NMSU: Standard Fluorescent References http://www.chemistry.nmsu.edu/Instrumentation/Fluorescence_Stds.

html

New Mexico State University

College of Arts and Sciences

Department of Chemistry and Biochemistry

Standard Fluorescent Reference Set

Molecular fluorescence spectroscopy is a sensitive
and often selective technique, but unlike absorption
spectrophotometry it is not an absolute technique
and the instrument requires calibration before every
series of measurements. This is most easily
achieved by using a stable reference material which
should absorb and emit at similar wavelengths to
the samples of interest. Use of the general purpose
fluorescent reference material set type 6BF (Starna) enables the day to
day stability of instruments to be measured.
The 6BF reference materials are not standards with specific values, but
a set of six stable fluorescent materials in a polymethylmethacrylate
matrix with which, the stability and precision of the instrument can be
monitored. There are four blocks with broad band spectra, which cover
the normally used UV and visible region of the spectrum, with
considerable overlap.

Two other blocks are included which contain materials suitable for
illustrating the selectivity of the technique as well as checking
instrument resolution and wavelength calibration.

Consists of six polymer blocks containing seven fluorescent

compounds whose spectra cover a broad spectral
range with emission maxima from 330 to 582 nm and
excitation maxima from 290 to 562 nm. This wide
spectral range enables the user to select a reference
material with broadly similar spectral properties to
those of the analyte, ensuring a reasonable spectral
overlap and measurable signals without changing important
parameters such as slit widths and wavlength settings.

Each block is produced with optically polished surfaces on six sides,

with dimensions of 12.5 x 12.5 x 45 mm to fit the standard cell
holders used in spectrofluorimeters. spectra were recorded in the ratio
mode, using a Perkin Elmer LS 50B Luminescence Spectrometer. The

1 de 2 06/09/2010 08:38
NMSU: Standard Fluorescent References http://www.chemistry.nmsu.edu/Instrumentation/Fluorescence_Stds.html

spectral band width for both excitation and emission

monochromators, was 2.5 nm. Data points were recorded at 0.5 nm
intervals. These spectra are specific to this instrument and the
operating conditions used. Other instruments, or different operating
conditions will produce spectra with maxima which may vary both in
terms of relative magnitude and wavelength. The corrected Spectra
were recorded using a Perkin Elmer MPF 44-B and should relate to
others recorded in a similar manner.

The following table describes the six standard materials and shows
their fluorescence spectra.

Sanple Molar Excitation Emission

Compound
Number Concentration Wavelength Wavelength

Anthracene 1 x 10-5 360 402

1
Napthalene 6 x 10-5 290 330
2 Ovalene 2 x 10-7 342 482
3 p -Terphenyl 5 x 10-7 295 338
4 Tetraphenylbutadiene 3 x 10-7 348 422
5 Compound 601 1 x 10-6 440 475
6 Rhodamine B 2 x 10-7 562 573

Return to Instrumentation or Teaching Instruments

Copyright 2005 NMSU Board of Regents

2 de 2 06/09/2010 08:38