Preparation and characterization of calixarene-coated capillaries for capillary electrophoresis

Preparation and characterization of calixarene-coated capillaries for capillary electrophoresis (CE) were exemplified with p-allylcalix[4]arene (pACX4) which was immobilized to the fused silica surface using gamma-methacryloxypropyl-trimethoxysilane (gamma-MAPS) as linking agent. Successful gamma-ACX4 coating was suggested by the greatly decreased electroosmotic flow (EOF), due to the introduction of phenolic hydroxyl groups on the inner surface of the capillaries. A slight slope of EOF versus pH at pH <8 would help make the separation reproducible. The coated columns also featured a low ultraviolet (UV) absorption background and long lifetime (> 6 months at 4相似文献   

Capillary electrophoresis using core‐based hyperbranched polyethyleneimine (CHPEI) static‐coated capillaries

With unique 3‐D architecture, the application of core‐based hyperbranched polyethyleneimine (CHPEI), as a capillary coating in capillary electrophoresis, is demonstrated by manipulation of the electroosmotic mobility (EOF). CHPEI coatings (CHPEI5, M_w ≈? 5000 and CHPEI25, M_w ≈? 25 000) were physically adsorbed onto the inner surface of bare fused‐silica capillary (BFS) via electrostatic interaction of the oppositely charged molecules by rinsing the capillaries with different CHPEI aqueous solutions. The EOF values of the coated capillaries were measured over the pH range of 4.0–9.0. At higher pH (pH >6) the coated capillary surface possesses excess negative charges, which causes the reversal of the EOF. The magnitudes of the EOF obtained from the coated capillaries were three‐fold lower than that of BFS capillary. Desirable reproducibility of the EOF with % RSD (n = 5) ? 2 was obtained. Effect of ionic strength, stability of the coating (% RSD = 0.3) and the dependence of the EOF on pH (% RSD = 0.5) were also investigated. The CHPEI‐coated capillaries were successfully utilized to separate phenolic compounds, B vitamins, as well as basic drugs and related compounds with reasonable analysis time (<20 min) and acceptable migration‐time repeatability (<0.7% RSD for intra‐capillary and <2% RSD for inter‐capillary).     

Influencing electroosmotic flow and selectivity in open tubular electrochromatography by tetrakis(pentafluorophenyl)porphyrin as capillary wall modifier

A physically adsorbed and covalently bonded porphyrin derivative, 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin, H₂TPFPP, has been used as a fused-silica capillary wall modifier in open tubular capillary electrochromatography (OT-CEC), and its influence on the electroosmotic flow (EOF) velocity and on the selectivity of OT-CEC separations of a set of model aromatic carboxylic acids has been tested. Whereas most of the coatings of this category bring about an increase in selectivity with a concomitant slow down of the EOF, H₂TPFPP coating, depending on pH of the background electrolyte used, resulted both in decreasing of EOF at pH 8.5 by 5% and in increasing of EOF by 10–43% at pH 6 and 5, respectively. The separation efficiency and the resolution of aromatic carboxylic acids separation in coated capillaries, namely in that one with covalent coating, were better than in the bare fused-silica capillary. The perspectives of H₂TPFPP as capillary wall modifier are visualized in introducing well defined electroosmotic properties of materials used for miniaturized separation channels preparation in chip-based electromigration devices.     

