ABSTRACT
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.
ABSTRACT
Capillary electrophoresis (CE) and related techniques yield highly efficient separations while requiring only minute amounts of sample. Thus, these techniques are amenable to analyses of complex samples in diverse matrices and in situations where sample is extremely limited. The constraints of on-column detection generally result in poor detection limits and have reduced the overall application of CE. One logical approach to increased sensitivity in CE detection has been the development of chemiluminescence (CL)-based detectors. The current state of post-column detector development, CL applications, and limitations of the technique are reported herein.
