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.     

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.     

Affinity monolithic capillary columns for glycomics/proteomics: 1. Polymethacrylate monoliths with immobilized lectins for glycoprotein separation by affinity capillary electrochromatography and affinity nano-liquid chromatography in either a single column or columns coupled in series

In this report, microcolumn separation schemes involving monolithic capillary columns with immobilized lectins, and relevant to nanoglycomics/nanoproteomics were introduced. Positive and neutral monoliths based on poly(glycidyl methacrylate-co-ethylene dimethacrylate) were designed for achieving lectin affinity chromatography (LAC) by nano-LC and CEC. The positive monoliths (i.e., monoliths with cationic sites) afforded relatively high permeability in nano-LC but lack predictable EOF magnitude and direction, while neutral monoliths provided a good compromise between reasonable permeability in nano-LC and predictable EOF in CEC. Lectin affinity nano-LC permitted the enrichment of classes of different glycoproteins having similar N-glycans recognized by the immobilized lectin, whereas lectin affinity CEC provided the simultaneous capturing and separation of different glycoproteins due to differences in charge-to-mass ratio. Also, this investigation demonstrated for the first time the coupling of lectin capillary columns in series (i.e., tandem columns) for enhanced separation of glycoproteins by LAC using the CEC modality. Furthermore, in the coupled columns format, glycoforms of a given glycoprotein were readily separated.     

Hybrid silica monolithic column for capillary electrochromatography with enhanced cathodic electroosmotic flow

A hybrid silica monolithic stationary phase for RP CEC was prepared by in situ co-condensation of (3-mercaptopropyl)-trimethoxysilane (MPTMS), phenyltriethoxysilane (PTES), and tetraethoxysilane (TEOS) via a sol-gel process. The thiol groups on the surface of the stationary phase were oxidized to sulfonic acids by peroxytrifluoroacetic acid. The introduced sulfonic acid moieties on the monoliths were characterized by a strong and relatively stable EOF in a broad pH range from 2.35 to 7.0 in CEC. Aromatic acids and neutral compounds can be simultaneously separated in this column under cathodic EOF. The CEC column exhibited a typical RP chromatographic mechanism for neutral compounds due to the introduced phenyl groups.     

Hybrid organic-inorganic phenyl monolithic column for capillary electrochromatography

A novel hybrid organic-inorganic silica-based monolithic column possessing phenyl ligands for reversed-phase (RP) capillary electrochromatography (CEC) is described. The monolithic stationary phase was prepared by in situ co-condensation of tetraethoxysilane (TEOS) with phenyltriethoxysilane (PTES) via a two-step catalytic sol-gel procedure to introduce phenyl groups distributed throughout the silica matrix for chromatographic interaction. The hydrolysis and condensation reactions of precursors were chemically controlled through pH variation by adding hydrochloric acid and dodecylamine, respectively. The structural property of the monolithic column can be easily tailored through adjusting the composition of starting sol solution. The effect of PTES/TEOS ratios on the morphology of the created stationary phases was investigated. A variety of neutral and basic analytes were used to evaluate the column performance. The CEC columns exhibited typical RP chromatographic retention mechanism for neutral compounds and had improved peak shape for basic solutes.     

Practical aspects of using methacrylate-ester-based monolithic columns in capillary electrochromatography

Methacrylate-ester-based monoliths containing quaternary ammonium groups were prepared in situ in capillary columns and in simultaneous experiments in vials, employing thermal initiation. The chromatographic properties of the monoliths were determined with capillary electrochromatography (CEC), and their morphology was studied with mercury-intrusion porosimetry on the bulk materials. Materials with different, well repeatable pore-size distributions could be prepared. A satisfactory column-to-column and run-to-run repeatability was obtained for the electro-osmotic mobility, the retention characteristics (k-values) and the efficiency on the columns prepared and tested in the CEC mode. A relatively high electro-osmotic flow was observed in the direction of the positive electrode. The electro-osmotic mobility was found to be influenced only marginally by mobile-phase parameters such as the pH, ionic strength, and acetonitrile content. The retention behavior of the monolithic columns was similar to that of columns packed with C18-modified silica particles. Columns could be prepared with optimum plate heights ranging from 6 microm for unretained compounds to 20 microm for well retained (k=2.5) polyaromatic hydrocarbons. However, for specific analytes a - still unexplained - lower chromatographic column efficiency was observed.     

