Influence of cetyltrimethylammonium bromide on phosphatidylcholine-coated capillaries

Large unilamellar vesicles of egg-phosphatidylcholine (eggPC), a naturally occurring phospholipid, were used in capillary electrophoresis (CE) for semi-permanent coating of fused silica capillaries. The stability of the phospholipid coating was tested at different cetyltrimethylammonium bromide (CTAB) concentrations with and without CaCl₂ present in the coating solution. The effect of physical factors influencing the coating stability (e.g. duration of the coating time, storage temperature of the coating solution) were also studied. Standing overnight in background electrolyte (BGE) solution did not alter the eggPC phospholipid coating noticeably. The performance of the coating was tested with a mixture of basic proteins (lysozyme, ribonuclease A and -chymotrypsinogen A). Highest efficiencies (over 200,000 plates m^–1) were achieved when the capillary was filled for 15 h with a liposome solution containing both CTAB and CaCl₂.Electronic Supplementary Material Supplementary material is available for this article at     

Preparation approaches of the coated capillaries with liposomes in capillary electrophoresis

The use of liposomes as coating materials in capillary electrophoresis has recently emerged as an important and popular research area. There are three preparation methods that are commonly used for coating capillaries with liposomes, namely physical adsorption, avidin–biotin binding and covalent coupling. Herein, the three different coating methods were compared, and the liposome-coated capillaries prepared by these methods were evaluated by studying systematically their EOF characterization and performance (repeatability, reproducibility and lifetime). The amount of immobilized phospholipids and the interactions between liposome or phospholipid membrane and neutral compounds for the liposome-coated capillaries prepared by these methods were also investigated in detail. Finally, the merits and disadvantages for each coating method were reviewed.     

Nanoparticle‐based capillary electroseparation of proteins in polymer capillaries under physiological conditions

Totally porous lipid‐based liquid crystalline nanoparticles were used as pseudostationary phase for capillary electroseparation with LIF detection of proteins at physiological conditions using unmodified cyclic olefin copolymer capillaries (Topas^®, 6.7 cm effective length). In the absence of nanoparticles, i.e. in CE mode, the protein samples adsorbed completely to the capillary walls and could not be recovered. In contrast, nanoparticle‐based capillary electroseparation resolved green fluorescent protein from several of its impurities within 1 min. Furthermore, a mixture of native green fluorescent protein and two of its single‐amino‐acid‐substituted variants was separated within 2.5 min with efficiencies of 400 000 plates/m. The nanoparticles prevent adsorption by introducing a large interacting surface and by obstructing the attachment of the protein to the capillary wall. A one‐step procedure based on self‐assembly of lipids was used to prepare the nanoparticles, which benefit from their biocompatibility and suspension stability at high concentrations. An aqueous tricine buffer at pH 7.5 containing lipid‐based nanoparticles (2% w/w) was used as electrolyte, enabling separation at protein friendly conditions. The developed capillary‐based method facilitates future electrochromatography of proteins on polymer‐based microchips under physiological conditions and enables the initial optimization of separation conditions in parallel to the chip development.     

Capillary electrophoresis of liposomes functionalized for protein binding

CE enabled assessing the attachment of hexa-histidine-tagged proteins to functionalized phospholipid liposomes. The liposomes were made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, phosphatidyl-ethanolamine, cholesterol and distearoyl-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) in a molar ratio of 29:26:40:5. The unilamellar vesicles, which had an average diameter of 170 nm, were labelled by inclusion of FITC-dextran for fluorescence detection. CE was carried out in poly(vinyl alcohol) (PVA)-coated capillaries at 25 degrees C with a BGE consisting of Tris-HCl (50 mM, pH 8.0). For conjugation of the liposomes with the proteins (soluble synthetic receptor fragments with molecular mass of 60 and 70 kDa, respectively), Ni(2+) was implanted into the vesicle surface by an anchor lipid containing a nitrilotriacetate acid (NTA) group as complexation agent for the metal ions. The difference in surface charge enabled the separation of the different species of interest by CE: plain vesicles, vesicles functionalised with Ni-NTA, vesicle-protein complexes and the species formed upon removal of the Ni-ions by complexation with EDTA. Loss of the Ni-ions resulted in the release of the proteins and the reappearance of the plain Ni-free NTA-liposome species in the electropherograms.     

