Inner Wall Of Fused-silica Capillary Research Articles

Graphene oxide and reduced graphene oxide as novel stationary phases via electrostatic assembly for open‐tubular capillary electrochromatography

We present here the application of graphene oxide (GO) and reduced graphene oxide (GOOH) sheet as novel stationary phases for open-tubular CEC (OTCEC) separation based on electrostatic assembly. The inner walls of a bare capillary column was first modified by ionic assembly of poly (diallyldimethylammonium chloride) (PDDA), and then negatively charged GO or GOOH was easily assembled on a positively charged interior walls of the capillary by electrostatic force. Scanning Electron Microscope images showed that GO and GOOH can still maintain sheet-layer-like structure when coated onto the capillary via electrostatic assembly. The chromatographic properties of the GO and GOOH coated columns were evaluated via OTCEC separations of various kinds of analytes, including three acid nitrophenol isomers, three basic nitroaniline isomers, and four neutral PAHs. Efficient separations of all the analytes were achieved with optimized buffer pH and organic additive. The reproducibility and stability of the GO or GOOH coated columns were investigated. Our results indicate the capability of application GO or GOOH sheet in OTCEC separation, which can be coated on the inner wall of fused-silica capillary via electrostatic assembly.

Polyamidoamine dendrimer as a spacer for the immobilization of glucose oxidase in capillary enzyme microreactor

Polyamidoamine dendrimer (PAMAM) is one of a number of dendritic polymers with precise molecular structure, highly geometric symmetry, and a large number of terminal groups. In this study, different generations of PAMAM (G0–G4) were introduced onto the inner wall of fused-silica capillaries by microwave irradiation and a new type of glucose oxidase (GOx) capillary enzyme microreactor was developed based on enzyme immobilization in the prepared PAMAM-grafted fused-silica capillaries. The optimal enzymolysis conditions for β- d-glucose in the microreactor were evaluated by capillary zone electrophoresis. In addition, the enzymolysis efficiencies of different generations of PAMAM–GOx capillary enzyme microreactor were compared. The results indicate that enzymolysis efficiency increased with increasing generations of PAMAM. The experimental results provide the possibility for the development and application of an online immobilized capillary enzyme microreactor.

Electrokinetic separation of peptides and proteins using a polyvinylamine‐coated capillary with UV and ESI‐MS detection

In order to accomplish the analysis of peptides and proteins by capillary electrophoresis, Lupamin, a high-molecular-weight linear polyvinylamine (PVAm) polymer, was introduced to modify the inner wall of fused-silica capillaries by physical absorption. Thanks to the high density of positively charged amino groups in Lupamin under acidic conditions, not only is a strong reversed electroosmotic flow generated in the coated capillary but the adsorption of analytes on the inner wall of the capillary is also efficiently eliminated. It has been demonstrated that the Lupamin-coated capillary can be used to advantage for the rapid analysis of amino acids, peptides, and proteins with good resolution and peak shape by capillary electrophoresis. In order to evaluate the basic feature of a Lupamin-coated capillary, electroosmotic flows generated by a Lupamin coating layer under different conditions including pH, coating time, concentration, and the composition of electrolytes on Lupamin-coated and uncoated capillaries were investigated. Furthermore, electrospray ionization-mass spectrometry (ESI-MS) detection was carried out for the analysis of amino acids and peptides.

Photochemical Patterning of Biological Molecules Inside a Glass Capillary

A simple way for photochemical patterning of biological molecules onto the inner wall of fused-silica capillary is described. The method is based on a modification of the inner capillary surface with photoactive benzophenone (BP) derivative. The UV irradiation at 365 nm of the capillary filled with a sample solution results in cross-linking of the solutes to the BP moiety via a stable covalent bond. As a proof of concept, oligonucleotides and proteins were arrayed inside the capillary using an inverted microscope as an irradiation device. We demonstrated that the capillary arrays produced in this way are functional and could be used in different bioassays including DNA hybridization, protein interaction studies, and immunoassays. Having a sensitivity comparable to the fluorophore-based assays in a planar format, the capillary array possesses several advantages including submicroliter sample volume and a short assay time. The capillary format should therefore be considered as a possible alternative to a planar format in a number of low-density array applications such as mutation detection and diagnostic immunoassays.

Moderation of the electroosmotic flow in capillary electrophoresis by chemical modification of the capillary surface with tentacle-like oligourethanes

The surface chemistry of the inner wall of fused-silica capillaries is one important means to control the magnitude as well as the direction of the electroosmotic flow and the adsorption activity. A method was developed to change the surface characteristics of fused-silica capillaries by binding tentacle-like oligourethane groups onto the inner surface. The electroosmotic flow at a buffer pH of 6–9 was reduced by 15 to 40% compared to that in a bare fused-silica tubing, dependent on the type of coating. Sample adsorption is diminished at the same time resulting in a separation of proteins with higher resolution and good migration time precision. At a pH below 4.5 the electroosmotic flow is reversed into the anodic direction, which offers further possibilities for the separation of positively charged analytes as demonstrated for the separation of aromatic and biogenic amines.

