Karin Cabrera

A New Monolithic-Type HPLC Column For Fast Separations

Summary The application of a new silica-based, monolithic-type HPLC-column for fast separations is presented. The column is prepared according to a new sol-gel process, which is based on the hydrolysis and polycondensation of alkoxysilanes in the presence of water soluble polymers. The method leads to “rods” made of a single piece of porous silica with a defined pore structure, i. e. macro- and mesopores. The main feature of silica rod columns is a higher total porosity, about 15% higher than of conventional particulate HPLC columns. The resulting column pressure drop is therefore much lower, allowing operation at higher flow rates including flow gradients. Consequently, HPLC analysis can be performed much faster, as it is demonstrated by various applications.

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.

SilicaROD™ — A new challenge in fast high-performance liquid chromatography separations

Abstract High performance liquid chromatography (HPLC) has become one of the most used methods for the analysis of compound mixtures in industry, especially for the quality control of products. Nowadays productivity is the major and dominant upcoming issue, i.e. the goal is to drastically reduce the analysis time and cost per analysis. The solution of the task is higher throughput and faster HPLC methods. Here we describe a new monolithic type of HPLC column, the SilicaROD™ column, which permits the fast HPLC separation of compound mixtures within a few minutes.

Determination of enantiomerization barriers by computer simulation of experimental elution profiles obtained by high-performance liquid chromatography on a chiral stationary phase

The enantiomers of oxazepam, chlorthalidone and phenylthiohydantoin-phenylalanine (PTH-phenylalanine) were separated by HPLC using chemically bonded β-cyclodextrin (ChiraDex) as chiral stationary phase. The obtained chromatograms showed typical plateaus between the peaks of the separated enantiomers, indicating enantiomerization, i.e. reversible enantiomer interconversion. Computer simulation of the experimental chromatographic elution profiles was employed for the determination of rate constants and corresponding enantiomerization barriers for the above compounds. The data were compatible with independently determined values, but a very strong dependence on the solvent was observed.

The performance of hybrid monolithic silica capillary columns prepared by changing feed ratios of tetramethoxysilane and methyltrimethoxysilane

The effect of a feed ratio of methyltrimethoxysilane (MTMS) to tetramethoxysilane (TMOS) was studied to improve the performance of a hybrid monolithic silica capillary column with 100-mum i.d. in HPLC in a range MTMS/TMOS (v/v)=10/90-25/75. The domain size was also varied by adjusting the amount of PEG to control permeability (K=2.8x10(-14)-6.9x10(-14)m(2)). Evaluation of the performance for those capillary columns following octadecylsilylation proved an increase in retention factor (k) and a decrease in steric selectivity alpha(triphenylene/ortho-terphenyl) with the increase in MTMS content in the feed. The effect of the feed ratio was also observed in porosity and hydrophobic property of the C18 stationary phase from the results of size exclusion chromatography (SEC) and reversed phase characterization. The monolithic silica capillary columns prepared under new preparation conditions were able to produce a plate height of 4.6-6.0microm for hexylbenzene in a mobile phase acetonitrile/water=80/20 at a linear velocity of 2mm/s. Consequently, it was possible to prepare hybrid monolithic silica capillary columns with higher performance than those reported previously while maintaining the retention factors in a similar range by reducing the MTMS/TMOS ratio and increasing the total silane concentration in feed.

Influence of temperature on chiral high-performance liquid chromatographic separations of oxazepam and Prominal on chemically bonded β-cyclodextrin as stationary phase

β-Cyclodextrin, chemically bonded to silica, was used as a chiral stationary phase for the HPLC separation of two chiral pharmaceuticals, oxazepam and Prominal. The influence of temperature on the separation of these two compounds was studied in detail. A decrease in temperature caused an increase in the retention for both compounds. Chiral separation of oxazepam was optimized by decreasing the temperature whereas that of Prominal was improved by increasing the temperature. Thermodynamic data reveal that the separation of Prominal is an interesting case of entropy-controlled separation whereas for oxazepam the expected enthalpy-controlled separation was observed. The enantiomerization of oxazepam that occurs during its separation can be suppressed by using low temperatures.

