Abstract
An automated method for determination of trace level amino acids in 2βΞΌL samples using onβcolumn sample preconcentration, gradient elution on 50βΞΌm inner diameter (i.d.) capillary columns packed with 5βΞΌm reversedβphase particles, and electrochemical detection is described. The 50βΞΌm i.d. capillary columns were efficiently coupled directly to an autosampler without increasing zone dispersion by preconcentrating derivatized amino acids at the head of the capillary column and minimizing gradient dwell volume. Using this system, the relative standard deviations (RSDs) of retention time for a 16 component amino acid mixture were between 0.2 and 0.8%. Using an automated microinjection method, as little as 0.3 ΞΌL of derivatized sample was consumed to perform a 0.25βΞΌL injection with peak area RSDs of 3.0β8.4%, allowing conservation of a majority of the derivatized sample for future analysis. Precision was improved to peak area RSDs of 1.8β4.1% when consuming 1.0 ΞΌL of sample per injection. Detection limits were <0.2 nM for most amino acids. The linear solvent strength theory was used to optimize the gradient and allowed resolution of 15 amino acids in 13 min. The final gradient separation was demonstrated to be selective for the neurotransmitter amino acids in the presence of 44 primary amines commonly found in physiological samples. The system was used to characterize amino acid secretion from single pancreatic islets of Langerhans under different physiological conditions with 2 min of temporal resolution.βΒ© 2001 John Wiley & Sons, Inc.βJ Micro Sep 13: 24β32, 2001