Influence of Lanthanum on Stern Layer Conductance in the Nanochannel.

In this report, high-frequency electric impedance spectroscopy was performed to investigate ionic transport through nanochannels. Special attention was focused on (i) conductance behaviors depending on the role of cation valence in three background electrolytes (XCln): monovalent 1-1 (K+ and Cl-), divalent 2-1 (Mg2+ and 2Cl-), and trivalent 3-1 (La3+ and 3Cl-), (ii) the effects of proton and bicarbonate ions on bulk and surface conductance, and (iii) the connected microchannel dimension (surface/height ratio aspect) within the nanochannel apparent conductance. The results highlight a net quantitative increase in surface silanol density and a strong decrease in surface ionization degree when lanthanum cations are employed. The results also demonstrate that La3+ strongly interacts with the silica surface, leading to negative values of standard free energy for ion-site interactions and chemical potential for ion-ion correlations in the Stern layer of -0.8 and -10.2 kT, respectively. We ascribed the evolution of surface charge density to the balance between the mole ratios of water molecules and adsorbed cations at equilibrium. We found that La3+ behaves as an acidic cation (Lewis conceptualization) that neutralizes the negative silica surface accompanying water molecule expulsion due to steric hindrance. This study constitutes a new contribution to ion-site interactions and to ion-ion correlation phenomena on the planar silica surface to explain charge inversion observation in micro-nanofluidic devices.

Electropreconcentration diagrams to optimize molecular enrichment with low counter pressure in a nanofluidic device.

Concentration polarization (CP)-based focusing electrokinetics nanofluidic devices have been developed in order to simultaneously detect and enrich highly diluted analytes on-a-chip. However, stabilization of focal points over long time under the application of the electric field remains as a technical bottleneck. If pressure-assisted preconcentration methods have been proposed to stabilize propagating modes at low inverse Dukhin number (1/Du≪1) , these recent protocols remain laborious for optimizing experimental parameters. In this paper, "electric field E/counter-pressure P" diagrams have been established during pressure-assisted electro-preconcentration of fluorescein as a model molecule. Such E/P diagram allows direct observation of the region for which the optimal counter-pressure P leads to a stable focusing regime. This region of stable focusing is shown to vary depending of the nanoslit length (100 µm < Lnanoslit < 500 µm) and the nature of the background electrolyte (KCl and NaCl). Longer nanoslits (500 µm) produce stabilization at low counter-pressure P, whereas NaCl offers a narrower region of stable focusing in the E/P diagram compared to KCl. Finally, the ability of such pressure-assisted protocol to concentrate negatively charged proteins has been tested with a more applicative protein, i.e., ovalbumin. The corresponding E/P diagram confirms the existence of the stable focusing regime at both low electric field E (≤20 V) and counter-pressure P (≤0.4 bar). With an enrichment factor as high as 70 after 2 min for ovalbumin at a concentration of 10 µM, such pressure-assisted nanofluidic electro-preconcentration protocol appears very promising to concentrate and detect biomolecules.

On-a-chip tryptic digestion of transthyretin: a step toward an integrated microfluidic system for the follow-up of familial transthyretin amyloidosis.

A microfluidic microreactor for trypsin mediated transthyretin (TTR) digestion has been developed as a step towards the elaboration of a fully integrated microdevice for the detection of a rare and disabling disease, the familial transthyretin amyloidosis (ATTR) which is related to specific TTR mutations. Therefore, an enzymatic microreactor coupled to an analytical step able to monitor the mutation of TTR on specific peptide fragments would allow an accurate monitoring of the treatment efficiency of ATTR. In this study, two types of immobilized trypsin microreactors have been investigated: a new miniaturized, microfluidic fluidized bed packed with trypsin functionalized magnetic particles (MPs), and a thiol-ene (TE) monolith-based chip. Their performances were first demonstrated with N-benzoyl-dl-arginine-4-nitroanilide hydrochloride BApNA, a low molecular weight substrate. High reaction yields (75.2%) have been reached within 0.6 min for the TE-based trypsin microreactor, while a lower yield (12.4%) was obtained for the micro-fluidized bed within a similar residence time. Transposition of the optimized conditions, developed with BApNA, to TTR digestion in the TE-based trypsin microreactor was successfully performed. We demonstrated that the TE-chip can achieve an efficient and reproducible digestion of TTR. This has been assessed by MS detection. In addition, TTR hydrolysis led to the production of a fragment of interest allowing the therapeutic follow-up of more than twenty possible ATTR mutations. High sequence coverage (90%), similar to those obtained with free trypsin, was achieved in a short time (2.4 min). Repeated experiments showed good reproducibility (RSD = 6.8%). These promising results open up the route for an innovative treatment follow-up dedicated to ATTR.

