Permanent gold nanoparticle coatings on polyelectrolyte multilayer modified capillaries for open-tubular capillary electrochromatography

This paper reports on a new strategy to coat fused silica capillaries through ionic adsorption of gold nanoparticles (AuNPs) on a polyelectrolyte multilayer (PEM) modified capillary wall. The coating was constructed in situ by alternating rinses with positively charged poly(diallydimethylammonium chloride), negatively charged poly(sodium-4-styrenesulfonate), and positively charged AuNPs. After self-assembly of n-octadecanethiol onto the surface of AuNPs, the modified capillary was investigated as a new medium for the separation of neutral analytes and proteins in open-tubular capillary electrochromatography (OT-CEC). The surface coverage of the capillary wall was increased using the high density of AuNPs which were dynamically capped with 4-dimethylaminopyridine (DMAP). The chromatographic performance of the column coated with positively charged AuNPs was remarkably improved compared with a column modified with negatively charged AuNPs. The coating was robust over more than 810 runs in this study and also showed high stability against 0.01 M NaOH, 0.01 M HCl, and electrolyte concentrations up to 70 mM. The run-to-run, day-to-day, and capillary-to-capillary reproducibilities of electroosmotic flow were satisfying with relative standard deviation values of less than 1% in all cases. The AuNP-coated PEM modified capillary column not only showed good performance for neutral analytes but also was suitable for the analysis of both basic and acidic proteins.     

Evaluation of migration behaviour of therapeutic peptide hormones in capillary electrophoresis using polybrene-coated capillaries

Modelling electrophoretic mobility as a function of pH can be simultaneously used for determination of ionization constants and for rapid selection of the optimum pH for separation of mixtures of the modelled compounds. In this work, equations describing the effect of pH on electrophoretic behaviour were used to investigate migration of a series of polyprotic amphoteric peptide hormones between pH 2 and 12 in polybrene-coated capillaries. Polybrene (hexadimethrin bromide) is a polymer composed of quaternary amines that is strongly adsorbed by the fused-silica inner surface, preventing undesired interactions between the peptides and the inner capillary wall. In polybrene-coated capillaries the separation voltage must be reversed, because of the anodic electroosmotic flow promoted by the polycationic polymer attached to the inner capillary wall. The possibility of using polybrene-coated capillaries for determination of accurate ionization constants has been evaluated and the optimum pH for separation of a mixture of the peptide hormones studied has been selected. Advantages and disadvantages of using bare fused-silica and polybrene-coated capillaries for these purposes are discussed.     

Capillary electrophoresis of synthetic peptide standards varying in charge and hydrophobicity

A mixture of eight structurally closely related synthetic peptides as capillary electrophoretic (CE) standards is introduced. The almost identical mass-to-charge ratio of the standards, coupled with their random-coil (i.e., no secondary structure) nature, offer a potent analytical test for CE to separate peptides varying only subtly in hydrophobicity. Parameters varied to effect a separation included background electrolyte concentration, temperature, applied voltage in capillary zone electrophoresis (CZE in uncoated capillaries), as well as the introduction of hydrophobic mechanisms to the separation either through the use of micelles or C8-coated capillaries. Our step-by-step approach culminated in an optimized combination of a CZE mechanism for separation of differently charged peptide groups (based on common mass-to-charge ratio) and an ion-pairing mechanism (effecting a separation within each group of identically charged peptides), which we have termed ion-interaction CZE or II-CZE. The study clearly shows how the peptide standards allow an excellent assessment of the resolving power of CE.     

