32 mg/mL of column volume, respectively) were comparable or superior to values that have been reported for various other monoliths. Chromatographic performance was also similar to that provided by a modified poly(GMA-ethylene glycol dimethacrylate) monolith. Separation of standard proteins was achieved under gradient elution conditions using these monolithic columns. Peak capacities of 34, 58 and 36 proteins were obtained with analysis times of 20–30 min. This work represents a successful attempt to prepare functionalized monoliths via direct copolymerization of monomers with desired functionalities. Compared to earlier publications, additional surface modifications were avoided and the PEGDA crosslinker helped to improve the biocompatibility of the monolithic backbone.     

Ring-opening metathesis polymerization-derived large-volume monolithic supports for reversed-phase and anion-exchange chromatography of biomolecules

Preparative-scale monolithic columns up to 433.5 mL in volume were prepared via transition metal-catalyzed ring-opening metathesis polymerization (ROMP) from norborn-2-ene (NBE) and trimethylolpropane-tris(5-norbornene-2-carboxylate) (CL) using the 1(st)-generation Grubbs initiator RuCl(2)(PCy(3))(2)(CHPh) (Cy = cyclohexyl) (1) in the presence of a macro- and microporogen, i.e. of 2-propanol and toluene. To prepare large-volume monoliths, bulk polymerizations were completed within borosilicate or PEEK column formats with diameters in the range of 3 to 49 mm. The pore structure of the large-volume monoliths was investigated by electron microscopy and inverse-size exclusion chromatography (ISEC), respectively. Monolithic columns with inner diameters (I.D.s) in the range of 10-49 mm were tested for the separation of a mixture of five proteins, i.e., insulin, cytochrome C, lysozyme, conalbumin, and β-lactoglobulin. Preparative separation of these proteins was achieved within less than 12 min in a 433.5 mL monolithic column by applying gradient elution in the RP-HPLC mode. Furthermore, weak and strong anion exchangers were prepared via post-synthesis grafting of bicyclo[2.2.1]hept-5-en-2-yl-methyl-N,N-dimethylammonium hydrochloride (4) and bicyclo[2.2.1]hept-5-en-2-ylmethyl-N,N,N-trimethylammonium iodide (5), respectively. The weak and strong anion exchangers were used for the preparative-scale separation of 5'-phosphorylated oligodeoxythymidylic acid fragments of d[pT](12-18) at pH values ranging from 5 to 9.     

Ring-opening metathesis polymerization-derived monolithic capillary columns for high-performance liquid chromatography. Downscaling and application in medical research

Monolithic capillary columns were prepared via ring-opening metathesis polymerization (ROMP) using norborn-2-ene (NBE) and 1, 4, 4a, 5, 8, 8a-hexahydro-1, 4, 5, 8-exo,endo-dimethanonaphthalene (DMN-H6) as monomers. The monolithic polymer was copolymerized with Grubbs-type initiator RuCl(2)(PCy(3))(2)(CHPh) and a suitable porogenic system within the confines of fused silica capillaries of different inner diameter (I.D.). The first part of the study focused on batch-to-batch reproducibility of ROMP-derived capillary monoliths. Capillary monoliths of 200 microm I.D. showed good reproducibility in terms of retention times, with relative standard deviations (RSD) of 1.9% for proteins and 2.2% for peptides. However, the separately synthesized capillary monoliths revealed pronounced variation in back pressure with RSD values of up to 31%. These variations were considerably reduced by cooling of the capillaries during polymerization. Using this optimized preparation procedure capillary monoliths of 100 and 50 microm I.D. were synthesized and the effects of scaling down the column I.D. on the morphology and on the reproducibility of the polymerization process were investigated. In the second part, the applicability of ROMP-derived capillary monoliths to a separation problem common in medical research was assessed. A 200 microm I.D. monolithic column demonstrated excellent separation behavior for insulin and various insulin analogs, showing equivalent separation performance to Vydac C4 and Zorbax C3-based stationary phases. Moreover, the high permeability of monoliths enabled chromatographic separations at higher flow rates, which shortened analysis time to about one third. For the analysis of insulin in human biofluid samples, enhanced sensitivity was achieved by using a 50 microm I.D. ROMP-derived monolith.     

