Polydopamine assisted fabrication of titanium oxide nanoparticles modified column for proteins separation by capillary electrochromatography

Development of a simple method for preparation of stable open tubular (OT) columns for proteins separation by capillary electrochromatography is still challenging. In this work, the titanium oxide (TiO₂) nanoparticles coated OT column was successfully prepared for separation of proteins by capillary electrochromatography. The polydopamine (PDA) film was first formed in the inner surface of a fused-silica capillary by the self-polymerization of dopamine under alkaline conditions. Then the TiO₂ coating was deposited onto the surface of pre-modified capillary with PDA by a liquid phase deposition process. The plentifully active hydroxyl groups in PDA coating can chelate with Ti⁴⁺ to boost the nucleation and growth of TiO₂ film. The as-prepared TiO₂ coated OT column was characterized by scanning electron microscopy and measurement of electroosmotic flow. Furthermore, the influence of liquid phase deposition time on the TiO₂ coating was investigated. The TiO₂ coated OT column was used for successful separation of two variants of β-lactoglobulin and eight glycoisoforms of ovalbumin. The column demonstrated good repeatability and stability. The relative standard deviations of migration times of proteins representing run-to-run, day-to-day, and column-to-column were less than 3.7%. Moreover, the application of the column was verified by successful separation of acidic proteins in egg white.     

Preparation of highly efficient monolithic silica capillary columns for the separations in weak cation-exchange and HILIC modes

Weak cation-exchange (WCX) and HILIC modes columns were prepared by on-column polymerization of acrylic acid on monolithic silica capillary columns modified with N-(3-triethoxysilylpropyl)methacrylamide anchor groups. The polymer-coated columns could be used for HILIC mode separation of pyridylamino (PA)-sugars and peptides including a tryptic digest of BSA, while for weak cation-exchange mode for the separation of proteins and nucleosides even at high linear velocity. The poly(acrylic acid) coated monolithic silica capillary columns showed greater retention toward PA-sugars than a polyacrylamide coated monolithic silica capillary columns prepared in the same manner. Proteins and nucleosides were separated effectively at pH 6.9 using the same column. The column provided fair permeability after the polymer-coating step. High-speed separation of proteins at u=4.66 mm/s with high efficiency was shown to be possible, while high-speed separation of nucleosides has achieved within one minute using the column at u=8.67 mm/s, suggesting that the column will be suitable for the second dimension separation of multidimensional HPLC systems.     

Preparation of highly efficient monolithic silica capillary columns for the separations in weak cation-exchange and HILIC modes

Weak cation-exchange (WCX) and HILIC modes columns were prepared by on-column polymerization of acrylic acid on monolithic silica capillary columns modified with N-(3-triethoxysilylpropyl)methacrylamide anchor groups. The polymer-coated columns could be used for HILIC mode separation of pyridylamino (PA)-sugars and peptides including a tryptic digest of BSA, while for weak cation-exchange mode for the separation of proteins and nucleosides even at high linear velocity. The poly(acrylic acid) coated monolithic silica capillary columns showed greater retention toward PA-sugars than a polyacrylamide coated monolithic silica capillary columns prepared in the same manner. Proteins and nucleosides were separated effectively at pH 6.9 using the same column. The column provided fair permeability after the polymer-coating step. High-speed separation of proteins at u = 4.66 mm/s with high efficiency was shown to be possible, while high-speed separation of nucleosides has achieved within one minute using the column at u = 8.67 mm/s, suggesting that the column will be suitable for the second dimension separation of multidimensional HPLC systems.     

Preparation and characterization of long alkyl chain methacrylate-based monolithic column for capillary chromatography

This paper describes the fabrication of long alkyl chain methacrylate monolithic materials for using as stationary phases in capillary liquid chromatography. Following deactivation of the capillary surface with 3-(trimethoxysilyl)propyl methacrylate (gamma-MAPS), monoliths were formed by co-polymerisation of stearyl methacrylate (SMA) with ethylene glycol dimethacrylate (EDMA) in the presence of the initiator AIBN and a mixture of porogens including iso-amyl alcohol and 1,4-butanediol. The monoliths were prepared in 100 microm i.d. capillaries and the composition of the polymerisation mixtures were optimised in terms of the ratio of SMA/EDMA, the porogen composition and ratio of porogen to monomers. As the porogen weight fraction decreased, the microglobules became smaller and as expected, the total porosity decreased. In order to determine the usability of such materials, the column permeability K was measured by pumping water through the columns at different linear flow rates. Good results were obtained when these capillaries were used to separate mixtures of weak acids, neutral and basic compounds.     