Preparation and Porous Property of C14-Monolithic Column for Capillary Electrochromatography

Capillary electrochromatography (CEC) has been performed with a series of C₁₄, methlyacrylamide based monolithic columns. These monoliths with different porosities were prepared by in-situ copolymerization in fused-silica capillaries. The porous properties of monoliths were further observed using scanning electron micrographs (SEM) and measured using a mercury porosimeter. The effect of various alcohols as porogens on porous structural properties and chromatographic behaviors were also investigated. The effects of organic additive, pH value and ionic strength in mobile phase on electroosmosis flow (EOF) and separation were further discussed. Meanwhile, the baseline separation of 6 neutral compounds can be well obtained. In addition, the monolithic column demonstrates the high column efficiency and satisfactory reproducibility.Acknowledgements We gratefully acknowledge the support of the National Natural Science Foundation of China.     

Nano-liquid chromatography and capillary electrochromatography hyphenated with mass spectrometry for tryptic digest protein analysis: A comparison

Nano-LC and CEC were studied for the separation of cytochrome c tryptic digest. The peptides mixture was analyzed using either a nano-LC commercial or a laboratory assembled instrumentation coupled with an IT-ESI-MS by using a nanospray interface. CEC experiments were carried out with a CE apparatus coupled with the IT-ESI-MS through a liquid junction interface. Analytes were separated utilizing C18 silica based stationary phases, of different properties and origin, silica derivatized with cyano groups and C18 monolithic material. The last column, just because the chemical composition (absence of charged/chargeable groups) was tested only using nano-LC. Best results mainly related to the highest number of peptides separated and column equilibration time were obtained by nano-LC employing the C18 stationary phase (detection of 20 peptides, coverage of 88%). Similar results were achieved using both commercial and laboratory assembled instrumentation. The use of CEC revealed a higher separation efficiency and shorter analysis time. However, the number of separated peptides were lower than those observed in nano-LC. In CEC the use of capillaries packed with cyanosilica particles offered better results; however, less satisfactory than those observed in the miniaturized LC technique. Provided the use of the same stationary phase and taking into account the driving forces, the two techniques can be considered complementary, offering different information related to the retention times of the studied peptides.     

Spherical ordered mesoporous silicas and silica monoliths as stationary phases for liquid chromatography

Ordered mesoporous silicas such as micelle-templated silicas (MTS) feature unique textural properties in addition to their high surface area (approximately 1000 m2/g): narrow mesopore size distributions and controlled pore connectivity. These characteristics are highly relevant to chromatographic applications for resistance to mass transfer, which has never been studied in chromatography because of the absence of model materials such as MTS. Their synthesis is based on unique self-assembly processes between surfactants and silica. In order to take advantage of the perfectly adjustable texture of MTS in chromatographic applications, their particle morphology has to be tailored at the micrometer scale. We developed a synthesis strategy to control the particle morphology of MTS using the concept of pseudomorphic transformation. Pseudomorphism was recognized in the mineral world to gain a mineral that presents a morphology not related to its crystallographic symmetry group. Pseudomorphic transformations have been applied to amorphous spherical silica particles usually used in chromatography as stationary phases to produce MTS with the same morphology, using alkaline solution to dissolve progressively and locally silica and reprecipitate it around surfactant micelles into ordered MTS structures. Spherical beads of MTS with hexagonal and cubic symmetries have been synthesized and successfully used in HPLC in fast separation processes. MTS with a highly connected structure (cubic symmetry), uniform pores with a diameter larger than 6 nm in the form of particles of 5 microm could compete with monolithic silica columns. Monolithic columns are receiving strong interest and represent a milestone in the area of fast separation. Their synthesis is a sol-gel process based on phase separation between silica and water, which is assisted by the presence of polymers. The control of the synthesis of monolithic silica has been systematically explored. Because of unresolved yet cladding problems to evaluate the resulting macromonoliths in HPLC, micromonoliths were synthesized into fused-silica capillaries and evaluated by nano-LC and CEC. Only CEC allows to gain high column efficiencies in fast separation processes. Capillary silica monolithic columns represent attractive alternatives for miniaturization processes (lab-on-a chip) using CEC.     

Performance limits of monolithic and packed capillary columns in high-performance liquid chromatography and capillary electrochromatography

A method is proposed for the comprehensive characterization and comparison of columns in the high-performance liquid chromatographic (HPLC) and capillary electrochromatographic (CEC) modes. Using this approach, column parameters such as the number of plates, the eddy-diffusion and mass-transfer contributions to peak broadening, the permeability, and the analysis time are incorporated in a single graph and a comparison in terms of efficiency and speed is obtained. The chromatographic performance of silica-based and polymer-based monolithic capillary columns is discussed and a comparison is made with the performance of packed columns. Also, the potential of ultra-high-pressure liquid chromatography is discussed in this context. In the HPLC mode, the best results were obtained with silica monoliths; in the CEC mode, the low-density methacrylate-ester-based monoliths showed the best performance.     