Effect of high pressure homogenization on the structure and the interfacial and emulsifying properties of ß-lactoglobulin.

The effect of high pressure homogenization (HPH) on the structure of ß-lactoglobulin (ß-lg) was studied by combining spectroscopic, chromatographic, and electrophoretic methods. The consequences of the resulting structure modifications on oil/water (O/W) interfacial properties were also assessed. Moderated HPH treatment (100 MPa/4 cycles) showed no significant modification of protein structure and interfacial properties. However, a harsher HPH treatment (300 MPa/5 cycles) induced structural transformation, mainly from ß-sheets to random coils, wide loss in lipocalin core, and protein aggregation via intermolecular disulfide bridges. HPH-modified ß-lg displayed higher surface hydrophobicity leading to a faster adsorption rate at the interface and an earlier formation of an elastic interfacial film at Cß-lg = 0.1 wt%. However, no modification of the interfacial properties was observed at Cß-lg = 1 wt%. At this protein concentration, the prior denaturation of ß-lg by HPH did not modify the droplet size of nanoemulsions prepared with these ß-lg solutions as the aqueous phases. A slightly increased creaming rate was however observed. The effects of HPH and heat denaturations appeared qualitatively similar, but with differences in their extent.

Chemical and Instrumental Approaches for Capillary Electrophoresis (CE)-Fluorescence Analysis of Proteins.

Capillary electrophoresis (CE) coupled to fluorescence detection is an invaluable technique for the quantitative analysis of proteins of interest in the field of clinical diagnosis and quality control of novel biotechnology products. The various chemical and instrumental approaches that have been reported to carry out such sensitive analysis are described in this paper. To illustrate the contribution of CE to the analysis of therapeutic proteins, a detailed protocol for impurities profiling of a recombinant antibody sample using CE-LEDIF is given.

Determination of the isomeric forms proportion of fluorogenic naphthalene-2,3-dicarboxaldehyde in a binary mixture of water:methanol using electrochemical methods.

The electrochemical response of the fluorogenic label naphthalene-2,3-dicarboxyaldehyde (NDA) in a binary mixture of water/methanol was characterized with cyclic voltammetry (CV) and differential pulse voltammetry (DPV) electrochemical techniques. Naphthalene-2,3-dicarboxyaldehyde does exist in three isomeric forms in aqueous solution: the unhydrated dialdehyde (DA), the acyclic monohydrated (MA) and the cyclic hemiacetal (HAC). The study underlines that the proportion of each of them varies according to the working pH. At low and high pH, the dialdehyde form is in larger proportion than the acyclic monohydrated form. Conversely at intermediate pH, the concentration of the acyclic form is in greater proportion than the dialdehyde form. These results allowed us to determine the optimal pH of 9 for which the labeling of biomolecules could be more efficient due to the base catalyzed regeneration of the unhydrated form. At this pH, the data processing from the analysis of measured currents and estimation of diffusion coefficients of each form according to the semi-empirical models of Wilke-Chang, Scheibel, Reddy-Doraiswamy and Lusis-Ratcliff allowed us to obtain the concentration of dialdehyde (0.28 mM), acyclic monohydrated (0.57 mM) and cyclic hemiacetal monohydrated (0.15 mM) forms starting from 1mM naphthalene-2,3-dicarboxyaldehyde.

Study of Surface Charge Instabilities by EOF Measurements on a Chip: A Real-Time Hysteresis and Peptide Adsorption Based Methodology.

This paper describes the measurement of the electroosmotic mobility (EOF) in a Wheatstone fluidic bridge (µFWB) as a direct probe of the surface instability. The variation of EOF known as one major contribution of the electrokinetic migration has been determined with a real-time measurement platform after different conditionings on chips. We also scan the pH of the background electrolytes with three different ionic strengths to evaluate the dependencies of the EOF as a function of the pH. A hysteresis methodology has been developed for probing the surface charge instabilities. EOF mobility has been recorded during on-a-chip electrophoresis to estimate the effect of such instability on the analytical performance. As expected, our experimental curves show that a decrease in the ionic strength increases the surface charge stability of the hybrid microchip. This result demonstrates that ionic exchanges between the surface and the fluid are clearly involved in the stability of the surface charge. With this original method based on real-time EOF measurement, the surface state can be characterized after hydrodynamic and electrophoresis sequences to mimic any liquid conditioning and separation steps. Finally, as a demonstrative application, isotherms of the adsorption of insulin have been recorded showing the change in surface charge by unspecific adsorption of this biomolecule onto the microfluidic channel's wall. These methodologies and findings could be particularly relevant to investigating various analytical pathways and to understanding the molecular mechanisms at solid/liquid interfaces.

