Ocean Optics S2000 - Manual (Page 92)

Absorbance Experiments
Absorbance spectra are a measure of how much light is absorbed by a sample. For most samples, absorbance is
linearly related to the concentration of the substance. The software calculates absorbance (
A λ ) using the
following equation:
S
λλ
- D
λλ
A
λλ
= - log
10
( )
R
λλ
- D
λλ
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 using OOIBase32, our spectrometer operating software,
follow these steps:
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.
2. 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. (This command merely stores a reference spectrum. You must select File | Save | Reference from the
menu to permanently save the spectrum to disk.) Storing a reference spectrum is requisite before the software
can calculate absorbance spectra.
3. 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. If you must turn off your light source to store a dark
spectrum, make sure to allow enough time for the lamp to warm up before continuing your experiment.) Take
the dark reading by clicking the store dark spectrum icon on the toolbar or selecting Spectrum | Store Dark
from the menu. (This command merely stores a dark spectrum. You must select File | Save | Dark from the
menu to permanently save the spectrum to disk.) Storing a dark spectrum is requisite before the software can
calculate absorbance spectra.
4. 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.
Experiment Tutorial: Absorbance
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