Evaluation of ring-opening metathesis polymerization (ROMP)-derived monolithic capillary high performance liquid chromatography columns (original) (raw)
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Journal of Chromatography A, 2006
Monolithic columns for capillary HPLC were prepared via ring-opening metathesis polymerization (ROMP) from cis-cyclooctene (COE), tris(cyclooct-4-enyl-1-oxy)methylsilane (CL) as monomers, 2-propanol and toluene as porogens and RuCl 2 (Py) 2 (IMesH 2 )(CHC 6 H 5 ) (Py = pyridine, IMesH 2 = 1,3-dimesityl-4,5-dihydroimidazolin-2-ylidene) as initiator within the confines of 200 m i.d. fused silica columns. For evaluation of the novel monolithic capillary HPLC columns, a protein standard consisting of six proteins in the molecular weight range of 5800-66000 g/mol, i.e. ribonuclease A, insulin, albumin, lysozyme, myoglobin and -lactoglobulin, was used. Reproducibility of synthesis was checked by determining the relative standard deviation (RSD) in retention times (t R ), which was found to be in the range of 2.9-3.9% for all analytes. Variations in polymer kinetics were realized by adding different amounts of free pyridine and had a significant influence on the monolith's morphology, the backpressure and retention times. On the contrary, variations in monomer content and COE to CL ratio showed only minor changes on these parameters. Long-term stability of 1000 runs at 50 • C showed excellent stability of the columns and no significant alteration in separation performance was observed in combination with slightly decreased retention times (approx. 1.6-7.2% for all analytes).
Journal of Chromatography A, 2008
Ring-opening metathesis polymerization-(ROMP) derived monoliths were prepared from 5-norborn-2enemethyl 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.
Journal of Chromatography A, 2006
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 m. 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.
Journal of Chromatography A, 2009
Monolithic capillary columns were prepared via electron beam triggered free radical polymerization within the confines of 0.2 and 0.1 mm I.D. capillary columns using ethyl methacrylate and trimethylolpropane triacrylate as monomers as well as 2-propanol, 1-dodecanol and toluene as porogenic system. The influence of column diameter on reproducibility and separation performance was investigated. For evaluation, a protein standard consisting of five proteins in the range of 5800-66 000 g mol −1 was used. Reproducibility was checked by determining the relative standard deviations in retention times, peak widths at half height, asymmetry and resolution. Excellent run-to-run reproducibility was found for both 0.2 and 0.1 mm I.D. columns; batch-to-batch reproducibility was good for both column types. In order to enhance the non-polar character of the monolithic columns, lauryl methacrylate-based capillary columns were prepared. These were successfully used for the separation of proteins and a cytochrome c digest.
ELECTROPHORESIS, 2007
We examined the use of monolithic capillary columns prepared via ring-opening metathesis polymerization (ROMP) for peptide separation in voltage-assisted capillary LC (voltage-assisted CLC). In order to demonstrate their potential for peptide separation, ROMPderived monoliths with RP properties were prepared. The preparation procedure of monoliths was transferred from ROMP monoliths optimized for CLC. ROMP monoliths were synthesized within the confines of 200 mm id fused-silica capillaries with a length of 37 cm. After optimization of the chromatographic conditions, the separation performance was tested using a well-defined set of artificial peptides as well as two peptidic mixtures resulting from a tryptic digest of BSA as well as a collagenase digest of collagen. ROMP monoliths showed comparable performance to other monolithic separation media in voltageassisted CLC published so far. Therefore, we conclude that by optimizing the composition of the ROMP monoliths as well as by using the controlled manner of their functionalization, ROMP monoliths bear a great potential in CLC and CEC.
Journal of Liquid Chromatography & Related Technologies, 2019
The performance of a 100 um i.d. open tubular column modified with linear co-polymer chains has been explored under low pressure operating conditions. A 512 mm fused-silica capillary was reacted with 3-chloropropyl isocyanate followed by sodium diethyldithiocarbamate. A copolymer layer was then immobilized on the inner surface via reversible addition-fragmentation transfer polymerization of styrene, methacrylic acid and N-phenylacrylamide. The thickness of the polymer layer was ca 1.5 mm. The column was evaluated for the separation of a mixture composed of three peptides (Trp-Gly, angiotensin I, bradykinin). The number of theoretical plates (N) as high as 72,100 plates/column (corresponding to 140,800/m, 7.10 mm plate height) was achieved. The column to column and day to day reproducibility of our columns was found better than 2.1%. For comparison purpose, similar chromatographic data for a selected analyte was also obtained with a commercial microcolumn of 0.5 mm id packed with a conventional C18 phase (5 mm), and 17 mm was the plate height obtained at the optimum flow rate. The van Deemter plots and kinetic plots were also obtained for comparison between our open tubular capillary column and the commercial packed column of a narrow id.
Journal of Chromatography A, 2011
A systematic study is reported on the performance of long monolithic capillary columns in gradient mode. Using a commercial nano-LC system, reversed-phase peptide separations obtained through UVdetection were conducted. The chromatographic performance, in terms of conditional peak capacity and peak productivity, was investigated for different gradient times (varying between 90 and 1320 min) and different column lengths (0.25, 1, 2 and 4 m) all originating from a single 4 m long column. Peak capacities reaching values up to n = 10 3 were measured in case of the 4 m long column demonstrating the high potential of these long monoliths for the analysis of complex biological mixtures, amongst others. In addition, it was found that the different column fragments displayed similar flow resistance as well as consistent chromatographic performance in accordance with chromatographic theory indicating that the chromatographic bed of the original 4 m long column possessed a structural homogeneity over its entire length.
Journal of Chromatography A, 2007
Monolithic silica capillary columns for hydrophilic interaction liquid chromatography (HILIC) were prepared by on-column polymerization of acrylic acid on monolithic silica in a fused silica capillary modified with anchor groups. The products maintained the high permeability (K = 5 × 10 −14 m 2 ) and provided a plate height (H) of less than 10 m at optimum linear velocity (u) and H below 20 m at u = 6 mm/s for polar solutes including nucleosides and carbohydrates. The HILIC mode monolithic silica capillary column was able to produce 10 000 theoretical plates (N) with column dead time (t 0 ) of 20 s at a pressure drop of 20 MPa or lower. The total performance was much higher than conventional particle-packed HILIC columns currently available. The gradient separations of peptides by a capillary LC-electrospray mass spectrometry system resulted in very different retention selectivity between reversed-phase mode separations and the HILIC mode separations with a peak capacity of ca. 100 in a 10 min gradient time in either mode. The high performance observed with the monolithic silica capillary column modified with poly(acrylic acid) suggests that the HILIC mode can be an alternative to the reversed-phase mode for a wide range of compounds, especially for those of high polarity in isocratic as well as gradient elution.
Journal of Chromatography A, 2008
Monolithic columns have been prepared via ring-opening metathesis polymerization using different monomers and crosslinkers, i.e. norborn-2ene, 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo,endo-dimethanonaphthalene, cyclooctene and tris(cyclooct-4-en-1-yloxy)methylsilane. 2-Propanol and toluene were used as macro-and microporogens. Alternatively, monolithic supports were realized via electron beam triggered free radical polymerization using trimethylolpropane triacrylate and ethylmethacrylate. Here, 2-propanol, 1-dodecanol and toluene were used as porogens. The three monolithic supports were structurally characterized by inverse size exclusion chromatography and investigated for their separation capabilities for a series of proteins. Separation efficiencies are discussed within the context of the different structural features of the monolithic supports and are compared to the separation data obtained on a commercial silica-based Chromolith ® RP-18e column.
Angewandte Chemie International Edition, 2000
Functionalized monolithic materials have been prepared by ring-opening metathesis copolymerization of norborn-2-ene (NBE) and 1,4a, within borosilicate columns in the presence of porogenic solvents such as toluene, methylene chloride, methanol, and 2-propanol. Grubbs-type initiators of the general formula Cl 2(PR3)2-Ru(dCHPh) (R ) phenyl, cyclohexyl) were used throughout. The resulting separation media possess microstructures consisting of microporous, spherical microglobules with a narrow microglobule size distribution. By variation of the polymerization conditions in terms of stoichiometry of the monomers, porogenic solvents, and temperature, microglobule diameters may be varied within a range of 2 ( 1 µm up to 30 ( 10 µm. Specific surface areas (σ) and inter-microglobule pore volumes ( z) may be altered within 60-210 m 2 /g and 2-50%, respectively. Nonfunctionalized separation media were successfully used for the separation of 10 different model proteins using reversed phase chromatography. Generally, high flow rates of up to 10 mL/min indicating fast mass transfer may be applied, resulting in fast and efficient separations. Functionalized continuous rods were synthesized by one additional synthetic step that takes advantage of the living character of the ROMP-based copolymerization. This "in situ" derivatization was achieved after the formation of the continuous rod by reacting the active, surface-bound initiator with functional, norborn-2-ene-and 7-oxanorborn-2-ene-based monomers such as endo,endo-7-oxabicyclo[2.2.1]hept-2-ene-5,6-dicarboxylic anhydride (I), (X), endo/exo-norborn-2-ene-5-ylmethylhydroxysiloxyl--cyclodextrin (XI), and endo/exo-7-oxanorborn-2-ene-5-carboxyl--cyclodextrin (XIII), by passing solutions thereof in dichloromethane and DMF over the rigid rod. A -cyclodextrin-functionalized continuous rod was successfully used for the enantioselective separation of proglumide ( -blocker).