Adsorptive BSA Coating Method for CE to Separate Basic Proteins

A novel and simple coating method was developed by coating bovine serum albumin (BSA) onto the inner surface of a fused-silica capillary, to avoid the adsorption of analytes during CE. The advantage presented here was that the coating process is more simple, fast, stable, and reproducible. The coated capillary avoided the adsorption of analytes onto the inner surface of a fused-silica capillary and might be a promising candidate for separation of complex biological samples with further development. Meanwhile, the efficiencies of the coated capillary were evaluated by EOF, chromatographic peak shape, and theoretical plate number (N m^?1) of RNase A. The optimal coating conditions were obtained from the results. The pH value of coating buffer PB was 4.2, the standing time was 12 h at 4 °C, and the coating concentration of BSA was 1.5 mg mL^?1. The stability of the coating on the inner wall of the capillary and the reproducibility of the coated capillaries were good. The theoretical plate number values of RNase A were over 1.3 × 10⁵ (N m^?1) in the coated capillary. After successive electrophoresis for 48 h using the coated capillary, the RSD values of EOF and the theoretical plate number were 4.14 % and 9.14 %, respectively. In addition, the RSD values of EOF and the theoretical plate number (N m^?1) in the coated capillaries were 13.19 % and 8.96 %, respectively. Finally, the coated capillary was successfully applied to separate the mixture of four basic proteins (RNase A, lysozyme, trypsin and myoglobin).     

Efficient and highly reproducible capillary electrophoresis-mass spectrometry of peptides using Polybrene-poly(vinyl sulfonate)-coated capillaries

The potential of capillaries noncovalently coated with a bilayer of oppositely charged polymers for the analysis of peptides by CE-MS was investigated. Bilayer coatings were produced by subsequently rinsing fused-silica capillaries with a solution of Polybrene (PB) and poly(vinyl sulfonate) (PVS). The PB-PVS coating showed to be fully compatible with MS detection causing no ionization suppression or background signals. The bilayer coating provided a considerable EOF at low pH, thereby facilitating the fast separation of peptides using a BGE of formic acid (pH 2.5). Under optimized CE-MS conditions, for enkephalin peptides high separation efficiencies were obtained with plate numbers in the range of 300,000-500,000. It is demonstrated that both the cancellation of the hydrodynamic capillary flow induced by the nebulizer gas and a sufficiently high-data acquisition rate are crucial for achieving these efficiencies. The overall performance of the CE-MS system using PB-PVS-coated capillaries was evaluated by the analysis of a tryptic digest of cytochrome c. The system provided an efficient separation of the peptide mixture, which could be effectively monitored by MS/MS detection allowing identification of at least 13 peptides within a time interval of 1.5 min. In addition, the PB-PVS coating proved to be very consistent yielding stable CE-MS patterns with highly favorable migration time reproducibilities (RSDs < 1% over a 3-day period).     

Efficient and reproducible analysis of peptides by capillary electrophoresis using noncovalently bilayer-coated capillaries

The usefulness of a noncovalent capillary coating consisting of two layers of oppositely charged polymers for the separation of peptides with capillary electrophoresis (CE) was studied. Capillaries were coated simply by subsequently flushing with solutions of 1% m/v Polybrene and 1% v/v poly(vinylsulfonate) (PVS) forming a bilayer, which showed to produce a strong and highly reproducible electroosmotic flow (EOF) at low pH. Using this coating in combination with a background electrolyte (BGE) containing sodium phosphate (pH 2.5) and 0.01% v/v PVS, initially broadened and overlapping peaks were obtained for some test peptides. By omitting the PVS from the BGE, the peak width and shape of the peptides improved resulting in baseline separation. A systematic study of the influence of the BGE composition showed that considerable further enhancement of the separation efficiency was achieved by increasing the ionic strength of the BGE. Using a BGE of 200 mM tris(hydroxymethyl)aminomethane (Tris)-phosphate (pH 2.5) plate numbers for the peptides were in the 300 000-600 000 range and the relative standard deviation of the peptide migration times was less then 0.3% (n = 5). The use of Tris-phosphate instead of sodium phosphate allowed the current to stay within acceptable limits when 30 kV was used as separation voltage. Overall, the bilayer coating showed a remarkable EOF repeatability, as well as long-term stability. Compared to bare fused-silica capillaries the intraday and interday repeatability of migration times was very favorable and coated capillaries could be used for over a month performing analyses with low and high ionic strength BGEs without any performance deterioration. The usefulness of the bilayer-coated capillaries for the analysis of positively charged peptides was demonstrated by the fast and efficient separation of various closely related enkephalins and the baseline separation of an isomeric peptide/peptoid couple exhibiting efficiencies of over 550 000 plates.     