Enantiomer separation by nonaqueous and aqueous capillary electrochromatography on cyclodextrin stationary phases

Native β‐ and γ‐cyclodextrin bound to silica (Chira Dex‐beta and Chira Dex‐gamma) were packed into capillaries and used for enantiomer separation by capillary electrochromatography (CEC) under aqueous and nonaqueous conditions. Negatively charged analytes (dansyl‐amino acids) were resolved into their enantiomers by nonaqueous CEC (NA‐CEC). The addition of a small amount of water to the nonaqueous mobile phase enhanced the enantioselectivity but increased the elution time. The choice of the background electrolyte (BGE) determined the direction of the electroosmotic flow (EOF). With 2‐(N‐morpholino) ethanesulfonic acid (MES) or triethylammonium acetate (TEAA) as BGE an inverse EOF (anodic EOF) was observed while with phosphate a cathodic EOF was found. The apparent pH (pH*), the concentration of the BGE, and the nature of the mobile phase strongly influenced the elution time, the theoretical plate number and the chiral separation factor of racemic analytes.

High-performance liquid chromatographic separation of fullerenes (C60 and C70) using chemically bonded γ-cyclodextrin as stationary phase

Abstract γ-Cyclodextrin chemically bonded to silica was used as a stationary phase for the HPLC separation of the two fullerenes, C 60 and C 70 . C 70 is much more strongly retarded than C 60 on this stationary phase. Chromatography on the corresponding unmodified silica showed no separation of the two fullerenes, indicating that the separation is due to the selective interaction with the γ-cyclodextrin moieties.

On‐line multi‐enzymatic approach for improved sequence coverage in protein analysis

The development of a new mixed bioreactor for proteomic studies based on trypsin and chymotrypsin is described. Trypsin and chymotrypsin were simultaneously bonded to an epoxy monolithic silica column (100 mmx4.6 mm id) in a one-step reaction via epoxy-groups. In order to compare the catalytic properties of the two enzymes in the isolated and in the multi-enzymatic approach, two other single enzyme bioreactors based on trypsin and chymotrypsin were prepared following the same immobilization protocol. The kinetic parameters of the multi-enzymatic bioreactor were derived and it was demonstrated that it retains the individual catalytic activity of the two enzymes. To prove the power of this experimental approach the new mixed bioreactor was integrated in an LC-ESI-MS/MS system for digestion, enrichment, separation and identification of the test protein insulin-like growth factor binding-protein 1 (IGFBP-1). The peptide map and protein sequence coverage obtained with the three bioreactors were compared. The results clearly indicate that the proposed multi-enzyme approach can reduce both digestion and analysis time, accelerate data interpretation and increase the confidence degree in protein identification.

On the use of mesophase pitch for the preparation of hierarchical porous carbon monoliths by nanocasting

Abstract A detailed study is given on the synthesis of a hierarchical porous carbon, possessing both meso- and macropores, using a mesophase pitch (MP) as the carbon precursor. This carbon material is prepared by the nanocasting approach involving the replication of a porous silica monolith (hard templating). While this carbon material has already been tested in energy storage applications, various detailed aspects of its formation and structure are addressed in this study. Scanning electron microscopy (SEM), Hg porosimetry and N2 physisorption are used to characterize the morphology and porosity of the carbon replica. A novel approach for the detailed analysis of wide-angle x-ray scattering (WAXS) from non-graphitic carbons is applied to quantitatively compare the graphene microstructures of carbons prepared using MP and furfuryl alcohol (FA). This WAXS analysis underlines the importance of the carbon precursor in the synthesis of templated porous carbon materials via the nanocasting route. Our study demonstrates that a mesophase pitch is a superior precursor whenever a high-purity, low-micropore-content and well-developed graphene structure is desired.