Current measurement of nonaqueous solvents: Applicatons to capillry electrophoresis and electrochromatography

The solvents acetonitrile, methanol, N, N-dimethylformamide, dimethyl sulfoxide, formamide, and deionized water were investigated for their ability to support current flow without added electrolyte. Using open tubular capillary electrophoresis, currents were measured to be in the nanoampere (10^?9 A) range for all solvents but formamide (10^?6 A). Comparisons with flow data showed no clear relationship between current and electroosmotic flow. Packed capillary columns (3-μm ODS) were used for separations using both pure solvent and hydrophobic dyes showed mild retention in pure ACN. A 16 polynuclear aromatic hydrocarbon (PAH) standard soluton was separated in 80/20 ACN/H₂O with reduced plate heights (h) between 2.8 and 3.1 for retained species. A separation of nine androstenediones was achieved using a 70/30 MeOH/H₂O mobile phase.     

Comparison of three modifications of fused‐silica capillaries and untreated capillaries for protein profiling of maize extracts by capillary electrophoresis

In this work, capillary electrophoresis was applied to protein profiling of fractionated extracts of maize. A comparative study on the application of uncoated fused‐silica capillaries and capillaries modified with hydroxypropylmethylcellulose, ω‐iodoalkylammonium salt and a commercially available neutral capillary covalently coated with polyacrylamide is presented. The coating stability, background electrolyte composition, and separation efficiency were investigated. It was found that for zeins separation, the most stable and efficient was the capillary coated with polyacrylamide. Finally, the usefulness of these methods was studied for the differentiation of zein fraction in transgenic and nontransgenic maize. Zeins extracted from maize standards containing 0 and 5% m/m genetic modification were successfully separated, but slight differences were observed in terms of the zein content. Albumin and globulin fractions were analyzed with the use of unmodified fused‐silica capillary with borate buffer pH 9 and the capillary coated with polyacrylamide with phosphate buffer pH 3. In the albumin fraction, additional peaks were found in genetically modified samples.     

Characterization of etched electrochemical detection for electrophoresis in micron inner diameter capillaries

An enhanced etched electrochemical (EC) detection technique has been developed for CE in micron inner diameter capillaries. The design improvements allow for better alignment between the capillary bore and the electrode. This new method involves utilizing a carbon fiber microelectrode and etching both the carbon fiber and the detection end of a micrometer-sized inner diameter capillary to limit dead volume and analyte diffusion at the amperometric EC detector. To understand the factors affecting enhanced detector efficiency, a detailed examination of the relationship between detector design and performance has been completed by exploring the effects of varying electrode diameter, tip shape, and size, in addition to the etch length of the capillary outlet. The enhanced detection provides peak efficiencies as high as 75000 theoretical plates and estimated detection limits as low as 40 nM for dopamine. This etched detection method should further facilitate volume-limited sample analysis by CE.     

Monomer surface modifications for rapid peptide analysis by capillary electrophoresis and capillary electrochromatography coupled to electrospray ionization-mass spectrometry

In this study, positively charged alkylaminosilyl monomers were used to modify the inner surface of fused silica capillaries, which subsequently were employed in capillary electrophoresis (CE) and capillary electrochromatography (CEC). The obtained surfaces yield a reversed electroosmotic flow (EOF) and have varying carbon chain lengths, that interact with the analytes and give chromatographic retention. The coating procedure is very simple and fast. The performance of the modified capillaries was evaluated regarding pH influence on EOF and chromatographic interactions. The experiments were conducted with UV and mass spectrometry (MS) and applied to the separation of various neuropeptides. The derivatized surfaces showed a linear (R(2) approximately 0.99) pH dependence with isoelectric points (pI) at 8.6-8.8. Rapid separations of peptide standards and a protein digest with efficiencies as high as 5 x 10(5) plates/m were performed.     