Enantioseparation by CE with vancomycin as chiral selector: Improving the separation performance by dynamic coating of the capillary with poly(dimethylacrylamide)

An approach for improving the separation performance of the enantioseparation by CE with vancomycin as chiral selector is described. In the present method, a solution of poly(dimethylacrylamide) (PDMA) was used for dynamic coating of the capillary wall to minimize the adsorption of vancomycin onto the capillary wall, and to depress the EOF. Compared with the bare fused-silica capillaries and the capillaries coated with the polycationic polymer hexadimethrine bromide (HDB), the PDMA-coated capillary displayed the best separation performance. The resulting coating could withstand hundreds of runs without losing its function. Moreover, a partial filling technique was applied to avoid interference in detection caused by the presence of vancomycin in the buffer. The separation time was shortened when a short-end-injection technique was applied. Several parameters such as buffer pH, vancomycin concentration and plug length of the vancomycin solution for the separation were optimized. Under the optimal conditions, all tested enantiomers, including FMOC amino acids derivatives, ketoprofen and fenoprofen, were baseline-separated in less than 4.2 min.     

Preparation of a sulfonated fused-silica capillary and its application in capillary electrophoresis and electrochromatography

In the present paper, two new methods, sol-gel and chemical bonding methods, were proposed for preparation of sulfonated fused-silica capillaries. In the sol-gel method, a fused-silica capillary was coated with the sol solution obtained by hydrolysis of 3-mercaptopropyltrimethoxysilane (MPTS) and tetramethoxysilane, and followed by age; while in the chemical bonding method, a capillary was chemically bonded directly with MPTS. Then, both the resulting capillaries were oxidized with an aqueous solution of hydrogen peroxide solution (H2O2) (30%, m/m) to obtain the sulfonated capillaries. The electroosmotic flow (EOF) for the sulfonated capillaries was found to remain almost constant within the studied pH range, and greater than that of the uncoated capillary. However, the coating efficiency of the capillary prepared by chemical bonding method was higher than that by sol-gel method, by comparing their magnitude of the EOF, the degree of disguise of the silanol and reproducibility of preparation procedure. The effects of the electrolyte's concentration and the content of methanol (MeOH) on the EOF were also studied. Especially, the study of the apparent pH (pH*) on the EOF in a water-MeOH system was reported. Finally, capillary electrophoretic separation of seven organic acids was achieved within 6.5 min under optimal condition using the chemically bonded sulfonated capillary. Moreover, separation of four alkaloids on the sulfonated capillary was compared with that on uncoated capillary in different conditions. Ion-exchange mechanism was found to play a key role for separation of these four basic analytes on the sulfonated capillary.     

Preparation and evaluation of non-bonded hyperbranched polymer-coated columns for capillary electrophoresis

A series of hyperbranched poly(amine-ester)s based on 1,1,1-trimethylolpropane, methyl acrylate and diethanolamine were synthesized and coated on the inner surface of the fused-silica capillaries by physical adsorption. The most effective coating was the seventh generation hyperbranched poly(amine-ester) coating, which reduced the electroosmotic flow (EOF) greatly and suppressed protein adsorption effectively. The high separation efficiencies for basic proteins were obtained and the coating had a good stability.     

Cationic starch derivatives as dynamic coating additives for protein analysis in capillary electrophoresis

Positively charged starch derivatives were used to modify the inner surface of fused-silica capillaries by addition to running buffer, which were subsequently employed in capillary electrophoresis (CE). Capillaries coated with the cationic starch derivatives were shown to generate a stable, reversed electroosmotic flow (EOF) in the investigated pH range of 3-9. The presented coating procedure was fast, based on a simple rinsing protocol where the polymer created a physically adsorbed, cationic polymer layer. Among the additives studied, a quaternary ammonium starch derivative showed a fast EOF mobility and effectively suppressed the adsorption of proteins. The intra- and inter-day reproducibility of the coating referring to the EOF mobility were satisfactory with relative standard deviation (RSD) of 0.27 and 1.67%, respectively. The coating enabled separation of some protein mixtures including basic proteins within l3 min with efficiencies up to 280,000 plates/m. In addition, this cationic starch derivative possessed a good solubility (about 100mg/mL), and it does not significantly contribute to the background adsorption in the UV region of 190-400 nm.     

