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Sub-Picomolar Fluorescein

Introduction
The SPEX FLUOROLOG spectrofluorometer is capable of detecting sub-picomolar ( < 1012 M) fluorescein according to the conventions established by the ASTM Committee E-13 on Molecular Spectroscopy (ANSI/ASTM E579-76). Other commercial manufacturers of spectrofluorometers specify their Minimum Detectable Concentration (MDC) somewhat differently. This technical note demonstrates how we achieve such a low MDC.

Experiment
The measurements were taken on a ing mode FLUOROLOG spectrofluorometer with a 450-W (521 nm) xenon lamp, and a cooled R928 photomultiplier operated at 900 V in the photon-counting mode. The bandpass was set to 4.0 nm on both excitation and emission spectrometers. Integration time was 11020 s, with a single scan and no smoothing. Excita-490 542.5 595 tion of the sample was at 480 nm. The acquisition Wavelength (nm)mode was S1/R1, that is, the emission signal was Figure 2. Raman spectrum of water. compensated by a reference quantum counter with voltage adjusted for a reading of 1.0 A. The scans At the highest wavelengths, at the extreme right were taken under ambient room conditions.

Results and Discussion
Figure 1 shows spectra of 400 fM (0.4 pM or 4 1013 M) fluorescein in 0.01-N NaOH, a blank of solely 0.01-N NaOH, and the subtracted spectrumof fluorescein without the solvent. The large peak near 520 nm clearly shows the presence of fluorescein at a 0.4-pM concentration.

At the highest wavelengths, at the extreme right of Figure 1, you can see the edge of the water Raman O-H stretch. A clearer graph of this region is shown in Figure 2, under 480-nm excitation. The Raman peak is inherently broad, centered at 575 nm, along with a weaker H-O-H bending mode near 521 nm. The bending mode is also visible in the solvent-containing spectra in Figure 1. This weak bending mode demonstrates the excellent sensitivity of the FLUOROLOG. In Figures 1 and 3, spectr
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