Hybrid phospholipid bilayer coatings for separations of cationic proteins in capillary zone electrophoresis

Protein separations in CZE suffer from nonspecific adsorption of analytes to the capillary surface. Semipermanent phospholipid bilayers have been used to minimize adsorption, but must be regenerated regularly to ensure reproducibility. We investigated the formation, characterization, and use of hybrid phospholipid bilayers (HPBs) as more stable biosurfactant capillary coatings for CZE protein separations. HPBs are formed by covalently modifying a support with a hydrophobic monolayer onto which a self‐assembled lipid monolayer is deposited. Monolayers prepared in capillaries using 3‐cyanopropyldimethylchlorosilane (CPDCS) or n‐octyldimethylchlorosilane (ODCS) yielded hydrophobic surfaces with lowered surface free energies of 6.0 ± 0.3 or 0.2 ± 0.1 mJ m^?2, respectively, compared to 17 ± 1 mJ m^?2 for bare silica capillaries. HPBs were formed by subsequently fusing vesicles comprised of 1,2‐dilauroyl‐sn‐glycero‐3‐phosphocholine or 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine to CPDCS‐ or ODCS‐modified capillaries. The resultant HPB coatings shielded the capillary surface and yielded reduced electroosmotic mobility (1.3–1.9 × 10^?4 cm² V^?1s^?1) compared to CPDCS‐ and ODCS‐modified or bare capillaries (3.6 ± 0.2 × 10^?4 cm² V^?1s^?1, 4.8 ± 0.4 × 10^?4 cm² V^?1s^?1, and 6.0 ± 0.2 × 10^?4 cm² V^?1s^?1, respectively), with increased stability compared to phospholipid bilayer coatings. HPB‐coated capillaries yielded reproducible protein migration times (RSD ≤ 3.6%, n ≥ 6) with separation efficiencies as high as 200 000 plates/m.     

氟西汀和西替利嗪对映体的双动态吸附毛细管电色谱手性分离

建立了以海美溴铵（hexadimethrine bromide,HDB)为阳离子表面活性剂，并以磺化β－环糊精（SO3－β－CD）为手性选择剂的双动态吸附毛细管电色谱；考察了背景电解质pH和浓度、环糊精种类和浓度对分离的影响，对分离条件进行了优化，并对手性识别机理进行了探讨；实验结果表明在含0．1g/L HDB和20mmol/L SO3-β-CD的20mmol/L Tris-H3PO4(pH3.00)的体系中，氟西汀和西替利嗪对映体在较短的时间内达到良好分离。     

Facile preparation of polysaccharide‐coated capillaries using a room temperature ionic liquid for chiral separations

In this study, the dissolution of polysaccharides into an ionic liquid was investigated and applied as a coating onto the capillary walls of a fused‐silica capillary in open‐tubular CEC. The coating was evaluated by examining the chiral separation of two analytes (thiopental, sotalol) with three cellulose derivatives (cellulose acetate, cellulose acetate phthalate, and cellulose acetate butyrate). Baseline separation of thiopental enantiomers was achieved by use of each polysaccharide coating (Rs: 7.0, 8.1, 7.1), while sotalol provided partial resolution (Rs: 0.7, 1.0, 0.9). In addition, reproducibility of the cellulose‐coated capillaries was evaluated by estimating the run‐to‐run and capillary‐to‐capillary RSD values of the EOF. Both stability and reproducibility were very good with RSD values of less than 7%.     

