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.     

Wall modified photonic crystal fibre capillaries as porous layer open tubular columns for in-capillary micro-extraction and capillary chromatography

Wall modified photonic crystal fibre capillary columns for in-capillary micro-extraction and liquid chromatographic separations is presented. Columns contained 126 internal parallel 4 μm channels, each containing a wall bonded porous monolithic type polystyrene-divinylbenzene layer in open tubular column format (PLOT). Modification longitudinal homogeneity was monitored using scanning contactless conductivity detection and scanning electron microscopy. The multichannel open tubular capillary column showed channel diameter and polymer layer consistency of 4.2 ± 0.1 μm and 0.26 ± 0.02 μm respectively, and modification of 100% of the parallel channels with the monolithic polymer. The modified multi-channel capillaries were applied to the in-capillary micro-extraction of water samples. 500 μL of water samples containing single μg L⁻¹ levels of polyaromatic hydrocarbons were extracted at a flow rate of 10 μL min⁻¹, and eluted in 50 μL of acetonitrile for analysis using HPLC with fluorescence detection. HPLC LODs were 0.08, 0.02 and 0.05 μg L⁻¹ for acenaphthene, anthracene and pyrene, respectively, with extraction recoveries of between 77 and 103%. The modified capillaries were also investigated briefly for direct application to liquid chromatographic separations, with the retention and elution of a standard protein (cytochrome c) under isocratic conditions demonstrated, proving chromatographic potential of the new column format, with run-to-run retention time reproducibility of below 1%.     

Hydrophobic-phase-modified fused-silica columns for capillary electrophoresis

Coated capillaries modified with a hydrophobic layer were developed. Linear hydrocarbons and ethylbenzene modified surfaces greatly improved the electrophoretic performance of the capillaries. The column efficiency for organic compounds reached as high as 327 000 theoretical plate numbers per meter on a 50 microm I.D. linear hydrocarbon (C6) surface treated fused-silica capillary column. This value did not change during 50 repeated analyses and the columns showed strong stability against 0.1 M NaOH and 0.1 M HCl. The relative standard deviation of the run-to-run, day-to-day, and capillary-to-capillary coating with hydrophobic layer showed values of < or =2.5%, and good reproducibility. The separations of four aromatic amines and six pharmacological amines at pH 2.5 is reported.     

Polyethyleneimine-bonded phases in the separation of proteins by capillary electrophoresis

A hydrophilic, positively charged, durable coating has been developed for capillary electrophoresis of macromolecules. Polyethyleneimine is adsorbed to the inner wall of fused silica capillaries and the adsorbed coating cross-linked into a stable layer. Capillaries of polyethyleneimine-coated silica gave unique separations owing to the reversal of electro-osmotic flow caused by the positively charged coating. The resulting coating was stable from pH 2-12 and could be used over a wide pH range without substantial change in electro-osmotic flow. High-molecular-weight polymers were needed to give thick coatings which mask silanol groups on the wall. Proteins were resolved quickly and efficiently with good recovery using capillaries of 50 cm in length.     

Practical considerations for preparing polymerized phospholipid bilayer capillary coatings for protein separations

Phosphorylcholine (PC) based phospholipid bilayers have proven useful as capillary coating materials due to their inherent resistance to non-specific protein adsorption. The primary limitation of this important class of capillary coatings remains the limited long-term chemical and physical stability of the coatings. Recently, a method for increasing phospholipid coating stability in fused silica capillaries via utilization of polymerized, synthetic phospholipids was reported. Here, we expand upon these studies by investigating polymerized lipid bilayer capillary coatings with respect to separation performance including run-to-run, day-to-day and column-to-column reproducibility and long-term stability. In addition, the effects of pH and capillary inner diameter on polymerized phospholipid coated capillaries were investigated to identify optimized coating conditions. The coatings are stabilized for protein separations across a wide range of pH values (4.0–9.3), a unique property for capillary coating materials. Additionally, smaller inner diameter capillaries (≤50 μm) were found to yield marked enhancements in coating stability and reproducibility compared to wider bore capillaries, demonstrating the importance of capillary size for separations employing polymerized phospholipid coatings.     

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.     

Poly-N-hydroxyethylacrylamide as a novel, adsorbed coating for protein separation by capillary electrophoresis.

