Wide-Bore Columns of Poly(Glycidyl Methacrylate-Co-Divinylbenzene)-Based Monolithic Beds for Reversed-Phase and Anion-Exchange Chromatographic Separation of Biomolecules.

Three monoliths based on poly(glycidyl methacrylate-co-divinylbenzene) were prepared in the confines of borosilicate glass columns (100 × 3 mm I.D.). The first monolith was applied for analysis of proteins by reversed-phase high-performance liquid chromatography. It furnished a fast base-line separation for four proteins (ribonuclease A, cytochrome c, α-lactalbumin and myoglobin) in <80 s, with optimum resolution range of 2.11-2.84, and extremely small values of peak width at half height with a range of 1.0-1.6 s. The second and third monoliths were surface-modified into weak and strong anion-exchangers, respectively, and were investigated for anion-exchange (AE) high-performance liquid chromatography of four proteins with acidic isoelectronic points (bovine carbonic anhydrase, conalbumin, ovalbumin and soybean trypsin inhibitor) and of 5-phosphorylated oligodeoxythymidylic acids fragments [d(pT)12-18]. The weak AE monolith experienced complete elution of the four proteins applying a basic Tris-HCl buffer (0.02 M, pH 8.9); however, the strong AE monolith established a base-line separation of these proteins in ~14 min. Both monoliths showed base-line separation of the seven fragments of d(pT)12-18 in ~6 min. The ion-exchange capacity determined by frontal and elemental analyses was comparable for the weak AE monolith (0.75 and 0.80 meq/g) and for the strong AE monolith (0.81 and 0.87 meq/g), respectively. Finally, a run-to-run and monolith-to-monolith reproducibility showed a relative standard deviation in retention time of d(pT)12-18 fragments of <2%.

Optimization of poly(methyl styrene-co-bis(p-vinylbenzyl)dimethylsilane)-based capillary monoliths for separation of low, medium, and high molecular-weight analytes.

Poly(methyl styrene-co-bis(p-vinylbenzyl)dimethylsilane)-based monolithic capillary columns were optimized for separation of low, medium, and high molecular-weight analytes. The morphology and consequently the chromatographic performance of these monoliths were tuned by changes in the volume-ratio of monomer to macroporogen, establishing good monolithic flow-through and retention pores. Two monoliths were prepared and analyzed by reversed-phase chromatographic separation of low molecular-weight analytes such as alkyl benzenes and ß-blockers, as well as medium and high molecular-weight analytes such as peptides and proteins, respectively. The microstructure was studied by scanning electron microscopy (SEM), and by inverse-size exclusion chromatography (ISEC). Monolith 1 demonstrated a high retention of alkyl benzenes, which coeluted from the column at the washing step of absolute acetonitrile; yet this monolith established a baseline separation of 9-peptide and 8-protein mixtures. Monolith 2 demonstrated efficient separation of the three analyte groups of different molecular weights. Six alkyl benzenes and five ß-blockers were base-line separated in less than 5 and 2min, respectively, with good resolution and very small values of peak width at half height. Moreover, a comparable performance of efficient separation of the 9-peptide mixture and a fast separation of 5- and 8-protein mixtures were achieved. Both monoliths were characterized by high mechanical strength, high permeability, and excellent reproducibility.

Temperature-independent Detection of Heteroduplex and Homoduplex Fragments Applying Poly(glycidylmethacryate-co-divinylbenzene-based) Monoliths Modified to Strong Anion-exchanger.

Monoliths of poly(glycidylmethacrylate-co-divinylbenzene) were prepared in the confines of presilanized borosilicate glass columns (100 × 3 mm I.D.). These monoliths were surface modified into strong anion-exchangers with hydrochloric acid (10%) and triethylamine, successively. The strong anion-exchanger established good separation of 5-phosphorylated oligodeoxythymidylic acids fragments [d(pT)(12-18)]. Moreover, heteroduplex and homoduplex fragments of a low-range mutation standard [of STS marker from the Y-chromosome (209 bp)] were separated at ambient and elevated temperatures using sodium phosphate buffer and a gradient former of sodium chloride, in anion-exchange high-performance liquid chromatography (AE-HPLC). This is a step forward for mutation detection as temperature-independent method, which is not the case in denatured ion-paired reversed-phase chromatography (D-IP-RP-HPLC), where mutation detection is temperature critical and might be bypassed if temperature changes slightly. Finally, reproducibility check from run-to-run and monolith-to-monolith showed a relative standard deviation (RSD) of less than 2%.

