Masayoshi Ohira

Sol–gel synthesis of macro–mesoporous titania monoliths and their applications to chromatographic separation media for organophosphate compounds

We have developed a method of independently tailoring the macro- and mesoporous structures in titania (TiO2) monoliths in order to achieve liquid chromatographic separations of phosphorous-containing compounds. Anatase TiO2 monolithic gels with well-defined bicontinuous macropores and microstructured skeletons are obtained via the sol-gel process in strongly acidic conditions using poly(ethylene oxide) as a phase separator and N-methylformamide as a proton scavenger. Aging treatment of the wet gels in the mother liquor at temperatures of 100-200 degrees C and subsequent heat treatment at 400 degrees C allow the formation and control of mesoporous structures with uniform pore size distributions in the gel skeletons, without disturbing the preformed macroporous morphology. The monolithic TiO2 rod columns with bimodal macro-mesoporous structures possess the phospho-sensitivity and exhibit excellent chromatographic separations of phosphorus-containing compounds.

Monolithic silica rod columns for high-efficiency reversed-phase liquid chromatography.

Chromatographic properties of a new type of monolithic silica rod columns were examined. Silica rod columns employed for the study were prepared from tetramethoxysilane, modified with octadecylsilyl moieties, and encased in a stainless-steel protective column with two polymer layers between the silica and the stainless-steel tubing. A 25 cm column provided up to 45,000 theoretical plates for aromatic hydrocarbons, or a minimum plate height of about 5.5 μm, at optimum linear velocity of ca. 2.3 mm/s and back pressure of 7.5 MPa in an acetonitrile-water (80/20, v/v) mobile phase at 40°C. The permeability of the column was similar to that of a column packed with 5 μm particles, with K(F) about 2.4×10(-14) m(2) (based on the superficial linear velocity of the mobile phase), while the plate height value equivalent to that of a column packed with 2.5 μm particles. Generation of 80,000-120,000 theoretical plates was feasible with back pressure below 30 MPa by employing two or three 25 cm columns connected in series. The use of the long columns enabled facile generation of large numbers of theoretical plates in comparison with conventional monolithic silica columns or particulate columns. Kinetic plot analysis indicates that the monolithic columns operated at 30 MPa can provide faster separations than a column packed with totally porous 3-μm particles operated at 40 MPa in a range where the number of theoretical plates (N) is greater than 50,000.

Semi-micro-monolithic columns using macroporous silica rods with improved performance.

Monolithic silica columns in semi-micro-format have been synthesized using poly(acrylic acid) as a phase-separation inducer via a sol-gel route. The absence of a thick skin layer accompanied by deformation of the micrometer-sized gelling skeletons on the outermost part of the macroporous silica rod contributed to improve the efficiency of monolithic silica columns as thick as 2.4 mm in diameter. The kinetic plot analysis revealed that monolithic silica columns with macropore diameter of 1 microm and skeleton thickness of 1 microm with decreased macroporosity behave similarly to columns packed with 3 microm particles with slightly lower back pressure.

Efficiency of short, small-diameter columns for reversed-phase liquid chromatography under practical operating conditions

Prototype small-size (1.0mm I.D., 5cm long) columns for reversed-phase HPLC were evaluated in relation to instrument requirements. The performance of three types of columns, monolithic silica and particulate silica (2μm, totally porous and 2.6μm, core-shell particles) was studied in the presence of considerable or minimal extra-column effects, while the detector contribution to band broadening was minimized by employing a small size UV-detector cell (6- or 90nL). A micro-LC instrument having small system volume (<1μL) provided extra-column band variance of only 0.01-0.02μL(2). The three columns generated about 8500 theoretical plates for solutes with retention factor, k>1-3 (depending on the column), in acetonitrile/water mobile phase (65/35=vol/vol) at 0.05mL/min, with the instrument specified above. The column efficiency was lower by up to 30% than that observed with a 2.1mm I.D. commercial column. The small-size columns also provided 8000-8500 theoretical plates for well retained solutes with a commercial ultrahigh-pressure liquid chromatography (UHPLC) instrument when extra-column contributions were minimized. While a significant extra-column effect was observed for early eluting solutes (k<2-4, depending on column) with methanol/water (20/80=vol/vol) as weak-wash solvent, the use of methanol/water=50/50 as wash solvent affected the column efficiency for most analytes. The results suggest that the band compression effect by the weak-wash solvent associated with partial-loop injection may provide a practical means to reducing the extra-column effect for small-size columns, while the use of an instrument with minimum extra-column effect is highly desirable.