Novel monolithic poly(p-methylstyrene-co-bis(p-vinylbenzyl)dimethylsilane) capillary columns for biopolymer separation

Hydrophobic organo-silane based monolithic capillary columns were prepared by thermally initiated free radical polymerisation within the confines of 200 microm i.d. fused silica capillaries. A novel crosslinker, namely bis(p-vinylbenzyl)dimethylsilane (BVBDMS), was copolymerised with p-methylstyrene (MS) in the presence of 2-propanol and toluene, using alpha,alpha'-azoisobutyronitrile (AIBN) as initiator. Monolithic capillary columns, differing in the total monomer, microporogen content and microporogen nature were fabricated and the chromatographic efficiency of each monolith, regarding the separation of proteins, peptides and oligonucleotides, was evaluated and compared. Changes in monolith morphology were monitored by scanning electron microscopy (SEM). Porosity and specific surface areas of the supports were studied by means of mercury intrusion porosimetry and BET measurements, respectively. Pressure drop vs. flow rate measurements proved the prepared poly(p-methylstyrene-co-bis(p-vinylbenzyl)dimethylsilane) (MS/BVBDMS) monoliths to be mechanically stable and swelling propensity (SP) factors of 0.78-1.10 indicate high crosslinking homogeneity.     

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.     

Polypyrazolinones from aromatic di(propynoic ester)s and aromatic dihydrazines

Novel polypyrazolinones with inherent viscosities ranging from 0.12 to 0.44 dL/g were prepared by the Michael-type nucleophilic addition-cyclization of various dihydrazines with 3,3′-(1,3- or 1,4-phenylene)bis(ethyl propynoate) (1,3- or 1,4-PEP) and 3,3′-(1,4-phenylene)bis(phenyl propynoate) (1,4-PPhP) in N-methylpyrrolidone (NMP) solution at 25–110°C. The polymers exhibited moderate thermal stability with initial weight loss in air about 200°C and in nitrogen about 300°C (TGA). No apparent T_g′s were observed by DSC analysis. The synthesis and characterization of the polypyrazolinones is discussed.     

Capillary electrochromatography with monolithic stationary phases: 1. Preparation of sulfonated stearyl acrylate monoliths and their electrochromatographic characterization with neutral and charged solutes

A novel monolithic stationary phase having long alkyl chain ligands (C17) was introduced and evaluated in capillary electrochromatography (CEC) of small neutral and charged species. The monolithic stationary phase was prepared by the in situ copolymerization of pentaerythritol diacrylate monostearate (PEDAS) and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a ternary porogenic solvent consisting of cyclohexanol/ethylene-glycol/water. While AMPS was meant to support the electroosmotic flow (EOF) necessary for transporting the mobile phase through the monolithic capillary, the PEDAS was introduced to provide the nonpolar sites for chromatographic retention. Monolithic columns at various EOF velocities were readily prepared by conveniently adjusting the amount of AMPS in the polymerization solution as well as the composition of the porogenic solvent. The monolithic stationary phases thus obtained exhibited reversed-phase chromatography behavior toward neutral solutes and yielded a relatively strong EOF. For charged solutes (e.g., dansyl amino acids), nonpolar as well as electrostatic interaction/repulsion with the monoliths were observed in addition to electrophoretic migration. Therefore, for charged solutes, selectivity and migration can be readily manipulated by changing various parameters including the nature of the monolith and the composition of the mobile phase (e.g., pH, ionic strength and organic modifier). Ultrafast separation on the time scale of seconds of 17 different charged and neutral pesticides and metabolites were performed using short capillary columns of 8.5 cm x 100 microm ID.     

Polymeric cation-exchange monolithic columns containing phosphoric acid functional groups for capillary liquid chromatography of peptides and proteins

Two different monoliths, both containing phosphoric acid functional groups and polyethylene glycol (PEG) functionalities were synthesized for cation-exchange chromatography of peptides and proteins. Phosphoric acid 2-hydroxyethyl methacrylate (PAHEMA) and bis[2-(methacryloyloxy)ethyl] phosphate (BMEP) were reacted with polyethylene glycol diacrylate (PEGDA) and polyethylene glycol acrylate (PEGA), respectively, in 75-μm i.d. UV-transparent fused-silica capillaries by photo-initiated polymerization. The hydrophobicities of the monoliths were evaluated using propyl paraben under reversed-phase conditions and synthetic peptides under ion-exchange conditions. The resulting monoliths exhibited lower hydrophobicities than strong cation-exchange monoliths previously reported using PEGDA as cross-linker. Dynamic binding capacities of 31.2 and 269 mg/mL were measured for the PAHEMA–PEGDA and BMEP–PEGA monoliths, respectively. Synthetic peptides were eluted from both monoliths in 15 min without addition of acetonitrile to the mobile phase. Peak capacities of 50 and 31 were measured for peptides and proteins, respectively, using a PAHEMA–PEGDA monolith. The BMEP–PEGA monolith showed negligible hydrophobicity. A peak capacity of 31 was measured for the BMEP–PEGA monolith when a 20-min salt gradient rate was used to separate proteins. The effects of functional group concentration, mobile phase pH, salt gradient rate, and hydrophobicity on the retention of analytes were investigated. Good run-to-run [relative standard deviation (RSD) < 1.99%] and column-to-column (RSD < 5.64) reproducibilities were achieved. The performance of the monoliths in ion-exchange separation of peptides and proteins was superior to other polymeric monolithic columns reported previously when organic solvents were not added to the mobile phase.     