Improved electrochemical detection of a transthyretin synthetic peptide in the nanomolar range with a two-electrode system integrated in a glass/PDMS microchip.

An alternative to a three-electrode set-up for electrochemical detection and analysis in microfluidic chips is described here. The design of the electrochemical sensor consists of the surface of the glass substrate covered with a PDMS block which bears the microfluidic channels. A band microelectrode which acts as a working electrode surrounded by a large counter electrode is obtained at the micrometric level to propose a simple and efficient sensing area for on-a-chip analysis. The counter-electrode with a surface area about 22-fold greater than the working-microelectrode can also be considered as a pseudo reference since its current density is low and thus limits the potential variations around the rest potential. To this purpose, the [Fe(III)(CN)6]³â»/[Fe(II)(CN)6]4⁻ redox couple was used in order to set a reference potential at 0 V since both electrodes were platinum. The electrochemical microchip performance was characterized using differential pulse voltammetric (DPV) detection and quantification of the optically multi-labelled transthyretin synthetic peptide mimicking a tryptic fragment of interest for the diagnosis of familial transthyretin amyloidosis (ATTR). The limit of detection of the peptide by the working microelectrode was 25 nM, a value 100-fold lower than the one reported with conventional capillary electrophoresis coupled with laser-induced fluorescence under the same analytical conditions.

Derivatization strategies for CE-LIF analysis of biomarkers: Toward a clinical diagnostic of familial transthyretin amyloidosis.

We report three derivatization strategies for CE analysis with LIF detection (CE-LIF) of two synthetic peptides mimicking the wild and mutated fragments of interest for the diagnosis of familial transthyretin amyloidosis. The precapillary derivatization of the peptides with three optical tags, 5-carboxytetramethylrhodamin succinimidyl ester (TAMRA-SE), naphtalene-2,3-dicarboxyaldehyde (NDA), and 3-(2-furoyl)quinoline-2-carboxyaldehyde (FQ) has been investigated by CE-LIF detection and MS. Results provide evidence that high reaction yields have been reached whereas the multitagging phenomenon has occurred for both NDA and TAMRA-SE labeling procedures. The derivatization and electrokinetic separation of a mixture of the two peptides of interest for the pathology diagnosis (22-aa peptides that differ only from one amino acid) were achieved using both approaches. The highest resolution with a value of 2.5 was obtained with TAMRA-SE labeled derivatives whereas NDA gave the best detection sensitivity (LOD of 2.5 µM). The validation of the developed methods showed a good linearity (R ≥ 0.997) between the peak area of the labeled derivatives and the peptide concentration for both NDA and FQ labeling procedures. The intraday RSDs of A and the migration times were less than 3.8 and 2.2%, respectively.

A microdevice for parallelized pulmonary permeability studies.

We describe a compartmentalized microdevice specifically designed to perform permeability studies across a model of lung barrier. Epithelial cell barriers were reproduced by culturing Calu-3 cells at the air-liquid interface (AIC) in 1 mm² microwells made from a perforated glass slide with an embedded porous membrane. We created a single basolateral reservoir for all microwells which eliminated the need to renew the growth medium during the culture growth phase. To perform drug permeability studies on confluent cell layers, the cell culture slide was aligned and joined to a collection platform consisting in 35 µL collection reservoirs connected at the top and bottom with microchannels. The integrity and functionality of the cell barriers were demonstrated by measurement of trans-epithelial electrical resistance (TEER), confocal imaging and permeability assays of ¹4C-sucrose. Micro-cell barriers were able to form confluent layers in 1 week, demonstrating a similar bioelectrical evolution as the Transwell systems used as controls. Tight junctions were observed throughout the cell-cell interfaces, and the low permeability coefficients of ¹4C-sucrose confirmed their functional presence, creating a primary barrier to the diffusion of solutes. This microdevice could facilitate the monitoring of biomolecule transport and the screening of formulations promoting their passage across the pulmonary barrier, in order to select candidates for pulmonary administration to patients.

