Tohru Ikegami

Monolithic silica columns for HPLC, micro-HPLC, and CEC

Two types of monolithic silica columns derivatized to form an ODS phase, one prepared in a fused silica capillary (SR-FS) and the other prepared in a mold and clad with an engineering plastic (poly-ether-ether-ketone) (SR-PEEK), were evaluated. The column efficiency and pressure drop were compared with those of a column packed with 5-μm ODS-silica particles and of an ODS-silica monolith prepared in a mold and wrapped with PTFE tubing (SR-PTFE). SR-FS gave a lower pressure drop than a column packed with 5-μm particles by a factor of 20, and a plate height of 20 μm at a linear velocity below 1 mm/s. SR-PEEK showed higher flow-resistance than the other monolithic silica columns, but they still showed a minimum plate height of 8-10 μm and a lower pressure drop than popular commercial columns packed with 5-μm particles. The evaluation of SR-FS columns in a CEC mode showed much higher efficiency than in a pressure-driven mode.

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.

Monolithic silica columns for high-efficiency separations by high-performance liquid chromatography.

Generation of a large number of theoretical plates was attempted by capillary HPLC. Monolithic silica columns having small skeletons (ca. 2 microm) and large through-pores (ca. 8 microm) were prepared by a sol-gel method in a fused-silica capillary (50 microm I.D.), and derivatized to C18 phase by on-column reaction. High external porosity (>80%) and large through-pores resulted in high permeability (K= 1.2 x 10(-2) m2). The monolithic silica column in the capillary produced a plate height of about 12 microm in 80% acetonitrile at a linear velocity of 1 mm/s. Separation impedance, E value, was found to be as low as 200, that was about an order of magnitude lower than reported values for conventional columns packed with 5 microm particles. Reproducibility of preparation within +/- 15% was obtained for column efficiency and for pressure drop. It was possible to generate 100,000 plates by using a 130-cm column at very low pressure (<7 kg/cm2). A considerable decrease in column efficiency was observed at high linear velocity, and for solutes with large retention factors due to the slow mobile-phase mass transfer in the large through-pores. The monolithic silica columns, however, showed performance beyond the limit of conventional particle-packed columns in HPLC under favorable conditions.

On-column concentration of bisphenol A with one-step removal of humic acids in water

An efficient extraction method for bisphenol A from environmental water including contaminants was developed using surface selective localization of functional group, on a polymeric separation device. The polymer utilized in this study was prepared through a kind of molecular imprinting technique, namely fragment imprinting effect utilizing a pseudo-template molecule (p-tert.-butylphenol) instead of bisphenol A. The concentration of bisphenol A onto the polymer device prepared, up to 1000 times concentration from environmental water including contaminants (humic acids), was achieved very easily with interesting exclusion effect for humic acids. The results obtained in this study suggest that molecular imprinting with the pseudo-template molecule is quite an effective way for selective concentration of the diluted target molecule from other contaminants including similar functional group with the target molecule.

Hydrophilic Interaction Chromatography Using a Meter-Scale Monolithic Silica Capillary Column for Proteomics LC-MS

A meter-scale monolithic silica capillary column modified with urea-functional groups for hydrophilic interaction liquid chromatography (HILIC) was developed for highly efficient separation of biological compounds. We prepared a ureidopropylsilylated monolithic silica capillary column with a minimum plate height of 12 μm for nucleosides and a permeability of 2.1 × 10(-13) m(2), which is comparable with the parameters of monolithic silica-C18 capillary columns. Over 300,000 theoretical plates were experimentally obtained in HILIC with a 4 m long column at 8 MPa; this is the best result yet reported for HILIC. A 2 m long ureidopropylsilylated monolithic silica capillary column was utilized to develop a HILIC mode LC-MS system for proteomics applications. Using tryptic peptides from human HeLa cell lysate proteins, we identified the comparable numbers of peptides and proteins in HILIC with those in reversed-phase liquid chromatography (RPLC) using a C18-modified monolithic silica column when shallow gradients were applied. In addition, approximately 5-fold increase in the peak response on average was observed in HILIC for commonly identified tryptic peptides due to the high acetonitrile concentration in the HILIC mobile phase. Since HILIC mode LC-MS shows orthogonal selectivity to RPLC mode LC-MS, it is useful as a complementary tool to increase proteome coverage in proteomics studies.

