Optical Spectroscopy Instrumentation Design, Quality Assurance, and Control: Bench-Top Fluorimetry - Aquatic Organic Matter Fluorescence - page 183

as the measurement, S m ( ) . If the step sizes are different between these two files then an
interpolation needs to be ascertained in order to fill in the “missing data points.” For exam-
ple: if Ex Co − ( λ is measured with 2 nm step size then there is a data point every 2 nm. If
the sample S m ( ) is measured at 0.5 nm step size over the same spectral range then there
are four times as many data points in the S m file as there are in the Ex Co − file, meaning
that a simple division of one file with the other becomes problematic. This issue is negated
by interpolating between the data points in the Ex Co − file to make an equivalent number
of data points of the same file length. Often this interpolation is a straight line between the
existing points, but more complex functions are also sometimes used. This problem applies
equally to using a reference detector and also to the emission channel correction.
In some ways the process of using the reference detector is quite similar to that of the
correction file. The main difference is that we make the correction measurement “live” with
every excitation scan. We need to know what the spectral response of the reference detec-
tor is so that we can correct for the reference detector itself. If the spectral response of the
reference detector is, RefDet
−

( λ , and the measured signal from the reference detector is

RefSi − ( λ , then the Ex Cor

( λ = RefSi − ( λ / RefDet ( ) λ Thus the corrected excitation − −

spectrum is:

( ) = ( ) ( ) ( )

Sc Ex λ S λ / ExCor λ or Sc Ex λ

− m − −

= ( ) ( )

( )

   

/ RefSig λ

S λ * Ref Det

m − −

(5.13)

Figure 5.21 provides examples of corrected and uncorrected excitation spectra.

5.4.19 Correcting Emission Signal Channels

The spectral correction of the emission channel is necessary to provide the “true” emission
spectrum of the sample because both the analyzing monochromator and detector each have
a spectral response. The normal situation is to measure this correction function once and
store the result in a file for use during a measurement. The correction file is measured using
a calibrated tungsten source operated under very strict constant current conditions (often
in conjunction with a calibrated deuterium source for UV correction). The tungsten source
has a spectral emission file that corresponds to the light intensity at a certain distance from
the source. The International Commission on Illumination (CIE) is responsible for publish-
ing technical data of all the well known standard illuminants. The CIE publishes relative
spectral power distribution data of such illuminants, and for a tungsten-filament source
the data will cover 380 nm to 780 nm in increments of 5 nm ( www.cie.co.at ) . However,
because the emission spectrum is smooth, interpolating this data to provide higher resolu-
tion data is possible.
The spectral power distribution of the tungsten lamp is used to generate a spectral emis-
sion file, W am _ () . If the measured signal through the emission channel is Em Si − ( λ ,
then the correction file Em Cor
−

( λ = Em Si − ( λ / W amp

( ) . Note that the spectral

_