Optically Gated Capillary Electrophoresis of o-Phthalaldehyde/β-Mercaptoethanol Derivatives of Amino Acids for Chemical Monitoring

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Abstract
Optically gated capillary electrophoresis (CE) of amino acids derivatized with o-phthalaldehyde/β-mercaptoethanol (OPA/β-ME) was explored as a means to monitor amino acids with high temporal resolution. In agreement with a theoretical model described herein, 98% of a given concentration of OPA/β-ME derivatives can be photobleached by a few milliwatts of the 350-nm line of an argon ion laser with just 0.7-ms exposure times in 5-μm-i.d. capillaries. The low background from such high photobleaching efficiency allows detection limits in the low-nanomolar range for all amino acids tested. The short injection times possible with optical gating allow separation efficiencies of nearly 200 000 plates to be achieved in less than 1 s under ideal conditions. Under mock in vivo conditions, separations were slower and had lower efficiency due to reduced electroosmotic flow associated with the high salt content. To demonstrate chemical monitoring, the optically gated CE system was interfaced to two different sampling probes with on-line derivatization with OPA/β-ME. With microdialysis sampling, the optically gated CE system could assay the sample stream every 2 s but actual temporal resolution for monitoring was limited by band broadening in the dialysis probe to ∼12 s. Optically gated CE was also interfaced to a 10-μm-i.d. sampling capillary that continuously pulled samples into the separation capillary at 6.5 nL/min. This direct sampling probe allowed monitoring of multiple amino acids with 10-s temporal resolution with several advantages compared to microdialysis including improved detection limits and spatial resolution.