Characterization of Pore Structure in Biologically Functional Poly(2-Hydroxyethyl Methacrylate) - Poly(Ethylene Glycol) Diacrylate (PHEMA-PEGDA)

A copolymer composed of poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(ethylene glycol) diacrylate (PEGDA) (PHEMA-PEGDA) is structurally versatile. Its structure can be adjusted using the following porogens: water, sucrose, and benzyl alcohol. Using phase separation technique, a variety of surface architectures and pore morphologies were developed by adjusting porogen volume and type. The water and sucrose porogens were effective in creating porous and cytocompatible PHEMA-PEGDA scaffolds. When coated with collagen, the PHEMA-PEGDA scaffolds accommodated cell migration. The PHEMA-PEGDA scaffolds are easy to produce, non-toxic, and mechanically stable enough to resist fracture during routine handling. The PHEMA-PEGDA structures presented in this study may expedite the current research effort to engineer tissue scaffolds that provide both structural stability and biological activity.     

Preparation and characterization of long alkyl chain methacrylate-based monolithic column for capillary chromatography

This paper describes the fabrication of long alkyl chain methacrylate monolithic materials for using as stationary phases in capillary liquid chromatography. Following deactivation of the capillary surface with 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS), monoliths were formed by co-polymerisation of stearyl methacrylate (SMA) with ethylene glycol dimethacrylate (EDMA) in the presence of the initiator AIBN and a mixture of porogens including iso-amyl alcohol and 1,4-butanediol. The monoliths were prepared in 100 μm i.d. capillaries and the composition of the polymerisation mixtures were optimised in terms of the ratio of SMA/EDMA, the porogen composition and ratio of porogen to monomers. As the porogen weight fraction decreased, the microglobules became smaller and as expected, the total porosity decreased. In order to determine the usability of such materials, the column permeability K was measured by pumping water through the columns at different linear flow rates. Good results were obtained when these capillaries were used to separate mixtures of weak acids, neutral and basic compounds.     

Analysis of recombinant human growth hormone by capillary electrophoresis with bilayer-coated capillaries using UV and MS detection

The characterization of recombinant human growth hormone (rhGH; somatropin) by capillary electrophoresis (CE) with UV-absorbance and mass spectrometric (MS) detection using capillaries noncovalently coated with polybrene (PB) and poly(vinyl sulfonic acid) (PVS) is demonstrated. Compared with bare fused-silica capillaries, PB-PVS coated capillaries yielded more favorable migration-time reproducibilities and higher separation efficiencies. Optimal separation conditions for the bilayer-coated capillaries comprised a background electrolyte (BGE) of 400 mM Tris phosphate (pH 8.5) yielding migration-time R.S.D.s of less than 1.0% and plate numbers above 300,000 for intact rhGH. The protein was also analyzed using the CE method described in the European Pharmacopoeia (Ph. Eur.) monograph. The pharmacopoeial method gave much longer analysis times (22 min versus 8 min), lower resolution and plate numbers, and consecutive shifts in migration time for rhGH, indicating possible interactions between the protein and the inner capillary wall. Due to stable migration times obtained with the coated capillaries, reliable profiling and quantification of rhGH and its byproducts in time was possible. Analysis of thermally degraded rhGH revealed the formation of two main degradation products. CE-mass spectrometry (MS) of this sample, using a PB-PVS coated capillary and a BGE of 75 mM ammonium formate (pH 8.5), suggests that these products are desamido forms of rhGH. Analyses of expired rhGH preparations with CE-UV and CE-MS indicated the presence of both deamidation and oxidation products.     

Analysis of bile acids in serum and bile by capillary gas-liquid chromatography

Various liquid phases for glass capillary columns have been evaluated for gas-liquid chromatographic analysis of methyl ester trimethylsilylether derivatives of bile acids from serum and bile. Bile acid analysis is rapid and exhibits high separation efficiency with a 20 X 0.3 mm glass capillary column whose internal surface is covered with a crystal layer of barium carbonate and coated with polyethyleneglycol 20000 as liquid phase according to Grob et al.     

