Bacterial surface layer proteins as a novel capillary coating material for capillary electrophoretic separations

A novel concept for stable coating in capillary electrophoresis, based on recrystallization of surface layer proteins on hydrophobized fused silica capillaries, was demonstrated. Surface layer protein A (SlpA) from Lactobacillus acidophilus bacteria was extracted, purified and used for coating pre-silanized glass substrates presenting different surface wettabilities (either hydrophobic or hydrophilic). Contact angle determination on SlpA-coated hydrophobic silica slides showed that the surfaces turned to hydrophilic after coating (53 ± 5°), due to a protein monolayer formation by protein-surface hydrophobic interactions. Visualization by atomic force microscopy demonstrated the presence of a SlpA layer on methylated silica slides displaying a surface roughness of 0.44 ± 0.02 nm. Additionally, a protein layer was visualized by fluorescence microscopy in methylated silica capillaries coated with SlpA and fluorescein isothiocyanate-labeled. The SlpA-coating showed an outstanding stability, even after treatment with 20 mM NaOH (pH 12.3). The electroosmotic flow in coated capillaries showed a partial suppression at pH 7.50 (3.8 ± 0.5 10⁻⁹ m² V⁻¹ s⁻¹) when compared with unmodified fused silica (5.9 ± 0.1 10⁻⁸ m² V⁻¹ s⁻¹). To demonstrate the potential of this novel coating, the SlpA-coated capillaries were applied for the first time for electrophoretic separation, and proved to be very suitable for the isotachophoretic separation of lipoproteins in human serum. The separations showed a high degree of repeatability (absolute migration times with 1.1–1.8% coefficient-of-variation (CV) within a day) and 2–3% CV inter-capillary reproducibility. The capillaries were stable for more than 100 runs at pH 9.40, and showed to be an exceptional alternative for challenging electrophoretic separations at long-term use.     

Stable neutral double hydrophilic block copolymer capillary coating for capillary electrophoretic separations

Quaternized diblock copolymer, poly(N‐methyl‐2‐vinylpyridinium iodide‐block‐ethylene oxide), was successfully used as a neutral, dynamic coating to suppress the electroosmotic flow. The block copolymer consisted of two polymers that were linked covalently together. The cationic block (poly(N‐methyl‐2‐vinylpyridinium iodide)) was bound efficiently to the negatively charged capillary wall via electrostatic interactions, and the hydrophilic block (ethylene oxide) stabilized the system and created a neutral capillary surface with ultralow electroosmotic flow (+2.0 ± 4.5 × 10^?10 m²/Vs). The main advantages of the coating were simple and fast preparation, easy regeneration and automation, and stable electroosmotic flow. To emphasize the potential of this type of coating its stability was measured at a wide pH range demonstrating a high stability in the pH range of 4.0–10.5 and lifetime up to 8 days. The successful studies carried out with beta‐blockers, basic proteins, and lipoproteins proved the suitability of the coating for the separation of different sized analytes. Furthermore, the neutral coating developed is useful in a wide range of protein analysis and biological interaction studies under physiological condition.     

Rapid determination of globin chains in red blood cells by capillary electrophoresis using INSTCoated fused‐silica capillary

A laboratory‐made INSTCoated fused‐silica capillary has been newly used for CE separation of four mixtures of proteins in sodium phosphate BGEs at pH 3.0 and 2.5, respectively. The obtained separation efficiencies range from 145 000 theoretical plates per meter for myoglobin to 1 216 000 m^?1 for lysozyme. A total of 49–89% of the number of theoretical plates was obtained in a commercial polyvinyl alcohol coated capillary compared to the INSTCoated capillary under the same experimental conditions, 0–86% was obtained in a laboratory polyacrylamide‐coated capillary, and only 0–6% was obtained in an uncoated fused‐silica capillary. The RSD values for the intraday repeatability for an INSTCoated capillary were 0.1–1.0% (migration time) and 0.3–2.4% (peak area); RSD values for the interday repeatability in the same capillary are 0.6–1.4% (migration time) and 2.4–5.5% (peak area); RSD values for interday repeatability between different capillaries equaled 1.7–2.1% (migration time) and 2.8–10.9% (peak area). The INSTCoated capillary has been further used for rapid determination of globin chains isolated from red blood cells. A separation of α and β chains prepared from adult blood has been completed in 3 min with a peak resolution of 1.3, and the separation of α and ^Gγ chains prepared from newborn blood took 3 min with a peak resolution of 3.6.     

