Wall Of Fused-silica Capillary Research Articles

Modifying last layer in polyelectrolyte multilayer coatings for capillary electrophoresis of proteins

Protein adsorption on the inner wall of the fused silica capillary wall is an important concern for capillary electrophoresis (CE) analysis since it is mainly responsible for separation efficiency reduction. Successive Multiple Ionic-polymer Layers (SMIL) are used as capillary coatings to limit protein adsorption, but even low residual adsorption strongly impacts the separation efficiency, especially at high separation voltages. In this work, the influence of the chemical nature and the PEGylation of the polyelectrolyte deposited in the last layer of the SMIL coating was investigated on the separation performances of a mixture of four model intact proteins (myoglobin (Myo), trypsin inhibitor (TI), ribonuclease a (RNAse A) and lysozyme (Lyz)). Poly(allylamine hydrochloride) (PAH), polyethyleneimine (PEI), ε-poly(L-lysine) (εPLL) and α-poly(L-lysine) (αPLL) were compared before and after chemical modification with polyethyleneglycol (PEG) of different chain lengths. The experimental results obtained by performing electrophoretic separations at different separation voltages allowed determining the residual retention factor of the proteins onto the capillary wall via the determination of the plate height at different solute velocities and demonstrated a strong impact of the polycationic last layer on the electroosmotic mobility, the separation efficiency and the overall resolution. Properties of SMIL coatings were also characterized by quartz microbalance and atomic force microscopy, demonstrating a glassy structure of the films.

Covalent cationic copolymer coatings allowing tunable electroosmotic flow for optimization of capillary electrophoretic separations

Electroosmotic flow (EOF) plays a pivotal role in optimization of capillary electrophoresis (CE) separations of (bio)molecules and (bio)particles. EOF velocity is directly related to analysis time, peak resolution and separation efficiency. Here, we report a concept of charged polymer coatings of the inner fused silica capillary wall, which allows anodic EOF with mobility ranging from 0 to ∼(30–40) × 10−9 m2V−1s−1. The capillary wall is modified by covalently bound cationic copolymer poly(acrylamide-co-(3-acrylamidopropyl)trimethylammonium chloride) (PAMAPTAC) containing variable ratio of the charged monomer in the 0–60 mol. % interval. The EOF mobility showed minor variability with composition of background electrolyte (BGE) and pH in the 2–10 interval. The coatings were evaluated by CE-UV and nanospray CE-MS in the counter-EOF arrangement for a series of basic drug molecules in acetic acid based acidic BGE. Tunable EOF velocity was demonstrated as a useful tool for optimization of peak resolution, separation efficiency and migration time of analytes. Electrostatic repulsion of positively charged capillary surface was shown as beneficial for suppression of analyte adsorption, notably for hydrophobic cationic analytes.

Multifunction covalent coatings for separation of amino acids, biogenic amines, steroid hormones, and ketoprofen enantiomers by capillary electrophoresis and capillary electrochromatography

AbstractIn this study, the preparation process of multifunctional coating based on imidazole and β‐cyclodextrin has been developed. The synthesis includes several steps: activation of fused silica capillary walls via alkali, then process of silylation and finally functionalization with imidazole and β‐cyclodextrin derivative. The latter step was carried over 22 h at 100°C at 750 mg/mL concentration of p‐toluenesulfonyl‐β‐cyclodextrin. It was established that the coatings thus obtained are stable within pH 2–7 range but decompose in alkaline medium. The stationary phase developed in this study allows simultaneous separation of hydrophobic and hydrophilic analytes. Chiral separation of ketoprofen enantiomers was also achieved with the newly developed covalent coating. It was found that covalent coating based on imidazole and β‐cyclodextrin exceeded the dynamic coatings of similar structure in terms of repeatability, analysis time and the limits of detection.

Coherent Optical Transduction of Suspended Microcapillary Resonators for Multi-Parameter Sensing Applications.

