Fluorescent Reagents to Improve the Analytical Infrastructure of Capillary Electrophoretic Separations

Two types of fluorescent molecules had been designed and synthesized to improve the analytical infrastructure of capillary electrophoretic separations. First, a hydrophilic version of the permanently cationic acridine-based fluorophore, HEG2Me2-DAA was synthesized. HEG2Me2-DAA has a lambda^ex max of 490 nm which matches the 488 nm line of the commonly used argon ion laser. The emission spectra of HEG2Me2-DAA are pH-independent. HEG2Me2-DAA was used in capillary electrophoresis with an aqueous background electrolyte and was found to be free of the detrimental peak tailing of the acridine orange-based fluorophore that was caused by adsorption on the inner wall of the fused silica capillary. Bovine serum albumin was labeled with excess of the designed amine reactive reagent and the lowest concentration at which the tagged bovine serum albumin was tested was 15 nM. Chicken ovalbumin was also labeled with FL-CA-PFP and analyzed by capillary isoelectric focusing (cIEF) with LIF detection. The pI values of the tagged proteins shifted in the alkaline direction by about 0.02 compared to the pI values of the non-tagged proteins. A tri-functional probe intended to enable selective enrichment and selective detection of a variety of molecules (e.g., natural products, pharmaceuticals, inhibitors, etc.) was also designed and synthetized by combining FL-CA with biotin and an azide group in a "proof-of-principle" level experiment.

In cIEF, the profile of the pH gradient can only be determined with the help of pI markers. A large set of pyrene-based fluorescent pI markers was rationally designed to cover the pI range 3 to 10. To prove the feasibility of the proposed synthetic approach, the subgroup of the pI markers having the greatest structural complexity was synthesized and characterized. The classical zone electrophoretic pI determination methods failed due to severe chromatographic retention of the APTS based pI markers on the capillary wall. Exploratory work was done to design a new pI value determination method that combines the advantages of the immobilized pH gradient technology of the OFFGEL instrument and the carrier-ampholyte-based IEF technology. The method aspects of cIEF have also been improved in this work. The new segmented loading method yielded a more linear pH gradient than the previously known cIEF methods. To exploit a unique property of the newly developed fluorescent pI markers, we used them as pyrene-based ampholytic carbohydrate derivatizing reagents. The pI4 carbohydrate derivatization reagent proved advantageous over 8-aminopyrene-1,3,6-trisulfonic acid (APTS): the pI4 conjugates have higher molar absorbance at 488 nm than the APTS conjugates and become detectable in positive ion mode of MS affording better detection sensitivity.