Advanced off-column technologies for capillary electrophoresis : microfluidic deposition, two-dimensional separations, and surface-enhanced Raman scattering-based detection

Capillary electrophoresis (CE) Is a widely accepted technique for performing high-efficiency liquid separations. The utility of CE separations could be increased by comprehensively depositing the capillary effluent onto a planar substrate, thus essentially preserving the separation to allow the application of off-column technologies. Electrospray is a convenient technique for continuously transferring column effluent from capillary-toplanar format. Conditions are optimized to produce a narrow (~ 20 pm) liquid filament (electrofilament), which is capable of depositing spatially focused bands with track widths that are routinely 100-200 pm. A fiber optic-based laser-induced fluorescence cell is employed to monitor the separation on-column while the separated bands are deposited onto a moving substrate. The electrofilament (EF) technique is evaluated based on its ability to deposit spatially focused bands, which preserve the on-column separation performance.

The EF technique was used to couple CE with thin-layer chromatography (TLC) to demonstrate a convenient technique for performing two-dimensional microseparatlons. The TLC plate stores the separation from the CE capillary in the first dimension and then serves as the stationary phase for the separation in the second dimension. To demonstrate this technique, dansylated derivatives of select amino acids are separated in the first dimension by micellar electrokinetic chromatography, and in the second dimension their enantiomers are separated by reversed-phase TLC using a mobile phase that contains cyclodextrins as a chiral reagent. Prior to TLC development, off-column CE efficiencies of 130,000 to 190,000 plates per meter were obtained. The enantiomers of four DNS-amino acids are baseline resolved and a significant improvement in peak capacity over the one dimensional separation is demonstrated.

Surface-enhanced Raman scattering (SERB) is employed to obtain distinctive spectra for compounds that are separated by CE and EF deposited onto planar SERS-active substrates. A simple method is described that explains how to prepare SERS-active substrates by depositing a silver-colloid solution onto frosted-glass microscope slides. Scanning electron micrographs reveal a layered coating of fairly uniform-sized, 100-nm silver nanoparticles with interstitial spaces ranging from a few to tens of nanometers. The test compounds used to demonstrate this technique include compounds of biological significance: benzyloxyresorufin, riboflavin, and resorufin. Characteristic spectra with major Raman bands exhibiting signal-to- noise of greater than 3 were obtained for a 3.2-nL injection of 10"® M (706 fg) resorufin. Forming a self-assembled monolayer on the substrate increases the sensitivity of the SERS technique and decreases the on-substrate broadening of deposited bands.