Counterflow isotachophoresis in a monolithic column

This study describes stationary counterflow isotachophoresis (ITP) in a poly(acrylamide‐co‐N,N′‐methylenebisacrylamide) monolithic column as a means for improving ITP processing capacity and reducing dispersion. The flow profile in the monolith was predicted using COMSOL's Brinkman Equation application mode, which revealed that the flow profile was mainly determined by monolith permeability. As monolith permeability decreases, the flow profile changes from a parabolic shape to a plug shape. An experimental monolithic column was prepared in a fused‐silica capillary using an ultraviolet‐initiated polymerization method. A monolithic column made from 8% (wt.) monomer was chosen for the stationary counterflow ITP experiments. Counterflow ITP in the monolithic column showed undistorted analyte zones with significantly reduced dispersion compared to the severe dispersion observed in an open capillary. Particularly, for r‐phycoerythrin focused by counterflow ITP, its zone width in the monolithic column was only one‐third that observed in an open capillary. These experiments demonstrate that stationary counterflow ITP in monoliths can be a robust and practical electrofocusing method.     

Characterization of some physical and chromatographic properties of monolithic poly(styrene-co-divinylbenzene) columns

Monolithic capillary columns were prepared by copolymerization of styrene and divinylbenzene inside a 200 microm i.d. fused silica capillary using a mixture of tetrahydrofuran and decanol as porogen. Important chromatographic features of the synthesized columns were characterized and critically compared to the properties of columns packed with micropellicular, octadecylated poly(styrene-co-divinylbenzene) (PS-DVB-C18) particles. The permeability of a 60 mm long monolithic column was slightly higher than that of an equally dimensioned column packed with PS-DVB-C18 beads and was invariant up to at least 250 bar column inlet pressure, indicating the high-pressure stability of the monolithic columns. Interestingly, monolithic columns showed a 3.6 times better separation efficiency for oligonucleotides than granular columns. To study differences of the molecular diffusion processes between granular and monolithic columns, Van Deemter plots were measured. Due to the favorable pore structure of monolithic columns all kind of diffusional band broadening was reduced two to five times. Using inverse size-exclusion chromatography a total porosity of 70% was determined, which consisted of internodule porosity (20%) and internal porosity (50%). The observed fast mass transfer and the resulting high separation efficiency suggested that the surface of the monolithic stationary phase is rather rough and does not feature real pores accessible to macromolecular analytes such as polypeptides or oligonucleotides. The maximum analytical loading capacity of monolithic columns for oligonucleotides was found to be in the region of 500 fmol, which compared well to the loading capacity of the granular columns. Batch-to-batch reproducibility proved to be better with granular stationary phases compared to monolithic stationary phase, in which each column bed is the result of a unique column preparation process.     

Highly efficient monolithic silica capillary columns modified with poly(acrylic acid) for hydrophilic interaction chromatography

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 x 10(-14)m(2)) and provided a plate height (H) of less than 10 microm at optimum linear velocity (u) and H below 20 microm at u=6mm/s for polar solutes including nucleosides and carbohydrates. The HILIC mode monolithic silica capillary column was able to produce 10000 theoretical plates (N) with column dead time (t(0)) of 20s 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.     

Short communication performance of octadecylsilylated monolithic silica capillary columns of 530 microm inner diameter in HPLC

Monolithic silica capillary columns were successfully prepared in a fused silica capillary of 530 microm inner diameter and evaluated in HPLC after octadecylsilylation (ODS). Their efficiency and permeability were compared with those of columns pakked with 5-microm and 3-microm ODS-silica particles. The monolithic silica columns having different domain sizes (combined size of through-pore and skeleton) showed 2.5-4.0-times higher permeability (K= 5.2-8.4 x 10(-14) m2) than capillary columns packed with 3-mm particles, while giving similar column efficiency. The monolithic silica capillary columns gave a plate height of about 11-13 microm, or 11 200-13 400 theoretical plates/150 mm column length, in 80% methanol at a linear mobile phase velocity of 1.0 mm/s. The monolithic column having a smaller domain size showed higher column efficiency and higher pressure drop, although the monolithic column with a larger domain size showed better overall column performance, or smaller separation impedance (E value). The larger-diameter (530 microm id) monolithic silica capillary column afforded a good peak shape in gradient elution of proteins at a flow rate of up to 100 microL/min and an injection volume of up to 10 microL.     

