Ocean Optics CHEM2000 - Manual (Page 24)

3: Experiment Tutorial
► Procedure
To take an absorbance measurement:
1. Select Scope under Mode of Operation in the software display area. Make sure the signal is on
scale. Adjust acquisition parameters so that the peak intensity of the reference signal is about
3500 counts. Take a reference spectrum by first making sure nothing is blocking the light path
going to your spectrometer. The analyte you want to measure must be absent while taking a
reference spectrum. Take the reference reading by clicking the Reference button in the software
display area. (This command merely stores a reference spectrum. To save a spectrum, you must
select File | Save Spectral Values from the menu.) Storing a reference spectrum is required
before the software can calculate absorbance spectra.
2. While still in Scope Mode, take a dark spectrum by first completely blocking the light path going
to your spectrometer. (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 Dark button in the software
display area. (This command merely stores a dark spectrum. To save a spectrum, you must select
File | Save Spectral Values from the menu.) Storing a dark spectrum is requisite before the
software can calculate absorbance spectra.
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 select Absorbance under Mode of Operation in
the software display area. Click on the Scan button in the display area to take a scan. If Single is
selected, only one scan will be taken. If Continuous is selected, the spectrometer will
continuously take scans until you click on the Stop button. To save the spectrum, select File |
Save Spectral Values from the menu.
Note
If at any time any sampling variable changes – including integration period, averaging,
boxcar smoothing, distance from light source to sample, etc. – you must store a new
reference and dark spectrum.
Transmission Experiments
Transmission is the percentage of energy passing through a system relative to the amount that passes
through the reference. Transmission Mode is also used to show the portion of light reflected from a
sample. Transmission and reflection measurements require the same mathematical calculation. The
transmission is expressed as a percentage (
%T λ ) relative to a standard substance (such as air).
18 PRELIMINARY DRAFT 105-00000-CHM-02-0405