Characterization of micro and nanoparticle mass has become increasingly relevant in a wide range of fields, from materials science to drug development. The real-time analysis of complex mixtures in liquids demands very high mass sensitivity and high throughput. One of the most promising approaches for real-time measurements in liquid, with an excellent mass sensitivity, is the use of suspended microchannel resonators, where a carrier liquid containing the analytes flows through a nanomechanical resonator while tracking its resonance frequency shift. To this end, an extremely sensitive mechanical displacement technique is necessary. Here, we have developed an optomechanical transduction technique to enhance the mechanical displacement sensitivity of optically transparent hollow nanomechanical resonators. The capillaries have been fabricated by using a thermal stretching technique, which allows to accurately control the final dimensions of the device. We have experimentally demonstrated the light coupling into the fused silica capillary walls and how the evanescent light coming out from the silica interferes with the surrounding electromagnetic field distribution, a standing wave sustained by the incident laser and the reflected power from the substrate, modulating the reflectivity. The enhancement of the displacement sensitivity due to this interferometric modulation (two orders of magnitude better than compared with previous accomplishments) has been theoretically predicted and experimentally demonstrated.

Poly-4-vinylpiridinium nanosponges as modifiers of the electrophoretic systems for the charged analytes separation

Novel hydrophilic ionic polymer nanoparticles based on the N-alkylated hypercrosslinked poly-4-vinylpyridine (nanosponges ‒ NS ) have porous structure, pH-independent positive charge and contain the aromatic rings. These nanosponges have not yet been studied in the capillary electrophoresis mode, but their characteristics suggest the possibility of creating the fused silica capillary walls coating for the separation of anionic and cationic analytes. In the current research, the authors, for the first time, have proposed the approaches to the formation of coatings based on the nanosponges with a molecular weight of 400 and 10 kDa. It was established that the stability of such coatings could be achieved by the introduction of NS into the background electrolyte. The pH range of background electrolytes for the subsequent electrophoretic experiments was determined to be from 4 to 9. The NS-based coatings were compared with the previously investigated coatings based on the crosslinked polystyrene nanoparticles ( NPs ), functionalized with the quaternary ammonium groups. Capillaries modified with NS (400 kDa) were tested for the electrophoretic separations of wide range of analytes: carboxylic acids, aminoacids, fluoroquinoline antibiotics, biogenic amines, and proteins. The separation selectivity of carboxylic acids on the NS-modified capillaries was different from the separation selectivity on the capillaries modified with NPs, functionalized with quaternary ammonium groups. NS affect the separation selectivity of aminoacids by interacting with these analytes, while for fluoroquinoline antibiotics NS act as an agent generating the reversed electroosmotic flow ( EOF ). The modification of fused silica capillary walls by NS with molecular mass of 400 kDa prevented the sorption of biogeniс amines and lyzocyme protein during their electrophoretic separation. Keywords : capillary electrophoresis, nanoparticles, coatings of fused silica capillary walls DOI: http://dx.doi.org/10.15826/analitika.2019.23.2.006 (Russian) D.A. Polikarpova 1 , D.V. Makeeva 1 , L.A. Kartsova 1 , V.A. Davankov 2 , L.A. Pavlova 2 1 Saint-Petersburg State University, Institute of Chemistry, Universitetskii pr. 26., St. Petersburg, Petergof, 198504, Russian Federation 2 A.N. Nesmeyanov Institute of Organoelement Compounds of Russian Academy of Sciences, Vavilova St. 28, Moscow, 119334, Russian Federation

Nano-sized anion-exchangers as a stationary phase in capillary electrochromatography for separation and on-line concentration of carboxylic acids