Preparation and characterization of monolithic column by grafting pH-responsive polymer

A novel modified monolithic column with pH-responsive polymer chains was prepared by grafting methacrylic acid onto the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith. The grafting polymerization was achieved in an in situ manner which was performed by pumping methacrylic acid directly through an acidic hydrolysis monolithic column using potassium peroxydisulfate initiated free-radical polymerization. The grafted monolithic column was demonstrated to be the pH-responsive to the pore structure and the chromatographic characterization. The permeability of the column and the retention factors of five benzene homologues decreased due to the conformational changes of the polymer chains when the pH of mobile phase increased from 4.5 to 7.5. Furthermore, the modified monolithic column was used as the pH-responsive stationary phase and exhibited an excellent separation of four basic proteins.     

Coupled affinity-hydrophobic monolithic column for on-line removal of immunoglobulin G, preconcentration of low abundance proteins and separation by capillary zone electrophoresis

A butyl methacrylate-co-ethylene dimethacrylate (BuMA-co-EDMA) monolith was synthesized by UV initiated polymerization at the inlet end of a 75 microm I.D. fused silica capillary that had been previously coated with a protein compatible polymer, poly(vinyl)alcohol. The monolith was used for on-line preconcentration of proteins followed by capillary electrophoresis (CE) separation. For the analysis of standard proteins (cytochrome c, lysozyme and trypsinogen A) this system proved reproducible. The run-to-run %RSD values for migration time and corrected peak area were less than 5%, which is typical of CE. As measured by frontal analysis using lysozyme as solute, saturation of a 1cm monolith was reached after loading 48 ng of protein. Finally, the BuMA-co-EDMA monolithic preconcentrator was coupled to a protein G monolithic column via a zero dead volume union. The coupled system was used for on-line removal of IgG, preconcentration of standard proteins and CE separation. This system could be a valuable sample preparation tool for the analysis of low abundance proteins in complex samples such as human serum, in which high abundance proteins, e.g., human serum albumin (HSA) and immunoglobulin G (IgG), hinder identification and quantification of low abundance proteins.     

Effects of inner diameter of monolithic column on separation of proteins in capillary-liquid chromatography

Polymer monolithic columns with I.D. between 100 and 320 microm were prepared by in-situ polymerization of styrene and divinylbenzene in fused silica capillaries. The effects of monolithic column I.D. on the separation of proteins in reversed-phase capillary-liquid chromatography under gradient elution were systemically studied. The loading capacity was positively proportional to the volume of the stationary phase. It was found that the smaller diameter columns showed better performance for protein separation. The minimum plate height decreases from 34.99 microm (320 microm I.D. column) to 5.39 microm (100 microm I.D. column) for a retained protein. After studying the three parameters of the Van Deemter equation, it was interpreted that the smaller diameter can provide less flow resistance and the better performance may also be improved by the increasing of the effective diffusion. This conclusion was also supported by the data of separation permeability and breakthrough curves.     

Polymetacrylate and hybrid interparticle monolithic columns for fast separations of proteins by capillary liquid chromatography

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 microm. 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.     

Silica particles encapsulated poly(styrene-divinylbenzene) monolithic stationary phases for micro-high performance liquid chromatography

In the paper we demonstrate a new approach for the preparation and application of continuous silica bed columns that involve encapsulation (entrapment) of functionalized silica microparticles, which can be used as packing material in micro high performance liquid chromatography (micro-HPLC) and capillary electrochromatography (CEC). Like traditional packed columns, these capillaries possess characterized silica particles that offer high phase ratio and narrow pore size distribution leading to high retention and separation efficiency, respectively. More importantly, immobilization of the microparticles stabilizes the separation bed and eliminates the need for retaining frits. The developed capillary columns were fabricated in exactly the same way as a packed capillary column (slurry packing) but with an additional entrapment step. This immobilization of the packed bed was achieved by in situ polymerization of styrene and divinylbenzene in presence of decanol as a porogen and azobisisobutyronitrile as thermal initiator. Silica particles with different particle sizes and pore sizes ranging from 60 to 4000 A were studied. In addition different modified silica was used, including C-18 reversed phase, anion exchange and chiral stationary phases. Efficient separation of polyphenolic compounds, peptides, proteins and even DNA mutation were achieved using the developed technique depending on the properties of the silica particles used (particles pore size). For example, using 3 microm ProntoSIL C-18 particles with 300 A pore size, separation efficiencies in the range of 120,000-200,000 plates/m were obtained for protein separation, in a 6 cm x 200 microm i.d. capillary column. Using encapsulated silica C-18 with 1000 A pore size, separation of DNA homo and hetero duplexes were achieved under denaturing HPLC conditions for mutation detection. In addition, nucleotides were separated using anion exchange material encapsulated with poly(styrene-divinylbenzene) (PS/DVB), which indicated that the chromatographic properties of the silica packing material were still active after polymerization. The prepared capillary columns were found to be stable and could easily be operated continuously up to a pressure of 350 bar without column damage and capillary can be cut to any desired length.     