Development of rigid bidisperse porous microspheres for high-speed protein chromatography

Development of a high-performance stationary phase is an essential demand for high-speed separation of proteins by liquid chromatography. Based on a novel porogenic mode, that is, using superfine granules of calcium carbonate as solid porogen and a mixture of cyclohexanol and dodecanol as liquid porogen, a rigid spherical biporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) matrix has been prepared by radical suspension-polymerization. The epoxide groups of the matrix were modified with diethylamine to afford the ionizable weak base 1-N,N-diethylamino-2-hydeoxypropy functionalities that are required for ion exchange chromatography. Results from scanning electron microscopy and mercury intrusion porosimetry measurements revealed that the matrix contained two families of pores, that is, micropores (10-90 nm) and macropores (180-4000 nm). Furthermore, the biporous medium possesses specific surface area as high as 91.3 m(2)/g. Because of the presence of the macropores that provided convective flow channels for the mobile phase, the dynamic adsorption capacity was found to be as high as 54.6 mg/g wet bead at 300 cm/h, approximately 63.2% of its static capacity. In addition, the column efficiency and dynamic binding capacity decreased only slightly with mobile-phase flow rate in the range of 300-3000 cm/h. These properties made the packed bed with the bidisperse porous matrix suitable for high-speed protein chromatography.     

Octadecylated silica monolith capillary column with integrated nanoelectrospray ionization emitter for highly efficient proteome analysis

An improved strategy for the preparation of octadecylated silica monolith capillary column with high homogeneity was proposed. Column performance was evaluated by nanoscale HPLC. The design for constructing an integrated nanoelectrospray emitter on the octadecylated silica monolith capillary column was first introduced. In comparison with the separated configuration where the emitter is connected to monolithic capillary column by the aid of a zero dead volume union, the integrated capillary column has the inherent advantage of the minimized extracolumn volume thus providing improved separation quality. The performance of the integrated monolithic capillary column was evaluated by separation of BSA tryptic digest, and peak capacity of 313 with a 30-cm column was obtained. The high separation performance allowed highly confident identification of 662 distinct proteins through assignment of 1933 unique peptides by analysis of tryptic digest of 0.5 mug of Saccharomyces cerevisiae proteins. The higher separation efficiency by a 60-cm monolithic capillary column increased the proteome coverage with identification of 1323 proteins through assignment of 5501 unique peptides over 400-min gradient elution.     

Separation of DNA fragments and single strand conformation polymorphism analysis in bare capillaries using poly(acrylamide–dimethylacrylamide) as a separation medium

A short chain poly(acrylamide–dimethylacrylamide) (PADMA) was synthesized in aqueous phase using isopropanol as a chain transfer agent, and was characterized according to the chemical composition and molecular mass. This polymer can form a stable dynamic coating on the inner surface of the capillary, thereby suppressing the electroosmotic flow and DNA–capillary wall interaction. The sieving medium has low viscosity and capillary filling with this medium and medium replacement were conveniently carried out by commercial capillary electrophoresis instruments. The effects of components and concentration of copolymers on the separation of DNA fragments were investigated. Highly efficient separation of DNA fragments, successful single strand conformation polymorphism (SSCP) analysis and good reproducibility of the migration time were obtained in bare capillaries using these copolymers as sieving media. Our preliminary results demonstrate that PADMA will become an alternative matrix for DNA separation by capillary electrophoresis.     

Preparation and characterization of a new open‐tubular capillary column for enantioseparation by capillary electrochromatography

In order to use the enantioseparation capability of cationic cyclodextrin and to combine the advantages of capillary electrochromatography (CEC) with open‐tubular (OT) column, in this study, a new OT‐CEC, coated with cationic cyclodextrin (1‐allylimidazolium‐β‐cyclodextrin [AI‐β‐CD]) as chiral stationary phase (CSP), was prepared and applied for enantioseparation. Synthesized AI‐β‐CD was characterized by infrared (IR) spectrometry and mass spectrometry (MS). The preparation conditions for the AI‐β‐CD‐coated column were optimized with the orthogonal experiment design L₉(3⁴). The column prepared was characterized by scanning electron microscopy (SEM) and elemental analysis (EA). The results showed that the thickness of stationary phase in the inner surface of the AI‐β‐CD‐coated columns was about 0.2 to 0.5 μm. The AI‐β‐CD content in stationary phase based on the EA was approximately 2.77 mmol·m^?2. The AI‐β‐CD‐coated columns could separate all 14 chiral compounds (histidine, lysine, arginine, glutamate, aspartic acid, cysteine, serine, valine, isoleucine, phenylalanine, salbutamol, atenolol, ibuprofen, and napropamide) successfully in the study and exhibit excellent reproducibility and stability. We propose that the column, coated with AI‐β‐CD, has a great potential for enantioseparation in OT‐CEC.     