Vesicles as pseudostationary phase for enantiomer separation by capillary electrophoresis

The suitability of noncovalently bilayer-coated capillaries for the analysis of proteins by capillary electrophoresis (CE) at medium pH was investigated. Fused-silica capillaries were coated simply by successively flushing with a polybrene (PB) and a poly(vinyl sulfonate) (PVS) solution. A protein test mixture was used to evaluate the performance of the coated capillaries. Comparisons with bare fused-silica capillaries were made. Several background electrolytes (BGEs) were tested in combination with the PB-PVS coating, showing that optimum performance was obtained for the proteins using high BGE concentrations. With a 300 mM Tris phosphate buffer (pH 7.0), good plate numbers (150,000-300,000), symmetrical peaks, and favorable migration-time repeatabilities (RSDs below 0.8%) were obtained for the proteins. Using bare fused-silica capillaries, the protein peaks were significantly broadened and the migration-time RSDs often exceeded 5%. It is concluded that the PB-PVS coating effectively minimizes adverse protein adsorption and provides a very stable electroosmotic flow (EOF). We also investigated the potential of a commercially available bilayer coating (CEofix) for protein analysis. It is demonstrated that with this coating, good plate numbers and peak symmetries for proteins can be achieved when the CEofix BGE ("accelerator") is replaced by a common BGE such as sodium or Tris phosphate. Apparently, the negatively charged polymer present in the "accelerator" interacts with the proteins causing band broadening. The utility of the bilayer coatings is further illustrated by the separation of proteins such as interferon-alpha 2b, myoglobin and carbonic anhydrase, by the analysis of a degraded insulin sample in time, and by the profiling of the glycoprotein ovalbumin. In addition, it is demonstrated that even in the presence of concentrations of human serum albumin in the sample of up to 60 mg/mL, the PB-PVS coating still provides reproducible protein separations of good performance.     

Transient isotachophoresis of highly saline trace metals under strong electroosmotic flow conditions

Transient isotachophoresis (TITP) is usually performed under low-electroosmotic flow (EOF) conditions using a coated capillary or a low pH background electrolyte. We used a bare fused-silica capillary for TITP stacking of anionic complexes of some heavy metals under high-EOF conditions (pH 9.0). The sample component chloride as a leading electrolyte induced stacking by an isotachophoretic mechanism and the complexing agent 4-(2-pyridylazo) resorcinol (PAR) acted as a terminating electrolyte. The optimized background electrolyte was composed of 150 mM N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, 127 mM triethylamine, and 0.1 mM PAR at pH 9.0. The strong EOF at pH 9.0 pulled the analytes against their mobilities toward the outlet side, allowing a separation in the normal polarity mode. The stacking efficiency, reproducibility, analysis time, and sample loading capacity in coated and bare capillaries were compared. The stacking efficiency and reproducibility were higher and the analysis time was shorter in the coated capillary. However, a larger volume of a sample could be injected in the bare capillary to achieve detection limits comparable to those for the coated one without compromising the resolution between the analyte peaks. The limits of detection (S/N = 3) were in the sub-ppb range for the selected metals (Fe2+, 0.3 ppb; Ni2+, 0.16 ppb; and Zn2+, 0.8 ppb) in a standard saline sample with 250 mM NaCl matrix. The proposed method was successfully applied to the analysis of reference urine samples and human urine samples.     

