Dendritic Mesoporous Silica Nanoparticles with Abundant Ti4+ for Phosphopeptide Enrichment from Cancer Cells with 96% Specificity.

Selective enrichment and sensitive detection of phosphopeptides are of great significance in many bioapplications. In this work, dendritic mesoporous silica nanoparticles modified with polydopamine and chelated Ti4+ (denoted DMSNs@PDA-Ti4+) were developed to improve the enrichment selectivity of phosphopeptides. The unique central-radial pore structures endowed DMSNs@PDA-Ti4+ with a high surface area (362 m2 g-1), a large pore volume (1.37 cm3 g-1), and a high amount of chelated Ti4+ (75 µg mg-1). Compared with conventional mesoporous silica-based materials with the same functionalization (denoted mSiO2@PDA-Ti4+) and commercial TiO2, DMSNs@PDA-Ti4+ showed better selectivity and a lower detection limit (0.2 fmol/µL). Moreover, 2422 unique phosphopeptides were identified from HeLa cell extracts with a high specificity (>95%) enabled by DMSNs@PDA-Ti4+, better than those in previous reports.

Facile Preparation of Titanium(IV)-Immobilized Hierarchically Porous Hybrid Monoliths.

Hierarchically porous materials have become a key feature of biological materials and have been widely applied for adsorption or catalysis. Herein, we presented a new approach to directly prepare a phosphate-functionalized hierarchically porous hybrid monolith (HPHM), which simultaneously contained mesopores and macropores. The design was based on the copolymerization of polyhedral oligomeric vinylsilsesquioxanes (vinylPOSS) and vinylphosphonic acid (VPA) by adding degradable polycaprolactone (PCL) additive. The phosphate groups could be directly introduced into the hybrid monoliths. This approach was simple and time-saving, and overcame the defect of a rigorous, complex process for preparing traditional Ti4+-immobilized metal ion affinity chromatography (IMAC) materials. The specific surface area of an optimal hybrid monolith could reach 502 m2/g obtained by nitrogen adsorption/desorption measurements, which originated from the degradation of PCL. Meanwhile, the characterization of scanning electron microscopy (SEM) and mercury intrusion porosimetry (MIP) also suggested that the macropores existed in the hybrid monoliths. The size of macropores could be controlled by the content of PCL in the polymerization mixture. The prepared Ti4+-IMAC HPHMs exhibited high adsorption capacity (63.6 mg/g for pyridocal 5'-phosphatemonohydrate), and excellent enrichment specificity (tryptic digest of ß-casein/BSA at a molar ratio of 1:1000) and sensitivity (tryptic digest of 5 fmol of ß-casein). Moreover, the Ti4+-IMAC HPHMs provided effective enrichment ability of low-abundance phosphopeptides from human serum and HeLa cell digests.

Preparation of Polypropylene Spin Tips Filled with Immobilized Titanium(IV) Ion Monolithic Adsorbent for Robust Phosphoproteome Analysis.

In this study, we developed a Ti(IV) monolithic spin tip for phosphoproteome analysis of a minute amount of biological sample for the first time. The surface of polypropylene pipet tip was activated by the photoinitiator benzophenone under UV light radiation followed by polymerization of ethylene glycol methacrylate phosphate and bis-acrylamide in the tip to form a porous monolith with reactive phosphate groups. The as-prepared tips grafted with monolithic adsorbent were then chelated with titanium(IV) ion for phosphopeptide enrichment. It was found that the tips enabled fast and efficient capture of phosphopeptides from microscale complex samples. The monolithic tip was demonstrated to have a detection limit as low as 5 fmol ß-casein tryptic digest, along with an exceptionally high specificity to capture phosphopeptides from complex tryptic digest mixed with an unphosphorylated protein and a phosphorylated protein at a molar ratio up to 1000:1. When the tip was applied to enrich phosphopeptides from 5 µg of tryptic digest of complex HeLa cell proteins, 1185 high confidence of phosphorylated sites were successfully identified with the specificity as high as 92.5%. So far, this is the most sensitive phosphoproteomics analysis using a standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) system for proteome-wide phosphorylation analysis in mammalian cells.

Polyacrylamide gel with switchable trypsin activity for analysis of proteins.