The synthesis of chloropropyl-functionalized silica hybrid monolithic column with modification of N,N-dimethyl-N-dodecylamine for capillary electrochromatography separation

A chloropropyl-functionalized silica (CP-silica) hybrid monolithic column was synthesized within the confines of a capillary via the sol–gel process using tetramethoxysilane (TMOS) and (3-chloropropyl)-trimethoxysilane (CPTMS) as the precursors. The resulting CP-silica hybrid monolith inside the capillary showed homogeneous macroporous morphology and was well attached to the inner wall of the capillary. The obtained CP-silica hybrid monolithic capillary column demonstrated the inherent hydrophobic property and could be applied as a reversed-phase stationary phase in CEC directly. Due to the great chemical reactivity of the incorporated chloro groups on the hybrid silica monolithic matrix, the chloropropyl moieties on the surface of the hybrid silica monolith matrix could be conveniently further modified by a tertiary amine of N,N-dimethyl-N-dodecylamine (DMDA) via the nucleophilic substitution reaction at 70 °C to introduce a dodecyl groups (C12) onto the CP-silica hybrid monolithic matrix. The resulting C12-silica hybrid monolithic column not only demonstrated the significantly enhanced hydrophobic property in the separation of alkylbenzenes in reversed-phase capillary electrochromatography (RP-CEC), but also the strong electroosmotic flow (EOF) in a wide pH range. Five alkylbenzenes could be baseline separated in 3 min with column efficiency ranging from 189 700 to 221 000 N/m with a 70% ACN running buffer in CEC.     

Preparation of a Monolith with Covalently Bound Bovine Serum Albumin for Capillary Electrochromatography

Capillary electrochromatography (CEC) is important for applications in enantiomer separation. The problems associated with column fabrication bring a challenge in developing monoliths with ease of preparation, robustness of separation, enhanced mass transfer, and lower pressure drop. In this research, the covalent binding of proteins on to a monolithic matrix was investigated to overcome the drawback of loss and/or denaturing of the biomolecules from physical adsorption and encapsulation method. A chitosan/silica hybrid monolith was prepared and a protein, bovine serum albumin, was covalently immobilized on the column. The prepared monolith was evaluated using the enantioseparation of D,L-tryptophan by CEC. It was found that separation of tryptophan enantiomers with a resolution of 2.44 was achieved by using 20 mmol L^?1 phosphate buffer at pH 7.5. A higher chitosan concentration was also proven to be of possible use in the synthesis with the aid of acetic acid as the solvent. The much shorter retention time and increased separation ability demonstrate the advantages of capillary column under investigation.     

Voltage-assisted capillary LC of peptides using monolithic capillary columns prepared by ring-opening metathesis polymerization

We examined the use of monolithic capillary columns prepared via ring-opening metathesis polymerization (ROMP) for peptide separation in voltage-assisted capillary LC (voltage-assisted CLC). In order to demonstrate their potential for peptide separation, ROMP-derived monoliths with RP properties were prepared. The preparation procedure of monoliths was transferred from ROMP monoliths optimized for CLC. ROMP monoliths were synthesized within the confines of 200 microm id fused-silica capillaries with a length of 37 cm. After optimization of the chromatographic conditions, the separation performance was tested using a well-defined set of artificial peptides as well as two peptidic mixtures resulting from a tryptic digest of BSA as well as a collagenase digest of collagen. ROMP monoliths showed comparable performance to other monolithic separation media in voltage-assisted CLC published so far. Therefore, we conclude that by optimizing the composition of the ROMP monoliths as well as by using the controlled manner of their functionalization, ROMP monoliths bear a great potential in CLC and CEC.     

Preparation and applications of hybrid organic-inorganic monoliths: a review

This review presents an overview of the properties of hybrid organic-inorganic monolithic materials and summarizes the recent developments in the preparation and applications of these hybrid monolithic materials. Hybrid monolithic materials with porosities, surface functionalities, and fast dynamic transport have developed rapidly, and have been used in a wide range of applications owing to the low cost, good stability, and excellent performance. Basically, these materials can be divided into two major types according to the chemical composition: hybrid silica-based monolith (HSM) and hybrid polymer-based monolith (HPM). Compared to the HPM, HSM monolith has been attracting most wide attentions, and it is commonly synthesized by the sol-gel process. The conventional preparation procedures of two type's hybrid organic-inorganic monoliths are addressed. Applications of hybrid organic-inorganic monoliths in optical devices, capillary microextraction (CME), capillary electrochromatography (CEC), high performance liquid chromatography (HPLC), and chiral separation are also reviewed.     

