Methacrylate monolithic columns of 320 microm I.D. for capillary liquid chromatography

Monolithic capillary columns (320 microm I.D.) were prepared for capillary liquid chromatography (CLC) by radical polymerization of butylmethacrylate (BMA) and ethylenedimethacrylate (EDMA) in the presence of a porogen solvent containing propan-1-ol, butane-1,4-diol and water. The influence of the contents of the porogen solvent and EDMA in the polymerization mixture on the monolith porosity and column efficiency was investigated. The composition of the polymerization mixture was optimized to attain a minimum HETP of the order of tens of microm for test compounds with various polarities. The separation performance and selectivity of the most efficient monolithic column prepared was characterized by van Deemter curves, peak asymmetry factors and Walters hydrophobicity and silanol indices. It was demonstrated that the 320-microm I.D. monolithic column exhibited CLC separation performance similar to that observed for 100- and 150-microm I.D. monolithic columns reported in the literature; moreover, the 320-microm I.D. column was easier to operate in CLC and exhibited a higher sample loadability.     

Monolithic silica columns with various skeleton sizes and through-pore sizes for capillary liquid chromatography

Reduction of through-pore size and skeleton size of a monolithic silica column was attempted to provide high separation efficiency in a short time. Monolithic silica columns were prepared to have various sizes of skeletons (approximately 1-2 microm) and through-pores (approximately 2-8 microm) in a fused-silica capillary (50-200 microm I.D.). The columns were evaluated in HPLC after derivatization to C18 phase. It was possible to prepare monolithic silica structures in capillaries of up to 200 microm I.D. from a mixture of tetramethoxysilane and methyltrimethoxysilane. As expected, a monolithic silica column with smaller domain size showed higher column efficiency and higher pressure drop. High external porosity (> 80%) and large through-pores resulted in high permeability (K = 8 x 10(-14) -1.3 x 10(-12) m2) that was 2-30 times higher than that of a column packed with 5-mirom silica particles. The monolithic silica columns prepared in capillaries produced a plate height of about 8-12 microm with an 80% aqueous acetonitrile mobile phase at a linear velocity of 1 mm/s. Separation impedance, E, was found to be as low as 100 under optimum conditions, a value about an order of magnitude lower than reported for conventional columns packed with 5-microm particles. Although a column with smaller domain size generally resulted in higher separation impedance and the lower total performance, the monolithic silica columns showed performance beyond the limit of conventional particle-packed columns under pressure-driven conditions.     

Short communication performance of octadecylsilylated monolithic silica capillary columns of 530 microm inner diameter in HPLC

Monolithic silica capillary columns were successfully prepared in a fused silica capillary of 530 microm inner diameter and evaluated in HPLC after octadecylsilylation (ODS). Their efficiency and permeability were compared with those of columns pakked with 5-microm and 3-microm ODS-silica particles. The monolithic silica columns having different domain sizes (combined size of through-pore and skeleton) showed 2.5-4.0-times higher permeability (K= 5.2-8.4 x 10(-14) m2) than capillary columns packed with 3-mm particles, while giving similar column efficiency. The monolithic silica capillary columns gave a plate height of about 11-13 microm, or 11 200-13 400 theoretical plates/150 mm column length, in 80% methanol at a linear mobile phase velocity of 1.0 mm/s. The monolithic column having a smaller domain size showed higher column efficiency and higher pressure drop, although the monolithic column with a larger domain size showed better overall column performance, or smaller separation impedance (E value). The larger-diameter (530 microm id) monolithic silica capillary column afforded a good peak shape in gradient elution of proteins at a flow rate of up to 100 microL/min and an injection volume of up to 10 microL.     

Chemically modified chiral monolithic silica column prepared by a sol-gel process for enantiomeric separation by micro high-performance liquid chromatography

In this work a new type of chiral monolith silica column was developed for the chiral separation by micro high-performance liquid chromatography (micro-HPLC). The chiral monolith column with a continuous skeleton and a large through-pore structure was prepared inside a capillary of 100 microm I.D. by a sol-gel process, and chemically modified with chiral selectors, such as L-phenylalaninamide, L-alaninamide and L-prolinamide, on the surface of the monolithic silica column. Based on the principle of ligand exchange, these chiral monolithic columns were successfully used for the separation of dansyl amino acid enantiomers, as well as hydroxy acid enantiomers by micro-HPLC. The chromatographic conditions, the enantioselectivity and the performance of columns are discussed.     