Trypsin was immobilized on a variety of materials to improve digestion efficiency. However, because the immobilized trypsin will digest proteins during electrophoresis, direct immobilization of active trypsin in polyacrylamide gel will compromise the protein separation. To overcome this problem, here we report a novel polyacrylamide gel with switchable trypsin activity. It was prepared by copolymerization of the PEG-trypsin-aprotinin complex during the gel-casting step. Because the inhibitor aprotinin binds strongly with trypsin at alkaline pH, this novel gel does not display hydrolytic activity during electrophoresis. After electrophoresis, the activity of trypsin embedded in gel could be recovered by simply washing away the bound inhibitor at a low pH. It was demonstrated that this unique switchable activity design allowed high resolution of the complex protein mixture during electrophoresis and highly efficient digestion of the separated proteins in situ in the gel after electrophoresis.

Preparation of epoxy-functionalized hierarchically porous hybrid monoliths via free radical polymerization and application in HILIC enrichment of glycopeptides.

Owing to their multiscale pore size regimes and unique properties, the materials with hierarchically porous structures have become an important family of functional materials in recent years. They have been applied from energy conversion and storage, catalysis, separation to drug delivery, etc. The synthesis of them is difficult by the need to employ multiple templates and take complicated steps. Herein, we successfully prepared epoxy-functionalized hierarchically porous hybrid monoliths (HPHMs) with micro/meso/macro-structures in an easy way. Firstly, a bulk monolithic material was formed via free radical polymerization between polyhedral oligomeric vinylsilsesquioxanes (vinylPOSS) and allyl glycidyl ether (AGE) in the presence of polycaprolactone (PCL). Then PCL was degraded with hydrochloric acid solution, and the epoxy-functionalized HPHM was obtained. This approach was very simple and suitable for large-scale preparation. Hybrid monoliths with different specific surface area (from 5.4 to 636.7 m2/g) were prepared by adjusting the mole ratio of vinylPOSS to AGE and the content of PCL. The results of several characterization methods, including nitrogen adsorption/desorption measurements, scanning electron microscopy (SEM) and mercury intrusion porosimetry (MIP), showed that these materials contained not only micropores and mesopores but also macropores. The materials were further modified with penicillamine to be used as hydrophilic interaction chromatography (HILIC) adsorbents for enriching N-glycopeptides in IgG and serum protein tryptic digests. Up to 23 N-glycopeptides were identified from IgG digest, and 385 N-glycopeptides and 283 N-glycosylation sites were identified from human serum digest.

Facile preparation of microporous organic polymers functionalized macroporous hydrophilic resin for selective enrichment of glycopeptides.

A macroporous adsorption resin (MAR) with ∼10 µm diameter was synthesized by seed-swelling polymerization and further modified with a layer of microporous organic polymers (MOP) by "one-pot" solvothermal reaction. The resulting MAR@MOP exhibited high specific surface area of 131.3 m2/g, which was higher than that of pristine MAR (57.8 m2/g). The contact angle also decreased from 58.8° (MAR) to 24° (MAR@MOP), indicating that the MOP was successfully grafted onto the surface of MAR. The chemical composition of MAR@MOP was confirmed by Fourier-transform infrared spectroscopy, 13C NMR and element analysis. The enrichment efficiency of MAR@MOP to glycopeptides was demonstrated by trapping N-linked glycopeptides from tryptic digests of human immunoglobulin G (IgG), horseradish peroxidase (HRP) and bovine fetuin. Furthermore, 879 unique N-glycosylation sites in 811 unique glycopeptides sequence mapped to 516 N-glycosylated proteins were identified in three replicate analyses of proteins extracted from mouse liver. Therefore, this hydrophilic MOP-coated adsorbent would be applied in the enrichment and identification of low-abundance N-linked glycopeptides in complicated biological samples.

Fast preparation of hybrid monolithic columns via photo-initiated thiol-yne polymerization for capillary liquid chromatography.