Preparation of linear polyacrylamide-coated capillaries: Study of the polymerization process and its effect on capillary electrophoresis performance

The effect of different parameters controlling the characteristics of linear polyacrylamide coatings deposited on the inner wall of fused-silica capillaries and their influence on capillary electrophoresis (CE) performance of these coated columns is investigated. To carry out this study, a reproducible procedure to obtain capillaries with similar extent of modification of the surface silanols with 7-oct-1-enyltrimethoxisilane was first approached. Next the polymer attachment to the silica wall, via covalent linkage to the silyl reagent grafted onto the silica, was investigated. In this way, by using columns with a similar silylation extent, differences in CE performance observed among capillaries coated under diverse conditions could be assigned to the characteristics of the polyacrylamide layer. It is demonstrated that the characteristics and reproducibility of these polymeric coatings depend on the adequate control of both the temperature of polymerization and the degassing of the polymerizing dissolutions used. More interestingly, it is also demonstrated that the quantities of monomer (acrylamide), initiator (ammonium persulfate) and activator ( N, N, N′, N′-tetramethylethylenediamine), and the ratio among them used in the preparation of the coating polymer have a large influence on the performance of CE columns. The optimum conditions for preparing the polyacrylamide coatings are discussed. The applicability of these linear polyacrylamide-coated capillaries to the separation of basic and acidic proteins in free zone CE is demonstrated. Besides, the use of these coated columns in capillary gel electrophoresis for the separation of DNA fragments is shown.

Treatments of fused-silica capillaries and their influence on the electrophoretic characteristics of these columns before and after coating

Making coated columns in capillary electrophoresis (CE) is a laborious task involving many preparation steps such as etching, leaching, dehydration, silylation and coating of the inner wall of fused-silica capillaries. In this work we demonstrate, by testing more than 250 columns, that it is possible to follow up the influence of the different steps on both the electrophoretic behavior of CE columns and the reproducibility of their preparation. This study was done by carrying out triplicate measurements of electroosmotic flow values from columns at four different pH values after each step. The effectiveness of the coatings was also investigated by injecting a test-group of basic proteins. It is demonstrated that etching the columns with sodium hydroxide followed by a leaching treatment with hydrochloric acid provides higher reproducibility than by either leaching or etching alone. Dehydration of the capillary tubing affects the yield of the subsequent silylation reaction while the best results seem to be obtained by dehydrating the columns overnight at 160°C. The silylation degree achieved is demonstrated to also depend on the time of reaction and the concentration of silyl reagent. Moreover, a conclusive demonstration about the effect of both silylation and polymerization reactions on the final coating performance is given.

Capillary electrophoresis of peptides and proteins at neutral pH in capillaries covalently coated with polyethyleneimine.

Two procedures for the derivatization of the inner wall of fused-silica capillaries for the analysis of peptides and proteins by capillary electrophoresis (CE) at neutral pH are presented. In the first procedure, polyethyleneimine (PEI) is covalently attached to the capillary wall. In the second procedure, PEI is additionally cross-linked. We present analysis of standard peptides and proteins by CE using the coated capillaries. These coatings will have application for the separation of protein complexes at neutral pH, prior to analysis by electrospray mass spectrometry.

Kinetic study on the cis-trans isomerization of peptidyl-proline dipeptides

The equilibrium and kinetic parameters of cis-trans interconversion of dipeptides containing peptidyl-proline moiety were investigated using the in-column incubation method with both CZE and HPLC and the ad hoc dissolution method. The use of the latter was possible because the conformational make-up of the solid peptides, and consequently of their ad hoc solution, was sufficiently different from that of the solution at equilibrium. This method with 1H-NMR and CZE analyzes yielded very similar results for the cis-trans isomerization of Phe-Pro in aqueous sodium borate, pH 8.4 at 10°C with an average value of 0.34 and 6.6·10 −5 s −1 for the equilibrium and rate constant, respectively. The in-column incubation method is performed by CZE or HPLC so that the conformers are separated in the first half of the column and then incubated in column where they interconvert and reach equilibrium. Subsequent separation in the second half of the column yielded four peaks. Thus by measuring the conformer composition as a function of the reaction time, the rate constant can be evaluated. The forward rate constant of 1.42·10 −4 s −1 determined by the CZE in-column incubation method for Phe-Pro isomerization at 10°C was twice of the value obtained by the ad hoc dissolution method. It is believed that the inner wall of fused-silica capillaries has a catalytic effect on the isomerization. Computer simulation was also employed to gain further insight on the catalytic activity of the capillary inner wall on such isomerization. Whereas the experimental and simulation profiles of Phe-Pro in aqueous borate buffer, pH 8.4, with a 37 cm long capillary were in excellent agreement, a four times faster interconversion rate had to be used to match the experimental profile obtained with a 57 cm long capillary under otherwise identical conditions. The catalytic effect of the octadecyl silica stationary phase on the isomerization was confirmed by the in-column incubation method with HPLC. The overall rate of the cis-trans isomerization of Phe-Pro, which entails the reaction both on the stationary phase and in the mobility phase, was about six times faster at 0°C than the rate measured by NMR in free solution using the mobile phase containing 65% (v/v) sodium phosphate, pH 6.5, and 35% (v/v) methanol. The results presented here serve as a caveat that the effect of the wall in CZE or the stationary phase in HPLC on the reaction cannot be ignored.