Measurement of ATPase activity of immobilized myosin heads

Myosin, heavy meromyosin, and myosin subfragment-1 (S-1) were immobilized on the inner surfaces of glass capillary tubes, the inside walls of which had been coated with nitrocellulose. The ATPase activities of the immobilized proteins were measured using radiolabeled ATP and electrophoretic separation of the reaction products. The activity was proportional to the amount of immobilized protein. Activation by actin of the ATPase was also observed.     

Separation of polyprenol and dolichol by monolithic silica capillary column chromatography

We attempted an analysis of naturally occurring polyprenol and dolichol using a monolithic silica capillary column in HPLC. First, the separation of the polyprenol mixture alone was performed using a 250 x 0.2 mm inner diameter (ID) octadecylsilyl (ODS)-monolithic silica capillary column. The resolution of the separation between octadecaprenol (prenol 18) and nonadecaprenol (prenol 19) exceeded by >or=2-fold the level recorded when using a conventional ODS-silica particle-packed column (250 x 4.6 mm ID) under the same elution conditions. Next, the mixture of the prenol type (polyprenol) and dolichol type (dihydropolyprenol) was subjected to this capillary HPLC system, and the separation of each homolog was successfully achieved. During the analysis of polyprenol fraction derived from Eucommia ulmoides leaves, dolichols were found as a single peak, including all-trans-polyprenol and cis-polyprenol previously identified. This sensitive high-resolution system is very useful for the analysis of compounds that are structurally close to polyprenols and dolichols and that have a low content.     

Separation of enantiomers by open capillary electrochromatography on polysiloxane-bonded permethyl-beta-cyclodextrin

The separation of enantiomers by open capillary electrochromatography (o-CEC) using Chirasil-Dex as chiral stationary phase (CSP) is reviewed. In Chirasil-Dex, permethylated beta-cyclodextrin is linked via a single octamethylene spacer to polydimethylsiloxane. The CSP is coated and thermally immobilized onto the internal surface of a fused-silica column (i.d. 50 microm). Employing a single open-tubular column coated with Chirasil-Dex, a unified enantioselective approach can be realized using the four common chromatographic techniques: o-GC, o-SFC, o-LC and o-CEC. The chiral stationary phase Chirasil-Dex can be combined with a charged cyclodextrin derivative, which is added into the mobile phase. In the resulting dual chiral recognition system, enhancement of enantioselectivity (matched case) or compensation of enantioselectivity (mismatched case) are observed. The overall enantioselectivity is dependent on the sense of enantioselectivity of the selectors chosen and their influence on the electrophoretic and electroosmotic migration of the enantiomers of a selectand. The feasibility to couple chiral o-CEC and ESI/MS is demonstrated for trace analysis of enantiomeric drugs in body fluids.     

Bottom‐up proteome analysis of E. coli using capillary zone electrophoresis‐tandem mass spectrometry with an electrokinetic sheath‐flow electrospray interface

The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE‐ESI‐MS/MS. The separation was performed in a 60‐cm‐long linear polyacrylamide‐coated capillary with a 0.1% v/v formic acid separation buffer. An electrokinetic sheath‐flow electrospray interface was used to couple the separation capillary with an Orbitrap‐Velos operating in higher‐energy collisional dissociation mode. Each CZE‐ESI‐MS/MS run lasted 50 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom‐up analysis of prokaryote proteomes.     

