Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
4-18
Note:
The excita-
tion range is 240–
600 nm; the emis-
sion range is
290–850 nm.
Correcting data
Introduction
Collecting accurate information about the fluorescent or phosphorescent properties of a
sample depends upon several factors:
•
Equipment specifications
•
Sample characteristics
•
Timing considerations.
To ensure that the spectra collected indicate the actual properties of the sample and not ex-
ternal conditions, data often must be corrected. To
correct
data means to subtract informa-
tion from the data not directly related to the properties of the sample.
Gratings, detectors,
and other spectrometer components have response characteristics that vary as a function of
wavelength. These characteristics may be superimposed on
spectra, thereby yielding a potentially misleading trace. For
accurate intensity comparisons, such as those required for
quantum-yield determinations, response characteristics must
be eliminated. Supplied with the instrument are sets of
excitation and emission correction factors to eliminate
response characteristics. These files
1
,
xcorrect
and
mcorrect
, are included with the software; copy them to
your hard disk.
Items that may be convoluted into a
spectrum
Ways to remove these artifacts
Fluctuations caused by the light source
Monitoring lamp output using the
reference detector,
R
, and using the
signal ratio
S/R
to correct lamp profile
or temporal fluctuations
Influence of the sample holder
Running a blank scan (which is then
subtracted from the sample scan)
System hardware (e.g., optics, detectors).
Using radiometric correction factors
To use radiometric correction factors, either:
•
Select the ones supplied with the program, or
•
Select a set generated at your facility during or after acquisition, discussed in the
following section. Acquiring radiometric correction factors is explained in Chaper
11:
Producing Correction Factors.
Blank Subtraction
and
Dark Offset
functions are described in the DataMax manual.
1
Filenames include a three-letter extension. For the sake of clarity, we have omitted the extensions in this section. Refer to the
software manual for specifics regarding extensions.
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