Although several approaches have been developed to fabricate hybrid monoliths, it would still take a few hours to finish the formation of monoliths. Herein, photo-initiated thiol-yne polymerization was first adopted to in situ fabricate hybrid monoliths within the confines of UV-transparent fused-silica capillary. A silicon-containing diyne (1,3-diethynyltetramethyl-disiloxane, DYDS) was copolymerized with three multithiols, 1,6-hexanedithiol, trimethylolpropane tris(3-mercaptopropionate) and pentaerythriol tetrakis(3-mercaptopropionate), by using a binary porogenic system of diethylene glycol diethyl ether (DEGDE)/poly(ethylene glycol) (PEG200) within 10 min. Several characterizations of three hybrid monoliths (assigned as I, II and III, respectively) were performed. The results showed that these hybrid monoliths possessed bicontinuous porous structure, which was remarkably different from that via typical free-radical polymerization. The highest column efficiency of 76,000 plates per meter for butylbenzene was obtained on the column I in reversed-phase liquid chromatography (RPLC). It was observed that the efficiencies for strong-retained butylbenzene were almost close to those of weak-retained benzene, indicating a retention-independent efficient performance of small molecules on hybrid column I. The surface area of this hybrid monolith was very small in the dry state (less than 10.0 m2/g), and the chromatographic behavior of hybrid monolithic columns would be possibly explained by radical-mediated step-growth process of thiol-yne polymerization. Finally, the column I was applied for separation of BSA tryptic digest by cLC-MS/MS, indicating satisfactory separation ability for complicated samples.

[Fabrication of acylsemicarbazide-based porous organic polymer for selective enrichment of glycopeptides].

An acylsemicarbazide-based porous organic polymer (POP) was facilely prepared by the polymerization of benzene-1,3,5-tricarbohydrazide (BTZ) and 1,4-phenylene diisocyanate (PDI). The physical properties of as-synthesized material were characterized by Fourier-transform infrared spectroscopy (FT-IR), solid-state cross polarisation magic angle spinning carbon-13 nuclear magnetic resonance (CP-MAS 13C NMR), nitrogen adsorption/desorption measurement, water contact angle and so on. The specific surface area was 166 m2/g, and the water contact angle was 46.4°, exhibiting hydrophilic property of the porous material. Thus, POP-1 was tried to be used for glycopeptides enrichment from tryptic digest of standard protein and complex biosamples in hydrophilic mode. The 19 typical N-linked glycopeptides were identified from 500 fmol tryptic digest of immunoglobulin G (IgG) from human serum by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, meanwhile, 1919 unique glycopeptides with 1350 N-glycosylation sites from 605 different N-linked glycoproteins were identified from 100 µ g mouse liver tryptic digest by capillary liquid chromatography (cLC)-MS/MS analysis. These results demonstrated the great potential of POP-1 for glycoproteome analysis.

Synthesis of polymeric monoliths via thiol-maleimide polymerization reaction for highly efficient chromatographic separation.

One-step thiol-maleimide polymerization reaction was firstly adopted for direct preparation of polymeric monoliths via alkaline-catalyzed reaction of 4,4'-bis(maleimidophenyl)methane (BMI) and trimethylolpropane tris(3-mercaptopropionate) (3SH)/pentaerythriol tetra(3-mercaptopropionate) (4SH) in the presence of a small amount of triethylamine (TEA). The polymerization could be performed within 3h, which was faster than thermal-initiated free radical polymerization. Two kinds of monoliths, poly(BMI-co-3SH) (marked as I) and poly(BMI-co-4SH) (marked as II), were characterized with scanning electron microscopy (SEM), attenuated total reflection Fourier-transformed infrared spectroscopy (ATR-FTIR), thermal gravimetric analysis (TGA) and mercury intrusion porosimetry (MIP). Satisfactory chromatographic separation ability and column efficiency were gained for analysis of small molecular compounds such as alkylbenzenes, polynuclear aromatic hydrocarbons (EPA 610) and phenols in reversed-phase capillary liquid chromatography (cLC). High column efficiency (180,500N/m) for butylbenzene was acquired on poly(BMI-co-3SH) column I-2, which was higher than those on most reported polymeric monoliths. A retention-independent efficient performance of small molecules was obtained by plotting of plate height (H) of alkylbenzenes versus the linear velocity (u). A term values in van Deemter equation of I-2 (1.72-0.24µm) and poly(BMI-co-4SH) column II-2 (5.28-4.14µm) were smaller than those of traditional organic/hybrid monoliths. Finally, as a practical application, 53 and 2184 unique peptides from the tryptic digests of bovine serum albumin (BSA) and HeLa cell proteins were positively identified with poly(BMI-co-3SH) monolith in cLC-MS.

Hydrogen bond based smart polymer for highly selective and tunable capture of multiply phosphorylated peptides.