Ring-opening metathesis polymerization-derived monolithic strong anion exchangers for the separation of 5'-phosphorylated oligodeoxythymidylic acids fragments

Ring-opening metathesis polymerization (ROMP) derived monoliths were prepared from 5-norborn-2-enemethyl bromide (NBE-CH(2)Br) and tris(5-norborn-2-enemethoxy)methylsilane ((NBE-CH(2)O)(3)SiCH(3)) within the confines of surface-silanized borosilicate columns (100 mm × 3 mm I.D.), applying Grubbs' first generation benzylidene-type catalyst [RuCl(2)(PCy(3))(2)(CHPh)]. Two monoliths of the same recipe were converted into strong anion-exchangers applying two different approaches. Monolith I was prepared by a two-step reaction of the poly(NBE-CH(2)-Br) moieties with diethyl amine forming a weak-anion exchanger followed by reaction (quaternization) with ethyl iodide. Monolith II was prepared via a single-step reaction of the poly(NBE-CH(2)-Br) moieties with triethyl amine. The resulting monolithic anion-exchangers prepared demonstrated a good aptitude for the anion-exchange separation of single-stranded nucleic acids (ss-DNA). However, monolith II showed superior separation efficiency compared to monolith I indicated by sharper analyte peaks and better resolution values for the 5'-phosphorylated oligodeoxythymidylic acids fragments. On monolith II, the seven fragments of [d(pT)(12-18)] were baseline separated in less than 9 min. The influence of the buffer pH on the separation efficiency was studied applying a phosphate (0.05 mol/L, pH 7 and 8) and Tris-HCl buffer (0.05 mol/L, pH 9), respectively.     

Polymetacrylate and hybrid interparticle monolithic columns for fast separations of proteins by capillary liquid chromatography

Preparation of organic polymer monolithic columns in fused silica capillaries was aimed at fast gradient separation of proteins. For this purpose, polymerization in situ procedure was optimized, using ethylene dimetacrylate and butyl metacrylate monomers with azobisisobutyronitrile as initiator of the polymerization reaction in presence of non-aqueous porogen solvent mixtures composed of 1-propanol and 1,4-butanediol. The separation of proteins in totally monolithic capillary columns was compared with the chromatography on a new type of "hybrid interparticle monolithic" capillary columns, prepared by in situ polymerization in capillary packed with superficially porous spherical beds, 37-50 microm. The "hybrid" columns showed excellent stability and improved hydrodynamic flow properties with respect to the "totally" monolithic capillary columns. The separation selectivity is similar in the two types of columns. The nature of the superficially porous layer (bare silica or bonded C18 ligands) affects the separation selectivity less significantly than the porosity (density) of the monolithic moiety in the interparticle space, controlled by the composition of the polymerization mixture. The retention behaviour of proteins on all prepared columns is consistent with the reversed-phase gradient elution theory.     

Preparation of low flow-resistant methacrylate-based monolithic stationary phases of different hydrophobicity and the application to rapid reversed-phase liquid chromatographic separation of alkylbenzenes at high flow rate and elevated temperature

Low flow-resistant alkyl methacrylate-based monolithic stationary phases of different hydrophobicity were constructed for reversed-phase capillary liquid chromatography by thermally initiated radical polymerization of respective methacrylate ester monomer with different alkyl chain (C2, C4, C6, C12, C18) and ethylene glycol dimethacrylate (EDMA) in a 250 microm i.d. fused silica capillary. The hydrophobicity was basically controlled by changing the length and/or the density of the alkyl-chain, while the composition and the ratio of porogenic solvent were adjusted to obtain highly permeable rigid monoliths with adequate column efficiency. Among the prepared monolithic stationary phases, C18-methacrylate monoliths polymerized from a binary porogenic solvent of isoamyl alcohol and 1,4-buthandiol exhibited the most promising performance in terms of hydraulic resistance and column efficiency. The pressure drops of 20-cm long monolithic columns were below approximately 0.4 MPa at a normal linear velocity of 1mm/s (a flow rate of 3 microL/min), and the numbers of theoretical plates for alkylbenzenes mostly exceeded 3000 plates/20 cm. The produced monolithic columns had good mechanical strength for high pressure and temperature, and could be properly operated even at a temperature of 80 degrees C and at a pressure of at least 33 MPa. At 80 degrees C, the theoretical plate numbers reached 6000 plates/20 cm because of the enhanced mass transfer. Due to the novel hydraulic resistance and mechanical strength, the separation time could be reduced 120-fold simply by raising the flow rate and column temperature.     