Novel cationic coating agent for protein separation by capillary electrophoresis†

A novel positively charged surfactant N‐dodecyl‐N,N‐dimethyl‐(1,2‐propandiol) ammonium chloride was used for the dynamic coating of the inner wall of a silica capillary. This paper covers the evaluation of dynamic coating and study of the influence of the analysis conditions for the magnitude and direction of electroosmotic flow as well as for the effective and selective separation of chosen proteins (ribonuclease A, cytochrome c, lysozyme, and myoglobin). The concentration of 0.1 mM of N‐dodecyl‐N,N‐dimethyl‐(1,2‐propandiol) ammonium chloride enabled the reversal of the electro‐osmotic flow, however, to separate basic as well as neutral proteins the higher concentration of the studied surfactant was necessary. The final conditions for the separation of studied proteins were set at 100 mM sodium acetate pH 5.5 with 10.0 mM of the studied surfactant. The results were also compared with those of two commercially available cationic surfactants, cetyltrimethylammonium bromide and dodecyltrimethylammonium bromide. Additionally, the developed method for protein separation was applied for the determination of lysozyme in a cheese sample. The limits of detection and quantification of lysozyme were 0.9 and 3.0 mg/L, respectively. The mean concentration of lysozyme found in the cheese sample was 167.3 ± 10.3 mg/kg.     

DNA capillary electrophoresis using poly(vinyl alcohol). I. Inner capillary coating

Two new methods of inner capillary coating with poly(vinyl alcohol) (PVAL) have been investigated and evaluated by performing DNA capillary electrophoresis (CE) using PVAL as a separation medium and by measuring the electroosmotic flow (EOF) mobility. The treatment of capillaries with a silanol-group modified PVAL (PVAL-Si) has been found to give good coating effects for improving the resolution of DNA CE and for reducing the EOF. This coating must be effectively achieved by combining the adsorptive property of PVAL chains onto silica with the reaction between the silanol groups of PVAL-Si and the silica surface. The adsorption of PVAL onto silica has been observed by using atomic force microscopy (AFM) for PVAL-Si as well as for a nonmodified PVAL as a control. The coating with PVAL that links to the capillary wall surface with more hydrolytically stable bonding, -Si-C-, has been formed by performing the Grignard reaction, followed by in-capillary polymerization of vinyl acetate (VAc) and hydrolysis. This coating has been found to be effective for improving the resolution of DNA CE and for reducing the EOF.     

Preparation and evaluation of stable coating for capillary electrophoresis using coupled chitosan as coated modifier

A coated capillary modified with a coupled chitosan (COCH) was developed by using a simple and fast (60 min) process that could be easily automated in capillary electrophoresis instrument. The COCH coating was achieved by first attaching chitosan to the capillary inner wall, and then coupling with glutaraldehyde, and rinsing chitosan again to react with glutaraldehyde. The COCH coating was stable and showed amphoteric character over the pH range of 1.8-12.0. When the pH value was lower than 4.5, the capillary surface possessed positive charges, which caused a reversal in the direction of the electroosmotic flow (EOF). The normal EOF direction could be obtained when the pH value was higher than 4.5. The COCH coating showed strong stability against 0.1 mol/L HCl, 0.1 mol/L NaOH and other solvents compared with conventional chitosan coating. The relative standard deviation of the run-to-run, day-to-day and capillary-to-capillary coating was all below 2% for the determination of EOF. The COCH-modified capillary was applied to acidic and basic proteins analyses and high efficiency could be attained. The comparison between unmodified capillary, chitosan-modified and COCH-modified capillary for the separation of real sample, extract from Elaphglossum yoshinagae with water, was also studied. Better results could be obtained on COCH-modified capillary than the other two capillaries.     

