Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
11-1
11: Producing Correction
Factors
Introduction
Collecting accurate information about the fluorescent or phosphorescent properties of a
sample depends upon several factors: equipment, sample, and timing. To ensure that
the spectra are indicative of the actual sample properties and not of external conditions,
data often must be corrected. To
correct
data means to remove information from the
data not directly related to the properties of the sample. Specifically, several items that
may appear mixed into a spectrum are:
•
Fluctuations caused by the light source.
•
Influence of the sample holder.
•
System features.
Corrections are made for each of these potential problems by using radiometric correc-
tion factors, running a blank scan (which is then subtracted from the sample scan), and
using the software’s
Auto Zero
function (which closes the programmable excitation
shutter, records the background dark counts for 10 s, and automatically subtracts this
value from the data as they are acquired).
Blank
and
Auto Zero
functions are described
in the system’s software manual.
Gratings, detectors and other spectrometer components have response characteristics
that are functions of wavelength. These characteristics are superimposed on spectra,
and may yield a potentially misleading trace. For accurate intensity comparisons, such
as those required for quantum-yield determinations, spectrometer-response characteris-
tics must be eliminated.
Supplied with your instrument are sets of excitation and emission correction factors de-
signed to eliminate response characteristics. These files
1
,
xcorrect
and
mcorrect
,
are included with the software and should be copied to the hard disk. These files must
be in the same directory as the data to be corrected. The excitation correction range is
from 220–600 nm, and the correction range for emission spectra is from 300–850 nm.
Instructions for use of correction factors are in Chapter 4. This chapter only describes
the steps for
generating
correction factors. Perform this procedure
only
when the grat-
ings or detectors have been replaced with those of different specifications than the
original hardware.
1
Filenames include a three-letter extension. For the sake of clarity, the extensions are omitted in this manual. Refer to the soft-
ware manual for specifics regarding extensions.
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