In-line coupling of an aptamer based miniaturized monolithic affinity preconcentration unit with capillary electrophoresis and Laser Induced Fluorescence detection.

A composite 30-cm capillary was prepared. The head of the capillary was a 1.5-cm original and miniaturized aptamer-based monolithic affinity support that was in-line coupled to the end of the capillary used for capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The device was used for the preconcentration, separation and quantification of ochratoxin A (OTA) as a test solute. The 1.5-cm preconcentration unit consists of a fritless affinity monolithic bonded with 5'-SH-modified oligonucleotide aptamers. A vinyl spacer was used for thiol-ene photoclick chemistry with a 5min irradiation at 365nm. Photografting allowed to confine the binding reaction to the desired silica monolithic segment, upstream the empty section of the CE capillary using an UV mask. The photografting procedure was optimized preparing 10-cm capillary monoliths for nano-LC. The retention factors of cationic solutes in ion-exchange nano-LC allowed to follow the aptamer binding on the monolith. The reproducibility of the photografting process was satisfactory with inter-capillary variation lower than 10%. The aptamer bonding density can be increased by successive graftings of 100µM aptamer concentration solution (5pmol/cm/grafting). The optimal conditions to successfully perform the in-line coupling (preconcentration, elution and separation of OTA) with the composite capillary were adjusted depending on individual requirements of each step but also insuring compatibility. Under optimized conditions, OTA was successfully preconcentrated and quantified down to 0.1pg (percolation of 2.65µL of a 40ng/L OTA solution). A quantitative recovery of OTA (93±2%) was achieved in a single elution of 30pg percolated OTA amount. The reproducibility of the overall process was satisfactory with a relative standard deviation lower than 10% with negligible non-specific adsorption. This device was applied for the preconcentration and analysis of OTA in beer and wine at the ppb level within a total analysis time of 30min.

Versatile ene-thiol photoclick reaction for preparation of multimodal monolithic silica capillary columns.

This paper presents a photografting process of monolithic silica capillary columns based on the ene-thiol click chemistry. This study is performed on a "generic" vinyl-functionalized silica monolith (Hmin 6±1µm). The photoclick reaction is investigated using different thiol monomers (octadecanethiol, cysteine and sodium mercaptoethanesulfonate) to prepare capillary columns dedicated to various chromatographic modes (reversed-phase, HILIC and strong cation exchange). Whatever the monomer used, the photografting reaction is achieved in less than 5min with a relatively high thiol monomer content. This allows preparing highly retentive and efficient monolithic columns while avoiding polymerization and/or column clogging. In addition to the aforementioned properties (duration, versatility, efficiency), this photo-triggered chemical reaction allows addressing several appropriate surface functionalizations inside a single monolithic column in order to prepare nanovolume multimodal capillary columns. A multimodal biphasic monolithic column with a 1cm length cation-exchange segment followed by a 9cm length reversed-phase segment (SCX-RP) is prepared through two successive photografting reactions using a UV-mask to localize the reactions. This multimodal biphasic column is investigated using a model sample for the selective fractionation and separation of cationic and neutral compounds and is applied to the on-line preconcentration and separation of ß-blockers.

"Thiol-ene" photoclick chemistry as a rapid and localizable functionalization pathway for silica capillary monolithic columns.

Herein, we report the "ene-thiol" photografting of 1-octadecanethiol onto vinyl pre-functionalized silica monolith to prepare clicked reversed-phase silica monolithic columns with high permeability and performances. The experimental conditions (concentration of thiol in solution, irradiation duration) are optimized with respect to highest retention and methylene selectivity, i.e. to the highest surface coverage of the monolith. It is demonstrated that an irradiation duration of 5min is enough with a 0.8M concentration of 1-octadecanethiol in solution or that it may be replaced by successive irradiations at a lower ODT concentration (0.19M) with renewing of the solution between the illuminations. Retention factors as high as those obtained with standard silanization are reached while keeping the intrinsic monolith permeability and efficiency (160,000plates/m in nano-LC at 0.7mm/s). The absence of polymerization, in the "ene-thiol" version, is demonstrated. Indeed, the steric selectivity of our clicked-material is characteristic of monolayer-like functionalized silica and significantly lower than the steric selectivity measured on polymeric-like functionalized silica.