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.

Ring-opening metathesis polymerization-derived monolithic anion exchangers for the fast separation of double-stranded DNA 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 (100×3 mm I.D.), applying Grubbs' first generation benzylidene-type catalyst [RuCl(2)(PCy(3))(2)(CHPh)]. Monoliths were converted into weak anion exchangers via reaction with diethyl amine. The resulting monolithic anion exchangers demonstrated a very good potential for the anion-exchange separation of nucleic acids applying a phosphate buffer (0.05 mol/L, pH 7) and NaCl (1.0 mol/L) as a gradient former. Fast and efficient separations, indicated by sharp and highly symmetric analyte peaks, were established. Except for the 267 and 298 base pair fragments, the eleven fragments of a ds-pUC18 DNA Hae III digest were baseline separated within ∼8 min. Nineteen fragments of a ds-pBR322 Hae III digest were separated within ∼12 min. There, only the 192 and 213 base pair fragments and the 458, 504 and 540 base pair fragments coeluted. A ds-pUC18 DNA Hae III digest was used as a control analyte in evaluating the influence of organic additives on the mobile phase such as methanol and acetonitrile on nucleic acid separation. Methanol, and even better, acetonitrile improved the separation efficiency and shortened the analysis time.

Wide-bore P-methylstyrene-co-dimethylbis(p-vinylbenzyl) silane Based-monoliths Columns for Separation of Peptides and Proteins.

Wide-bore monolithic columns of p-methylstyrene-co-dimethylbis(p-vinylbenzyl)silane were prepared within glass columns (100 × 3 mm I.D.). Monolithic columns differing in the amount of initiator were fabricated. The pressure drop vs. flow-rate measurements and the relatively low swelling propensity factor indicated the good cross-linking homogeneity and good mechanically stability. The chromatographic performance of monoliths prepared was assessed by analysis of protein and peptide mixtures. Five proteins were separated in less than 2 minutes. Moreover, a mixture of 9 peptides was separated in less than 11 minutes, aside from the co-elution of vasopressin [arg8] and methionine enkephalin.

Fast separation of low molecular weight analytes on structurally optimized polymeric capillary monoliths.

The optimization of recently introduced tetrakis(4-vinylbenzyl)silane (TVBS)-derived capillary monoliths to suit the fast separation of small molecules was accomplished by simultaneous changes in both the stoichiometry of the polymerization mixture and in the polymerization temperature. Four types of monoliths were prepared by varying the polymerization temperature within 60 and 65 degrees C as well as by changing the monomer content between 15.0 and 20.0 wt.%. Changes in the porosity and morphology of the capillary columns were studied applying inverse size exclusion chromatography (ISEC), nitrogen sorption (BET), and scanning electron microscopy (SEM). All monoliths were used for the reversed-phase separation of various sets of low molecular weight analytes such as alkyl benzenes, aryl amines, carboxylic acids, and a mixture of phenols and carbonyl compounds. Comparable efficiency and performance towards the different sets of analytes were observed. With optimized monolith structures, the fast separation of six-alkyl benzenes was accomplished within less than 2 min. This was made possible by a high linear solvent flow (10mm/s) at low column pressure drops (<15 MPa).

Highly cross-linked polymeric capillary monoliths for the separation of low, medium, and high molecular weight analytes.