A novel chiral inorganic mesoporous silica used as a stationary phase in GC

Chiral mesoporous silica (CMS) has attracted widespread attention because of some unique properties, such as high surface area, uniformly structured nanoscale cavities, and excellent chemical and thermal stability. In this work, we report the utilization of a CMS as the stationary phase for the separation of racemates in gas chromatography (GC). A CMS‐coated capillary column was fabricated by a dynamic coating method. Eighteen racemates belonging to different classes of organic compounds were separated on this column, including chiral alcohols, aldehydes, ketones, organic acids, halohydrocarbons, alkenes, alcohol amines, epoxides, and amino acid derivatives. In addition, linear alkanes, alcohols, and aromatic hydrocarbons have also been resolved.     

Hydroxyethylcellulose as an effective polymer network for DNA analysis in uncoated glass microchips: optimization and application to mutation detection via heteroduplex analysis

The nature of the sieving matrix for DNA fragment separation is of immense importance in capillary and microchip electrophoresis. The chemical nature of the surface of the capillary or microchannel wall is equally as important, particularly with DNA electrophoresis where a substantial electroosmotic flow (EOF) may be detrimental to the separation. Although DNA analysis has been carried out successfully in both coated and uncoated capillaries, analysis of unpurified polymerase chain reaction products has been carried out primarily with covalently coated surfaces, especially with microchip electrophoresis. In this report, double-stranded (ds) DNA fragment analysis using hydroxyethylcellulose (HEC) buffered in 1xTris-borate-EDTA is demonstrated both in uncoated capillaries and in microchips. EOF was suppressed 20% in the presence of 1.5% HEC, and the effectiveness of HEC as a polymer for dsDNA fragment analysis was dependent on the pH, with pH 8.6 being optimal. Using separation efficiency (number of theoretical plates) and resolution to gauge the effectiveness of a variety of polymers for the capillary separation of dsDNA fragments in the size range 60-587bp, HEC was found to be comparable in performance to polydimethylacrylamide (PDMA), and superior to linear polyacrylamide and polyethylene oxide for DNA analysis. With respect to longevity and robust performance, HEC could be used effectively in an uncoated capillary for more than 40 runs and for more than 90 runs (without replenishing the polymer) in an uncoated microchip. Application of the optimized HEC conditions is demonstrated through its ability to facilitate heteroduplex analysis.     

On-line combination of monolithic immobilized pH gradient-based capillary isoelectric focusing and capillary zone electrophoresis via a partially etched porous interface for protein analysis

An integrated platform consisting of monolithic immobilized pH gradient-based capillary isoelectric focusing (M-IPG CIEF) and capillary zone electrophoresis (CZE) coupled by a partially etched porous interface was established. Since carrier ampholytes (CAs) were immobilized on monolith in M-IPG CIEF to form a stable pH gradient, subsequent depletion of CAs at the interface to prevent the interference on CZE separation and detection were avoided. Moreover, a partially etched porous capillary column, which was facile for fabrication and durable for operation, was exploited as the interface to combine M-IPG CIEF and CZE. The RSD values in terms of the migration time for M-IPG CIEF separation, transfer protein from the first dimension to the second dimension, and CZE separation, were 2.4%, 3.9% and 2.3%, respectively. With a 6-protein mixture as the sample, two-dimensional capillary electrophoresis (2D-CE) separation was successfully completed within 116 min, yielding a peak capacity of ～200 even with minute sample amount down to 5.0 μg/mL. The limit of detection was 0.2 μg/mL. In addition, proteins extracted from milk were used to test the performance of such a 2D-CE separation platform. We expect that such a novel 2D-CE system would provide a promising tool for protein separation with high throughput and high peak capacity.     

Separation of ursodeoxycholic acid from its isomeric mixture using core–shell molecular imprinting polymer

In order to separate ursodeoxycholic acid (UDCA) from its isomeric mixture, the molecular imprinting polymers (MIPs) were synthesized by using core–shell emulsion polymerization. In the porous imprinting polymer, ursodeoxycholic acid was used as imprinting molecule, acrylamide (AM) and α-methacrylic acid (MAA) were functional monomers, and CaCO₃ was used for the porogen in the polymerization to obtain large pore. Characterization of the MIP structure with IR spectra demonstrated the expected MIPs. Through adsorption and selectivity assays, AM as the functional monomer showed better separation efficiency than MAA, and nonspecific and specific adsorption capacities of MIP with AM were 43.52 and 13.93 mg/g, respectively. The separation factor of MIP with AM for UDCA was 2.20. Furthermore, MIP with AM could be applied to separate UDCA from the isomeric mixture by column chromatography successfully.