Preparation and Evaluation of Long Chain Alkyl Methacrylate Monoliths for Capillary Chromatography

This work describes the fabrication of long chain alkyl methacrylate monolithic materials for use as stationary phases in capillary liquid chromatography. After capillary inner wall surface activation with 3-(trimethoxysilyl)propyl methacrylate, monoliths were formed by copolymerization of either lauryl or stearyl methacrylate (LMA or SMA) with ethylene dimethacrylate (EDMA) as crosslinker, in the presence of azobisisobutyronitrile (AIBN) as initiator and a mixture of porogenic solvents including water, 1-propanol and 1,4-butanediol. The composition of the polymerization mixture was changed in terms of monomer, crosslinker and porogen ratio composition, in order to compare the influence of these parameters. The monoliths were prepared in 320 ??m i.d. and 200 mm length capillaries. The column morphology was characterized by optical microscopy and scanning electron microscopy (SEM). Total porosity and permeability of each column were calculated using uracil as unretained material by measuring the pressure drop across the columns as a function of linear velocity. The microglobule average size for each column was also determined using Hagen?CPoiseuille equation and compared with the SEM images. As expected, a decrease of the porogen to monomer ratio corresponded to smaller microglobules and a lower total porosity. The columns were then chromatographically evaluated; good results were obtained when these capillaries were used to separate mixtures of phenols, aromatics and drug compounds.     

Development and in situ synthesis of monolithic stationary phases for electrochromatographic separations

Organic monolithic stationary phases have been synthesized in UV-transparent fused-silica capillaries, which have been used as test format of microfabricated device channels. The columns have been prepared by in situ polymerization of butyl acrylate, lauryl acrylate, 1,3-butanediol diacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a ternary porogenic solvent. The resulting stationary phases have been tested in capillary electrochromatography and exhibited reversed-phase chromatography behavior toward neutral solutes. Van Deemter plots of phenylureas and polycyclic aromatic hydrocarbons, selected as model analytes, have been determined to study the influence of various polymerization and separation parameters on properties of the monoliths. The amount of AMPS and the nature of monomers in the polymerization solution have been thus adjusted. It has been observed that the ionic strength of the mobile phase may affect significantly the efficiency of the separation. The effect of the percentage of acetonitrile in the mobile phase on efficiency and permeability of the organic monoliths has also been investigated. Efficiencies greater than 300,000 plates/m have been obtained with the test compounds. Stability and reproducibility have been extensively studied.     

Affinity chromatography with monolithic capillary columns. II. Polymethacrylate monoliths with immobilized lectins for the separation of glycoconjugates by nano-liquid affinity chromatography

Monolithic capillary columns with surface bound lectin affinity ligands were introduced for performing lectin affinity chromatography (LAC) by nano-liquid chromatography (nano-LC). Two kinds of polymethacrylate monoliths were prepared, namely poly(glycidyl methacrylateco-ethylene dimethacrylate) and poly(glycidyl methacrylate-co-ethylene dimethacrylate-co-[2-(methacryloyloxy)ethyl]trimethyl ammonium chloride) to yield neutral and cationic macroporous polymer, respectively. Two lectins including concanavalin (Con A) and wheat germ agglutinin (WGA) were immobilized onto the monolithic capillary columns. The neutral monoliths with immobilized lectins exhibited lower permeability under pressure driven flow than the cationic monoliths indicating that the latter had wider flow-through pores than the former. Both types of monoliths with immobilized lectins exhibited strong affinity toward particular glycoproteins and their oligosaccharide chains (i.e., glycans) having sugar sequences recognizable by the lectin. Due to the strong binding affinity, the monoliths with surface bound lectins allowed the injection of relatively large volume (i.e., several column volumes) of dilute samples of glycoproteins and glycans thus allowing the concentration of the glycoconjugates and their subsequent isolation and detection at low levels (approximately 10(-8) M). To further exploit the lectin monoliths in the isolation of glycoconjugates, two-dimensional separation schemes involving LAC in the first dimension and reversed-phase nano-LC in the second dimension were introduced. The various interrelated methods established in this investigation are expected to play a major role in advancing the sciences of "nano-glycomics".