Aaron T. Timperman

Fluorescence detection of proteins and amino acids in capillary electrophoresis using a post-column sheath flow reactor.

A simple laser-induced fluorescence detection method for proteins and amino acids in capillary electrophoresis is reported. A sheath flow cell is utilized as a post-column reactor for fluorescence derivatization of proteins and amino acids by addition of o-phthaldialdehyde-2-mercaptoethanol to the sheath fluid. With the use of a 50 microns I.D. capillary, the limits of detection for carbonic anhydrase are 0.73 nM or 1.8 amol which represents a five- and two-fold improvement, respectively, over the best results previously reported for post-column detection. In addition, separation efficiencies up to 8.07 x 10(5) are achieved and the detector response is linear over three-orders of magnitude. These results demonstrate that mixing is adequate and the reaction kinetics are rapid enough to provide sensitive detection with this approach. Also, because this post-column derivatization scheme requires no instrumental changes to a typical sheath flow cell detector, the system can be used for detection of pre-column labeled analytes and for native fluorescence detection.

Investigation of zone migration in a current rectifying nanofluidic/microfluidic analyte concentrator.

A simple microfluidic device that uses a nanocapillary membrane (NCM) to connect a microfluidic channel and solution reservoir is capable of rectifying ionic current and enrichment of ionic species. Application of a potential induces concentration polarization (CP), which creates ion-depleted and ion-enriched zones on opposite sides of the permselective NCM. A force balanced (FB) enriched zone forms at the interface of the bulk buffer solution and depleted CP zone in the off state or the low-current case. After polarity reversal, the migration of a FB enriched zone of anionic tracer is imaged. By decreasing the solution volume at the microchannel and NCM interface, the response time of the current rectifier is decreased and elution of the zone of anionic tracer is achieved. The decrease in response time is most dramatic for the on to off state transition. For this transition, the response time decreases from approximately 50 to approximately 1 s. The decrease in response time for the off to on state is not as dramatic and is characterized by the time from polarity reversal to current peak, which decreased from 84 to 21 s. The features in the I-t plots can be accounted for with schematics of the zone migration that show the migration of depleted CP and enriched CP zones. Together, the fluorescent images and the I-t plots provide the foundation for schematics that describe the zone elution following polarity reversal. These results provide an improved understanding of the zone migration and current rectification in nanofluidic-microfluidic interfaces with symmetric nanochannels.

Tutorial review. Capillary electrophoresis with wavelength-resolved fluorescence detection

Single-channel fluorescence detectors provide extremely low limits of detection for analytes separated by capillary electrophoresis (CE), but little structural or diagnostic information. In wavelength-resolved fluorescence detection, complete fluorescence emission spectra are acquired for every analyte separated by CE. The fluorescence spectral information has been used for DNA sequencing and to identify the tyrosine and tryptophan content of peptides. In addition, a variety of diagnostic uses for the fluorescence information exist, including monitoring excitation source stability and the pH, organic content and impurities in the running buffer. The popularity of spectrally resolved fluorescence detection in CE is expected to increase just as diode-array detection has become relatively common for ultraviolet-visible absorbance.

Charge-coupled device based fluorescence detection in capillary electrophoresis

Abstract Capillary electrophoresis (CE) is currently one of the highest efficiency small volume separation methods available, and laser induced fluorescence (LIF) detection the most sensitive CE detection method. The design and characteristics of a unique charge-coupled device based CE/LIF system are described that acquires simultaneous fluorescence emission spectra from components separated by CE. The limit of detection for this multichannel detection system is below 10-13 M (less than 100 molecules) for sulforhodamine 101. The advantages of obtaining the wavelength resolved data as opposed to more conventional single channel data are outlined. Two methods for enhancing the ability to attach a fluorescent tag to low concentration peptide samples are presented, including the fabrication of a preconcentrator/reactor at the inlet tip of the capillary and the use of a UV laser with the fluorogenic derivatising agent fluorescamine.