Investigating of labelling and detection of transthyretin synthetic peptide derivatized with naphthalene-2,3-dicarboxaldehyde.

Labelling and detection of a synthetic peptide (PN) mimicking a tryptic fragment of interest for the diagnosis of familial amyloidal polyneuropathy have been investigated optically and electrochemically. We decided to covalently label naphtalene-2,3-dicarboxyaldehyde (NDA), a fluorogenic and electroactive molecule on PN. First, the optimization of the labelling chemical reaction was performed by capillary electrophoresis coupled with laser induced fluorescence detection (CE-LIF). The analytical parameters such as separation efficiency and peak area were considered to propose this optimized derivatization reaction. The results obtained allowed us to establish the pH and ionic strength of the derivatization buffer, the molar ratio between NDA and PN and the reaction time of the labelling. Optimal conditions are obtained when [NDA]/[PN]=40, buffer pH of 9, buffer ionic strength of 70 mM and reaction time of 15 min. Second, differential pulse voltammetry (DPV) and cyclic voltammetry (CV) were also used to characterize NDA-labelled PN and different electroinactive amino acids (histidine, lysine, serine, threonine) which are in the PN sequence. The electrochemical detection experiments demonstrated that the labelled biomolecules could be also easily detected at low concentration. Moreover, the derivatization reaction could be followed to describe more precisely the labelling process of these biomolecules. Optimal conditions for labelling are obtained when [NDA]total/[CN(-)] ratio =1 and [NDA]total/[amino acid or peptide]=100 with a buffer having a pH=9 on a glassy carbon electrode. In all cases, an obvious oxidation peak for the N-2-substituted-1-cyanobenz-[f]-isoindole derivative (CBI) has been observed at 0.5-0.7 V/SCE. The multi-labelling of PN and lysine were shown with DPV. We presumed this result to occur because of the shouldered shape of the DPV peak shape. These experiments confirm that NDA can be used as a derivative agent for PN, allowing for electrochemical and fluorescence detections with a limit of detection of labelled PN estimated at 0.2 µM and 5 µM, respectively.

Computational study of velocity profile obtained in microfluidic channel bearing a fluidic transistor: toward highly resolved electrophoretic separation.

The present work is a computational study of velocity profiles in microfluidic channels bearing field flow effect transistors (FFET). In particular, this work investigates perturbations and distortions of the sample band during electrophoretic transport in a rectangular separation channel. The EOF heterogeneity and its induced pressure render the predictions of the analytical performances rather complex. In this context, we propose a systematic numerical inquiry that focuses on the distribution of the velocities for several geometries and EOF modulations. We compare the calculated parabolic velocity profiles to the bare glass microchips. Here, the reported parabolic velocity profiles are coherent with recent experimental results that have been published elsewhere. From the presented equations, in such active hybrid microfluidic chip that integrates a FFET gate layer, separation can be optimized by playing on the gate coverage ratio. The flow fields obtained from analytical models allow further investigations about the efficiency and resolution during electrophoresis. The resulting induced pressure gradient and the associated band broadening underline the need to optimize the resolution in the detriment of the efficiency in such active microfluidic chips.

A new CZE method for profiling human serum albumin and its related forms to assess the quality of biopharmaceuticals.

We present a new CZE method, which uses a polyethylene oxide-coated capillary to separate native HSA from more than five of its structural variants. These variants include oxidized, truncated, and cysteinylated forms of HSA which can all be found in biopharmaceutical products. Both CE and MS confirmed the high degree of heterogeneity of HSA preparations. Recovery studies demonstrated that adsorption of HSA on the capillary was significantly reduced under the conditions we developed, which led to a satisfactory repeatability (RSD for migration times and relative peak areas were less than 0.2 and 7.0%, respectively). Assignment of the main peaks was attempted using in vitro degraded/stressed HSA. We used our method to test batch-to-batch comparability and detected slight quantitative differences in the proportion of native HSA in batches produced from different fractionation methods.

Stabilization mechanism of oil-in-water emulsions by ß-lactoglobulin and gum arabic.

Natural biopolymer stabilized oil-in-water emulsions were formulated using ß-lactoglobulin (ß-lg), gum arabic (GA), and ß-lg:GA solutions as an alternative to synthetic surfactants. Emulsions using these biopolymers and their complexes were formulated varying the biopolymer total concentration, the protein-to-polysaccharide ratio, and the emulsification protocol. This work showed that whereas ß-lg enabled the formulation of emulsions at concentration as low as 0.5 (w/w)%, GA allowed to obtain emulsions at concentrations equal to or higher than 2.5 (w/w)%. In order to improve emulsion stability, ß-lg and GA were complexed through strong attractive electrostatic interactions. GA solution had to be added to previously prepared ß-lg emulsions in order to obtain stable emulsions. Interfacial tension and interfacial rheological measurements allowed a better understanding of the possible stabilizing mechanism. ß-lg and GA both induced a very effective decrease in interfacial tension and showed interfacial elastic behaviour. In the mixed system, ß-lg adsorbed at the interface and GA electrostatically bound to it, leading to the formation of a bi-layer stabilized emulsion. However, emulsion stability was not improved compared to ß-lg stabilized emulsion, probably due to depletion or bridging flocculation.

A quantitative CE method to analyse tau protein isoforms using coated fused silica capillaries.

We evaluated the potential of CE to analyse different isoforms of unphosphorylated recombinant tau protein and for separating one phosphorylated tau from the respective unphosphorylated protein. Different capillary coatings such as polyacrylamide, poly-(ethylene oxide) and polybrene (PB) were evaluated to overcome the poor efficiencies obtained with fused-silica capillary. Although peak asymmetry values were quite similar for the three investigated coatings, the peak efficiencies were 35-fold and 5-fold higher with PB coating than with polyacrylamide and poly(ethylene oxide) coatings, respectively. The recovery percentage (over 97%) was satisfactory and confirmed the efficacy of PB coating to limit the adsorption of tau protein to capillary walls. Moreover, PB coating produced higher repeatability for migration times (RSD values <1.2%) in comparison to the neutral coatings. The potential of PB-modified capillary in producing high resolutive separations of one phosphorylated tau isoform from its unphosphorylated counterpart and of a mixture of phosphorylated and unphosphorylated tau peptides was demonstrated with 50 mM phosphate buffer pH 3.0. The separation of unphosphorylated tau isoform 352 (Tau-352) from Tau-352 phosphorylated in vitro by the mitogen-activated protein kinase ERK2, was accomplished in less than 15 min.

Determination of binding constants of vasoactive intestinal peptide to poly(amidoamine) dendrimers designed for drug delivery using ACE.

The purpose of the present paper was to study at physiological pH the affinity between vasoactive intestinal peptide (VIP) and four poly(amidoamine) dendrimers (PAMAMs) designed for drug delivery. Therefore, a fast and reproducible CE method was first developed to analyze the strongly basic peptide. To allow an accurate determination of binding constant (K) values, the ability to suppress peptide adsorption onto the silica capillary of nonpermanent coatings (poly(ethylene oxide) (PEO), low and medium relative molecular masses poly(diallyldimethylammonium chloride) (PDDA)) or poly(acrylamide) permanent coating (PAA) was evaluated. Very good intraday repeatability of VIP migration times and peak areas (0.1-0.6 and 2.9-4.9% RSD, respectively) was obtained using two of the investigated coatings (PEO and PDDA with medium molecular mass). ACE combined with these dynamic coatings was then employed to evaluate K between VIP and two amine-terminated PAMAM dendrimers of generation 2 and 5 (G2.NH2, G5.NH2) and two carboxyl-terminated PAMAM derivatives of generation 2 and 5 (G2.COOH, G5.COOH). Binding constant of (6.7 +/- 1.1) x 10(4)/M could be determined for the couple VIP/G5.NH2, while no affinity was evidenced between VIP and all other dendrimers investigated. These results suggest that G5.NH2 might be an interesting carrier for the delivery of VIP.

Fluorescent detection of peptides and amino acids for capillary electrophoresis via on-line derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole.

An in-capillary derivatization of amino acids and peptides with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed for their subsequent capillary electrophoretic analysis with laser-induced fluorescence detection (lambda (ex)=488 nm). The in-capillary derivatization was achieved in zone-passing mode by introducing successive plugs of sample and NBD-F into a fused silica capillary previously equilibrated with an alkaline borate buffer. To prevent NBD-F hydrolysis and to achieve a reliable derivatization, NBD-F was prepared daily in absolute ethanol and a plug of absolute ethanol was introduced between the sample and NBD-F reagent plugs. Various parameters influencing the derivatization efficiency were investigated and the optimum conditions were as follows: background electrolyte (BGE), 20 mM borate buffer (pH 8.8); introduction time, 4 s for sample and 2 s for NBD-F; molar ratio of NBD-F/sample, above 215; temperature, 45 degrees C for amino acids and 35 degrees C for peptides; applied voltage, +15 kV. The validation of the in-capillary derivatization method under optimal conditions showed a good linearity between the heights of the derivative peaks and the concentrations of the amino acids. The intra-day relative standard deviations of the migration times and the peak heights were less than 1.3% and 4.6%, respectively. The efficient derivatization and separation of a mixture of valine, alanine, glutamic acid and aspartic acid were achieved using this technique. Peptides such as buccaline and beta-protein fragment 1-42 could also be derivatized using the developed in-capillary derivatization procedure.