Monolithic stationary phases for fast ion chromatography and capillary electrochromatography of inorganic ions

The focus of this review is on current developments in monolithic stationary phases for the fast analysis of inorganic ions and other small molecules in ion chromatography (IC) and capillary electrochromatography (CEC), concentrating in particular on the properties of organic (polymer) monolithic materials in comparison to inorganic (silica-based) monoliths. The applicability of these materials for fast IC is discussed in the context of recent publications, including the range of synthesis and modification procedures described. While commercial monolithic silica columns already show promising results on current IC instrumentation, polymer-based monolithic stationary phases are currently predominantly used in the capillary format on modified micro-IC systems. However, they are beginning to find application in IC particularly under high pH conditions, with the potential to replace their particle-packed counterparts.     

Novel preparation of organic polymer-based monolithic column by microwave irradation

Microwave irradiation was firstly attempted for the preparation of organic-based monoliths of poly（styrene-divinylbenzene- methacrylic acid）, which single step in situ polymerization was carried out during 15 min. The colunm permeability, electrophoretic and chromatographac behaviors were comparatively evaluated using pressure-assisted CEC, GEC and low pressure-driven separation modes. The largest theoretical plates for the preparing column could be close to 18,0000 plates/m for thiourea in the mode of p-CEC. It provided a viable alternative to traditional initiation means for the perparation of monolithic capillary columns.     

Metal organic framework–organic polymer monolith stationary phases for capillary electrochromatography and nano-liquid chromatography

In this study, metal organic framework (MOF)–organic polymer monoliths prepared via a 5-min microwave-assisted polymerization of ethylene dimethacrylate (EDMA), butyl methacrylate (BMA), and 2-acrylamido-2-methylpropane sulfonic acid (AMPS) with the addition of various weight percentages (30–60%) of porous MOF (MIL-101(Cr)) were developed as stationary phases for capillary electrochromatography (CEC) and nano-liquid chromatography (nano-LC). Powder X-ray diffraction (PXRD) patterns and nitrogen adsorption/desorption isotherms of these MOF–organic polymer monoliths showed the presence of the inherent characteristic peaks and the nano-sized pores of MIL-101(Cr), which confirmed an unaltered crystalline MIL-101(Cr) skeleton after synthesis; while energy dispersive spectrometer (EDS) and micro-FT-IR spectra suggested homogenous distribution of MIL-101(Cr) in the MIL-101(Cr)–poly(BMA–EDMA) monoliths. This hybrid MOF–polymer column demonstrated high permeability, with almost 800-fold increase compared to MOF packed column, and efficient separation of various analytes (xylene, chlorotoluene, cymene, aromatic acids, polycyclic aromatic hydrocarbons and trypsin digested BSA peptides) either in CEC or nano-LC. This work demonstrated high potentials for MOF–organic polymer monolith as stationary phase in miniaturized chromatography for the first time.     

Recent development of monolithic stationary phases with emphasis on microscale chromatographic separation

The column technologies play a crucial role in the development of new methods and technologies for the separation of biological samples containing hundreds to thousands compounds. This review focuses on the development of monolithic technology in micro-column formats for biological analysis, especially in capillary liquid chromatography, capillary electrochromatography and microfluidic devices in the past 5 years (2002-2007) since our last review in 2002 on monoliths for HPLC and CEC. The fabrication and functionalization of monoliths were summarized and discussed, with the aim of presenting how monolithic technology has been playing as an attractive tool for improving the power of existing chromatographic separation processes. This review consists of two parts: (i) the recent development in fabrication of monolithic stationary phases from direct synthesis to post-functionalization of the polymer- and silica-based monoliths tailoring the physical/chemical properties of porous monoliths; (ii) the application of monolithic stationary phases for one- and multi-dimensional capillary liquid chromatography, fast separation in capillary electro-driven chromatography, and microfluidic devices.     

One‐pot synthesis of (3‐sulfopropyl methacrylate potassium)‐silica hybrid monolith via thiol‐ene click chemistry for CEC

A novel (3‐sulfopropyl methacrylate potassium)‐silica hybrid monolithic column for CEC has been prepared by a simple one‐pot approach based on efficient thiol‐ene click chemistry. In this process, the polycondensation of hydrolyzed alkoxysilanes and in situ click reaction of vinyl groups on 3‐sulfopropyl methacrylate potassium and thiol groups on the precondensed siloxanes simultaneously occurred in a pretreated capillary. Homogeneous monolithic matrix with large through‐pores tightly bonded to the inner wall of the capillary was shown by optical microscope and SEM. The minimum plate height of this hybrid monolithic column was determined as 3.9 μm for thiourea. Anilines, alkylbenzenes, and phenols were well separated on this hybrid monolithic column by CEC, which indicated typical reversed‐phase and cation‐exchange chromatographic retention mechanisms of the column.