Direct sample injection for capillary electrophoretic determination of organic acids in cerebrospinal fluid

Organic acids in cerebrospinal fluid (CSF) are potential diagnostic markers for neurological diseases and metabolic disorders. A capillary electrophoretic (CE) method for the direct analysis, i.e., without any sample preparation, of six organic acids in CSF was developed. A capillary coating consisting of a triple layer of charged polymers (polybrene-dextran sulfate-polybrene) was used in combination with a negative separation voltage, providing fast and efficient analysis of acidic compounds. Separation conditions, such as background electrolyte (BGE) concentration and pH were optimized, and the influence of albumin and sodium chloride was systematically studied using a set of test compounds. With injection volumes of ca. 44 nL, plate numbers of up to ca. 150,000 were obtained with a BGE of 200 mM sodium phosphate (pH 6.0). It appeared that high sodium chloride concentrations in the sample hardly affected the peak width and shape of the organic acids, most probably due to transient isotachophoresis effects occurring in the sample zone. Adverse effects of CSF proteins, which frequently compromise the CE performance, could be effectively minimized by the triple layer coating in combination with rinses of 0.1 M hydrochloric acid. Overall, the developed CE system allowed direct injections of CSF samples, yielding good separation efficiencies and stable migration times (RSDs < 2%) for organic acids. Validation of the method with artificial and real CSF samples showed good linear responses (r > 0.99), and LODs for the organic acids were in the range of 2–8 μg/mL when applying UV detection. RSDs for migration times and peak areas were <2% and <7%, respectively. The applicability of the CE system is shown for the determination of organic acids in CSF samples.     

Synthesis and application of dehydroabietylisothiocyante as a new chiral derivatizing agent for the enantiomeric separation of chiral compounds by capillary electrophoretic

A new chiral derivatizing reagent, dehydroabietylisothiocyante (DHAIC), was synthesized and used for the enantiomeric separation of chiral compounds in capillary electrophoresis (CE). The synthetic route to obtain DHAIC is described. The separation conditions for the chiral separation of several chiral compounds, such as protein amino acids and chiral drug DOPA were optimized. Best results for the chiral separation of DHAIC derivatized amino acids and DOPA were obtained in a running buffer consisted of 50 mM borate (pH 9.5), 5 mM sodium dodecyl sulphate (SDS) and 20% acetonitrile for amino acids and 60 mM Na₂HPO₄ (pH 8.0), 17 mM SDS and 25% acetonitrile for DOPA. Under the conditions studied, chiral separation of five amino acids including Ser, Val, Ala, Thr, Cys and a chiral drug DOPA as their diastereomeric DHAIC derivatives has been achieved by micellar electrokinetic chromatography (MEKC).     

A modified supported bilayer/diblock polymer--working towards a tunable coating for capillary electrophoresis

A surfactant bilayer/diblock polymer coating was previously developed for the separation of proteins. The coating consisted of a mixture of the cationic surfactant dioctadecyldimethylammonium bromide (DODAB) and the neutral polymer poly-oxyethylene (POE) 40 stearate (Journal of Chromatography A 1130 (2006) 265–271). Herein an improved method of generating DODAB/POE stearate coatings is demonstrated, which yields more predictable EOF, more stable coatings, greater average efficiencies and easier method development. In this sequential preparation method the DODAB is first flowed through the capillary, followed by a flow of the POE stearate (sequential method). A tunable EOF (−2.40 to −0.17 × 10⁻⁴ cm²/Vs) is achieved by varying the POE chain length (8, 40 and 100 oxyethylene units). Mixtures of POE 8 and POE 40 stearate enabled continuous variation in EOF from −2.44 to −0.42 × 10⁻⁴ cm²/Vs. Separations of basic proteins yielded efficiencies of 760 000–940 000 plates/m. Coatings formed using the sequential method were more stable over a larger number of runs (%RSD for migration times: 0.7–1.0% over 30 runs) than those formed using the original mixed method (%RSD: 2.4–4.6% over 14 runs). The ability to tune the EOF is important in maximizing the resolution of analytes with similar electrophoretic mobilities. Histone proteins are separated on a sequentially coated capillary with resolution of nine possible subtypes. Acidic proteins are separated on a sequentially coated capillary at pH 6.4.     

