Roderic O. Cole

Bile salt surfactants in micellar electrokinetic capillary chromatography. Application to hydrophobic molecule separations

Abstract Bile salt surfactants are used in the micellar electrokinetic capillary chromatography (MECC) separation of various hydrophobic compounds. The use of methanol in the mobile phase allows the separation of previously intractable compounds including polyaromatic hydrocarbons. The effects of methanol on critical micelle concentration is investigated for sodium dodecyl sulfate (SDS) and the bile salt sodium cholate. It is determined that the unique structure of the bile salt micelle is much more tolerant to the addition of organic solvents than SDS, thereby increasing the scope of applications of MECC to include hydrophobic compounds.

Use of Native and Chemically Modified Cyclodextrins for the Capillary Electrophoretic Separation of Enantiomers

Capillary zone electrophoresis is shown to be useful for the separation of select enantiomers via the use of mobile phases containing cyclodextrins (CDs). Both native and chemically modified CDs are utilized herein. Parameters important in achieving enantiomeric separations are CD type, concentration, and mobile phase pH. In addition, rapid (1 minute) enantiomeric separations are demonstrated. Experimental parameters important in attaining short analysis times are discussed.

Stable Phases for Capillary Electrophoresis

Abstract Three phases for use in capillary electrophoresis, two bonded hydrophobic phases and one bonded hydrophilic phase, were characterized as to stability over time at neutral pH, effect on electroosmotic flow velocity, effect on electroosmotic flow velocity changes with pH, and ability to provide protein separations not possible with untreated silica under the same conditions. All columns demonstrated stability comparable to bare silica at pH 7.0, over more than 50 continuous runs. the hydrophobic phases reduced the electroosmotic flow by 37 to 43% and minimized the change in electroosmotic flow velocity with changing pH over a pH range of 3 to 10. Protein separations were achieved from pH 6 to 9.3. Changes in pH over the above range were used to optimize these separations without changing electroosmotic flow characteristics.

Factors influencing performance in the rapid separation of aflatoxins by micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MECC) is applied to the high-speed analysis of aflatoxins. Baseline separation of the four common aflatoxins G(1), G(2), B(1) and B(2), is accomplished in less than 30 sec. Small (25 mum) internal diameter capillaries are found to be critical in maintaining high efficiency under rapid MECC separation conditions. Van Deemter-like plots are generated in order to study the effects of capillary diameter and organic solvent on efficiency under high electric field conditions. Other experimental parameters affecting efficiency are investigated, including buffer concentration, surfactant concentration, and detector time constant. Simple on-column laser-based fluorescence detection, employing helium-cadmium laser radiation at 325 nm for excitation, allows for limits of detection in the range of 0.05-0.9 femtomoles injected for underivatized aflatoxins. Considerations important in the analysis of aflatoxins in real matrices are presented.

Evaluation of a sheath flow cuvette for postcolumn fluorescence derivatization of DNA fragments separated by capillary electrophoresis.

The investigation and evaluation of the sheath flow cell as a reaction chamber to postcolumn fluorescently derivatize DNA fragments separated by capillary electrophoresis is described herein. Use of the sheath flow cell arrangement facilitates the mixing of the intercalating dye, ethidium bromide (EB), and the effluent from the separation capillary by diffusion without a high degree of band dispersion. Theoretical plate counts of >1 × 10(6) are reported with the postcolumn derivatization technique, and resolution of all of the fragments in a φx-174-HaeIII digest is achieved. Optimization of experimental parameters such as flow rate, position of the detection zone, and EB concentration is examined. A limit of detection in the low nanograms-per-milliliter range with a linear dynamic range over 3 orders of magnitude is reported for a sample of φx-174-HaeIII digest. Evaluation of postcolumn derivatization for the investigation of DNA-protein interactions is demonstrated. The integrity of a DNA-trp-repressor protein interaction is maintained with the postcolumn approach but is compromised when EB is added to the running buffer.

A SHEATH FLOW CELL-BASED LASER INDUCED FLUORESCENCE DETECTION SYSTEM WITH PRE-IMAGING SPATIAL FILTERING FOR REDUCED DIMENSION SEPARATION TECHNIQUES

The construction, performance, and practical implementation of a rugged device for laser induced fluorescence (LIF) detection in capillary electrophoresis (CE) and liquid chromatography (LC) techniques is described. This device is based on a sheath flow cell (SFC) arrangement with attributes that include ruggedness, simplicity, ease of use, low cost, intra-day reproducibility, and small size. With this design, a pre-imaging mode of spatial filtering is employed to effectively reduce the contributions of both wall scatter and wall fluorescence to the background signal levels. A comparison of the pre-imaging spatial filtering and traditional methods of far-field spatial filtering is performed. This SFC system has been designed specifically for use in the UV spectral region, which can be very advantageous since it broadens the scope of the possible applications of LIF; however, fluorescence of the flow cell walls is particularly problematic in the UV region. The effect of flow cell geometry, slit widths, and detector position on fluorescent background levels are shown. The device has been applied to the fluorescence detection of three dansyl amino acids for both CE and capillary LC techniques. Efficiencies on the order of >105 plates/meter and 104 plates/meter are shown for CE and capillary LC techniques respectively. A comparison is made with CE between on-column and post-column detection demonstrating the latter preserves the integrity of a chromatographic separation.