The production of packed capillaries using a novel pressurised ultrasound device

Summary This paper introduces a novel, highly effective method of producing packed capillaries for use in capillary electrochromatography (CEC) or microbore HPLC. It is our opinion that CEC offers significant advantages for future separation systems particularly with MS detection and these methods will assist the development of the capillary production technology.     

Coupled fused silica capillaries for rapid capillary zone electrophoresis of proteins

A novel two-dimensional electrophoretic system for the control of electroosmosis in capillary zone electrophoresis has been developed and evaluated for rapid separations of proteins. The system comprises uncoated and polyether-coated fused silica capillaries coupled in series. An equation relating the average electroosmotic flow velocity in the coupled capillaries to the intrinsic electroosmotic velocities of the connected segments and their corresponding lengths has been derived and verified experimentally. This approach has the advantage of enabling the electroosmotic flow to be tuned independently of the applied voltage. As a consequence, rapid protein analysis at relatively low field strength was achieved without sacrificing the high separation efficiencies obtained with surface-modified capillaries.     

Phospholipid-lysozyme coating for chiral separation in capillary electrophoresis

A phospholipid coating with lysozyme as chiral recognition reagent permeated into the phospholipid membrane was developed for the chiral capillary electrophoretic (CE) separation of D- and L-tryptophan. As a kind of carriers, coated as phospholipid membranes onto the inner wall of a fused-silica capillary, liposomes are able to interact with basic proteins such as lysozyme, which may reside on the surface of the phospholipid membrane or permeate into the middle of the membrane. The interaction results in strong immobilization of lysozyme in the capillary. Coatings prepared with liposomes alone did not allow stable immobilization of lysozyme into the phospholipid membranes, as seen from the poor repeatability of the chiral separation. When 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide (M1C4) was applied as a first coating layer in the capillary, the electroosmotic flow (EOF) was effectively suppressed, the phospholipid coating was stabilized, and the lysozyme immobilization was much improved. The liposome composition, the running buffer, and the capillary inner diameter all affected the chiral separation of D- and L-tryptophan. Coating with 4 mM M1C4 and then 1 mM phosphatidylcholine (PC)/phosphatidylserine (PS) (80:20 mol%), with 20 mM (ionic strength) Tris at pH 7.4 as the running buffer, resulted in optimal chiral separation with good separation efficiency and resolution. Since lysozyme was strongly permeated into the membrane of the phospholipids on the capillary surface, the chiral separation of D- and L-tryptophan was achieved without lysozyme in the running buffer. The effects of different coating procedures and separation conditions on separation were evaluated, and the M1C4-liposome and liposome-lysozyme interactions were elucidated. The usefulness of protein immobilized into phospholipid membranes as a chiral selector in CE is demonstrated for the first time.     

The production of packed capillaries using a novel pressurised ultrasound device

Summary This paper introduces a novel, highly effective method of producing packed capillaries for use in capillary electrochromatography (CEC) or microbore HPLC. It is our opinion that CEC offers significant advantages for future separation systems particularly with MS detection and these methods will assist the development of the capillary production technology.     

Capillary electrophoresis method to determine siRNA complexation with cationic liposomes

Small interfering RNA (siRNA) inducing gene silencing has great potential to treat many human diseases. To ensure effective siRNA delivery, it must be complexed with an appropriate vector, generally nanoparticles. The nanoparticulate complex requires an optimal physiochemical characterization and the complexation efficiency has to be precisely determined. The methods usually used to measure complexation in gel electrophoresis and RiboGreen^® fluorescence‐based assay. However, those approaches are not automated and present some drawbacks such as the low throughput and the use of carcinogenic reagents. The aim of this study is to develop a new simple and fast method to accurately quantify the complexation efficiency. In this study, capillary electrophoresis (CE) was used to determine the siRNA complexation with cationic liposomes. The short‐end injection mode applied enabled siRNA detection in less than 5 min. Moreover, the CE technique offers many advantages compared with the other classical methods. It is automated, does not require sample preparation and expensive reagents. Moreover, no mutagenic risk is associated with the CE approach since no carcinogenic product is used. Finally, this methodology can also be extended for the characterization of other types of nanoparticles encapsulating siRNA, such as cationic polymeric nanoparticles.     