Rapid detection of perchlorate in groundwater using capillary electrophoresis

Summary Perchlorate is a groundwater contaminant originating from facilities that manufacture and test solid rocket fuel. A new technology, capillary electrophoresis, has the potential to measure perchlorate rapidly and inexpensively in water samples. With its speed and simplicity, this method would complement existing methods. The perchlorate anion is routinely detected in water samples using high performance ion exchange chromatography, a very sensitive yet time consuming and expensive method. In this work, the parameters for detection of perchlorate are optimized to permit detection of 0.400 mgL⁻¹ perchlorate in a standard solution. The usefulness of this technology is demonstrated for measuring perchlorate in several ground-water samples from the Western United States. The results demonstrate that CE can be used to rapidly screen environmental samples for perchlorate at intermediate to high levels (greater than 0.400 mgL⁻¹). This technique allows faster, easier screening of potential contamination sites and could complement the use of ion exchange chromatography for groundwater testing.     

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).     

Development and validation of a capillary electrophoretic method for the determination of amiodarone and desethylamiodarone

Summary A simple, sensitive and rapid capillary electrophoretic method has been developed for the separation and quantification of amiodarone and its metabolite, desethylamiodarone. The compounds were separated in a capillary of 45 cm effective length and 75 μm i.d., by use of an applied voltage of 25 kV and an electrolyte containing 15mm ADA buffer (pH 7.5), 10mm SDS, and 70% (v/v) acetonitrile. The selectivity, precision, linearity, range, sensitivity, and robustness of the method were good. The applicability of the assay was demonstrated by analyzing these drugs in serum. Electrokinetic injection with field-amplified sample-stacking was used to increase sensitivity. The limit of detection of the serum assay was 6.46 ng mL⁻¹ and the precision 3.7%.     

A covalent capillary coating of diazoresin and polyglycerol dendrimer for protein analysis using capillary electrophoresis

Overcoming proteins adsorption on the inner surface of capillary has attracted increasing attention recently. By using the unique photochemistry reaction of diazoresin (DR), a new covalent capillary coating was prepared on the fused‐silica capillary through layer‐by‐layer self‐assembly of DR with polyglycerol (PG) dendrimer. The separation performance of covalently DR/PG‐dendrimer coated capillary noticeably exceeded the bare capillary and the noncovalently linked DR/PG‐dendrimer capillary. A baseline separation of lysozyme, myoglobin, bovine serum albumin, and ribonuclease A was achieved using CE within 20 min. Besides, the covalently linked DR/PG‐dendrimer coating has the remarkable stability and reproducibility. Especially, compared with the traditional method which use highly toxic and moisture‐sensitive silane coupling agent, this method seems to be a simple and environmental friendly way to prepare the covalently coated capillaries for CE.     

Capillary electrophoretic determination of ammonia using headspace single-drop microextraction

A new method involving headspace single-drop microextraction (SDME) and capillary electrophoresis (CE) is developed for the preconcentration and determination of ammonia (as dissolved NH₃ and ammonium ion). An aqueous microdrop (5 μL) containing 1 mmol/L H₃PO₄ and 0.5 mmol/L KH₂PO₄ (as internal standard) was used as the acceptor phase. Common experimental parameters (sample and acceptor phase pH, extraction temperature, extraction time) affecting the extraction efficiency were investigated. Proposed SDME-CE method provided about 14-fold enrichment in about 20 min. The calibration curve was linear for concentrations of NH₄⁺ in the range from 5 to 100 μmol/L (R² = 0.996). The LOD (S / N = 3) was estimated to be 1.5 μmol/L of NH₄⁺. Such detection sensitivity is high enough for ammonia determination in common environmental and biological samples. Finally, headspace SDME was applied to determine ammonia in human blood, seawater and milk samples with spiked recoveries in the range of 96-107%.     

Determination of pKa values by capillary zone electrophoresis with a dynamic coating procedure

CZE allows to measure the acidic dissociation constant (pKa) of many drug substances. However, determining the EOF intensity may be time-consuming, especially at a low pH. In order to overcome this drawback, a dynamic coating procedure of the capillary was carried out to increase microEOF, and thus to reduce the analysis time. In addition, this coating procedure enhanced migration time stability. The effective mobilities of 15 compounds were measured at different pH, producing pK'a values dependent on BGE ionic strength. The latter values were corrected with the activity coefficient to obtain a "true" pKa value. The 15 investigated compounds were (i) five acids: namely, salicylic acid, benzoic acid, ketoprofen, phenobarbital, and phenol, (ii) four bases: lidocaine, propafenone, propranolol, and quinine, (iii), five ampholytes: sulfanilamide, sulfabenzamide, sulfadimethoxine, sulfadoxine, and sulfisoxazole, and (iv) one zwitterion: cetirizine. The range of determined pKa values was between 1.2 and 11.2, and close to the pKa values available from the literature.     

