Characterization of hydroxypropylmethylcellulose (HPMC) using comprehensive two-dimensional liquid chromatography.

Various hydroxyl-propylmethylcellulose (HPMC) polymers were characterized according to size and compositional distributions (percentage of methoxyl and hydroxyl-propoxyl substitution) by means of comprehensive two-dimensional liquid chromatography (LC×LC) using reversed-phase (RP) liquid chromatography in the first dimension and aqueous size-exclusion chromatography (aq-SEC) in the second dimension. RP separation was carried out in gradient-elution mode applying 0.05% TFA in water and 1-propanol, while 0.05% TFA in water was used as mobile phase in aqueous SEC. A two-position ten-port switching valve equipped with two storage loops was used to realize LC×LC. Detection of HPMC was accomplished by charged-aerosol detection (CAD). Data processing to visualize chromatograms was carried out using Matlab software. The significant influence of the LC×LC temperature on (the retention of) HPMC was studied using a column oven which allowed accurate temperature control. Due to the phenomenon of thermal gelation, which is a result of methyl and hydroxypropyl substitution of anhydroglucose units from the cellulose backbone, we were able to obtain additional, specific information on compositional characteristics of various HPMC samples. As the retention behaviour of gelated and non-gelated polymer proved to be different, the fraction of the polymer that is gelated in the chromatographic column could be monitored at different temperatures. Moreover, the temperature at which half of the polymer is gelated could be correlated with the cloud-point temperature. As a result, differences in inherent cloud points of modified cellulose can be used as a further distinguishing property in "temperature-responsive" LC×LC.

Influence of the polymerisation time on the porous and chromatographic properties of monolithic poly(1,2-bis(p-vinylphenyl))ethane capillary columns.

In order to elucidate the effect of the polymerisation time on the morphology of styrene based monolithic support materials, continuous poly(1,2-bis(p-vinylphenyl))ethane (BVPE) rods were synthesised in 1.0ml glass vials by thermally initiated free radical polymerisations of BVPE in the presence of porogens (toluene, decanol) and a,a'-azoisobutyronitrile (AIBN) as initiator at 65 degrees C for different polymerisation times (60, 90, 150, 300 and 600min). Porosity parameters like pore-size-distribution and total porosity were investigated by mercury intrusion porosimetry, while the specific surface area of the BVPE monolithic supports was determined by N(2)-adsorption (BET) measurements. An untypical bimodal pore-size-distribution comprising a high fraction of both mesopores (2-50nm) and macropores (mainly flow-channels in the micrometer range) was observed as a result of the stepwise decrease of the polymerisation time. In consequence of the significant changes of the pore-size-profile, shortening the polymerisation time also resulted in enhanced total porosity due to enlarged flow-channel diameters and increased surface area according to the presence of a considerable amount of mesopores. Results upon the porosity profile of the support are further confirmed by SEM images of monoliths polymerised for different time periods. Since mesoporosity and high surface area of the chromatographic support material play key roles in the interaction and thus retention of low-molecular-weight compounds, polymerisation time should also affect the chromatographic properties and applicability of these polymers. To study the influence of the polymerisation time towards the separation efficiency of small molecules on BVPE capillary columns (200microm I.D., 8cm), a mixture of homologous alkylbenzenes was chosen for column evaluation. In accordance with the observations of the porous properties of BVPE stationary phases, the rapid and high resolution separation of a range of low-molecular-weight compounds on monolithic BVPE supports were successfully realised. The methodical reduction of the polymerisation time has been demonstrated to be a simple and effective tool to tailor the porous properties of organic monoliths to provide novel polymer-based stationary phases with porous properties adequate for the rapid and high resolution chromatography of small organic molecules.

Monolithic poly(1,2-bis(p-vinylphenyl)ethane) capillary columns for simultaneous separation of low- and high-molecular-weight compounds.

Monolithic poly(1,2-bis(p-vinylphenyl)ethane (BVPE)) capillary columns were prepared by thermally initiated free radical polymerisation of 1,2-bis(p-vinylphenyl)ethane in the presence of inert diluents (porogens) and alpha,alpha'-azoisobutyronitrile (AIBN) as initiator. Polymerisations were accomplished in 200 microm ID fused silica capillaries at 65 degrees C and for 60 min. Mercury intrusion porosimetry measurements of the polymeric RP support showed a broad bimodal pore-size-distribution of mesopores and small macropores in the range of 5-400 nm and flow-channels in the mum range. N(2)-adsorption (BET) analysis resulted in a tremendous enhancement of surface area (101 m(2)/g) of BVPE stationary phases compared to typical organic monoliths (approximately 20 m(2)/g), indicating the presence of a considerable amount of mesopores. Consequently, the adequate proportion of both meso- and (small) macropores allowed the rapid and high-resolution separation of low-molecular-weight compounds as well as biomolecules on the same monolithic support. At the same time, the high fraction of flow-channels provided enhanced column permeability. The chromatographic performance of poly(1,2-bis(p-vinylphenyl)ethane) capillary columns for the separation of biomolecules (proteins, oligonucleotides) and small molecules (alkyl benzenes, phenols, phenons) are demonstrated in this article. Additionally, pressure drop versus flow rate measurements of novel poly(1,2-bis(p-vinylphenyl)ethane) capillary columns confirmed high mechanical robustness, low swelling in organic solvents and high permeability. Due to the simplicity of monolith fabrication, comprehensive studies of the retention and separation behaviour of monolithic BVPE columns resulted in high run-to-run and batch-to-batch reproducibilities. All these attributes prove the excellent applicability of monolithic poly(1,2-bis(p-vinylphenyl)ethane) capillary columns for micro-HPLC towards a huge range of analytes of different chemistries and molecular sizes.

Derivatized graphitic nanofibres (GNF) as a new support material for mass spectrometric analysis of peptides and proteins.

Graphitic nanofibres (GNFs), 100-200 nm in diameter and 5-20 microm in length have been modified in order to yield different affinities (Cu2+ and Fe3+ loaded immobilized metal affinity chromatography (IMAC) as well as cation and anion exchange materials) for the extraction of a range of biomolecules by their inherited hydrophobicity and the hydrophilic chemical functionalities, obtained by derivatization. Modified GNFs have for the first time been employed as carrier materials for protein profiling in material-enhanced laser desorption/ionization (MELDI) for the enrichment and screening of biofluids. For that purpose, the derivatized GNF materials have comprehensively been characterized regarding surface area, structural changes during derivatization, IMAC, as well as ion exchange and protein-loading capacity and recovery. GNF derivatives revealed high protein-binding capacity (2,000 microg ml(-1) for insulin) and ideal sensitivities, resulting in a detection limit of 50 fmol microl(-1) (for insulin), which is crucial for the detection of low abundant species in biological samples. Compared to other MELDI carrier materials, sensitivity was enhanced on GNF derivatives, which might be ascribed to the fact that GNFs support desorption and ionization mechanisms and by absorbing laser energy in addition to matrix.