Ocean Optics OOIBase16 - Manual (Page 12)

10
Doing an Experiment
By now, your spectrometer and sapling optics should be set-up for your application. Confirm that the
parameters for your setup are satisfactory. Assuming that you have followed the necessary steps and started
OOIBase, your spectrometer is already acquiring data. With no light in the spectrometer you should see a
fluctuating trace on the bottom of the graph. If you turn on your lamp, you should see the graph trace rise
with increasing light intensity.
Now you are ready to take your measurements. There are three basic spectral measurements from which to
choose: absorbance, transmission, and relative irradiance. Certain measurements require specific
components. Your current setup might not be the best configuration for all measuring functions. Contact an
Ocean Optics Applications Scientist for options.
For each measurement, a reference and dark spectrum must first be made. After taking a reference and a
dark spectrum, you may take as many absorbance, transmission, or relative irradiance measurement scans
as you want.
Absorbance Measurement
Absorbance spectra are a measure of how much light is absorbed by a sample. Absorbance spectra are
formed by radiation that has penetrated a material medium. For most samples, absorbance is linearly related
to the concentration of the substance. The software calculates absorbance using the following equation:
S λ - D λ
A λ = - log
10
(
R λ - D λ
)
S λ = sample intensity of each wavelength, D λ = dark intensity of each wavelength,
R λ = reference intensity of each wavelength, λ = each wavelength
Absorbance can also be expressed as proportional to the concentration of the substance interacting with the
light. 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
View | Scope Mode. Make sure the signal is on scale. The peak intensity of the reference signal should
be about 3500 counts. (See
Integration Time on page 10.) 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 File | Store Reference Spectrum.
2. While still in Scope Mode, take a Dark Spectrum by first completely blocking the light path going to
your sample. Do not turn off the light source. Take the dark reading by clicking the Store Dark
Spectrum icon on the toolbar or selecting File | Store Dark Spectrum.
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
View | Absorbance Mode. To save the spectrum, click the Save icon on the toolbar or select File |
Save Sample Spectrum.
If at any time any sampling variable changes -- integration time, averaging, smoothing, distance
from light source to sample, etc. -- you must store a new reference and dark spectrum.