Surface initiated polymerization of a cationic monomer on inner surfaces of silica capillaries: Analyte separation by capillary electrophoresis versus polyelectrolyte behavior

[2‐(Methacryloyl)oxyethyl]trimethylammonium chloride was successfully polymerized by surface‐initiated atom transfer radical polymerization method on the inner surface of fused‐silica capillaries resulting in a covalently bound poly([2‐(methacryloyl)oxyethyl]trimethylammonium chloride) coating. The coated capillaries provided in capillary electrophoresis an excellent run‐to‐run repeatability, capillary‐to‐capillary and day‐to‐day reproducibility. The capillaries worked reliably over 1 month with EOF repeatability below 0.5%. The positively charged coated capillaries were successfully applied to the capillary electrophoretic separation of three standard proteins and five β‐blockers with the separation efficiencies ranging from 132 000 to 303 000 plates/m, and from 82 000 to 189 000 plates/m, respectively. In addition, challenging high‐ and low‐density lipoprotein particles could be separated. The hydrodynamic sizes of free polymer chains in buffers used in the capillary electrophoretic experiments were measured for the characterization of the coatings.     

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.     

New carrier electrolytes for the separation of chlorophenols by capillary electrophoresis

Optimum conditions for the separation of positional isomers of chlorophenols by capillary zone electrophoresis (CZE) were established. The behavior of five volatile electrolytes (L-cysteic acid, 3-amino-1-propanesulfonic acid, aminomethanesulfonic acid, diethylmalonic acid, and ammonium acetate) was compared. The best performance based on low electrophoretic current and high separation efficiency was obtained for diethylmalonic acid as working electrolyte. The influence of pH on the separation, using both uncoated fused-silica capillaries and modified capillaries (NaAMPS from EKT) with anionic coating, was discussed. Moreover, the effect of electrolyte concentration and applied voltage using fused-silica capillaries was studied. The optimum CZE conditions that allowed the separation of 16 chlorophenols were 20 kV, 30 mM diethylmaIonic acid, pH 7.25, and uncoated fused-silica capillary. Figures of merit such as run-to-run and day-to-day precision, linearity, and limits of detection were calculated.     

New Approach to the Formation of Physically Adsorbed Capillary Coatings Consisting of Hyperbranched Poly(Ethylene Imine) with a Maltose Shell to Enhance the Separation of Catecholamines and Proteins in CE

Core–shell-type polymers based on a hyperbranched (hb) poly(ethylenimine) core and a shell with a variable maltose content were applied as coating materials for fused silica capillaries. A new, simple, fast, and reproducible way of modifying the capillary walls through the physical adsorption of the core–shell-type polymers using a Cu²⁺ support was developed. The coating created by this method was found to be very stable compared to the coating created using a solution of the polymer only. Capillaries modified with the core–shell-type polymers were tested by applying them to the electrophoretic separation of catecholamines and proteins. The modified capillaries showed high efficiencies (up to 800,000 theoretical plates per meter for lysozyme) and separation selectivities. The highest efficiency was achieved using capillaries modified with the polymer containing the lowest content of maltose in the shell and the most accessible positively charged core. Various online concentration techniques were also tested as a means to lower detection limits further, making it possible to analyze proteins in biological fluids (saliva) as well as catecholamines in human urine after SPE using activated alumina.     

Capillary electrophoretic separation of toxic drugs using a polyacrylamide-coated capillary

Summary Capillary electrophoresis (CE) has recently become an attractive approach for the analysis of pharmaceuticals. In this study, capillary electrophoretic separation of anxiolytic drugs, including barbiturates and benzodiazepines, was carried out using polyacrylamide (PAA)-coated capillaries. The surface of the capillary inner wall was coated with a neutral layer, and separation was performed in the absence of electroosmotic flow (EOF). Both charged and neutral solutes were separated in the presence of sodium dodecyl sulfate (SDS) above its critical micelle concentration (CMC) in the running buffer. This kind of CE method provided fast and efficient separation of a total of 24 kinds of toxic drugs in a mixture. In addition, the analysis of toxic drugs in body fluids was attempted after the sample preparation using liquid-liquid extraction or solid-phase microextraction (SPME).     

