Poly(Glycidyl Methacrylate-Divinylbenzene) Monolithic Capillary as a Stationary Phase for the Reversed-Phase Chromatographic Separation of Proteins (original) (raw)
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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.
Monolithic Disk for the Fast Chromatographic Separation
Indonesian Journal of Chemistry, 2010
Poly(styrene/divinylbenzene) (PS/DVB) monolithic disk was prepared by in situ free-radical copolymerization of styrene and divinylbenzene in the presence of decanol and tetrahydrofuran as porogens. PS/DVB monolithic disks were produced in two different lengths 1.5 mm and 3 mm. The disks were used in reversed phase chromatography of proteins with 0.2 % trifuoroacetic acid (TFA) and 0.2 % TFA in acetonitrile as mobile phase A and B, respectively. The effect of gradient rate, flow rate, temperature and disk length on the separation of proteins were also studied. PS/DVB monolithic disks allow the rapid separation of proteins in reversed phase chromatography.
Asian Journal of Chemistry, 2014
Capillary liquid chromatography has been one of the most important recent developments in separation and analysis technology. Capillary liquid chromatography offers several advantages over conventional normal chromatography. The advantages include increased chromatographic resolution, higher efficiency, lower sample and solvent consumption, the ability to analyze and isolate rare compounds of interest, greater mass sensitivity and ease of on-line connection to a mass spectrometer 1-3. Traditionally, capillary liquid chromatography uses fused silica capillaries prepared with a variety of stationary phases. This technique appears to be very promising for separating a wide variety of analytes for different applications 4-10. However, the successful development of this technique is closely related to the technical challenges associated with manufacturing the column. Monolithic columns have quickly become extremely popular and they have attracted increasing interest as separation media for all chromatographic methods. The unique structure of the monoliths and their ease of preparation offer improved chromatographic performance and favorable properties that
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:
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, 2006
In the present work, an orthogonal two-dimensional (2D) capillary liquid chromatography (LC) method for fractionation and separation of proteins using wide range pH gradient ion exchange chromatography (IEC) in the first dimension and reversed phase (RP) in the second dimension, is demonstrated. In the first dimension a strong anion exchange (SAX) column subjected to a wide range (10.5-3.5) descending pH gradient was employed, while in the second dimension, a large pore (4000Å) polystyrene-divinylbenzene (PS-DVB) RP analytical column was used for separation of the protein pH-fractions from the first dimension. The separation power of the off-line 2D method was demonstrated by fractionation and separation of human plasma proteins. Seventeen pH-fractions were manually collected and immediately separated in the second dimension using a column switching capillary RP-LC system. Totally, more than 200 protein peaks were observed in the RP chromatograms of the pHfractions. On-line 2D analysis was performed for fractionation and separation of ten standard proteins. Two pH-fractions (basic and acidic) from the first dimension were trapped on PS-DVB RP trap columns prior to back-flushed elution onto the analytical RP column for fast separation of the proteins with UV/MS detection.
Analytical Chemistry, 2002
Monolithic capillary columns were prepared by transition metal-catalyzed ring-opening metathesis copolymerization of norborn-2-ene and 1,4,4a,5,8,8a-hexahydro-1,4,5,8exo,endo-dimethanonaphthalene inside a silanized 200µm-i.d. fused-silica capillary using a mixture of toluene and 2-propanol as porogen and Cl 2 (PCy 3 ) 2 Ru(dCHPh) as initiator. The synthesized columns allowed the rapid and highly efficient separation of single-and double-stranded nucleic acids by ion-pair reversed-phase high-performance liquid chromatography and of proteins by reversedphase high-performance liquid chromatography. Compared to 3-mm-i.d. analytical columns synthesized from an identical polymerization mixture, a considerable improvement in the peak widths at half-height of oligonucleotides in the order of 60-80% was obtained. Significant differences in morphology between the capillary column, where the surface of the monolith was rather soft and rugulose, and the analytical column, where the surface was very sharp and smooth, were observed, most probably due to differences in polymerization kinetics. The synthesized monoliths were successfully applied to the separation of the diastereomers of phosphorothioate oligodeoxynucleotides. To confirm the identity of the eluting compounds on the basis of their intact molecular masses, the chromatographic separation system was on-line hyphenated to electrospray ionization mass spectrometry.
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 Biochemical and Biophysical Methods, 2007
There is a demand of novel high resolution separation media for separation of complex mixtures, particularly biological samples. One of the most flexible techniques for development of new separation media currently is synthesis of the continuous bed (monolithic) stationary phases. In this study the capillary format gradient stationary phases were formed using continuous bed (monolith) polymerization in situ. Different reversedphase stationary phase gradients were tailored and their resolution using capillary liquid chromatography and capillary electrochromatography at isocratic mobile phase conditions was evaluated. It is demonstrated, that efficiency and resolution of the gradient stationary phases can be substantially increased comparing to the common (isotropic) stationary phases. The proposed formation approach of the gradient stationary phase is reproducible and compatible with the capillary format or microchip format separations. It can be easily automated for the separation optimizations or mass production of the capillary columns or chips.