Electrochromatography with a 2.7 mm inner diameter monolithic column

Monolithic columns of 2.7 mm I.D. have been prepared and used in electrochromatography (EC) separation. Although capillary electrochromatography (CEC) has higher separation efficiency, it displays some shortcomings, such as limited sample loadability and restricted concentration detectability etc. In this paper, we investigate the feasibility of EC separation with millimeter diameter monolithic columns. By using a designed preparation method of monolithic column packed with about 150 microm quartz sand, the effect of Joule heating can be reduced, and the processes of frit making and column packing can be avoided. The concentration detectability of the EC is improved comparing with that of CEC. Moreover, the separation efficiency of 52,000 plates/m was achieved with a 70 mm length and 2.7 mm I.D. monolithic column.     

Ring-opening metathesis polymerization-derived monolithic capillary columns for high-performance liquid chromatography. Downscaling and application in medical research

Monolithic capillary columns were prepared via ring-opening metathesis polymerization (ROMP) using norborn-2-ene (NBE) and 1, 4, 4a, 5, 8, 8a-hexahydro-1, 4, 5, 8-exo,endo-dimethanonaphthalene (DMN-H6) as monomers. The monolithic polymer was copolymerized with Grubbs-type initiator RuCl(2)(PCy(3))(2)(CHPh) and a suitable porogenic system within the confines of fused silica capillaries of different inner diameter (I.D.). The first part of the study focused on batch-to-batch reproducibility of ROMP-derived capillary monoliths. Capillary monoliths of 200 microm I.D. showed good reproducibility in terms of retention times, with relative standard deviations (RSD) of 1.9% for proteins and 2.2% for peptides. However, the separately synthesized capillary monoliths revealed pronounced variation in back pressure with RSD values of up to 31%. These variations were considerably reduced by cooling of the capillaries during polymerization. Using this optimized preparation procedure capillary monoliths of 100 and 50 microm I.D. were synthesized and the effects of scaling down the column I.D. on the morphology and on the reproducibility of the polymerization process were investigated. In the second part, the applicability of ROMP-derived capillary monoliths to a separation problem common in medical research was assessed. A 200 microm I.D. monolithic column demonstrated excellent separation behavior for insulin and various insulin analogs, showing equivalent separation performance to Vydac C4 and Zorbax C3-based stationary phases. Moreover, the high permeability of monoliths enabled chromatographic separations at higher flow rates, which shortened analysis time to about one third. For the analysis of insulin in human biofluid samples, enhanced sensitivity was achieved by using a 50 microm I.D. ROMP-derived monolith.     

Optimization of binary porogen solvent composition for preparation of butyl methacrylate monoliths in capillary liquid chromatography

Butyl methacrylate monolithic columns in 320 microm i.d. fused silica capillaries for reversed-phase capillary liquid chromatography were prepared by radical polymerization initiated thermally with azobisisobutyronitrile (AIBN). Polymerization mixture contained butyl methacrylate (BMA) as the function monomer and ethylene dimethacrylate (EDMA) as the crosslinking agent with 1,4-butanediol and 1-propanol as a binary porogen solvent. Ratio of 1,4-butanediol to 1-propanol in the porogen solvent was optimized regarding the monolithic column efficiency and performance. Total porosity, column permeability, separation impedance, Walters hydrophobicity index, retention factors, peak asymmetry factors, height equivalents to a theoretical plate and peak resolutions were used for characterization of the prepared monolithic columns. The polymerization mixture consisting of 17.8% of BMA, 21.8% of EDMA, 18.0% of 1,4-butanediol, 42.0% of 1-propanol and 0.4% AIBN generated monolithic columns of the best performance having a sufficient permeability and the lowest separation impedance. It was also demonstrated that monolithic columns of this composition exhibited good preparation reproducibility and an excellent pressure resistance when applied in capillary liquid chromatography.     