We present the polymer poly-N-hydroxyethylacrylamide (PHEA) (polyDuramide) as a novel, hydrophilic, adsorbed capillary coating for electrophoretic protein analysis. Preparation of the PHEA coating requires a simple and fast (30 min) protocol that can be easily automated in capillary electrophoresis instruments. Over the pH range of 3-8.4, the PHEA coating is shown to reduce electroosmotic flow (EOF) by about 2 orders of magnitude compared to the bare silica capillary. In a systematic comparative study, the adsorbed PHEA coating exhibited minimal interactions with both acidic and basic proteins, providing efficient protein separations with excellent reproducibility on par with a covalent polyacrylamide coating. Hydrophobic interactions between proteins and a relatively hydrophobic poly-N,N-dimethylacrylamide (PDMA) adsorbed coating, on the other hand, adversely affected separation reproducibility and efficiency. Under both acidic and basic buffer conditions, the adsorbed PHEA coating produced an EOF suppression performance comparable to that of covalent polyacrylamide coating and superior to that of adsorbed PDMA coating. The protein separation performance in PHEA-coated capillaries was retained for 275 consecutive protein separation runs at pH 8.4, and for more than 800 runs at pH 4.4. The unique and novel combination of hydrophilicity and adsorptive coating ability of PHEA makes it a suitable wall coating for automated microscale analysis of proteins by capillary array systems.     

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.     

Rapid analysis of spent fixing solutions by capillary electrophoresis

Summary A capillary electrophoretic method for the determination of thiosulphate, bromide, Fe(III)-EDTA chelate and free EDTA in spent fixing solutions has been developed. Free EDTA was complexed with Ni(II) ions prior to analysis. The optimised separations were carried out in a fused silica capillary (57 cm×75 μm I.D.) filled with a borate buffer (100mmol L⁻¹ borate, 0.2 mmol L⁻¹ tetradecyltrimethylammonium hydroxide, pH 8.5; applied voltage, −30kV) using direct UV detection at 214 nm. All four anions were well separated in less than 4 min. The method was applied to the rapid monitoring of spent fixing solutions.     

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.     

A fully automated linear polyacrylamide coating and regeneration method for capillary electrophoresis of proteins

Surface modification of the inner capillary wall in CE of proteins is frequently required to alter EOF and to prevent protein adsorption. Manual protocols for such coating techniques are cumbersome. In this paper, an automated covalent linear polyacrylamide coating and regeneration process is described to support long‐term stability of fused‐silica capillaries for protein analysis. The stability of the resulting capillary coatings was evaluated by a large number of separations using a three‐protein test mixture in pH 6 and 3 buffer systems. The results were compared to that obtained with the use of bare fused‐silica capillaries. If necessary, the fully automated capillary coating process was easily applied to regenerate the capillary to extend its useful life‐time.     

Determination of artificial sweeteners by capillary electrophoresis with contactless conductivity detection optimized by hydrodynamic pumping

The common sweeteners aspartame, cyclamate, saccharin and acesulfame K were determined by capillary electrophoresis with contactless conductivity detection. In order to obtain the best compromise between separation efficiency and analysis time hydrodynamic pumping was imposed during the electrophoresis run employing a sequential injection manifold based on a syringe pump. Band broadening was avoided by using capillaries of a narrow 10 μm internal diameter. The analyses were carried out in an aqueous running buffer consisting of 150 mM 2-(cyclohexylamino)ethanesulfonic acid and 400 mM tris(hydroxymethyl)aminomethane at pH 9.1 in order to render all analytes in the fully deprotonated anionic form. The use of surface modification to eliminate or reverse the electroosmotic flow was not necessary due to the superimposed bulk flow. The use of hydrodynamic pumping allowed easy optimization, either for fast separations (80 s) or low detection limits (6.5 μmol L⁻¹, 5.0 μmol L⁻¹, 4.0 μmol L⁻¹ and 3.8 μmol L⁻¹ for aspartame, cyclamate, saccharin and acesulfame K respectively, at a separation time of 190 s). The conditions for fast separations not only led to higher limits of detection but also to a narrower dynamic range. However, the settings can be changed readily between separations if needed. The four compounds were determined successfully in food 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.     

Ethylbenzene-modified fused-silica columns for capillary electrophoresis

Summary Coated capillaries modified with a hydrocarbon layer have been developed. Modification of the surface with ethylbenzene greatly improved the electrophoretic performance of the capillaries. The column efficiency for basic organic compounds was as high as 378000 theoretical plates per meter on a 50 μm i.d. ethylbenzene-surface-treated fused-silica capillary column. This value did not change during 25 replicate analyses and the capillary columns were very stable against continuous treatment for 30 h with buffer of pH 10 and treatment for 3 h with HCl of pH 1 and NaOH of pH 12. The relative standard deviation of run-to-run, day-to-day, and capillary-to-capillary for coating with the ethylbenzene layer was<2.6%, and reproducibility was good. The separation of four aromatic amines and six pharmacological amines at pH 2.5 is reported.     