Nano-sized anion-exchangers (NSAE) are promising materials in electrophoretic separation methods due to their high ion-exchange capacity, large surface-to-volume ratios, high adhesion to the quartz surface and pH-independent positive charge. In current research we describe a simple approach for NSAE synthesis, which includes two-step grinding of macroanionite followed by centrifugation. The synthesized stable aqueous suspension of NSAE particles was applied as physically adsorbed modifier of fused-silica capillary walls for CEC separation of carboxylic acids. We proposed fast and simple approach to formation of NSAE-based stationary phase on the internal fused-silica surface, which included 15 min rinsing of the capillary with diluted water suspension of NSAE. Formed physically-adsorbed coating turned out to be extremely stable in a wide range of pH (from 2 to 10). NSAE modified capillaries provided high separation efficiency (N = 148–732 *103 t.p./m) and selectivity (Rs = 1.2–5.7) of carboxylic acids. Simultaneous application of NSAE-modified capillaries with various on-line concentration techniques (such as field amplified sample stacking and field amplified sample injection) provided both low detection limits (up to 1–3 ng/mL) and high separation selectivity of carboxylic acids. It was useful for their quantitative determination in wines samples. Physically-adsorbed coatings based on NSAE exhibit higher selectivity and lower detection limits compared to commonly used dynamic modifier of fused-silica capillary walls - cetyltrimethylammonium bromide. NSAE-based coatings do not require equilibrium sustaining to maintain the surface coverage. It makes them appropriate for CE-MS application.

Highly fluorinated polymers with sulfonate, sulfamide and N,N-diethylamino groups for the capillary electromigration separation of proteins and steroid hormones.

Highly fluorinated polymers are promising materials in separation methods due to their combination of high chemical and thermally stability, hydro- and oleophobicity, and weak intermolecular forces. However, application of these polymers in chromatography is limited because of their low solubility in aqueous-organic solvents. In our research, the highly fluorinated water soluble polymers with -SO3- N(Et)4+ , -SO2 NH2 , and -N(Et)2 terminal groups were synthesized and applied as additives to the background electrolyte for the separation of steroid hormones and proteins by capillary electromigration methods. It is shown that highly fluorinated polymers can be used both as pseudo-stationary phases in electrokinetic chromatography for high separation efficiency (N∼200×103 ) and selectivity (α∼1.1) of uncharged analytes (e.g., steroid hormones), and as dynamic modifiers of fused silica capillary walls. The highest separation efficiency (N∼1×106 ) and selectivity (α∼1.3) of steroid hormones was achieved by combination of sodium dodecyl sulfate and fluoropolymer with sulfonate groups in background electrolyte with pH 2. Dynamic wall coatings based on fluoropolymer with -SO2 NH2 (which are easier and faster to create and wash off) exhibit significantly higher separation efficiency and selectivity compared to capillary electrochromatography on capillary columns based on polymethacrylate polymers.

A star-shaped poly(2-methyl-2-oxazoline)-based antifouling coating: Application in investigation of the interaction between acetaminophen and bovine serum albumin by frontal analysis capillary electrophoresis

In this work, an antifouling capillary modified with star-shaped poly(2-methyl-2-oxazoline)-based copolymer was used to study the interaction between acetaminophen (APAP) and bovine serum albumin (BSA) by frontal analysis capillary electrophoresis (FACE). The star-shaped copolymer, poly(ethylene imine)-graft-poly(2-methyl-2-oxazoline) (PEI-g-PMOXA), was immobilized onto the fused-silica capillary inner wall via dopamine-assisted co-deposition strategy, yielding a PEI-g-PMOXA/polydopamine (PDA)-coated antifouling capillary, i.e., an antifouling capillary coated with the PEI-g-PMOXA/PDA co-deposited film. Electroosmotic flow (EOF) mobility of the PEI-g-PMOXA/PDA-coated capillary was almost zero in a wide pH range (3.0–10.0), while the EOF mobility of bare capillary was much larger and increased significantly with pH increasing. When the PEI-g-PMOXA/PDA-coated capillary was exploited to separate a protein mixture including cytochrome c, lysozyme, ribonuclease A and α-chymotrypsinogen A, the theoretical plate numbers were of five orders of magnitude which were about ten-fold higher over those obtained with bare capillary; in addition, the RSD values of migration time were mostly less than 0.7% (30 consecutive runs) which were much smaller than those of bare capillary (c.a. 5.7%). The protein-resistant PEI-g-PMOXA/PDA-coated capillary was then used to investigate the interaction between APAP and BSA by FACE, the binding constant and number of binding sites at 25°C and pH 7.4 (Tris/HCl buffer of 25mM) were 1.39×104M−1 and 1.08, respectively, which were comparable to the results determined by fluorescence spectroscopic measurement (3.18×104M−1 and 1.19, respectively).