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.

Preparation and full characterization of a micro-immunoaffinity monolithic column and its in-line coupling with capillary zone electrophoresis with Ochratoxin A as model solute.

A micro-immunoaffinity monolithic column (µIAC) was developed and in-line coupled with capillary zone electrophoresis in a fully automated way with Ochratoxin A as test solute. The in-line micro-immunoaffinity columns based on monolithic methacrylate polymers (EDMA-GMA) were prepared in situ at the inlet end of a PTFE coated fused silica capillary by UV initiated polymerization and subsequently grafted with antibodies. These µIACs were thoroughly characterized. The synthesis of the polymeric support was first demonstrated to be reproducible in terms of permeability, surface properties and efficiency. The antibodies immobilization was then studied by a new original hydrodynamic method (ADECA) allowing the in situ quantitative determination (at a miniaturized scale) of the total amount of immobilized antibodies. The combination of this measurement with the binding capacity of the µIAC allowed, for the first time, the in situ determination of immobilized antibody activity. A total of 260 ± 15 ng (1.6 ± 0.1 pmol) of IgG antibodies/cm in 75 µm i.d. monolithic column (i.e. 18 µgmg(-1)) was obtained with (anti-Ochratoxin A/Ochratoxin A) as antibody/antigen model. 40% of the immobilized antibodies remain active corresponding to a binding capacity of 1.2 ± 0.2 pmol antigen/cm (i.e. 600 pg/cm of our test solute OTA), a very high capacity when dealing with trace analysis and with regard to the detection limits (30 pg and 0.5 pg with UV and LIF detection, respectively). The recovery yields were quantitative with negligible non-specific adsorption and allow analysis of diluted samples (1 ngmL(-1)) for a percolated volume of 10 µL. It was also demonstrated that despite the progressive denaturation of antibodies consecutive to the elution step, the binding capacity of the µIAC remained high enough to implement at least 15 consecutive analyses with the same column and in a fully automated way.

In situ characterization of antibody grafting on porous monolithic supports.

The efficient immobilization of antibodies on monolithic support is one of the most critical steps when preparing immunoaffinity supports. In this work, the ADECA (amino density estimation by colorimetric assay) method was adapted to tridimensional supports (in a dynamic mode) and proved to be efficient to characterize the antibodies grafting efficiency on 15.3±0.9mg porous glycidyl methacrylate (GMA)-co-ethylene dimethacrylate (EDMA) monolithic columns. The amount of grafted antibodies measured in situ on the monolith by ADECA (8.2±0.2µg of antibodies per milligram of monolith) was consistent with values obtained by bicinchoninic acid assay (BCA) after crushing the monolith. ADECA was shown to be less time-consuming and more versatile than BCA. The ADECA method was further implemented to thoroughly study and optimize the antibody grafting conditions (influence of pH and kinetics of the grafting step) on GMA-based monoliths and to check the covalent nature of the antibody/surface linking and its stability. Using the total amount of grafted antibodies and the amount of recognized antigen, we found that 65±6% of antibodies were able to capture their antigen. Finally, the grafting of Fab and F(ab')(2) fragments demonstrated that no significant improvement of the global binding capacity of the monolith was obtained.

Improvement of chromatographic performances of in-situ synthesized hybrid C8 silica monoliths by reduction of structural radial heterogeneities.

Several modifications of a previously described protocol are proposed to improve the performances of in-situ synthesized C(8) hybrid silica monoliths. Our attention was focused on reducing the sources of radial heterogeneity that may be responsible for the poor efficiencies observed in the hydrodynamic elution mode. It was demonstrated that a decrease in the temperature of the capillary during the filling step equally to that of the polymerization mixture (0 degrees C), associated with a decrease of the gelation temperature to 20 degrees C along with a new pre-treatment of the capillary's internal walls [with a mixture of tetraethoxysilane (TEOS)/EtOH (1/3, v/v)] allows (i) increasing the radial homogeneity of the monolith, thus further enhancing the performances in the nano-liquid chromatography (nano-LC) mode, (ii) improving the capillary to capillary reproducibility in terms of permeability and efficiencies. In fact, the average minimum plate height H(min) was lowered from 24 to 14 microm and the capillary-to-capillary reproducibility of the synthesis was widely improved by factors two and three of reduction on the calculated standard deviation, respectively for both the efficiency in the nano-LC mode and the permeability. At last, the improved radial homogeneity and anchoring of the synthesized monoliths allowed increasing the inner diameter of the capillary (up to 150 microm) without any significant loss in efficiency. Finally, long term stability of the as-obtained monolithic stationary phases in terms of retention and efficiency was studied. In addition, the evaluation of their chromatographic behaviour was also achieved with the Tanaka test and the results were compared to those already published for commercial monoliths (Chromolith) as well as for particulate stationary phases.