Preparation and evaluation of zirconia-coated silica monolith for capillary electrochromatography

Silica monoliths were fabricated inside fused-silica capillaries. Then the monolithic columns were coated with membrane-like zirconia. The zirconia-coated silica monoliths exhibited different EOF behavior comparing with that of bare silica monoliths. The magnitude and direction could be manipulated by changing the running buffers. Due to the amphoteric characteristic of zirconia, the silica monoliths with zirconia surface facilitate the separation of basic compounds. Aromatic amines and alkaloids were separated without obvious peak tailing. The zirconia surface was easily modified with octadecylphosphonic acid for the separation of neutral compounds. Column efficiency as high as 90,000 and 80,000 m⁻¹ was obtained for beberine and naphthalene, respectively. Furthermore, the zirconia coating increased the stability of the monolithic columns. Even after being exposed to severe condition, there was no apparently efficiency decrease for the test samples.     

Single-run separation of cationic, anionic, and polyanionic compounds by CE-ESI-MS

A method for a single-run separation of cationic, anionic, and polyanionic compounds by CE hyphenated to ESI MS (CE-ESI-MS) is described. One of the main issues for coupling CE to MS with an ESI source consists in maintaining an electric contact for the electrophoretic separation. This condition is only performed if a liquid flow arising from the separation capillary is directed to the needle, making it coupling-compatible. This latter situation is incompatible with the separations of polyanionic compounds of higher electrophoretic mobility (in absolute value) than the electroosmotic mobility, performed in bare fused-silica capillaries under a negative polarity. In this study, several alternative approaches were evaluated to circumvent this difficulty, and applied to the setup of the CE-MS separation of a mixture containing both cationic and polyanionic compounds, which are synthesis intermediates of contrast agents for medical imaging. Eventually, the detection of the cationic and anionic compounds in a single run could be obtained by either using neutrally coated polymethylsiloxane (DB-1) capillaries and simultaneously applying a negative voltage polarity and a pressure allowing to compensate for the residual cathodic EOF or by dynamically modifying the inner wall of a bare fused-silica capillary with a polycationic polymer (hexadimethrine bromide) and using it afterwards under negative voltage polarity.     

Quaternary ammonium substituted agarose as surface coating for capillary electrophoresis

A novel positively charged polymer of quaternary ammonium substituted agarose (Q-agarose) has been synthesized and explored for use as a coating in capillary electrophoresis. The fast and simple coating procedure is based on a multi-site electrostatic interaction between the polycationic agarose polymer and the negatively charged fused-silica surface. By simply flushing fused-silica capillaries with hot polymer solution a positively charged, hydrophilic deactivation layer is achieved. The polymer surface provides an intermediate electroosmotic flow of reversed direction, over a range of pH 2-11, compared to unmodified fused-silica. The coating procedure was highly reproducible with an RSD of 4%, evaluated as the electroosmotic flow mobility for 30 capillaries prepared at 10 different occasions. The application of Q-agarose coated capillaries in separation science was investigated using a set of basic drugs and model proteins and peptides. Due to the intermediate electroosmotic flow generated, the resolution of basic drugs could be increased, compared to using bare fused-silica capillaries. Moreover, the coating enabled separation of proteins and peptides with efficiencies up to 300.000 plates m(-1).     

A well-defined diblock copolymer of poly-(ethylene oxide)-block-poly (4-vinylpyridine) for separation of basic proteins by capillary zone electrophoresis

A series of well-defined diblock copolymers, poly(ethylene oxide)-block-poly(4-vinylpyridine) (PEO-b-P4VP) used as physical coating of capillaries, were synthesized by atom transfer radical polymerization (ATRP). EOF measurement results showed that all synthesized PEO-b-P4VP diblock copolymer-coated capillaries in this report could suppress EOF effectively compared to the bare fused-silica capillaries, and efficient separations of basic proteins were achieved. The effects of the molecular weight of P4VP block in PEO-b-P4VP and buffer pH on the separation of basic proteins for CE were investigated in detail. Moreover, the relationships between morphologies of PEO-b-P4VP diblock copolymers in buffer, which were studied by transmission electron microscopy, and the separation efficiencies of basic protein with PEO-b-P4VP diblock copolymers coatings were discussed.     

