Dimethylditetradecylammonium bromide (2C14DAB) as a self-assembled surfactant coating for detection of protein–dye complexes by CE-LIF

Abstract

A self-assembled column coating for capillary electrophoresis in conjunction with laser-induced fluorescence detection (CE-LIF) has been evaluated for the separation and quantitation of protein–dye complexes. This semi-permanent coating, composed of dimethylditetradecyl-ammonium bromide (2C14DAB), is inexpensive and easily assembled onto the column and it allows for better peak resolution and greater control over electroosmotic flow. The versatility of long-chained surfactant coatings was determined particularly with respect to their use with fluorescent probes, different pH buffers, and different proteins. Studies were performed to determine the stability of the coating under various pH and buffer conditions. Red-1c, a red luminescent squarylium dye, was used for on-column protein labeling concurrently with the surfactant coating and LIF detection. Protein–Red-1c complexes were excited with a 650-nm diode laser and their emission detected by a photomultiplier tube with a 664-nm filter. A comparison of pre-column labeling and on-column labeling of a two-model protein system (human serum albumin and β-lactoglobulin A) revealed higher efficiencies and greater sensitivities for both proteins using on-column labeling and coated columns. A linear relationship between peak height and protein concentration was obtained by CE-LIF for this on-column labeling method with 2C14DAB-coated columns and the Red-1c probe.