Rapid identification of pathogenic bacteria by capillary electrophoretic analysis of rRNA genes

Molecular diagnosis is playing an increasingly important role in the rapid detection and identification of pathogenic organisms in clinical samples. The genetic variation of ribosomal genes in bacteria offers an alternative to culturing for the detection and identification of these organisms. Here 16S rRNA and 16S-23S rRNA spacer region genes were chosen as the amplified targets for single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) capillary electrophoresis analysis and bacterial identification. The multiple fluorescence based SSCP method for the 16S rRNA gene and the RFLP method for the 16S-23S rRNA spacer region gene were developed and applied to the identification of pathogenic bacteria in clinical samples, in which home-made short-chained linear polyacrylamide (LPA) was used as a sieving matrix; a higher sieving capability and shorter analysis time were achieved than with a commercial sieving matrix because of the simplified template preparation procedure. A set of 270 pathogenic bacteria representing 34 species in 14 genera were analyzed, and a total of 34 unique SSCP patterns representing 34 different pathogenic bacterial species were determined. Based on the use of machine code to represent peak patterns developed in this paper, the identification of bacterial species becomes much easier.     

Continuous on-line derivatization and determination of amino acids by a microfluidic capillary electrophoresis system with a continuous sample introduction interface

An automatic, rapid and continuous on-line derivatization system coupled to microfluidic capillary electrophoresis (CE) for the determination of amino acids using o-phthaldialdehyde/N-acetyl-l-cysteine (OPA/NAC) as the derivative agents has been developed. By on-line derivatization, amino acids were automatically and reproducibly converted to the UV-absorbing derivatives, which were separated by capillary zone electrophoresis (CZE). Optimization of derivatization and separation condition was carried out to achieve both good sensitivity and separation efficiency. The separation could be achieved within 4 min and sample throughput rate can reach up to 16 h⁻¹. The repeatability (defined as relative standard deviation, R.S.D.) was 2.56, 2.85, 3.24 and 3.60% with peak area evaluation and 2.93, 3.12, 4.20 and 4.91% with peak height evaluation for arginine (Arg), phenylalanine (Phe), serine (Ser) and glycine (Gly), respectively. The limits of detection (S/N=3) were 10.46, 13.14, 34.39 and 44.79 μmol/l for Arg, Phe, Ser and Gly, respectively. Major advantages of the proposed method include improved precision and efficient automation of the derivatization by the FI system and the enhanced sampling frequencies by the combined FI-CE system.     

A semipermanent coating for preventing protein adsorption at physiological pH in kinetic capillary electrophoresis

Protein adsorption to the inner capillary wall hinders the use of kinetic capillary electrophoresis (KCE) when studying noncovalent protein-ligand interactions. Permanent and dynamic capillary coatings have been previously reported to alleviate much of the problems associated with protein adsorption. The characteristic limitations associated with permanent and dynamic coatings motivated us to look at a third type of coating - semipermanent. Here, we demonstrate that a semipermanent capillary coating, designed by Lucy and co-workers, comprised of dioctadecyldimethylammonium bromide (DODAB) and polyoxyethylene (POE) stearate, greatly reduces protein adsorption at physiological pH - a necessary requirement for KCE. The coating (i) does not inhibit protein-DNA complex formation, (ii) prevents the adsorption of the analytes, and (iii) supports an electoosmotic flow required for many applications of KCE. The coating was tested in three physiological buffers using a well-known DNA aptamer and four proteins that severely bind to bare silica capillaries as standards. For every protein, a condition was found under which the semipermanent coating effectively suppresses protein adhesion. While no coating can completely prevent the adsorption of all proteins, our findings suggest that the DODAB/POE stearate coating can have a broad impact on CE at large, as it prevents the absorption of several well studied, highly adhesive proteins at physiological pH.