Separation of acidic and basic compounds in capillary electrochromatography with polymethacrylate-based monolithic columns

Methacrylate-based monolithic columns with electroosmotic flow (EOF) or very weak EOF are prepared by in situ copolymerization in the presence of a porogen in fused-silica capillaries pretreated with a bifunctional reagent. Satisfactory separations of acidic and basic compounds on the column with EOF at either low or high pH are achieved, respectively. With sulfonic groups as dissociation functionalities, sufficient EOF mobility still remains as high as 1.74 x 10(-4) cm2 s(-1) V(-1) at low pH. Under this condition, seven acidic compounds are readily separated within 5.7 min. Moreover, at high pH, the peak shape of basic compounds is satisfactory without addition of any masking amines into running mobile phase since the secondary interaction between the basic compounds and the monolithic stationary phase are minimized at high pH. Reversed-phase mechanism for both acidic and basic compounds is observed under investigated separation conditions. In addition, possibilities of acidic and basic compound separations on a monolithic column with extremely low EOF are discussed.     

Polymeric ionic liquid-coated capillary for capillary electrophoresis

A new application of the polymeric ionic liquid (PIL) in capillary electrophoresis is reported. Poly(1-vinyl-3-butylimidazolium bromide) was physically adsorbed on silica capillary as the simple and effective coating for capillary electrophoresis (CE) analysis, in which the PIL is not present in the background electrolyte. The electroosmotic flow (EOF) of the PIL-coated capillary as compared with that of the bare fused-silica capillary shows a different dependence on electrolyte pH values. The EOF is reversed over a wide pH range from 3.0 to 9.0 and shows good repeatability. It is also found that the coated capillary has a good tolerance to some organic solvents, 0.1 M NaOH and 0.1 M HCl. The PIL-coated capillary has been employed in different areas. Both the basic proteins and anionic analytes can be well separated by PIL-coated capillaries in a fast and easy way. The PIL-coated capillary is also able to separate organic acid additives in a grape juice. The results showed that this type of coating provides an alternative to the CE separation of anions and basic proteins.     

超支化聚合物化学键合毛细管电泳柱的制备及其对蛋白质的分离行为

分别合成了以三羟甲基丙烷和季戊四醇为核的超支化聚(胺-酯),并对其进行了红外测定、羟值测定、粘度测定等表征。采用化学键合方法将其涂于毛细管内壁,并测定涂层柱的电渗流以及对碱性蛋白质的分离能力,结果表明,涂层柱能有效地抑制碱性蛋白质在毛细管内壁上的吸附,大大降低电渗流;以三羟甲基丙烷为核的超支化聚(胺-酯)涂层柱的塔板数达105/m,而以季戊四醇为核的超支化聚(胺-酯)涂层柱的分离柱效更高,塔板数达107/m。实验结果表明这两类涂层柱都具有较好的分离效果和稳定性。     

Capillary electrophoresis using copolymers of different composition as physical coatings: a comparative study

In this work, a comparative study on the use of different polymers as physically adsorbed coatings for CE is presented. It is demonstrated that the use of ad hoc synthesized polymers as coatings allows tailoring the EOF in CE increasing the flexibility of this analytical technique. Namely, different polymers were synthesized at our laboratory using different percentages of ethylpyrrolidine methacrylate (EpyM) and N,N-dimethylacrylamide (DMA). Thus, by modifying the percentage of EpyM and DMA monomers it is possible to manipulate the positive charge of the copolymer, varying the global electrical charge on the capillary wall and with that the EOF. These coated capillaries are obtained by simply flushing a given EpyM-DMA aqueous solution into bare silica capillaries. It is shown that by using these coated capillaries at adequate pHs, faster or more resolved CE separations can be achieved depending on the requirements of each analysis. Moreover, it is demonstrated that these coated capillaries reduce the electrostatic adsorption of basic proteins onto the capillary wall. Furthermore, EpyM-DMA coatings allow the reproducible chiral separation of enantiomers through the partial filling technique (PFT). The EpyM-DMA coated capillaries are demonstrated to provide reproducible EOF values independently of the pH and polymer composition with%RSD values lower than 2% for the same day. It is also demonstrated that the coating procedure is reproducible between capillaries. The compatibility of this coating protocol with CE in microchips is discussed.