Ultratrace liquid chromatography/mass spectrometry analysis of large peptides with post-translational modifications using narrow-bore poly(styrene-divinylbenzene) monolithic columns and extended range proteomic analysis

This paper describes approaches to optimize the chromatographic performance for our recently developed LC-MS platform, extended range proteomic analysis (ERPA), for comprehensive protein characterization at the ultratrace level. Large digested peptide fragments up to 10 kDa (e.g., from lysyl endopeptidase digestion) with or without modifications were well separated with high resolution using narrow bore (20 and 50 microm I.D.) poly(styrene-divinylbenzene) (PS-DVB) monolithic columns constructed by in situ solution polymerization. Importantly, the macroporous structure of the monolithic columns facilitated mass transport of large peptides with improved recovery relative to small pore size reversed-phase packings. High sequence coverage (>95%), including identification of phosphorylated and glycosylated particles was achieved for beta-casein and epidermal growth factor receptor (EGFR) at the 4 and 20 fmol levels per injection, respectively, using the 20 microm I.D. PS-DVB monolithic column. For peptides with greater ionization efficiency, the detection limit could be lowered to approximately 400 zmol. Typically, the separation system produced a peak capacity of approximately 200 for a 10 cm column. This paper demonstrates that narrow-bore monolithic columns are suitable for high sensitivity and high-resolution separation of large peptide fragments by LC-MS analysis.     

Fabrication of a poly(styrene-octadecene-divinylbenzene) monolithic column and its comparison with a poly(styrene-divinylbenzene) monolithic column for the separation of proteins

In this paper, a poly(styrene-octadecene-divinylbenzene) (PS-OD-DVB) monolithic column was prepared in one step by introducing a C18 carbon chain as monomer. N,N-Dimethylformamide and decanol served as porogens to make a homogeneous polymerization mixture in a fused silica capillary (320 microm inner diameter). Its physical and chromatographic properties were compared with those of poly(styrene-divinylbenzene) (PS-DVB) monolithic column, which was also fabricated by in-situ polymerization in a fused silica capillary with the same inner diameter. Six standard proteins were used to evaluate the columns and their potential application for the separation of human hemoglobin was also discussed. It was shown that the PS-OD-DVB and PS-DVB monoliths appeared to have similar efficiency for rapid separation of six proteins within 3.5 min. The PS-OD-DVB monolith was found to have higher loading capacity and higher resolution for the separation of alpha and beta chains of hemoglobin because of the introduction of C18 carbon chains, and shows great potential for the separation of bio-macromolecules.     

Silica-based monolithic columns with mixed-mode reversed-phase/weak anion-exchange selectivity principle for high-performance liquid chromatography

This article describes the synthesis, chromatographic characterization, and performance evaluation of analytical (100 x 4.6 mm id) and semipreparative (100 x 10 mm id) monolithic silica columns with mixed-mode RP/weak anion-exchange (RP/WAX) surface modification. The monolithic RP/WAX columns were obtained by immobilization of N-(10-undecenoyl)-3-aminoquinuclidine onto thiol-modified monolithic silica columns (Chromolith) by a radical addition reaction. Their chromatographic characterization by Engelhardt and Tanaka tests revealed slightly lower hydrophobic selectivities than C-8 phases, as well as higher polarity and also improved shape selectivity than RP-18e silica rods. The surface modification enabled separation by both RP and anion-exchange chromatography principles, and thus showed complementary selectivities to the RP-18e monoliths. The mixed-mode monoliths have been tested for the separation of peptides and turned out to be particularly useful for hydrophilic acidic peptides, which are usually insufficiently retained on RP-18e monolithic columns. Compared to a corresponding particulate RP/WAX column (5 microm, 10 nm pore diameter), the analytical RP/WAX monolith caused lower system pressure drops and showed, as expected, higher efficiency (e.g. by a factor of about 2.5 lower C-term for a tetrapeptide). The upscaling from the analytical to semipreparative column dimension was also successful.     

Electron beam triggered, free radical polymerization-derived monolithic capillary columns for high-performance liquid chromatography

Monolithic capillary columns were prepared via electron beam triggered free radical polymerization within the confines of 0.2 and 0.1mm I.D. capillary columns using ethyl methacrylate and trimethylolpropane triacrylate as monomers as well as 2-propanol, 1-dodecanol and toluene as porogenic system. The influence of column diameter on reproducibility and separation performance was investigated. For evaluation, a protein standard consisting of five proteins in the range of 5800-66,000 g mol(-1) was used. Reproducibility was checked by determining the relative standard deviations in retention times, peak widths at half height, asymmetry and resolution. Excellent run-to-run reproducibility was found for both 0.2 and 0.1mm I.D. columns; batch-to-batch reproducibility was good for both column types. In order to enhance the non-polar character of the monolithic columns, lauryl methacrylate-based capillary columns were prepared. These were successfully used for the separation of proteins and a cytochrome c digest.     