Multisite phosphorylation is an important and common mechanism for finely regulating protein functions and subsequent cellular responses. However, this study is largely restricted by the difficulty to capture low-abundance multiply phosphorylated peptides (MPPs) from complex biosamples owing to the limitation of enrichment materials and their interactions with phosphates. Here we show that smart polymer can serve as an ideal platform to resolve this challenge. Driven by specific but tunable hydrogen bonding interactions, the smart polymer displays differential complexation with MPPs, singly phosphorylated and non-modified peptides. Importantly, MPP binding can be modulated conveniently and precisely by solution conditions, resulting in highly controllable MPP adsorption on material surface. This facilitates excellent performance in MPP enrichment and separation from model proteins and real biosamples. High enrichment selectivity and coverage, extraordinary adsorption capacities and recovery towards MPPs, as well as high discovery rates of unique phosphorylation sites, suggest its great potential in phosphoproteomics studies.Capture of low-abundance multiply phosphorylated peptides (MPPs) is difficult due to limitation of enrichment materials and their interactions with phosphates. Here the authors show, a smart polymer driven by specific but tunable hydrogen bonding interactions can differentially complex with MPPs, singly phosphorylated and non-modified peptides.

Preparation of open tubular capillary columns by in situ ring-opening polymerization and their applications in cLC-MS/MS analysis of tryptic digest.

An open tubular (OT) column (25 µm i.d.) was prepared by in situ ring-opening polymerization of octaglycidyldimethylsilyl polyhedral oligomeric silsesquioxanes (POSS-epoxy) with 4-aminophenyl disulfide (APDS) in a binary porogenic system of ethanol/H2O. It was found that porogenic composition played an important role in the formation of OT stationary phases. The ratio of ethanol/H2O at 6/1 (v/v) would lead to the fabrication of hybrid monoliths, while the ratio of ethanol/H2O at 13/1 (v/v) would result in the synthesis of OT phases. In addition, the effects of precursor content and reaction duration on the thickness of OT stationary phases were investigated. Either lower precursor content or shorter reaction duration would produce thinner layer of OT column. The repeatability of OT columns was evaluated through relative standard deviation (RSD%) with benzene as the analyte. The run-to-run, column-to-column and batch-to-batch repeatabilities were 1.7%, 4.8% and 5.6%, respectively, exhibiting satisfactory repeatability of the OT column. Then tryptic digest of mouse liver proteins was used to evaluate the performance of the resulting OT columns (25 µm i.d. × 2.5 m in length) by cLC-MS/MS analysis, demonstrating their potential in proteome analysis.

Facile synthesis of zwitterionic polymer-coated core-shell magnetic nanoparticles for highly specific capture of N-linked glycopeptides.

Highly selective and efficient capture of glycosylated proteins and peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. Recently, hydrophilic interaction liquid chromatography (HILIC)-based functional materials have been extensively utilized for glycopeptide enrichment. However, the low amount of immobilized hydrophilic groups on the affinity material has limited its specificity, detection sensitivity and binding capacity in the capture of glycopeptides. Herein, a novel affinity material was synthesized to improve the binding capacity and detection sensitivity for glycopeptides by coating a poly(2-(methacryloyloxy)ethyl)-dimethyl-(3-sulfopropyl) ammonium hydroxide (PMSA) shell onto Fe3O4@SiO2 nanoparticles, taking advantage of reflux-precipitation polymerization for the first time (denoted as Fe3O4@SiO2@PMSA). The thick polymer shell endows the nanoparticles with excellent hydrophilic property and several functional groups on the polymer chains. The resulting Fe3O4@SiO2@PMSA demonstrated an outstanding ability for glycopeptide enrichment with high selectivity, extremely high detection sensitivity (0.1 fmol), large binding capacity (100 mg g(-1)), high enrichment recovery (above 73.6%) and rapid magnetic separation. Furthermore, in the analysis of real complicated biological samples, 905 unique N-glycosylation sites from 458 N-glycosylated proteins were reliably identified in three replicate analyses of a 65 µg protein sample extracted from mouse liver, showing the great potential of Fe3O4@SiO2@PMSA in the detection and identification of low-abundance N-linked glycopeptides in biological samples.

Facile synthesis of guanidyl-functionalized magnetic polymer microspheres for tunable and specific capture of global phosphopeptides or only multiphosphopeptides.