Test compounds for detecting the silanol effect on the elution of ionized amines in reversed-phase LC

The effectiveness of several basic compounds for testing silica-based stationary phases was reviewed by applying them to recent columns for reversed-phase HPLC. Most octadecylsilylated (C18) stationary phases, prepared as a base-deactivated material from high-purity silica gel with endcapping, provided excellent peak shape and column efficiency for the bases including benzylamine and amitriptyline that once caused problems and were subsequently employed for testing silanol activities. However, a cyclic tertiary amine, dextrometorphan, was eluted as an acceptable peak from only a few columns at neutral pH. Such a more sensitive probe is expected to contribute to further improvement of the stationary phase for reversed-phase HPLC.

Development of a new cyano-bonded column for high-performance liquid chromatography

A unique cyano-bonded column for high-performance liquid chromatography was developed by chemically bonding cyanopropyl groups to the silica gel support, and its chromatographic performance was described. Eight cyanopropyl-bonded silica gels were prepared under different conditions. These packing materials were packed into a stainless steel column, the chromatographic properties and durability of which were investigated in both normal- and reversed-phase partition modes. The separating selectivity and the durability of cyanopropyl-bonded silica (CN) columns were dependent on the preparation conditions. Particularly, the relationship between the density of cyanopropyl groups on the surface of the silica gel and the irreversible adsorption of basic compounds was examined. Also, the experimental results indicated that endcapping resulted in the poor separating selectivity and durability of the CN columns.

Effect of pressure on the selectivity of polymeric C18 and C30 stationary phases in reversed-phase liquid chromatography. Increased separation of isomeric fatty acid methyl esters, triacylglycerols, and tocopherols at high pressure.

A high-density, polymeric C18 stationary phase (Inertsil ODS-P) or a polymeric C30 phase (Inertsil C30) provided improved resolution of the isomeric fatty acids (FAs), FA methyl esters (FAMEs), triacylglycerols (TAGs), and tocopherols with an increase in pressure of 20-70MPa in reversed-phase HPLC. With respect to isomeric C18 FAMEs with one cis-double bond, ODS-P phase was effective for recognizing the position of a double bond among petroselinic (methyl 6Z-octadecenoate), oleic (methyl 9Z-octadecenoate), and cis-vaccenic (methyl 11Z-octadecenoate), especially at high pressure, but the differentiation between oleic and cis-vaccenic was not achieved by C30 phase regardless of the pressure. A monomeric C18 phase (InertSustain C18) was not effective for recognizing the position of the double bond in monounsaturated FAME, while the separation of cis- and trans-isomers was achieved by any of the stationary phases. The ODS-P and C30 phases provided increased separation for TAGs and β- and γ-tocopherols at high pressure. The transfer of FA, FAME, or TAG molecules from the mobile phase to the ODS-P stationary phase was accompanied by large volume reduction (-30∼-90mL/mol) resulting in a large increase in retention (up to 100% for an increase of 50MPa) and improved isomer separation at high pressure. For some isomer pairs, the ODS-P and C30 provided the opposite elution order, and in each case higher pressure improved the separation. The two stationary phases showed selectivity for the isomers having rigid structures, but only the ODS-P was effective for differentiating the position of a double bond in monounsaturated FAMEs. The results indicate that the improved isomer separation was provided by the increased dispersion interactions between the solute and the binding site of the stationary phase at high pressure.

Development of a novel octadecyl-bonded silica column and evaluation of its reliability in chromatographic analysis

The chromatographic characterization of an octadecyl-bonded silica (ODS) column for high-performance liquid chromatography is described. In general, columns of the same type but obtained from various manufacturers give different chromatographic results, due to differences in the purity of column packings, the properties of the silica gel supports and the density of silanols on the surface of the silica gel. In order to solve this problem and to obtain a high performance ODS column, a series of methods with different samples and conditions were evaluated. The result is important for the optimization of conditions for the synthesis of ODS packings and for minimizing the deleterious effects arising from adsorption activity and metal impurity.

Comparison of the kinetic performance of different columns for fast liquid chromatography, emphasizing the contributions of column end structure

The kinetic performance of five chromatographic columns designed for fast liquid chromatography with different column packing materials - including fully porous (2.0 and 1.9μm particles), core-shell (2.6μm particles) or monolithic packings - with identical column dimensions (2.1×50mm) was tested. Since the tested monolithic column showed systematically better efficiency for early eluting compounds than the packed columns, an additional band broadening effect was suspected for the packed columns. The effects of the presence of the frits and the bed heterogeneity of the columns near the frits were characterized by a column-reversal method. It has been shown that significant differences - even 20-25% difference in efficiency - can exist between the two ends of the packed columns, while the monolithic column shows rather similar performance at either column end.

Characterization of an avidin-bonded column for direct injection in reversed-phase high-performance liquid chromatography

A denatured avidin-bonded column was suitable for use in the reversed-phase HPLC and complete recovery of serum protein over a wide range of pH. Retention property of the denatured avidin-bonded column was very nearly to a non-denatured avidin-bonded column already reported and, however, showed very high stability to organic solvent. A performance of the denatured avidin-bonded column was maintained even if 500 continuous injections of human serum (total 10 ml).