Preparation of high efficiency and highly retentive monolithic silica capillary columns for reversed-phase chromatography by chemical modification by polymerization of octadecyl methacrylate

High efficiency and highly retentive monolithic silica capillary columns were obtained by polymerization of octadecyl methacrylate using alpha,alpha'-azobis-isobutyronitrile (AIBN) as a free radical initiator. Hybrid type monolithic silica columns (25 cm total length x 200 microm I.D.) prepared from a mixture of tetramethoxysilane and methyltrimethoxysilane were used as a support. The effects of the monomer and the radical initiator concentrations in the reaction mixture were examined. The performance of the columns was tested in terms of column efficiency and retention behavior by using alkylbenzenes and a few other compounds as solutes and compared with that of hybrid monolithic silica columns modified with octadecylsilyl-(N,N-diethylamino)silane (ODS-DEA). Highly retentive monolithic silica columns were obtained by polymerization at high monomer concentrations. Although a decrease in column efficiency was observed with the increase in the monomer concentration in a feed mixture, an improvement in efficiency was achieved (a plate height value lower than 10 microm) by increasing an initiator concentration without significant variations in column retention properties. Results obtained by polymerization using other monomers are also presented to demonstrate the applicability of the preparation method.     

Cyclodextrin-modified monolithic columns for resolving dansyl amino acid enantiomers and positional isomers by capillary electrochromatography

We describe beta- and gamma-cyclodextrins (beta- and gamma-CD)-modified monolithic columns prepared by sol-gel process and chemical modifications. The monolithic silica column was fabricated inside a fused-silica capillary with 100 microm inner diameter by sol-gel process. The monolithic silica matrix was chemically modified chiral selectors of beta- or gamma-CDs with a spacer of 3-glycidoxypropyltrimethoxysilane by on-column reactions. Gamma-CD-modified monolithic column has successfully been applied for the separation of dansyl amino acid enantiomers. Beta-CD-modified monolithic column has been used for the separation of the positional isomers of o-, m-, and p-cresols and the enantioseparation of racemates of benzoin and several dansyl amino acids by capillary electrochromatography, respectively. For the separation of neutral positional isomers, a positive electric field was applied. However, for the separation of negatively charged analytes, a negative electric field was applied at the inlet of column. The separation efficiency of 5.0 x 10(4) theoretical plates/m for dansyl-L-threonine was obtained at electric field strength of -300 V/cm in the mobile phase of 50 mM 2-(N-morpholino)ethanesulfonic acid (MES)-Tris/methanol (70/30) buffer at pH 7.0. L-enantiomers were eluted as the first peak. Scanning electron micrograph showed that monolithic columns have the morphology of continuous skeleton and large through-pores.     

Structure and performance of silica-based monolithic HPLC columns

Silica-based monolithic columns were prepared for HPLC with systematic variations of the tetramethoxysilane (TMOS) and polyethylene oxide (PEO) content as reactants in a sol-gel process accompanied by phase separation. The resulting monoliths showed differences in the macropore and silica skeleton diameter as well as in the corresponding domain sizes (the sum of macropore and skeleton diameter). All monoliths were synthesized with a diameter of 4.6 mm and cladded with a suitable polyaryletheretherketone (PEEK) polymer in a standardized and optimized manner for the subsequent chromatographic evaluation of the resulting monolithic HPLC columns. The columns were tested under normal phase conditions using n-heptane/dioxane (95:5 v/v) as a mobile phase and 2-nitroanisole as a test compound for the determination of separation efficiency and permeability. Two different sets of columns were prepared: the first one in which the amount of PEO was stepwise decreased to yield monoliths with identical macropore volumes and variations in the domain sizes. The second group of materials was synthesized adjusting both TMOS and PEO quantities to yield monolithic columns with identical macropore diameters of about 1.80 microm but different skeleton diameters and macropore volumes. The chromatographic results suggest that an increase in the column performance cannot be achieved by just arbitrarily decreasing the domain size of a given column. From a certain point of "downsizing" the monolithic structure a loss of structural homogeneity can be observed, which is apparently responsible for a lower chromatographic performance.     

