Monolithic Disk for the Fast Chromatographic Separation (original) (raw)
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Indonesian Journal of Chemistry, 2010
Capillary column with monolithic stationary phase was prepared from silanized fused-silica capillary of 200 µm I.D. by in situ free radical polymerization of divinylbenzene with glycidy methacrylate in the presence of decanol and tetrahydrofuran as porogens. The hydrodynamic and chromatographic properties of this monolith, such as backpressure at different flow-rate, pore size distribution, van Deemter plot and the effect of varying gradient-rate were investigated. Poly(glycidyl methacrylate-divinylbenzene) monolithic capillary has been used successfully for the reversed-phase chromatographic separation of proteins.
Journal of Separation Science, 2009
Original Paper 1 mm ID poly(styrene-co-divinylbenzene) monolithic columns for high-peak capacity one-and two-dimensional liquid chromatographic separations of intact proteins The LC performance of a 1650 mm polymer monolithic column format was demonstrated with high-peak capacity one-(1D) and offline two dimensional (2D) LC separations of intact proteins. After optimizing the RP 1D-LC conditions, including column temperature, flow rate and gradient time, a peak capacity of 475 was achieved within a 2-h analysis. The suitability of the monolithic column was also demonstrated for fast 1 min protein separations yielding 1 s peak widths determined at half peak height. In addition, an offline 2D-LC method was developed using the micro-fraction collection capabilities of the autosampler allowing automatic fractionation of intact proteins after the weak-ion-exchange (WAX) separation, and re-injection of the fractions onto the second-dimension RP monolithic column. The best peak capacity-to-analysis time ratio was obtained when applying 10 min seconddimension RP gradients. At optimized conditions, the WAX/6/RPLC separation of intact Escherichia coli proteins was performed within 6 h yielding a maximum theoretical peak capacity of 4880.
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.
Pharmaceutical Sciences Asia, 2020
A mixed-mode liquid chromatography PSDVB-PE solid support based on (R)-phenylephrine bonded non-porous polystyrene-divinylbenzene (PSDVB) was prepared and characterized for the fast separation of proteins. The support consisting of both anion-exchange and hydrophobic moieties was synthesized by bonding of the (R)-phenylephrine to the non-porous PSDVB particles via hydroboration, bromination and nucleophilic substitution reactions. The support with a surface coverage of 420 μmol/g was characterized with regard to its physical and chromatographic properties. The reverse phase and anion-exchange behaviors of this new stationary phase were investigated by injection of acidic, neutral and basic molecules at a wide pH range (pH 2 to 11.0) of eluents. The retention behaviors of the tested compounds (e.g. ethylparaben, benzoic acid, benzene and aniline) were studied by modifications of pH values and amounts of organic modifiers in the mobile phase in an isocratic elution. Fast separation of six proteins (e.g. cytochrome c, immunoglobulin G, ribonuclease A, human serum albumin, ovalbumin, and conalbumin) within 3 min showed many advantages over non-modified PSDVB support. Applications for separation of proteins in papaya latex, egg white, and human serum were demonstrated. Compare to the reverse phase column, the mixed-mechanism column provided a flexible and versatile method for fast separation of proteins.
Journal of Chromatography A, 2004
This report details the fabrication of polystyrene-based nano-LC monolithic columns for the separation of protein molecules. The report focuses on the practical advantages of monolithic columns when compared to conventional packed columns. Capillary columns were made to 100 and 50 m i.d. and used to analyse a mixture of proteins, these separations were then compared with a conventional protein phase under the same conditions. A second functionalised monolithic polystyrene-based column was also manufactured and compared for the analysis of proteins, under the same analytical conditions, to the standard non-functionalised bare polystyrene monolith. Nano-LC polystyrene monolithic columns were found to be advantageous to conventional phases for the analysis of protein molecules, with a one-step fabrication process, faster analysis times, lower limits of detection hence higher sensitivity.