Comparision of succinate- and phthalate-functionalized etched silica hydride phases for open-tubular capillary electrochromatography

Two open-tubular (OT) capillary electrochromatographic (CEC) columns were prepared by chemically bonding ionizable mono-(2-(methacryloyloxy)ethyl) succinate (MES) and phthalate-functionalized (MEP) ligands onto silica hydride-based phases through surface etching, silanization, and hydrosilation reactions, starting with a bare fused-silica tube. An analysis of the effect of performance of electrophoretic flow (EOF) on the changes in pH values, ionic strength, and the amount of acetonitrile modifiers helped to reveal that some silanol groups remained in the surface composite of the modified capillaries and to prove that MEP capillaries actually exerted greater EOF than MES ones. To explore the potential utilization of these two columns in various fields, three categories of samples, which spanned a wide range of polarities, were prepared and analyzed through many systematic trials of optimizing CEC conditions. For the separation of a mixture of nucleosides and thymine, guanine and adenine with purine uncleobases, which exhibit greater aromaticity than pyrimidine nucleobases, performed a higher retention in the MEP capillary through a π–π interaction than in the MES capillary. While four steroids were used as test samples, their migration order revealed that the MES stationary phase is hydrophilic in comparison with the MEP. An addition of methanol modifier (30%, v/v) into 10 mM borate buffer (pH 9.55 for MEP; pH 10.0 for MES) was necessary to accomplish a baseline separation of nine flavonoids in the MEP and MES capillaries. Studies on the elution order of these solutes revealed the presence of chromatographic activity in addition to electrophoretic migration. Especially in the MEP capillary, hydrophobic characteristics and π–π interactions with aromatic solutes were found and further improved to resolve an enantiomeric pair, catechin and epicatechin. Overall, the hydride-based stationary phases with ionizable ligands were successfully applied to the OT-CEC separations, and these results confidently propose an ideal route to the synthesis of a novel OT-CEC column.     

Modification of a Capillary for Electrophoresis by Electrostatic Self‐Assembly of an Enzyme for Selective Determination of the Enzyme Substrate

The integration of a separation capillary for capillary electrophoresis (CE) with an on‐column enzyme reaction for selective determination of the enzyme substrate is described. Enzyme immobilization is achieved by electrostatic assembly of poly(diallydimethylammonium chloride) (PDDA) followed by adsorption of a mixture of the negatively charged enzyme glucose oxidase (GOx) and anionic poly(styrenesulfonate) (PSS). The reaction of glucose with the GOx produces hydrogen peroxide which migrates the length of the capillary and is detected amperometrically at the capillary outlet. The enzyme reaction occurs during a capillary separation, allowing selective determination of the substrate in complex samples without the need for pre‐ or post‐separation chemical modification of the analyte. The enzyme reactor is found to have an optimal response to glucose when a 5 : 1 mixture of PSS:GOx is used. Under these conditions the limit of detection for glucose is found to be between 5.0×10^?4 and 1.3×10^?3 M, dependent upon the inner‐diameter of the capillary. The apparent Michaelis‐Menten constant for the enzyme reaction was determined to be 0.047 (±0.001) M and 0.0037 (±0.0007) M for a 50 and 10 μm inner‐diameter capillaries, respectively. These results indicate that the enzyme reaction is efficient, having enzyme kinetics similar to that of a reaction occurring in solution. This enzyme immobilization method was also applied to another enzyme, glutamate oxidase, yielding similar results.     

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.     

Investigation of the separability of thaumatin by capillary electrophoresis

The electrophoretic behaviour of the highly basic protein thaumatin was explored in strongly acid (pH 2) and mildly acid (pH 4.5) separation systems using both bare and coated fused silica capillaries. The separation selectivity for thaumatin I, thaumatin II, and for other sample constituents was insufficient for their baseline separation at pH 2 in an uncoated capillary because the separation efficiency was markedly lower than is common in the electrophoretic separations of proteins. A separation selectivity higher by up to one order of magnitude has been reached at pH 4.5. A pronounced asymmetry of zones, which impaired resolution at this pH, was effectively suppressed by coating of the capillary wall with a polymer. In fact, adsorption on the capillary coating always plays a contributory role whenever a good separation of thaumatin constituents is attained. This indicates that electrochromatographic separation systems based on capillaries coated with the layer of either cationic or hydrophilic uncharged polymer hold promise for the development of methods for thaumatin analysis.     