Advantages of ultra performance liquid chromatography over high-performance liquid chromatography: comparison of different analytical approaches during analysis of diclofenac gel

Miniaturization embracing instrumentation, column particle size, and column dimensions is one of the major current trends in separation techniques. This leads to shortening of analysis time and great savings in solvent consumption. Ultra performance liquid chromatography (UPLC) is one of the new developments in liquid chromatography. An ultra-high pressure system allows using of small particle-packed columns with small diameter, which has a positive effect on both system efficiency and analysis time. An analytical method for determination of the active substance diclofenac, the degradation product 1-(2,6-dichlorphenyl)-2-indolinone, and the preservatives methylparaben and propylparaben was used for testing and comparing LC systems. Various octadecylsilica-based analytical columns were examined. Acquity UPLC BEH C18 (2.1 x 50 mm, 1.7 microm) and (2.1 x 100 mm, 1.7 microm) were tested for UPLC. The following analytical columns were used in a test for HPLC: Purospher RP 18e (125 x 4.0 mm, 5 microm), Zorbax Eclipse XDB C18 (75 x 4.6 mm, 3.5 microm), Zorbax Eclipse SB C18 (50 x 4.6 mm, 1.8 microm), as was a monolithic column (Chromolith Performance RP-18e (100 x 4.6 mm). Results of a System Suitability Test (SST) were calculated and compared for each chromatographic peak. System efficiency and analysis duration were compared with regard to solvent consumption and system maintenance     

Conventional and narrow bore short capillary columns with cyclodextrin derivatives as chiral selectors to speed-up enantioselective gas chromatography and enantioselective gas chromatography-mass spectrometry analyses

The analysis of complex real-world samples of vegetable origin requires rapid and accurate routine methods, enabling laboratories to increase sample throughput and productivity while reducing analysis costs. This study examines shortening enantioselective-GC (ES-GC) analysis time following the approaches used in fast GC. ES-GC separations are due to a weak enantiomer-CD host-guest interaction and the separation is thermodynamically driven and strongly influenced by temperature. As a consequence, fast temperature rates can interfere with enantiomeric discrimination; thus the use of short and/or narrow bore columns is a possible approach to speeding-up ES-GC analyses. The performance of ES-GC with a conventional inner diameter (I.D.) column (25 m length x 0.25 mm I.D., 0.15 microm and 0.25 microm d(f)) coated with 30% of 2,3-di-O-ethyl-6-O-tert-butyldimethylsilyl-beta-cyclodextrin in PS-086 is compared to those of conventional I.D. short column (5m length x 0.25 mm I.D., 0.15 microm d(f)) and of different length narrow bore columns (1, 2, 5 and 10 m long x 0.10 mm I.D., 0.10 microm d(f)) in analysing racemate standards of pesticides and in the flavour and fragrance field and real-world-samples. Short conventional I.D. columns gave shorter analysis time and comparable or lower resolutions with the racemate standards, depending mainly on analyte volatility. Narrow-bore columns were tested under different analysis conditions; they provided shorter analysis time and resolutions comparable to those of conventional I.D. ES columns. The narrow-bore columns offering the most effective compromise between separation efficiency and analysis time are the 5 and 2m columns; in combination with mass spectrometry as detector, applied to lavender and bergamot essential oil analyses, these reduced analysis time by a factor of at least three while separation of chiral markers remained unaltered.     

Evaluation of frontal chromatograms

A novel pressure-balanced injection valve was evaluated for use with ultrahigh pressure liquid chromatography (UHPLC) at pressures up to 120 MPa (1,200 bar). Fused-silica capillaries (30-33 cm x 100 microm I.D.) packed with nonporous 1.5 microm isohexylsilane-modified (C6) silica particles were employed to study maximum pressure, injection reproducibility, injection time, and sample amount consumed for an injection. The new valve was more reproducible, convenient, and required much less sample than previously used injection systems. The effect of column diameter on efficiency and sensitivity was studied. The 100 microm I.D. columns demonstrated approximately 40% lower efficiency but 10-fold higher sensitivity than the 29 microm I.D. columns. Columns packed with nonporous C6 particles produced higher efficiencies than columns packed with a 1.5 microm porous octadecylsilane-modified (C18) material.     