Journal of Chromatography A, 2008
Poly(lauryl methacrylate-co-ethylene dimethacrylate) and poly(styrene-co-divinylbenzene) stationary phases in monolithic format have been prepared by thermally initiated free radical polymerization within polyimide chips featuring channels having a cross-section of 200 m × 200 m and a length of 6.8 cm. These chips were then used for the separation of a mixture of proteins including ribonuclease A, myoglobin, cytochrome c, and ovalbumin, as well as peptides. The separations were monitored by UV adsorption. Both the monolithic phases based on methacrylate and on styrene chemistries enabled the rapid baseline separation of most of the test mixtures. Best performance was achieved with the styrenic monolith leading to fast baseline separation of all four proteins in less than 2.5 min. The in situ monolith preparation process affords microfluidic devices exhibiting good batch-to-batch and injection-to-injection repeatability.
Journal of Chromatography A, 2015
A new spiral tube assembly was designed to improve the column capacity and partition efficiency for protein separation. This spiral tube assembly has greater column capacity than the original tubing because of an increase in radial grooves from 4 to 12 to accommodate more spiral layers and 12 narrow spots instead of 4 in each circular loop to interrupt the laminar flow that causes sample band broadening. Standard PTFE tubing (1.6 mm ID) and the modified flat-twisted tubing were used as the separation column. The performances of both assemblies were compared for separating 3 stable test proteins including cytochrome c, myoglobin, and lysozyme using a two phase aqueous-aqueous solvent system composed of polyethylene glycol 1000 (12.5% w/w) and dibasic potassium phosphate (12.5% w/w). All samples were run at 1,2, 3, and 5 mL/min at both 800 RPM and 1000 RPM. The separation of these three protein samples produced high stationary phase retentions at 1, 2, and 3 mL/min, yet separated efficiently at 5 mL/min in 40 minutes. After comparing the separation efficiency in terms of the peak resolutions, theoretical plate numbers, and separation times, it was determined that the flat-twisted tubing was more effective in separating these protein samples. In order to validate the efficacy of this novel assembly, a mixture of five protein samples (cytochrome c, myoglobin, ovalbumin, lysozyme, and hemoglobin) were separated, under the optimal conditions established with these 3 protein samples, at 1 mL/min with a revolution speed of 1000 RPM. There were high stationary phase retentions of around 60%, with effective separations, demonstrating the efficiency of the flat-twisted spiral tube assembly. The separation time of 6 hours was a limitation but can potentially be shortened by improving the strength of the column that will permit an increase in revolution speed and flow rate. This novel spiral separation column will allow rapid and efficient separation of mixtures with high yield of the constituent components.
Journal of separation science, 2015
Polymethacrylate-based monolithic capillary columns, prepared by γ-radiation-induced polymerization, were used to optimize the experimental conditions (nature of the organic modifiers, the content of trifluoroacetic acid and the column temperature) in the separation of nine standard proteins with different hydrophobicities and a wide range of molecular weights. Because of the excellent permeability of the monolithic columns, an ion-pair reversed-phase capillary liquid chromatography with high-resolution mass spectrometry method has been developed by coupling the column directly to the mass spectrometer without a flow-split and using a standard electrospray interface. Additionally, the high working flow and concomitant high efficiency of these columns allowed us to employ a longer column (up to 50 cm) and achieve a peak capacity value superior to 1000. This work is motivated by the need to develop new materials for high-resolution chromatographic separation that combine chemical stab...
Journal of Chromatography A, 2014
Influence of acid concentration in the mobile phase on protein separation was studied in a wide concentration range using trifluoroacetic acid (TFA) and formic acid (FA). At low, 0.001-0.01 (v/v%) TFA concentration and appropriate solvent strength proteins elute before the column's dead time. This is explained by the proteins having a structured, but relatively extended conformation in the eluent; and are excluded from the pores of the stationary phase. Above ca. 0.01-0.05 (v/v%) TFA concentration proteins undergo further conformational change, leading to a compact, molten globule-like structure, likely stabilized by ion pairing. Proteins in this conformation enter the pores and are retained on the column. The results suggest a pore exclusion induced separation related to protein conformation. This effect is influenced by the pH and type of acid used, and is likely to involve ion-pair formation. The TFA concentration needed to result in protein folding (and therefore to observe retention on the column) depends on the protein; and therefore can be utilized to improve chromatographic performance. Conformation change was monitored by circular dichroism spectroscopy and mass spectrometry; and it was shown that not only TFA but FA can also induce molten globule formation.