Capillary electrophoretic separation of amino acids: Fraction collection

A simple method is described which enables solutes to be collected at an electrically isolated exit after they have been separated by a free solution capillary electrophoretic system. The method is illustrated by the separation of dansyl amino acids using multiple separation capillaries.     

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.     

Capillary electrophoresis in a fused‐silica capillary with surface roughness gradient

The electro‐osmotic flow, a significant factor in capillary electrophoretic separations, is very sensitive to small changes in structure and surface roughness of the inner surface of fused silica capillary. Besides a number of negative effects, the electro‐osmotic flow can also have a positive effect on the separation. An example could be fused silica capillaries with homogenous surface roughness along their entire separation length as produced by etching with supercritical water. Different strains of methicillin‐resistant and methicillin‐susceptible Staphylococcus aureus were separated on that type of capillaries. In the present study, fused‐silica capillaries with a gradient of surface roughness were prepared and their basic behavior was studied in capillary zone electrophoresis with UV‐visible detection. First the influence of the electro‐osmotic flow on the peak shape of a marker of electro‐osmotic flow, thiourea, has been discussed. An antifungal agent, hydrophobic amphotericin B, and a protein marker, albumin, have been used as model analytes. A significant narrowing of the detected zones of the examined analytes was achieved in supercritical‐water‐treated capillaries as compared to the electrophoretic separation in smooth capillaries. Minimum detectable amounts of 5 ng/mL amphotericin B and 5 μg/mL albumin were reached with this method.     

Mobility-based selective on-line preconcentration of proteins in capillary electrophoresis by controlling electroosmotic flow

A simple method to perform selective on-line preconcentration of protein samples in capillary electrophoresis (CE) is described. The selectivity, based on protein electrophoretic mobility, was achieved by controlling electroosmotic flow (EOF). A short section of dialysis hollow fiber, serving as a porous joint, was connected between two lengths of fused silica capillary. High voltage was applied separately to each capillary, and the EOF in the system was controlled independently of the local electric field intensity by controlling the total voltage drop. An equation relating the EOF with the total voltage drop was derived and evaluated experimentally. On-line preconcentration of both positively charged and negatively charged model proteins was demonstrated without using discontinuous background electrolytes, and protein analytes were concentrated by approximately 60-200-fold under various conditions. For positively charged proteins, positive voltages of the same magnitude were applied at the free ends of the connected capillaries while the porous joint was grounded. This provided a zero EOF in the system and a non-zero local electric field in each capillary to drive the positively charged analytes to the porous joint. CE separation was then initiated by switching the polarity of the high voltage over the second capillary. For negatively charged proteins, the procedure was the same except negative voltages were applied at the free ends of the capillaries. Mobility-based selective on-line preconcentration was also demonstrated with two negatively charged proteins, i.e. beta-lactoglobulin B and myoglobin. In this case, negative voltages of different values were applied at the free ends of the capillaries with different values, which provided a non-zero EOF in the system. The direction of EOF was the same as that of the electrophoretic migration velocities of the protein analytes in the first capillary and opposite in the second capillary. By controlling the EOF, beta-lactoglobulin B, which has a higher mobility, could be concentrated over 150-fold with a 15 min injection while myoglobin, which has a lower mobility, was eliminated from the system.     

Separation of somatropin charge variants by multiple‐injection CZE with Polybrene/chondroitin sulfate A double‐coated capillaries

The performance of dynamic double‐coated fused‐silica capillaries with Polybrene and chondroitin sulfate A has been compared with uncoated fused‐silica capillaries for the determination of recombinant human growth factor (somatropin) charge variants. The separations were carried out under the same electrophoretic conditions as described in the European Pharmacopoeia, i.e. at pH 6.0 and 30°C. The coating significantly reduced the interactions between the proteins and the surface of the fused‐silica capillary. The first five separations performed in a new bare fused‐silica capillary were discarded because of very poor separation performance as a result of protein–surface interactions. There was an approximate twofold increase in the interday migration time precision (%RSD ≤ 6.5%) in the double‐coated capillaries. The method was successfully transferred to a multiple CZE mode where two samples were analyzed in a single electrophoretic run. The average purity of somatropin certified reference standard was 98.0% (%RSD ≤ 0.3%) determined by using uncoated and coated capillaries.     