Injection system for fast capillary electrophoresis based on pressure regulation with flow restrictors

A fast automated system for rapid electrophoretic separations in short conventional capillaries employing contactless conductivity detection is presented. The instrument is based on pneumatic pressurization and does not require a conventional pump. The required pressures and flow rates for the different steps of the injection and flushing processes are produced with the help of two flow restrictors. The device is implemented on a microfluidic breadboard with dimensions of ca. 13 × 20 cm and employs miniature valves. Nine inorganic cations, namely NH₄⁺_, K⁺, Na⁺, Ca²⁺, Mg²⁺, Mn²⁺, Sr²⁺, Li⁺, and Ba²⁺, could be separated in a capillary of 10 μm inner diameter and 6 cm effective length within 25 s. Following a reduction of the effective length to 4 cm, still five inorganic cations could be separated in a time span of 12 s. The repeatability of peak areas was better than 3.1 % and limits of detection between 3.5 and 5.5 μM were achieved.     

Development of a stability-indicating capillary electrophoresis method for norfloxacin and its inactive decarboxylated degradant

A simple, accurate, precise, rapid and sensitive stability-indicating capillary electrophoresis (CE) method was optimized and validated for the simultaneous determination of norfloxacin and its inactive decarboxylated degradant in pharmaceuticals. The univariant method was used to optimize electrophoretic factors including injection time, separation voltage and column temperature. Electrolyte concentration and pH were optimized using the factorial design and response surface methods. The optimum conditions obtained were: 10 mmol l^− 1 phosphate at pH 2.5, hydrodynamic injection time of 8 s at pressure 0.5 p.s.i., separation voltage 25 kV and column temperature 25 °C. The separation was carried out into a fused-silica capillary column (31.2 cm length × 50 μm i.d.) with detection at 301 and 285 nm for the intact drug and the degradant, respectively using a diode array detector. For both analytes, the method enjoys wide dynamic range (1-50 μg ml^− 1) with good detectability (limits of detection 0.11 μg ml^− 1). In addition, acceptable accuracy (recovery > 95%); and good repeatability and intermediate precision (RSD < 3.5%) were obtained.     

Fast capillary electrophoresis-time-of-flight mass spectrometry using capillaries with inner diameters ranging from 75 to 5 μm

Fast electrophoretic separations in fused silica capillaries (CE) coupled to time-of-flight mass spectrometry (TOF-MS) are presented. CE separations of the model analytes (epinephrine, norepinephrine, dopamine, histidine, and isoproterenol) under conditions of high electric field strengths of up to 1.25 kV cm⁻¹ are completed in 20 s. Coupling of CE with TOF-MS is accomplished using a coaxial sheath liquid electrospray ionization interface. The influence of parameters inherent to the interface and their effects, including suction pressure and dilution, are discussed. In addition to standard capillaries of 75 and 50 μm inner diameter (ID), separations in capillaries with IDs of 25, 15, and 5 μm have been successfully applied to this setup. The analytical performance is compared over this range of capillary dimensions, and both advantages and disadvantages are discussed.     

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.     

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.     

Polymethacrylate monolithic and hybrid particle-monolithic columns for reversed-phase and hydrophilic interaction capillary liquid chromatography

We prepared hybrid particle-monolithic polymethacrylate columns for micro-HPLC by in situ polymerization in fused silica capillaries pre-packed with 3–5 μm C₁₈ and aminopropyl silica bonded particles, using polymerization mixtures based on laurylmethacrylate–ethylene dimethacrylate (co)polymers for the reversed-phase (RP) mode and [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl) zwitterionic (co)polymers for the hydrophilic interaction (HILIC) mode. The hybrid particle-monolithic columns showed reduced porosity and hold-up volumes, approximately 2–2.5 times lower in comparison to the pure monolithic columns prepared in the whole volume of empty capillaries. The elution volumes of sample compounds are also generally lower in comparison to packed or pure monolithic columns. The efficiency and permeability of the hybrid columns are intermediate in between the properties of the reference pure monolithic and particle-packed columns. The chemistries of the embedded solid particles and of the interparticle monolithic moiety in the hybrid capillary columns contribute to the retention to various degrees, affecting the selectivity of separation. Some hybrid columns provided improved separations of proteins in comparison to the reference particle-packed columns in the reversed-phase mode. Zwitterionic hybrid particle-monolithic columns show dual mode retention HILIC/RP behaviour depending on the composition of the mobile phase and allow separations of polar compounds such as phenolic acids in the HILIC mode at lower concentrations of acetonitrile and, often in shorter analysis time in comparison to particle-packed and full-volume monolithic columns.