Journal of Chromatography A, 2017
We added some comments in the text relating to the new literature references #66 and 67. 26. The performance of the monoliths presented in impressive for the separation of small molecules, which is generally not achieved by other research groups. But the main question is: (1) why do your monoliths perform so well (is it better homogeneity or are there no surface diffusion/gel porosity effects and why not?) and (2) if the monoliths perform so much better for small molecules does this mean that the performance of large molecule separations is also better. Discussion on these aspects is very much appreciated! We have not addressed these points in the paper, especially the relationship, if any, between the observed efficiency for small and large (bio)molecules. At this point, any comment would be speculative and we are planning to deeply investigate the interesting subject in a near future. 27. General comment: improve figure quality (4, 5, 6, 9).-labels on x and y axis, sometimes the font/size, sometimes the content!.-check the units.-check if appropriate scale are used (van Veemter curves: x-axis 5-6 mm/s does not show data points, kinetic plots y axis should be adjusted (there is no data in 10-1 and 10-2 range and 10-4 to 10-5 range, so please do not show this range). Corrected 28. Grammar last 2 highlights should be improved. Corrected Reviewer #2: Summary This work encompasses synthesis of poly(lauryl methacrylate-co-1,6-hexanediol dimethacrylate) monolithic columns in capillary diameters (75 µm, 200 µm and 250 µm i.d.) with a view to developing suitable materials for reversed-phase LC separation of small molecules, and to reinforce the suitability of the approach by demonstrating biomolecule separations. The novelty of the work is in the pre-treatment of capillary to allow a "grafting-to" approach to be used for anchoring the -ray initiated monolithic co-polymer to the surface. While no direct comparison to columns prepared using conventional pre-treatment procedures has been made within this work, the material has been structurally characterized (IR, solid state NMR, cryogenic NMR & SEM), compared to a thermally-initiated equivalent (using AIBN), with a number of chromatographic experiments with small and large molecules also shown. Isocratic chromatographic comparison centers around evaluation of newly-synthesized materials against a column packed with 5 µm C18functionalized particles in identical dimensions to the monolithic columns. Some further examples of biomolecule separations are shown in the gradient mode with high-resolution MS (Orbitrap). The monolith prepared is primarily novel due to the pre-treatment process used. Previous publications from this group (refs [17] & [43]) contain extensive examples of peptide and protein separations including peak capacity calculations. Thus, the biomolecule work is of secondary value, except that the best material is shown to demonstrate very similar characteristics as the same stationary phase material synthesized with a conventional pre-treatment process from previous publications. However, the performance for small molecule separations is excellent and shows some behavior that is contrast to previously-published materials of similar origin. Specifically the retention-dependence of plate height seems to be almost non-existent, which is a major problem for most organic polymer monoliths used for analytical chromatography of small molecules. There are some details missing from technical aspects of the work that I believe need to be addressed before publication. This includes a full explanation of the correction of plate height data for the chromatographic comparison (including the strange behavior of the packed column in terms We changed the Highlights accordingly, emphasizing the performances for both small molecules and proteins Abstract Line 41: has "reproducibility" been assessed here? Unless a different user, location, or instrument was the focus of this part of the study, then it is "repeatability"-e.g. batch-to-batch, column-tocolumn, run-to-run, interday… etc. Please choose accordingly. Changed Line 42: again, "methacrylate-based" would be better here. Changed Line 64: "They are most commonly obtained by a single-step…" Changed Line 70: "…suitable for high efficiency separations of large biomolecules,…" Changed Line 73: "…preparation and, consequently, monolithic…" Lines 77-78: "…used to implement the sensitivity of large molecules…" does not make sense. Please revise. Changed Line 79: "The majority of recent developments have been…" Changed Line 84: "microwave-assisted polymerization process." Line 85: missing comma after "photo-induced" Changed Line 408: "…for high speed and high efficiency separations…" Changed Line 414: "inspection" Changed Lines 417-419: see later comments on retention-dependence. A useful reference for comparison is: