Simultaneous Online Electrokinetic Sample Cleanup and Preconcentration in Capillary Zone Electrophoresis of Fluorophore-Labeled N-Glycans

Glycans play an essential role in the proper functioning of proteins, making them an important analyte of interest in the biomedical and biopharmaceutical fields. One of the most effective ways to analyze complex carbohydrates is by capillary electrophoresis, especially when stacking methods are employed. In addition, coupling with other electrokinetic-based options, such as simultaneous removal of interfering molecules from the sample matrix, is of high importance as well. The online stacking part of the method introduced in this paper is well suited for the preconcentration of fluorophore-labeled glycans. After hydrodynamic injection of the APTS labeling reaction mixture, the electric field was applied with the cathode at the injection side; thus, the neutral and positively charged molecules migrated toward the cathode according to the superimposed upstream electroosmotic flow (EOF) and electric force, while their negatively charged counterparts accumulated at the boundary of the sample plug and the background electrolyte. The polarity was then switched with the anode at the injection side, generating downstream EOF to carry the negatively charged fluorophore-labeled glycans toward the detection zone, while the unreacted APTS molecules and other negatively charged species with high electrophoretic mobilities migrated out of the capillary at the injection end. In this way, both sample preconcentration and purification were simultaneously achieved at the beginning of the separation process, alleviating the need for sample cleanup before analysis. The proposed method utilized a simple 150 mM MOPS-KOH (pH 7.2) background electrolyte, which provided high enough EOF and adequate conductivity difference to readily accomplish this dual task of purification and stacking, achieving more than an order of magnitude higher signal intensity and LOD in the sub-pM level.