Optimization of the single-step synthesis of hybrid C(8) silica monoliths dedicated to nano-liquid chromatography and capillary electrochromatography.

Hybrid silica monoliths functionalized with octyl groups and dedicated to chromatographic separations in the reversed-phase mode were directly synthesized within capillaries according to the protocol described by Yan et al. [L.J. Yan, Q.H. Zhang, Y.Q. Feng, W.B. Zhang, T. Li, L.H. Zhang, Y.K. Zhang, J. Chromatogr. A 1121 (2006) 92]. Although these monoliths allowed reaching high efficiencies in capillary electrochromatography (CEC), serious limitations prohibited their application in nano-liquid chromatography (nano-LC). Such limitations observed as poor performances in the nano-LC mode and the lack of reproducibility of the synthesis were related to the longitudinal morphological inhomogeneities of the hybrid material along the capillary. Thus, several modifications were conducted in the synthesis protocol in order to improve the resulting morphology of the monolith making it suitable for nano-LC separations. The influence of several critical parameters (such as the addition temperature of the basic catalyst and the hydrolysis duration) on the textural and chromatographic properties had been extensively studied. It was found that a decrease (i.e. 0 degrees C) of the temperature addition of the basic catalyst associated with a shorter hydrolysis duration (1h instead of 6h) allowed (i) delaying the gelation time and consequently facilitating the capillary filling step, (ii) increasing the structural homogeneity of the hybrid monoliths, i.e. their chromatographic performances in nano-liquid chromatography also (iii) greatly improving the reproducibility of the synthesis within the capillary without impairing the material's carbon load, i.e. the incorporation of the less hydrolysable C(8) precursor. The resulting hybrid monoliths afforded retention factors comparable to that previously obtained for C(18) grafted silica monoliths and efficiencies that are the best ever recorded in nano-LC with hybrid monoliths and that are close to the ones achieved with grafted silica monoliths. In fact, this modified protocol allowed a significant improvement of the performances in nano-LC which could be observed by the decrease of the mean value of H(min) going from 123 microm (Yan's protocol) to 24 microm (modified protocol) for a same length of capillary (l = 8.5 cm). In addition, the reproducibility of the synthesis was greatly improved through a factor six of reduction on the calculated standard deviation of these efficiencies. The high permeability and longitudinal homogeneity of the synthesized monolith allowed increasing the capillary length (for example, a 75-cm capillary was conveniently filled with hybrid silica monolith) and the column could be eluted at a very low backpressure leading to chromatographic performances up to 40,000 plates. Finally, the good efficiencies in the nano-LC mode combined with the excellent performances already present in the CEC mode led to fast (less than 1 min) and high efficient separations in the pressurized capillary electrochromatography (p-CEC) mode.

Influence of the hydrothermal treatment on the chromatographic properties of monolithic silica capillaries for nano-liquid chromatography or capillary electrochromatography.

In the last decade, silica monolithic capillaries have focused more and more attention on miniaturized separation techniques like capillary electrochromatography (CEC), nano-liquid chromatography (nano-LC) and chip electrochromatography owing to their unique chromatographic properties and their simplified preparation compared with packed columns. They are synthesized according to a sol-gel multi-step process that includes, after a gelation step at 40 degrees C leading to the formation of the macropores network and the silica skeleton, a post-gelation step (hydrothermal treatment at 120 degrees C in basic medium) that allows to tailor the mesopores and finally a calcination or a washing step to remove remaining polymers. In order to reduce the synthesis time, the number of synthesis steps and above all the temperature synthesis, to adapt the synthesis of such silica monoliths in polymeric microsystem devices, we extensively studied the influence of the hydrothermal treatment and its duration on textural (pore size distribution) and chromatographic properties (retention, efficiency) of in situ-synthesized capillary monoliths in nano-LC and CEC. This study was performed on pure silica and octyl chains grafted silica monoliths. Untreated monoliths show small pores (<6 nm), whereas hydrothermally treated monoliths exhibit medium and large mesopores (8-17 nm). It was demonstrated that the hydrothermal treatment at 120 degrees C was not necessary for pure silica monolithic capillaries dedicated to normal phase liquid chromatography or hydrophilic interaction liquid chromatography (HILIC) and electrochromatography: the suppression of the hydrothermal treatment did not impair efficiencies in CEC and in nano-LC but contributed to increase in retention factors. Minimal plate heights of ca. 5 microm in CEC and 6 microm in nano-LC were obtained with or without hydrothermal treatment with bare silica. In the same way, the hydrothermal treatment was not necessary for grafted silica monoliths only dedicated to CEC. However, the results clearly indicate that the hydrothermal treatment becomes essential before grafting in order to preserve the efficiency of the monolithic silica capillaries dedicated to nano-LC: in this particular case, the suppression of the hydrothermal treatment leads approximately to a loss of a factor two in efficiency.

Separation of basic compounds by capillary electrochromatography on an X-Terra RP18 stationary phase.

In this work we demonstrate that the X-Terra RP18 stationary phase, specially designed for the analysis of basic compounds in liquid chromatography, may be successfully used in capillary electrochromatography. Although this packing material does not afford a sufficient electroosmotic flow with classical hydro-organic mobile phases, the addition of a surfactant that adsorbs onto the stationary phase allows to generate a sustainable electroosmosis flow (EOF), the direction of which depends on the charge of the surfactant. The way of manipulating the electroosmotic flow is described (nature and concentration of the added surfactant, proportion of the organic modifier in the mobile phase, pH). It is then demonstrated that high efficiencies can be reached with this packing material (up to 220,000 plates/m with a mean diameter particles of 3.5 microm) when it is operated at high linear velocities. Then the separations of different classes of compounds such as amphenicol antibiotics, macrolide antibiotics or basic test solutes with mobile phases with pH up to 10.8 are described. The influence of the addition of sodium dodcylsulfate (SDS) to the mobile phase on the retention is described and the selectivity of the X-Terra RP18 stationary phase is compared to that of a more traditional phase, i.e. Hypersil C18 stationary phase with SDS added to the mobile phase. However, it is shown that a good repeatability of the retention factors can only be obtained when the ionization of the compounds is totally suppressed since electrolysis of the buffered hydro-organic mobile phase occurs in the buffer reservoirs leading to a variation of the mobile phase pH and consequently to a modification of the ionization degree of the solutes having their pKa close to the mobile phase pH.

Ability of porous graphitic carbon to support electroosmotic flow in capillary electrochromatography.

The existence of a cathodic EOF (electroosmotic flow) in the case of a porous graphitic carbon (PGC) partially packed column has been demonstrated. Then, the ability of PGC to afford electroosmosis has been brought to the fore with a fully PGC packed column. Experimental data have shown that PGC particles are negatively charged and their electrophoretic mobility has been evaluated. In order to investigate the conditions of existence of EOF different mobile phases have been tested. An EOF occurs when the conductivity of the PGC packed column is larger than the conductivity of an empty fused-silica capillary operating in the same conditions i.e. when the PGC participates in the electric conduction. Since the local electric fields in the two segments of the column are different, an evaluation of the electroosmotic mobility is not possible and the effect of the operational parameters such as the composition of the mobile phase (acetonitrile ratio and total ionic strength) has been studied in term of electroosmotic velocity V(eo).

Separation of cardiac glycosides by micellar electrokinetic chromatography and microemulsion electrokinetic chromatography.

The interest of micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC) for the resolution of four cardiac glycosides is demonstrated. First, the influence of some parameters on the resolution of the solutes in MEKC such as the concentration of the surfactant, pH, addition of organic modifiers and urea is discussed. Then, results are compared with those obtained in MEEKC using different microemulsion compositions. Results indicate that MEEKC possesses several advantages over MEKC for the separation of relatively hydrophobic compounds such as digitalic compounds. First, microemulsions allow a better manipulation of the migration time window and of the retention of the solutes. Moreover, efficiency is improved with shorter analysis time.