Poly (2-methyl-2-oxazoline) coating by thermally induced immobilization for determination of bovine lactoferrin in infant formula with capillary electrophoresis

In this work, a one-step coating procedure by a simple annealing protocol of poly (2-methyl-2-oxazoline)-random-glycidyl methacrylate (PMOXA-r-GMA) copolymer was used to yield covalent and cross-linked PMOXA-based antifouling coating on a fused-silica capillary inner surface, which was used to determine the bovine lactoferrin (Lf) in infant formula by capillary electrophoresis with ultraviolet (CE-UV) detection. The X-ray photoelectron spectroscopy (XPS) confirmed that PMOXA-r-GMA could be bonded onto fused-silica capillary inner wall and stable electroosmotic flow (EOF) was obtained in the PMOXA-r-GMA coated capillary at pH 2.2–9.0. The separation of a mixture of four basic proteins indicated that the PMOXA-r-GMA coated capillary exhibited excellent separation efficiency for the basic proteins. Therefore, the PMOXA-r-GMA coated capillary was used to determine the quantity of Lf in infant formula. Under the optimal conditions, the peak area (A) and the concentration of Lf showed a good linear relationship within the range of 10–500μg/mL with a linear correlation coefficient of 0.9995. The limit of detection (LOD) and limit of quantitation (LOQ) for Lf were 5.0μg/mL and 16.7μg/mL, respectively. The recoveries at spiked concentrations of 0.20 and 0.40mg/mL Lf in infant formula were 97.1±5.5% – 97.8±5.1%. The determined values of Lf in infant formula samples with Lf were consistent with the nominal values, indicating that our CE method could be successfully applied to the quantitative analysis of Lf in commercial infant formula.

Layer-by-layer assembly of zeolite imidazolate framework-8 as coating material for capillary electrochromatography.

In this work, open-tubular capillary column coated with zeolite imidazolate framework-8 (ZIF-8) nanocrystals was prepared by a layer-by-layer method. The coating was formed by growing ZIF-8 nanocrystals on either bare fused silica capillary wall or the capillary column premodified with amino groups. The shape and the thickness of the coating formed by using these two methods were almost the same. However, the coverage of the ZIF-8 crystals on the bare fused silica capillary wall was higher than that on the capillary column premodified with amino groups. The ZIF-8 coated capillary column was evaluated for open-tubular capillary electrochromatography. The effect of pH value, buffer concentration, and applied voltage on the separation of phenols was investigated. Good separation of nine phenolic isomers was achieved because of the strong interaction between unsaturated Zn sites and phenols. The column performance for o-nitrophenol was as high as 208 860 plates m(-1) . The run-to-run, day-to-day, and column-to-column reproducibility of retention time and resolution for p-nitrophenol and o-nitrophenol were very good with RSDs of less than 6.5%.

Analytical mitigation of solute–capillary interactions in double detection Taylor Dispersion Analysis

Taylor Dispersion Analysis (TDA) in the presence of interactions between solutes and capillary walls yields inaccurate results for the diffusion coefficients of the solutes because the resulting concentration profiles are broadened and asymmetric. Whilst there are practical ways of mitigating these interactions, it is not always possible to eradicate them completely. In this paper, an analytical method of mitigating the effects of the adsorptions is presented. By observing the dispersion of the solute molecules at two detection points and using the expected relations between measured parameters, such as the standard deviations and peak amplitudes, the dispersive components of the profiles were isolated with a constrained fitting algorithm. The method was successfully applied to lysozyme and cytochrome C which adsorb onto fused silica capillary walls. Furthermore, this illustrates an advantage of using the fitting method for Taylor Dispersion Analysis.

One-dimensional approach to study kinetics of reversible binding of protein on capillary walls.