Enhanced capillary zone electrophoresis in cyclic olefin copolymer microchannels using the combination of dynamic and static coatings for rapid analysis of carnosine and niacinamide in cosmetics

Cosmetics that have medicinal effects, including anti-inflammatory and antioxidant, have become a daily care routine consumption. The peptide additives, such as carnosine and nicotinamide, were frequently used to realize these medicinal effects. To accomplish rapid and effective quantitation of carnosine and niacinamide in cosmetics, capillary zone electrophoresis was executed in cyclic olefin copolymer microchips having both dynamic and static coatings. The static coating of cyclic olefin copolymer microchannel was constructed from bovine serum albumin adsorption, immobilization, and active site closure, while the dynamic coating was formed by adding surfactant into running buffer of capillary zone electrophoresis. The static coating can improve the hydrophilicity of cyclic olefin copolymer surface and avoid nonspecific peptide adsorption. The dynamic coating of sodium dodecyl sulfate in running buffer proved to be useful in flow velocity adjustment and the column efficiency enhancement in the capillary zone electrophoresis separation channel of the cyclic olefin copolymer microchip device. A separation resolution up to 4.24 on the mixture of carnosine and nicotinamide was obtained. Moreover, an analysis method was established and applied to simultaneous carnosine and nicotinamide determination in a liquid whitening essence and a solid antiglycation pill, and the results were verified by comparison with high-performance liquid chromatography methods, indicating its potential in complex sample analysis.     

Performance of throughout in-capillary derivatization capillary electrophoresis employing an on-line sample and run buffer loading device

We report on the effect on performance of varying the length of the capillary during throughout in-capillary derivatization (TICD) capillary electrophoresis (CE). Performance was evaluated by on-line coupling with a sample and CE runbuffer loading device that was newly introduced for this study. The device was assembled with a low cost using two 5 mm inner diameter (ID) disposable polyethylene syringes. First, a sequence was manually formed consisting of a 200 microL run buffer solution plug, a 100 microL sample plug and another 200 microL run buffer solution plug. Each plug was separated from its neighbor by a 100 microL air plug. When each plug reached the injection point where both a platinum-wire anode and the end of the separation capillary tube were located, 340 V/cm separation voltage (electrophoresis voltage) and 34 V/cm injection voltage were applied to the capillary for 3 s. Then the analytes were derivatized during migration in 50 microm ID capillaries filled with 2 mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) in a 20 mM phosphate-borate buffer (pH 10), followed by separating and detecting of OPA derivatives by absorbance of 340 nm. Derivatization, separation, and detection were performed systematically using capillaries which varied in length from 5 to 80 cm. In the case of TICD-CE of a mixture containing 1 mM aspartic acid (Asp) and 20 mM m-nitorophenol (MNP) as a test solution, it was determined that peak area and peak width ratios of Asp to MNP did not depend on capillary length. Enantiomeric separations of DL-alanine (Ala) and Asp were examined using a run buffer consisting of a 45 microM beta-cyclodextrin (CD)-2 mM OPA/NAC-20 mM phosphate-borate buffer (pH 10). Even though the resolution of these enantiomeric pairs decreased with decreasing capillary length, as expected, the peaks corresponding to both enantiomeric amino acids were identified even when a 5 cm capillary was used. An 8-component amino acid mixture was also tested with 5 cm and 10 cm capillaries.     

Chiral separations by open tubular capillary electrokinetic chromatography.

The inner walls of 50 microns fused-silica capillaries are etched by ammonium hydrogendifluoride and then modified by the silanization/hydrosilation method with a chiral selector. Three different types of selectors were evaluated: lactone, beta-cyclodextrin and naphthylethylamine. Each of the bonded chiral stationary phases provided at least partial separation for one type of racemic solute. These results confirm that bonded organic moieties on the etched inner wall of a capillary can provided sufficient solute-bonded phase interactions to influence the retention of molecules driven through a capillary by electroosmosis or a combination of electroosmosis and electrophoretic mobility.