Preparation of poly(N-vinylpyrrolidone-co-pentaerythritol triacrylate) monolithic column for hydrophilic interaction chromatography

A method for the preparation of poly(N-vinylpyrrolidone-co-pentaerythritol triacrylate copolymerization)-based monolithic capillary column was reported for the separation of polar small molecular weight compounds with nano-liquid chromatography in hydrophilic interaction chromatography mode. The monolithic columns were prepared by in situ copolymerization of N-vinylpyrrolidone and a cross-linker pentaerythritol triacrylate in a binary porogenic agent consisting of methanol and water. The composition of the polymerization solution was systematically optimized in terms of column permeability, theoretical plate number, asymmetric factor, and retention factor. A typical hydrophilic chromatography retention mechanism was observed with a mobile phase composed of a high content of organic solvent. The preparation method is simple and robust, the precursor N-vinylpyrrolidone is chemically stable, cheap, and easily available. The N-vinylpyrrolidone-based hydrophilic interaction chromatography stationary phase displays satisfactory separation selectivity for a range of polar test analytes, including benzoic acid derivatives, nucleosides, and phenols.     

Simple 2D-HPLC using a monolithic silica column for peptide separation

Separation of peptides by fast and simple two-dimensional (2D)-HPLC was studied using a monolithic silica column as a second-dimension (2nd-D) column. Every fraction from the first column, 5 cm long (2.1 mm ID) packed with polymer-based cation exchange beads, was subjected to separation in the 2nd-D using an octadecylsilylated (C18) monolithic sillica column (4.6 mm ID, 2.5 cm). A capillary-type monolithic silica C18column (0.1 mm ID, 10 cm) was also employed as a 2nd-D column with split flow/injection. Effluentof the first dimension (1st-D) was directly loaded into an injector loop of 2nd-D HPLC. UV and MS detection were successfully carried out at high linear velocity of mobile phase at 2nd-D using flow splitting for the 4.6 mm ID 2nd-D column, or with directconnection of the capillary column to the MS interface. Two-minute fractionation inthe 1st-D, 118-second loading, and 2-second injection by the 2nd-D injector, allowed one minute for gradient separation in the 2nd-D, resulting in a maximum peak capacity of about 700 within 40 min. The use of a capillary column in solvent consumption and better MS detectability compared to a larger-sized column. This kind of fast and simple 2D-HPLC utilizing monolithic silica columns will be useful for the separation of complex mixtures in a short time.     

Reduced-bore monolithic silica column modified with C8-TEOS for reversed-phase electrochromatography

Monolithic silica columns of 2.7 mm ID were prepared and derivatized with C8-TEOS and TEOS by on-column sol-gel reaction. These C8 large diameter monolithic silica columns gave 21 000 theoretical plates for aromatic hydrocarbons in 60% acetonitrile and 40% Tris-HCI buffer. The surface areas as well as the separation reproducibility were improved on coating by the sol-gel approach. Joule heating was greatly reduced by using monolithic columns to which fine quartz sand had been added during column preparation. Since this is a preliminary investigation on a monolithic column with such a large inner diameter, the separation efficiency was not so high as that presently achieved in normal capillary electrochromatography (CEC). However, use of the columns improved sample loadability and concentration detectability of electrochromatography, and semi-preparative separations could be performed.     