Preparation and evaluation of packed capillary columns for the separation of nucleic acids by ion-pair reversed-phase high-performance liquid chromatography

Oligonucleotides and double stranded DNA fragments were separated in 200 microm I.D. capillary columns packed with micropellicular, octadecylated, 2.1 microm poly(styrene-divinylbenzene) particles by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). Both the length and the diameter of the connecting capillaries (150 x 0.020 mm I.D.) as well as the detection volume (3 nl) had to be kept to a minimum in order to maintain the high efficiency of this chromatographic separation system with peak widths at half height in the range of a few seconds. Three different types of frits, namely sintered silica particles, sintered octadecylsilica particles, and monolithic poly(styrene-divinylbenzene) (PS-DVB) frits were evaluated with respect to their influence on chromatographic performance. Best performance for the separation of oligonucleotides and long DNA fragments was observed with the PS-DVB frits, whereas the short DNA fragments were optimally resolved in columns terminated by octadecylsilica frits. The maximum loading capacity of 60 x 0.20 mm I.D. columns ranged from 20 fmol (7.7 ng) for a 587 base pair DNA fragment to 500 fmol (2.4 ng) for a 16-mer oligonucleotide. Lower mass- and concentration detection limits in the low femtomol and low nanomol per liter range, respectively, make capillary IP-RP-HPLC with UV absorbance detection highly attractive for the separation and characterization of minute amounts of synthetic oligonucleotides, DNA restriction fragments, and short tandem repeat sequences amplified by polymerase chain reaction.     

Characterization of some physical and chromatographic properties of monolithic poly(styrene-co-divinylbenzene) columns

Monolithic capillary columns were prepared by copolymerization of styrene and divinylbenzene inside a 200 microm i.d. fused silica capillary using a mixture of tetrahydrofuran and decanol as porogen. Important chromatographic features of the synthesized columns were characterized and critically compared to the properties of columns packed with micropellicular, octadecylated poly(styrene-co-divinylbenzene) (PS-DVB-C18) particles. The permeability of a 60 mm long monolithic column was slightly higher than that of an equally dimensioned column packed with PS-DVB-C18 beads and was invariant up to at least 250 bar column inlet pressure, indicating the high-pressure stability of the monolithic columns. Interestingly, monolithic columns showed a 3.6 times better separation efficiency for oligonucleotides than granular columns. To study differences of the molecular diffusion processes between granular and monolithic columns, Van Deemter plots were measured. Due to the favorable pore structure of monolithic columns all kind of diffusional band broadening was reduced two to five times. Using inverse size-exclusion chromatography a total porosity of 70% was determined, which consisted of internodule porosity (20%) and internal porosity (50%). The observed fast mass transfer and the resulting high separation efficiency suggested that the surface of the monolithic stationary phase is rather rough and does not feature real pores accessible to macromolecular analytes such as polypeptides or oligonucleotides. The maximum analytical loading capacity of monolithic columns for oligonucleotides was found to be in the region of 500 fmol, which compared well to the loading capacity of the granular columns. Batch-to-batch reproducibility proved to be better with granular stationary phases compared to monolithic stationary phase, in which each column bed is the result of a unique column preparation process.     

Capillary scale monolithic trap column for desalting and preconcentration of peptides and proteins in one- and two-dimensional separations

Monolithic columns based on poly-(styrene-divinylbenzene) (PS-DVB) were utilized both for preconcentration (in 10 mm x 0.20 mm I.D. format) and analytical separation (in 60 mm x 0.20 and 0.10 mm I.D. format) of peptides and proteins in column switching micro-scale high-performance liquid chromatography. A special holder for short monolithic preconcentration columns was designed and pressure durability tests approved long-term stability up to 400 bar. An 11-20% decrease in the average peak widths of nine peptides was obtained upon combining a preconcentration column with an analytical column as compared with a setup using an analytical column only. Trapping efficiency, especially for small and hydrophilic peptides, was optimized by using 0.10% heptafluorobutyric acid instead of 0.050% trifluoroacetic acid as solvent additive during sample loading. Using a 10 mm x 0.20 mm I.D. preconcentration column, loadabilities between 0.5 and 1.6 microg were determined by frontal analysis of proteins and bioactive peptides, respectively. A 100-fold concentration followed by direct on-line intact mass determination is demonstrated for diluted (3 micromolL(-1)) protein solutions. The applicability of the monolithic preconcentration column for multidimensional chromatography was tested by off-line two-dimensional separation, combining strong cation-exchange chromatography and ion-pair reversed-phase chromatography. Peptide identification data from digested protein mixtures demonstrated reproducibilities of 46-75% in triplicate analyses, and confident peptide identifications of low abundant peptides even in the presence of a 650-fold molar excess of high abundant peptides.