We introduce a method for kinetic characterization of reversible binding of protein onto the inner capillary wall. In essence, a short plug of the protein solution is propagated through the capillary by pressure, and the protein is detected at the distal capillary end. The signal versus time profile is fitted with a numerical model which uses the rate constants of adsorption, kad, and desorption, kde, as fitting parameters. The values of kad and kde which result in the best fit are considered to be the sought ones. We first used COMSOL multiphysics software to develop a numerical model with two-dimensional (2D) equations of mass transfer. Although 2D models in general can describe experiments more accurately than one-dimensional (1D) models, computing 2D models takes much more time (many hours to find two parameters: kad and kde). We used the fact that the capillary is narrow and long to develop a simplified model with 1D equations of mass transfer. Our comparison of the 1D and 2D models showed that the errors of the 1D approximation were less than 5%, whereas the computation of the 1D model was 100 times faster. We finally used the 1D approach to study kinetics of reversible binding of conalbumin to the uncoated fused-silica capillary walls. We determined kad, kde, and a diffusion coefficient, D. The obtained value of D is in excellent agreement with literature data which suggests that the values of kad and kde (for which there are no literature data) are also calculated correctly. Our approach for finding kad and kde will facilitate quantitative characterization of protein adsorption on capillary walls as well as properties of passivating materials used for capillary coating.

Photopolymerization of acrylamide as a new functionalization way of silica monoliths for hydrophilic interaction chromatography and coated silica capillaries for capillary electrophoresis

A simple, rapid and localizable photochemical process for the preparation of hydrophilic coated capillary and silica-based monolithic capillary columns is described. The process involves the free radical polymerization of acrylamide monomers onto acrylate pre-activated silica surface triggered by UV photoinitiation. The experimental conditions (monomer content, time of irradiation) were optimized on silica monolithic columns by monitoring the evolution of the chromatographic properties (retention, permeability, efficiency) in HILIC mode using a set of nucleosides as test solutes. Compared to thermal polymerization process, the photoinitiation allows the preparation of highly retentive and efficient HILIC monolithic columns in less than 10min of irradiation. This process was then successfully applied to the surface coating of fused silica capillary walls. In addition to its relative high stability and ability to reduce the electroosmotic flow, this polyacrylamide coating is localizable. Benefits of this localizable photochemical process are highlighted through the conception of an in-line integrated bimodal microseparation tool combining a SPE preconcentration step on a photografted silica monolith and an electrokinetic separation step in a polyacrylamide photopolymerized capillary section. Two neuropeptides are used as model solutes to illustrate the suitability of this approach.

Application of the copolymers containing sulfobetaine methacrylate in protein separation by capillary electrophoresis

This study describes the formation of highly efficient antiprotein adsorption random copolymer coating of poly(N,N-dimethylacrylamide-co-sulfobetaine methacrylate) (poly(DMA-co-SBMA)) on the fused-silica capillary inner wall. Firstly, the poly(DMA-co-SBMA)s with different feed ratio (SBMA/DMA) were synthesized via the reversible addition fragmentation chain transfer polymerization. And then, X-ray photoelectron spectroscopy (XPS) and water contact angle (CA) were used to investigate the composition and hydrophilicity of poly(DMA-co-SBMA) coating formed on the glass slide surfaces. CA measurements revealed that the poly(DMA-co-SBMA) coating became more hydrophilic with the increment of feed ratio (SBMA/DMA), and at the same time, the XPS results showed that the coating ability was also increased with the increment of feed ratio. Followed, the copolymer was applied to coat the fused-silica capillary inner wall, and the coated capillary was used to separate the mixture of proteins (lysozyme, cytochrome c, ribonuclease A, and α-chymotrypsinogen A) in a pH range from 3.0 to 5.0. Under the optimum conditions, an excellent separation of basic proteins with peak efficiencies ranging from 551,000 to 1509,000 N/m had been accomplished within 10 min. Furthermore, the effect of coating composition on protein separation was also investigated through the comparison of separation efficiency achieved by using bare, PSBMA- and poly(DMA-co-SBMA)-coated capillary, respectively.

