Nine orders of magnitude dynamic range: picomolar to millimolar concentration measurement in capillary electrophoresis with laser induced fluorescence detection employing cascaded avalanche photodiode photon counters

Anal Chem. 2011 Apr 1;83(7):2748-53. doi: 10.1021/ac103374x. Epub 2011 Mar 16.

Abstract

The dynamic range of capillary electrophoresis analysis is ultimately limited by molecular shot noise at low concentrations and by concentration-induced band broadening at high concentrations. We report a system that approaches these fundamental limits. A laser-induced fluorescence detector is reported that employs a cascade of four fiber-optic beam splitters connected in series to generate a primary signal and four attenuated signals, each monitored by a single-photon counting avalanche photodiode. Appropriate scaling of the signals from the five photodiodes produces a linear optical calibration curve for 5-carboxyl-tetramethylrhodamine from the concentration detection limit of 1 pM to the upper limit of 1 mM. Mass detection limits are 120 yoctomoles (70 molecules) injected into the instrument. The very-wide dynamic range instrument was used to study the metabolic products of the fluorescently labeled glycosphingolipid tetramethylrhodamine labeled GM1 (GM1-TMR) produced by single cells isolated from the rat cerebellum.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Calibration
  • Cerebellum / cytology
  • Cerebellum / metabolism
  • Electrophoresis, Capillary / instrumentation
  • Electrophoresis, Capillary / methods*
  • Glycolipids / isolation & purification
  • Glycolipids / metabolism
  • Lasers*
  • Limit of Detection
  • Photons*
  • Rats
  • Single-Cell Analysis
  • Spectrometry, Fluorescence / methods*

Substances

  • Glycolipids