Spatially focused deposition of capillary electrophoresis effluent onto surface-enhanced Raman-active substrates for off-column spectroscopy

Surface-enhanced Raman scattering (SERS) is employed to obtain distinctive spectra of compounds that are efficiently separated by capillary electropho resis (CE) and deposited onto planar SERS-active substrates. A simple metho d is described that explains how to prepare SERS-active substrates by depos iting a silver-colloid solution onto frosted-glass microscope slides, using a high-efficiency nebulizer. Scanning electron micrographs reveal a layere d coating of fairly uniform-sized, 100-nm silver nanoparticles with interst itial spaces ranging from a few to tens of nanometers. The on-column separa tion is monitored by laser-induced fluorescence, while electrofilament depo siting the CE effluent onto a moving SERS-substrate. Subsequently, the SERS spectra and off-column electropherograms are obtained with a simple confoc al Raman spectrometer. The test compounds used to demonstrate this techniqu e include compounds of biological significance: benzyloxyresorufin, ribofla vin, and resorufln. CE and Raman conditions are evaluated to determine thei r affects on the SERS signals. An average off-column efficiency of 100000 p lates/m and a signal reproducibility of 11% relative standard deviation wer e achieved. Characteristic spectra with major Raman bands exhibiting signal -to-noise ratios of greater than 3 were obtained for a 3.2-nL injection of 10(-6) M (706 fg) resorufin. Forming a self-assembled monolayer (SAM) on th e substrate increases the sensitivity of the SERS technique and decreases t he on-substrate broadening. Calibration plots for both plain- and SAM-SERS substrates are demonstrated.