Graphene oxide and reduced graphene oxide as novel stationary phases via electrostatic assembly for open‐tubular capillary electrochromatography

We present here the application of graphene oxide (GO) and reduced graphene oxide (GOOH) sheet as novel stationary phases for open-tubular CEC (OTCEC) separation based on electrostatic assembly. The inner walls of a bare capillary column was first modified by ionic assembly of poly (diallyldimethylammonium chloride) (PDDA), and then negatively charged GO or GOOH was easily assembled on a positively charged interior walls of the capillary by electrostatic force. Scanning Electron Microscope images showed that GO and GOOH can still maintain sheet-layer-like structure when coated onto the capillary via electrostatic assembly. The chromatographic properties of the GO and GOOH coated columns were evaluated via OTCEC separations of various kinds of analytes, including three acid nitrophenol isomers, three basic nitroaniline isomers, and four neutral PAHs. Efficient separations of all the analytes were achieved with optimized buffer pH and organic additive. The reproducibility and stability of the GO or GOOH coated columns were investigated. Our results indicate the capability of application GO or GOOH sheet in OTCEC separation, which can be coated on the inner wall of fused-silica capillary via electrostatic assembly.

Cross-linker effects on the separation efficiency on (poly)methacrylate capillary monolithic columns. Part I. Reversed-phase liquid chromatography

We synthesized 8 polymethacrylate monolithic capillary columns using laurylmethacrylate functional monomer and various cross-linking monomers differing in the polarity and size. The efficiency of monolithic columns for low-molecular compounds significantly improved with increasing number of repeat non-polar methylene groups in the cross-linker molecules, correlating with greater proportion of small pores with size less than 50nm. The best efficiency with HETP=25μm for alkylbenzenes was achieved for columns prepared using hexamethylene dimethacrylate (HEDMA). Columns prepared with polar (poly)oxyethylene dimethacrylate cross-linkers show also improved efficiency with increasing chain length and generally better performance in comparison to the (poly)methylene dimethacrylate cross-linkers of comparable size, however with less apparent effects of the chain lengths on the pore distribution. The monolithic columns prepared with tetraoxyethylene dimethacrylate (TeEDMA) showed the best efficiency of all the columns tested, corresponding to HETP=15μm (approx. 70,000 theoretical plates/m), show excellent column-to-column reproducibility with standard deviations of 2.5% in retention times, good permeability and low mass transfer resistance, so that is suitable for fast separation of low-molecular compounds in 2min or less. By modification of the fused-silica capillary inner walls pre-treatment procedure, very good long-term stability was achieved even in 0.5mm i.d. capillary format. The TeEDMA column can be also used for size-exclusion chromatography of lower non-polar synthetic polymers, whereas it is less suitable for separations of proteins than the HEDMA column.

Enantioseparation of organic acids of pharmaceutical interest using eremomycin as a chiral selector

Strong adsorption of eremomycin on the fused-silica capillary wall was used for separation of enantiomers by CE. The capillary with adsorbed chiral selector was shown to be easily prepared and has reproducible properties. The effect of the chiral selector concentration, pH and composition of the BGE, and applied voltage on enantioseparation of acidic compounds, such as profens and aromatic carboxylic acids, was investigated. Two native α-amino acids, aspartic acid and glutamic acid, were enantioseparated. Fourteen tested compounds (including amino acids) were baseline resolved. Good selectivity of separation (α>1.09) was achieved. The migration order of ibuprofen and ketoprofen enantiomers was determined. The procedures were proposed for the analysis of flurbiprofen and warfarin in pharmaceuticals. Linearity was achieved in the concentration range of 4.0×10(-5)-2.0×10(-3) M for flurbiprofen and 3.2×10(-6)-4.9×10(-6) M for warfarin. The detection limits were found to be about 1×10(-5) M for flurbiprofen and 1×10(-6) M for warfarin.