The highly selective and efficient capture of heterogeneous types of phosphopeptides is critical for comprehensive and in-depth phosphoproteome analysis, but it still remains a challenge since the lack of affinity material with large binding capacity and controllable specificity. Here, a new affinity material was prepared to improve the enrichment capacity and endue the tunable specificity by introducing guanidyl onto poly(glycidyl methacrylate) (PGMA) modified Fe3O4 microsphere (denoted as Fe3O4@PGMA-Guanidyl). The thick polymer shell endows the composite microsphere with large amount of guanidyl and is beneficial to enhancing the affinity interaction between phosphopeptides and the material. Interestingly, the Fe3O4@PGMA-Guanidyl possesses tunable enriching ability for global phosphopeptides or only multiphosphopeptides through simple regulation of buffer composition. The composite has large enrichment capacity (200 mg g(-1)), extremely high detection sensitivity (0.5 fmol), high enrichment recovery (91.30%), great specificity, and rapid magnetic separation. Moreover, the result of the application to capture of phosphopeptides from tryptic digest of nonfat milk has demonstrated the great potential of Fe3O4@PGMA-Guanidyl in detection and identification of low-abundance phosphopeptides of interest in biological sample.

A proteomic analysis of engineered tendon formation under dynamic mechanical loading in vitro.

Previous studies have demonstrated the beneficial effect of mechanical loading on in vitro tendon engineering. To understand the mechanism, human tenocytes and polyglycolic acid long fibers were used for in vitro tendon engineering in a bioreactor system for 12 weeks with and without dynamic loading. The engineered neo-tendons were subjected to proteomic analysis using mass spectrometry along with shotgun strategy. As expected, mechanical loading resulted in a more mature tendon tissue characterized by a firmer tissue texture and densely deposited matrices which formed longitudinally aligned collagen fibers in a highly compact fashion. In contrast, non-loaded neo-tendon revealed loosely and less deposited matrices in a relatively less organized pattern. Proteins isolated from two groups of tissues exhibited similar distribution of isoeletric point and molecular weight indicating the similarity and comparability of the tissue specimens. Further, proteomic analysis showed that total 758 proteins were identified from both groups with 194 and 177 proteins uniquely presented in loaded and non-loaded tendons, respectively. Comparison of loaded and non-loaded tendons revealed 195 significantly up-regulated proteins and 189 significantly down-regulated proteins. The differentially expressed proteins could generally be classified into the categories of extracellular matrix, intra-cellular signaling, cytoskeleton and inflammatory response. Among them, significantly up-regulated collagens I and VI, MMP-14, WNT5A, microfilament molecules and some inflammatory factors suggest that the possible mechanism for this particular biological phenomenon may involve increased production of tendon specific matrices, enhanced cross-link of collagens and other matrix molecules, proper matrix remodeling for tissue maturation and mechanotransduction (including non-canonical Wnt signal pathway) mediated other biological processes.

Improvement of proteome coverage using hydrophobic monolithic columns in shotgun proteome analysis.

Monolithic columns are widely used in shotgun proteome analysis. However, it is difficult to increase the separation capability and proteome coverage by using conventionally organic polymer-based monolithic column due to the difficulty of controlling homogeneity of the overall pore structure (both pores and microglobules), which leads to relatively low column efficiency. Therefore, we studied the effect of constitute and percentage of porogenic solvent, functional monomer, column length, and separation gradient on the peak capacity and proteome coverage by methacrylate-based reversed phase monolithic columns. It was demonstrated that the porous property of the hydrophobic monolith, which was mainly determined by the porogenic solvent, was crucial to the proteome coverage when similar methacrylate monomer was utilized and a ternary porogenic solvent was adopted to prepare C12 monolithic column with relatively homogeneous overall pore structure. It was also shown that high proteome coverage could be reliably obtained with online multidimensional separation using totally monolithic columns system with the length of analytical column at 85 cm and reversed phase separation gradient at 210 min.

Synthesis and characterization of a new boronate affinity monolithic capillary for specific capture of cis-diol-containing compounds.

Boronate affinity chromatography (BAC) is an important tool for specific capture and separation of cis-diol-containing compounds such as glycoproteins, RNA and carbohydrates. Only a few reports on monolithic column-based BAC have appeared. In this paper, boronate functionalized monolithic capillary column was synthesized by in situ free radical polymerization for the first time. The prepared column was first characterized in terms of morphology, pore properties, capacity and retention mechanisms. The column exhibited uniform open channel network and high capture capacity. Systematical investigation on the retention mechanism revealed that multiple intermolecular interactions occur between the analytes and the boronate affinity monolith, including boronate affinity, reversed-phase, cation-exchange and hydrogen bonding interactions, depending on the conditions used. In addition, the presence of Lewis base such as fluoride ion in the mobile phase was found to be favorable to the complexation between cis-diol-containing compounds with the boronic acid ligand under less basic conditions. On the basis of these fundamental investigations, the prepared monolithic column was then applied to the capture of adenosine and flavin adenine dinucleotide. The investigations in this study provide sound understanding not only on how to manipulate the separation selectivity through selection of appropriate mobile phase composition on the currently prepared columns but also on how to design next-generation columns with desired properties and functions.

