Ocean Optics S1024DW Install - Manual (Page 55)

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Absorbance Experiments
Absorbance spectra are a measure of how much light is absorbed by a sample. Absorbance spectra are
formed by radiation that has interacted with a medium. For most samples, absorbance is linearly related to
the concentration of the substance. The software calculates absorbance (
A λ ) using the following equation:
S λ - D λ
A λ = - log
10
(
R λ - D λ
)
where S is the sample intensity at wavelength
λ
, D is the dark intensity at wavelength
λ
, R is the
reference intensity at wavelength
λ
.
Absorbance can also be expressed as proportional to the concentration of the substance interacting with the
light (known as Beer’s Law). Common applications include the quantification of chemical concentrations
in aqueous or gaseous samples. To take an absorbance measurement:
1. Make sure you are in scope mode, by either clicking the scope mode icon on the toolbar, or selecting
Spectrum | Scope Mode from the menu. Make sure the signal is on scale. The peak intensity of the
reference signal should be about 3500 counts. Take a reference spectrum by first making sure nothing
is blocking the light path going to your sample. The analyte you want to measure must be absent while
taking a reference spectrum. Take the reference reading by clicking the Store Reference Spectrum
icon on the toolbar or selecting Spectrum | Store Reference from the menu.
2. While still in scope mode, take a dark spectrum by first completely blocking the light path going to
your sample. If possible, do not turn off the light source. Take the dark reading by clicking the store
dark spectrum icon on the toolbar or selecting Spectrum | Store Dark from the menu.
3. Begin an absorbance measurement by first making sure the sample is in place and nothing is blocking
the light going to your sample. Then choose the absorbance mode icon on the toolbar or select
Spectrum | Absorbance Mode from the menu. To save the spectrum, click the save icon on the
toolbar or select File | Save | Processed from the menu.
" " " "
If at any time any sampling variable changes -- including integration time, averaging,
boxcar smoothing, distance from light source to sample, etc. -- you must store a new
reference and dark spectrum.
A typical configuration for an absorbance experiment.