Highly rigid capillary monoliths with low swelling propensity were prepared within the confines of 200 microm ID fused silica capillaries via thermally induced free radical polymerization of tetrakis(4-vinylbenzyl)silane (TVBS) in the presence of 1-dodecanol and toluene. alpha,alpha'-Azobisisobutyronitrile (AIBN) was used as initiator. The resulting monoliths were optimized for the separation of low, medium, and high molecular weight analytes. The microstructure and porosity of the monoliths prepared were studied by SEM and inverse size-exclusion chromatography (ISEC). The porosity of the monolithic supports was tuned by varying the amount of initiator (i. e. AIBN) between 0.5 and 2 wt%. All monoliths were tested for a series of low molecular weight compounds including alkylbenzenes, amines, carboxylic acids, phenols, carbonyl compounds, and beta-blockers, as well as for the separation of medium molecular weight analytes such as peptides and high-molecular weight analytes such as proteins. Due to the microporous structure, the novel monoliths displayed high efficiency and performance particularly in the separation of low molecular weight analytes. Relevant chromatographic parameters including permeability, swelling propensity, and height equivalents to theoretical plates were determined.

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.

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.

Novel monolithic poly(phenyl acrylate-co-1,4-phenylene diacrylate) capillary columns for biopolymer chromatography.

Monolithic capillary columns were prepared by thermally initiated free radical polymerisation of phenyl acrylate (PA) and 1,4-phenylene diacrylate (PDA) in the confines of 200 microm I.D. fused silica capillaries. Polymerisation was performed in the presence of 2-propanol and tetrahydrofuran (THF) as inert diluents (porogens), using alpha,alpha'-azoisobutyronitrile (AIBN) as initiator. Morphology and porosity of the resulting monoliths were comprehensively studied by scanning electron microscopy (SEM), mercury intrusion porosimetry and inverse size-exclusion chromatography (ISEC). The novel poly(phenyl acrylate-co-1,4-phenylene diacrylate) (PA/PDA) monoliths showed high mechanical stability and were successfully applied to the separation of proteins and oligodeoxynucleotides, employing reversed-phase (RP) and ion-pair reversed-phase (IP-RP) conditions, respectively. Maximum loading capacities for cytochrome c and d(pT)(16) were evaluated and found to be in the region of 200 fmol. Batch-to-batch reproducibility was determined for three independently prepared PA/PDA monolithic capillary columns. Relative standard deviations (RSDs) of retention time (t(R)) of 0.7-1.6% for proteins and 0.2-2.5% for d(pT)(12-18) proved high reproducibility of the PA/PDA supports.

Comparison between monolithic conventional size, microbore and capillary poly(p-methylstyrene-co-1,2-bis(p-vinylphenyl)ethane) high-performance liquid chromatography columns Synthesis, application, long-term stability and reproducibility.

Novel monolithic supports (MS/BVPE) were prepared by thermally initiated free radical copolymerisation of p-methylstyrene (MS) and 1,2-bis(p-vinylphenyl)ethane (BVPE). The polymer was synthesised in fused silica capillaries (80 mm x 0.2 mm and 80 mm x 0.53 mm) and in borosilicate glass columns (90 mm x 1.0 mm and 90 mm x 3.0 mm) to yield different HPLC column designs. A comparison of those column dimensions regarding morphology as well as separation efficiency and applicability in bioanalysis is presented. The efficiency towards proteins as well as oligonucleotides was found to be considerably improved with decreasing column I.D. While a 5-protein mixture was baseline separated on all investigated column designs, the separation of small biomolecules like oligonucleotides or peptides on microbore and conventional size glass columns was strongly restricted in terms of resolution due to extensive peak broadening or the occurrence of peak asymmetry. Monolithic MS/BVPE capillary columns up to 0.53 mm I.D., however, proved to be applicable to the fractionation of the whole spectrum of biopolymers, including proteins, peptides, oligonucleotides as well as double-stranded DNA fragments. Due to the fact that reliable chromatography makes great demand on the robustness of the stationary phase, monolithic MS/BVPE capillaries were subjected to a comprehensive reproducibility study including run-to-run as well as batch-to-batch reproducibility.

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.