Functionalized magnetic micro- and nanoparticles: optimization and application to micro-chip tryptic digestion.

The preparation of an easily replaceable protease microreactor for micro-chip application is described. Magnetic particles coated with poly(N-isopropylacrylamide), polystyrene, poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(glycidyl methacrylate), [(2-amino-ethyl)hydroxymethylen]biphosphonic acid, or alginic acid with immobilized trypsin were utilized for heterogeneous digestion. The properties were optimized, with the constraint of allowing immobilization in a microchannel by a magnetic field gradient. To obtain the highest digestion efficiency, sub-micrometer spheres were organized by an inhomogeneous external magnetic field perpendicularly to the direction of the channel. Kinetic parameters of the enzyme reactor immobilized in micro-chip capillary (micro-chip immobilized magnetic enzyme reactor (IMER)) were determined. The capability of the proteolytic reactor was demonstrated by five model (glyco)proteins ranging in molecular mass from 4.3 to 150 kDa. Digestion efficiency of proteins in various conformations was investigated using SDS-PAGE, HPCE, RP-HPLC, and MS. The compatibility of the micro-chip IMER system with total and limited proteolysis of high-molecular-weight (glyco)proteins was confirmed. It opens the route to automated, high-throughput proteomic micro-chip devices.

In-capillary non-covalent labeling of insulin and one gastrointestinal peptide for their analyses by capillary electrophoresis with laser-induced fluorescence detection.

The potential of the commercially available dye sypro orange for in-capillary derivatization was evaluated for the detection of insulin and one gastrointestinal peptide (Arg-Arg-gastrin) by capillary electrophoresis with laser induced fluorescence (CE-LIF). The fluorescent emission intensity (lambda(ex) = 488 nm, lambda(em) = 610 nm) of this probe is very low in aqueous medium, and increases strongly in less polar solvent, e.g. methanol. The hydrophobic character of the two analyzed peptides is too low to induce sufficient interaction with the fluorescent probe for good sensitivity when the latter is alone in the background electrolyte. Thus, the potential of several neutral, zwitterionic, cationic and anionic surfactants to favor probe/peptide interactions has been evaluated. It was demonstrated that a borate buffer (pH 8.5) containing tetradecyltrimethylammonium bromide (TTAB) in sub-micellar conditions can be considered as the most suitable buffer for insulin CE-LIF analysis. In addition, the method showed a good linearity between insulin concentration and the peak area of the labeled insulin, allowing quantitative measurements. The sensitivity achieved so far is comparable with that achieved with UV absorption detection, but even at this level it is interesting for microchip analysis, in which fluorescence detection is much more commonly available than UV absorption detection.

In-capillary derivatization approach applied to the analysis of insulin by capillary electrophoresis with laser-induced fluorescence detection.

An in-capillary derivatization procedure of insulin for its subsequent capillary electrophoretic analysis (with laser-induced fluorescence detection) was developed. The in-capillary derivatization performed using the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in a borate buffer pH 8.9, was achieved by successive introduction of plugs of sample and AQC reagent followed by application of a voltage (30 kV). Derivatization reaction results from the differential transport velocities that permit the distinct zones to penetrate each other under the applied field. Reagent/sample molar ratio (Rm) and plug lengths ratio were shown to have an influence on the efficiency of the derivatization reaction. A single peak could be obtained with a high reagent/sample molar ratio (Rm > or = 68). The tagged derivative peak intensity and efficiency were improved when reagent solution time injection was at least twice higher than that of insulin sample. The validation of the method showed a good linearity between the corrected area of the derivative peak and insulin concentrations. The relative standard deviations of the migration times and the corrected areas obtained for the tagged derivative were 2.3 and 4.6%, respectively. An efficient derivatization and separation of a mixture of insulin and two glycated forms of insulin was obtained using the technique.