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.     

Nonaqueous capillary electrophoresis in coated capillaries: an interesting alternative for proteomic applications

This work brings together some contributions for the use of nonaqueous media for proteomic analysis, for both capillary electrophoresis (CE) separation and the preparation of tryptic digests. First, a ternary nonaqueous buffer consisting of 60/30/10 v/v methanol/acetonitrile/acetic acid with 12.5 mmol/L ammonium acetate was optimized for CE separation of the tryptic digest of lysozyme. Lysozyme was chosen as a model system for the protein digestion, which has also been prepared in an organic-rich medium with methanol/50 mmol/L NH(4)HCO(3), pH 8.0 (60/40 v/v). The separation results were compared to in silico (PeptideCutter program) digestion conditions, and high-efficiency peak separation (18 peaks) was obtained in 20 min with an electric field of 350 V/cm. In addition, we have evaluated the stability of a coated capillary with poly-N,N-dimethylacrylamide (60/30 cm total/effective length and 75 microm ID) for over 100 runs of tryptic digest with the nonaqueous background electrolyte solvent system. The migration times for ten selected peptide peaks presented 3-7% relative standard deviation.     

Internal electrolyte temperatures for polymer and fused-silica capillaries used in capillary electrophoresis

Polymers are important as materials for manufacturing microfluidic devices for electrodriven separations, in which Joule heating is an unavoidable phenomenon. Heating effects were investigated in polymer capillaries using a CE setup. This study is the first step toward the longer-term objective of the study of heating effects occurring in polymeric microfluidic devices. The thermal conductivity of polymers is much smaller than that of fused silica (FS), resulting in less efficient dissipation of heat in polymeric capillaries. This study used conductance measurements as a temperature probe to determine the mean electrolyte temperatures in CE capillaries of different materials. Values for mean electrolyte temperatures in capillaries made of New Generation FluoroPolymer (NGFP), poly-(methylmethacrylate) (PMMA), and poly(ether ether ketone) (PEEK) capillaries were compared with those obtained for FS capillaries. Extrapolation of plots of conductance versus power per unit length (P/L) to zero power was used to obtain conductance values free of Joule heating effects. The ratio of the measured conductance values at different power levels to the conductance at zero power was used to determine the mean temperature of the electrolyte. For each type of capillary material, it was found that the average increase in the mean temperature of the electrolyte (DeltaT(Mean)) was directly proportional to P/L and inversely proportional to the thermal conductivity (lambda) of the capillary material. At 7.5 W/m, values for DeltaT(Mean) for NGFP, PMMA, and PEEK were determined to be 36.6, 33.8, and 30.7 degrees C, respectively. Under identical conditions, DeltaT(Mean) for FS capillaries was 20.4 degrees C.     

Chromatographic behavior of packing materials for capillary electrochromatography with a coating of different immobilized polysiloxanes

Octadecyl silyl silica (ODS) phase coated with immobilized polysiloxanes (OV1701, SE-54, SE-30) were synthesized, their characteristics as capillary electrochromatography (CEC) column packing materials were studied. It was found that, although the polysiloxane coatings were different in polarity, the resulting packing materials showed the highest efficiencies when the respective coating ratios (polysiloxane:ODS, w/w) were all 20-30%. As expected, packing materials coated with different polysiloxanes resulted in different selectivity on solute pairs. Separations on these stationary phases were studied with different factors such as pH values and acetonitrile contents of the mobile phases. It was found that all these kind of stationary phases could resist basic mobile phase with a pH value as high as 11.6. Tests were made to analyze polar, basic drugs with CEC using the stationary phases.