High capacity organic monoliths for the simultaneous application to biopolymer chromatography and the separation of small molecules.

A method for controlling the mesoporous structure of monolithic organic copolymers is presented by systematic variation in polymerisation time, employing poly(p-methylstyrene-co-1,2-(p-vinylphenyl)ethane) (MS/BVPE) as a representative styrene system. Decreasing the time of polymerisation introduces a considerable fraction of mesopores (up to 20% of the total pore volume), while keeping the support permeability reasonable high ( approximately 1.3x10(-14)m(2)). Monolith structures, prepared in such a manner, enable efficient (typically around 70,000plates/m) and fast separation of low-molecular-weight compounds, whereas their performance towards biopolymers is comparable to column supports, fabricated according to typically used protocols (polymerisation time >12h and thus monomer conversion >98%). The polymerisation time is hence a valuable tool to tailor the fraction of support flow-channels, macropores as well as mesopores, which is shown dramatically to influence the chromatographic separation characteristics of the respective column. This way, the preferred applicability of organic (styrene) monolithic copolymers can be extended to the separation of small molecules beyond biopolymer chromatography.

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(glycidyl methacrylate-co-divinylbenzene) capillary columns functionalized to strong anion exchangers for nucleotide and oligonucleotide separation.

In the present work, poly(glycidyl methacrylate-co-divinylbenzene) monoliths were synthesized and further derivatized to obtain strong anion exchange supports. Capillary monoliths (65 x 0.2 mm id) were prepared in situ by copolymerization of glycidyl methacrylate and divinylbenzene, employing 1-decanol and tetrahydrofuran as porogens. The free epoxy groups were derivatized in a two step synthesis to obtain quaternary ammonium functionalities. On testing the pressure stability of the synthesized monolith, a highly linear dependence between flow rate and pressure drop was obtained, indicating the high stability of the material even at high flow rates. The morphology of the copolymer was investigated by scanning electron microscopy. Mercury intrusion porosimetry showed a narrow pore size distribution, having a maximum at 439 nm. On recording a van Deemter plot the number of theoretical plates per meter was found to be 59324. The produced strong anion exchange monoliths turned out to be highly suitable for the separation of nucleotides and oligonucleotides.

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.