Two-dimensional strong cation exchange/porous layer open tubular/mass spectrometry for ultratrace proteomic analysis using a 10 microm id poly(styrene- divinylbenzene) porous layer open tubular column with an on-line triphasic trapping column

This study expands the capabilities for ultratrace proteomic analysis of our previous work by incorporating on-line sample desalting using a triphasic (RP/strong cation exchange (SCX)/micro-SPE) trapping column connected to a 3.2 m x 10 microm id poly(styrene-divinylbenzene) (PS-DVB) porous layer open tubular (PLOT) column. To minimize extra sample handling steps, C18 RP packing was incorporated in the capillary tubing upstream of the SCX column for the on-line desalting. For the micro-SPE column, a 50 microm id PS-DVB monolithic column was positioned downstream of the SCX column. High-performance separation was achieved on the PLOT column at a mobile phase flow rate of 20 nL/min. The sensitivity and high resolution capability of the new multidimensional platform was evaluated using an in-gel tryptic digested sample of a cervical cancer (SiHa) cell line. For the injected amount of 1200 cells ( approximately 500 ng), over 2700 peptides covering greater than 850 unique proteins were identified from the triphasic SCX/PLOT/MS analysis of a single SDS gel section (>40 kDa). The 2-D LC/MS platform demonstrated good separation performance, such that more than 85% of the identified peptides were detected from only one salt fraction. In a triplicate analysis of the above >40 kDa gel section, 4497 peptides and 1209 unique proteins were identified when applying stringent filtering criteria, with a false-positive rate of 2.4%. When all three SDS-PAGE gel sections of the lysed SiHa cells were analyzed, 5047 peptides and 1857 unique proteins (false-positive rate 1.8%), including cancer-related proteins such as MAP kinases, were identified.     

Separation of parabens in capillary electrochromatography using poly(styrene-divinylbenzene-methacrylic acid) monolithic column

In this study, a series of poly(styrene-divinylbenzene-methacrylic acid) monolithic capillaries was used as the separation column of CEC for the analyses of parabens in commercial pharmaceutical and cosmetic products. The results showed that the chromatographic characteristics of these analytes were strongly affected by the preparation condition of the monolithic column including monomer content, porogenic solvent composition, and polymerization time. Baseline separations were markedly sped up by lowering the polymerization time without any obvious loss of resolution. Furthermore, mobile-phase composition (pH, ACN, and electrolyte concentration) was also able to effectively improve the separation behavior. Similar to the influence of lowering the polymerization time, retention times for all analytes were significantly shortened in the CEC method by decreasing the electrolyte concentration in the mobile phase.     

Study of a monolithic silica capillary column coated with poly(octadecyl methacrylate) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds

The performance of a monolithic silica capillary column coated with poly(octadecyl methacrylate) (ODM column) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds was studied, and the results were compared to those obtained by using a monolithic silica capillary column modified with octadecylsilyl-(N,N-diethylamino)silane (ODS column). Benzene and naphthalene derivatives, polycyclic aromatic hydrocarbons (PAHs), steroids, alkyl phthalates, and tocopherol homologues were used as test samples. In general, compounds with aromatic character, rigid and planar structures, and lower length-to-breadth ratios (more compacted structures) seem to have more preference for the polymer coated stationary phase (ODM). Compounds with acidic character have also a higher retention on ODM columns because of the presence of ester groups in the stationary phase. The polymer coated column allowed the separation of some PAHs, alkyl phthalates, steroids, and of beta- and gamma-tocopherol isomers which cannot be separated under the same conditions on ODS columns, while keeping similar column efficiency. These results allowed to suggest ODM columns as a good alternative to conventional ODS columns for reversed-phase liquid chromatography.     

Optimized preparation of poly(styrene-co- divinylbenzene-co-methacrylic acid) monolithic capillary column for capillary electrochromatography

Preparation of a poly(styrene-co-divinylbenzene-co-methacrylic acid) monolithic stationary phase for the use in capillary electrochromatography (CEC) has been improved by optimizing the polymerization conditions. It is observed that the reaction time strongly affects column efficiency, while the proportion of isooctane in porogen influences peak symmetry of some solutes seriously. The lifetime of the monolithic columns prepared mainly depends on the pH of buffers used. Reproducibility of electroosmotic flow (EOF) from batch to batch columns are lower than 2.8% relative standard deviation. Unlike other types of capillary electrochromatographic monoliths, a pH-dependent EOF was observed on this type of column. Separation of various types of compounds including aromatic hydrocarbons, hormones, anilines, basic pharmaceuticals, and peptides was achieved. The facile preparation and wide application of this monolithic column may make styrene-based polymer a potential stationary phase in CEC.     