Immobilized zirconium ion affinity chromatography for specific enrichment of phosphopeptides in phosphoproteome analysis.

Large scale characterization of phosphoproteins requires highly specific methods for purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. Enrichment of phosphopeptides from complex peptide mixtures by IMAC is a popular way to perform phosphoproteome analysis. However, conventional IMAC adsorbents with iminodiacetic acid as the chelating group to immobilize Fe(3+) lack enough specificity for efficient phosphoproteome analysis. Here we report a novel IMAC adsorbent through Zr(4+) chelation to the phosphonate-modified poly(glycidyl methacrylate-co-ethylene dimethacrylate) polymer beads. The high specificity of Zr(4+)-IMAC adsorbent was demonstrated by effectively enriching phosphopeptides from the digest mixture of phosphoprotein (alpha- or beta-casein) and bovine serum albumin with molar ratio at 1:100. Zr(4+)-IMAC adsorbent was also successfully applied for the analysis of mouse liver phosphoproteome, resulting in the identification of 153 phosphopeptides (163 phosphorylation sites) from 133 proteins in mouse liver lysate. Significantly more phosphopeptides were identified than by the conventional Fe(3+)-IMAC approach, indicating the excellent performance of the Zr(4+)-IMAC approach. The high specificity of Zr(4+)-IMAC adsorbent was found to mainly result from the strong interaction between chelating Zr(4+) and phosphate group on phosphopeptides. Enrichment of phosphopeptides by Zr(4+)-IMAC provides a powerful approach for large scale phosphoproteome analysis.

Recent progress in polar stationary phases for CEC.

This review summarizes most of the recent developments in the preparation and application of polar stationary phases for CEC covering the literature published since the year 2004. These polar stationary phases have been adopted for separation of analytes by the modes of packing column CEC, open-tubular CEC (o-CEC) and monolithic column CEC. Currently, development of o-CEC using biomolecules, such as protein and DNA, as the immobilized ligands is highlighted partly due to the simplicity of preparation. Furthermore, monolithic columns have been extended quickly, particularly inorganic materials-based monoliths, such as silica, zirconia, hafnium, etc., as an alternative to packed columns have been developed quickly.

Preparation and evaluation of rigid porous polyacrylamide-based strong cation-exchange monolithic columns for capillary electrochromatography.

A CEC monolithic column with strong cation-exchange (SCX) stationary phase based on hydrophilic monomers was prepared by in situ polymerization of acrylamide, methylenebisacrylamide, and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a complete organic binary porogenic solvent consisting of DMSO and dodecanol. The sulfonic groups provided by the monomer AMPS on the surface of the stationary phase generate an EOF from anode to cathode, and serve as an SCX stationary phase at the same time. The monolithic stationary phase exhibited normal-phase chromatographic behavior for neutral analytes. For charged analytes, electrostatic interaction/repulsion with the monolith was observed. The strong SCX monolithic column has been successfully employed in the electrochromatographic separation of basic drugs, peptides, and alkaloids extracted from natural products.

Monolithic silica capillary column with coated cellulose tris(3,5-dimethylphenylcarbamate) for capillary electrochromatographic separation of enantiomers.

Monolithic silica capillary columns were prepared by a sol-gel process in fused-silica capillaries with an inner diameter of 50 microm and were modified by coating of cellulose tris(3,5-dimethylphenylcarbamate). Influences of the factors in the modification process on enantiomer separations were investigated. The prepared columns were used to perform enantiomer separations by CEC. Fifteen and two pairs of enantiomers were separated under aqueous and nonaqueous mobile phases, respectively, and most of them were baseline-separated with very high column efficiencies. The Van Deemter curve was found flat under high linear velocity of the mobile phase, which indicated favorable kinetic properties of the prepared columns. Baseline separation of a pair of enantiomers was achieved in 90 s with high-column efficiency by short-end separation under high voltage.