Interactions of Proteins with the Acidic Components of the Electrolyte Solution and Their Role in the Performance of Separations by CZE

This paper reports the results of a study performed to investigate the dependence of the performance of protein separation by capillary zone electrophoresis (CZE) on the anionic component of the electrolyte solutions consisting of 20 mM N,N,N′,N′-tetramethyl-1,3-butanediamine (TMBD) titrated to either pH 4.0 or pH 6.5 with either a monoprotic or a polyprotic acid. With the exception of hydrochloric acid, the acids were selected among those commonly used as the constituents of the solutions employed for protein analysis by either HPLC or CZE. TMBD was chosen for its effectiveness at preventing the interactions of proteins with the inner wall of bare fused-silica capillaries. The performance of separations was evaluated using four basic model proteins having pI value and molecular mass ranging from 9.5 to 11.0 and from 12,400 to 25,000 Da, respectively. It is shown that the different acids used as the components of the background electrolyte solutions, all containing the same concentration of TMBD, affect to different extents both migration time and peak shape of the tested proteins. The performance displayed by the BGE containing phosphate ions is enhanced using TMBD in combination with diethylenetriamine, an aliphatic vicinal triamine having effective buffering capacity at pH 4.0 and capability at minimizing protein–capillary wall interactions. The reported experimental evidences are discussed based on the possible interactions that the phosphate ions are known to establish with both the protein molecules and the surface of bare fused-silica capillaries.

Polyethylene glycol-stabilized lipid disks as model membranes in interaction studies based on electrokinetic capillary chromatography and quartz crystal microbalance

Distearoylphosphatidylcholine (DSPC)/cholesterol/distearoylphosphatidylethanolamine (DSPE)–polyethylene glycol 5000 [PEG(5000)] lipid disks, mimicking biological membranes, were used as pseudostationary phase in partial filling electrokinetic capillary chromatography (EKC) to study interactions between pharmaceuticals and lipid disks. Capillaries were coated either noncovalently with a poly(1-vinylpyrrolidone)-based copolymer or covalently with polyacrylamide to mask the negative charges of the fused-silica capillary wall and to minimize interactions between positively charged pharmaceuticals and capillary wall. Although the noncovalent copolymer coating method was faster, better stability of the covalent polyacrylamide coating at physiological pH 7.4 made it more reliable in partial filling EKC studies. Migration times of pharmaceuticals were proportional to the amount of lipids in the pseudostationary phase, and partition coefficients were successfully determined. Because the capillary coatings almost totally suppressed the electroosmotic flow, it was not practical to use the EKC-based method for partition studies involving large molecules with low mobilities. Hence, the applicability of the biomembrane mimicking lipid disks for interactions studies with large molecules was verified by the quartz crystal microbalance technique. Biotinylated lipid disks were then immobilized on streptavidin-coated sensor chip surface, and interactions with a high-molecular-mass molecule, lysozyme, were studied. Cryo-transmission electron microscopy and asymmetrical flow field-flow fractionation were used to clarify the sizes of lipid disks used.

Covalent binding of phospholipid vesicles on fused silica capillaries for electrochromatography

A rapid method for covalent binding of phospholipids with primary amino groups on fused silica capillaries was developed. In the 8 hour coating procedure the reaction product of aminopropylsilylation of the silanol groups of the fused silica capillary reacts with glutaraldehyde giving an imidoaldehyde which then reacts with the primary amino group of the phospholipid. Various types of liposomes with a broad range of composition and concentration were tested, and the role of primary amino groups in lipids was clarified. Since only 2.5% of lipids with primary amino groups were needed for stable covalent binding of liposomes to the fused silica capillary wall, the method allowed fine-tuning of the lipid composition to mimic given in vivo conditions. The optimized coating showed good stability, with intra-day and inter-day repeatability of electroosmotic flow equal to 2.36% RSD (n = 15) and 3.56% RSD (n = 3), respectively. The capillary to capillary reproducibility was 5.66% RSD (n = 3). The thickness and other properties of the attached liposome layer were measured using quartz crystal microbalance (QCM) technique. The capillary surface with covalently attached liposomes acted as a high throughput assay for the determination of interactions between the phospholipid bilayer and the aqueous phase. A model set of structurally diverse drugs was separated in the liposome coated capillary. The retention factors and distribution values were used as indicators of affinity of the drugs toward liposomes.