Monolithic poly(ethylhexyl methacrylate-co-ethylene dimethacrylate) column with restricted access layers prepared via reversible addition-fragmentation chain transfer polymerization

The "living"/controlled radical polymerization has provided an opportunity in making a more homogeneous polymer, which is favorable for polymer-based monolithic column fabrication. To study its application in the preparation of separation material, a capillary poly(ethylhexyl methacrylate-co-ethylene dimethacrylate) monolithic column has been synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The correlation between the synthetic conditions and the polymer structures and separation performance was studied. The result indicated RAFT-mediated reaction provides condition for creating polymers with narrower pore size distribution and higher column efficiency compared with traditional polymerization. The "living" property of the RAFT polymerization was further utilized to graft hydrophilic polymer on the surface of poly(ethylhexyl methacrylate-co-ethylene dimethacrylate). The hydrophilic chain modified monolithic column has both abilities of protein exclusion and small hydrophobic compound retention. The result indicated that RAFT polymerization can be used for making multifunctional material. The restricted access monolithic material synthesized by this method can be used in biological sample analysis with HPLC direct injection.     

Study of triacontyl-functionalized monolithic silica capillary column for reversed-phase capillary liquid chromatography

A novel stationary phase triacontyl-functionalized monolithic silica capillary column was successfully prepared for reversed-phase capillary liquid chromatography. The performance of the monolithic silica capillary column coated with triacontyl chain for the separation of alkylbenzenes, xylene isomers, polycyclic aromatic hydrocarbons, and mixture of α- and β-carotenes was studied, which was compared to that using the monolithic silica capillary column coated with octadecyl chain. The comparison results showed that triacontyl-functionalized monolithic silica capillary column would be a promising media to be used for the separation of isomeric solutes with long chain in reversed-phase capillary liquid chromatography.     

Capillary high-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic columns and carbon fiber electrospray ionization emitters

Monolithic columns having long hydrocarbon chains were prepared by in-situ polymerization in capillary fused silica tubing. The capillary columns were coupled with a newly developed carbon fiber electrospray ionization (ESI) emitter for proteomic analysis using sheathless capillary HPLC-ESI mass spectrometry (MS). The sample loading capacity and chromatographic performance of the styrene-based monolithic column, which was prepared by photo-polymerization of octylstyrene (OS) and divinylbenzene (DVB) were compared with that of the methacrylate-based monolithic column composed of lauryl methacrylate (LMA) and ethylene dimethacrylate (EDMA). The sample loading ability of tryptic digested protein in poly-OS (POS)-DVB column was higher than that of poly-LMA (PLMA)-EDMA column, possibly due to the irregular and rugluous surface offering a greater surface area of POS-DVB stationary phase. The POS-DVB column also provided better separation efficiency in the separation of high concentration (10 microg) of tryptic digested albumin bovine serum (BSA). Due to the successful interface of a highly efficient monolithic column and a stable, durable carbon fiber emitter, low femtomole levels of peptides were successfully separated and identified in the presence of large amounts of tryptic digested protein.     

Enantiomeric separation of amino acids and nonprotein amino acids using a particle-loaded monolithic column

A solution is prepared of 5 microm silica particles modified with (S)-N-3,5-dinitrobenzoyl-1-naphthylglycine (particle 1) or (S)-N-3,5-dinitrophenylaminocarbonyl-valine (particle 2) suspended in liquid tetraethylorthosilicate, ethanol, and aqueous hydrochloric acid. This solution is injected under pressure into a 30 cm long, 75 microm inner diameter capillary column and heated for 1 h at 120 degrees C after which the modified particles are embedded in a monolithic column of sol gel. The packed column measures approximately 15 cm from the inlet to the window used to view the laser-induced fluorescence. Thirteen different amino acids and three nonprotein amino acids are derivatized with the fluorogenic reagent 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) before injection onto the column for capillary electrochromatographic separation. The enantiomeric separation of the monolithic column packed with particle 1 results in a resolution ranging from 1.14 to 4.45, whereas that packed with particle 2 results in a resolution ranging from 0.79 to 1.17. On the basis of resolution and amount of chiral packing material the enantiomeric separation obtained by capillary electrochromatography is judged to be superior to that obtained previously with high performance liquid chromatography (HPLC).