Preparation of uniformly sized polymeric separation media potentially suitable for small-scale high-performance liquid chromatography and/or capillary electrochromatography

Uniformly sized polymer particles were prepared by either a two-step swelling and polymerization method or a Shirasu porous glass (SPG) emulsification technique to compare their suitability as a uniformly sized packing material for small-scale high-performance liquid chromatography (HPLC) or capillary electrochromatography (CEC). The SPG emulsification technique afforded slightly worse size uniformity compared to the two-step swelling and polymerization technique. However, fairly nice spherical shapes with reproducible outward appearance and internal pore size distribution were obtained for both of 3- or 6-micron sized particles. On the other hand, the two-step swelling and polymerization method afforded broken particles with quite different outward appearances due to the polymeric porogen effects of seed polymer utilized as the shape template. In HPLC, the column packed with the 3-micron particles prepared with the SPG emulsification technique proved to have a fairly high column efficiency with good column stability, as assessed under repeated use in gradient elution. On the other hand, the column packed with the packing material obtained through the two-step swelling and polymerization method showed much poorer column stability, while initial column efficiency was compatible to that obtained using the SPG emulsification method. In addition, the packing material prepared with the SPG emulsification technique could be modified with ion-exchangeable monomers and showed fairly good column efficiency in the CEC mode.

Immobilized β-cyclodextrin-based silica vs polymer monoliths for chiral nano liquid chromatographic separation of racemates

The enantioselectivity of immobilized β-cyclodextrin phenyl carbamate-based silica monolithic capillary columns was compared to our previously described polymer counterpart. 2,3,6-Tris(phenylcarbamoyl)-β-cyclodextrin-6-methacrylate was used as a functional monomer for the preparation of β-cyclodextrin (β-CD)-based silica and polymer monoliths. The silica monoliths were prepared via the sol-gel technique in fused silica capillary followed by modification of the bare silica monoliths with an anchor group prior to polymerization with β-CD methacrylate using either 2,2-azobis(isobutyronitrile) or benzoylperoxide as radical initiators. On the other hand, the polymer monoliths were prepared via the copolymerization of β-CD methacrylate and ethylene glycol dimethacrylate in different ratios in situ in fused silica capillary. The prepared silica/polymer monoliths were investigated for the chiral separation of different classes of pharmaceuticals namely; α- and β-blockers, anti-inflammatory drugs, antifungal drugs, dopamine antagonists, norepinephrine-dopamine reuptake inhibitors, catecholamines, sedative hypnotics, diuretics, antihistaminics, anticancer drugs and antiarrhythmic drugs. Baseline separation was achieved for alprenolol, bufuralol, carbuterol, cizolertine, desmethylcizolertine, eticlopride, ifosfamide, 1-indanol, propranolol, tebuconazole, tertatolol and o-methoxymandelic acid under reversed phase conditions using mobile phase composed of methanol and water. The silica-based monoliths showed a comparative enantioselectivity to the polymer monoliths.

New silica monolith bonded chiral (R)-γ butyrolactone for enantioselective micro high-performance liquid chromatography

A single low-molecular mass chiral selector namely (R)-acryloyloxy-β-β-dimethyl-γ-butyrolactone has been bonded to a modified silica-based monolith to form a new brush-type chiral stationary phase for micro-high performance liquid chromatography (HPLC) separation.

Recent Progress in Monolithic Silica Columns for High-Speed and High-Selectivity Separations

Monolithic silica columns have greater (through-pore size)/(skeleton size) ratios than particulate columns and fixed support structures in a column for chemical modification, resulting in high-efficiency columns and stationary phases. This review looks at how the size range of monolithic silica columns has been expanded, how high-efficiency monolithic silica columns have been realized, and how various methods of silica surface functionalization, leading to selective stationary phases, have been developed on monolithic silica supports, and provides information on the current status of these columns. Also discussed are the practical aspects of monolithic silica columns, including how their versatility can be improved by the preparation of small-sized structural features (sub-micron) and columns (1 mm ID or smaller) and by optimizing reaction conditions for in situ chemical modification with various restrictions, with an emphasis on recent research results for both topics.

Effects of electrokinetic chromatography conditions on the structure and properties of polyallylamine-supported pseudo-stationary phase: A study by dynamic light scattering

The hydrophobic selectivity and electrophoretic mobility of polymeric pseudo-stationary phases supported by polyallylamine (PAA) were found to be affected by the degree of alkylation of PAA, the organic solvent content of the separation medium, and also by the concentration of the pseudo-stationary phase. The results obtained with dynamic light scattering, fluorescence spectroscopy, viscosity measurement and electrokinetic chromatography indicate that the PAA-supported pseudo-stationary phases assume swollen structures to expose more ionic groups to the aqueous medium at a lower concentration and at a higher organic solvent content, leading to the lower hydrophobic property and the greater electrophoretic mobility, which in turn result in the wider migration time window. Inadequate solvation of alkyl groups causes intermolecular aggregation of the polymeric carriers in aqueous buffer solutions, especially at high carrier concentrations. Dynamic light scattering measurement was shown to be particularly useful to elucidate the conformational change of the polymeric pseudo-stationary phase under various conditions.