Ocean Optics OOIPS2000 - Manual (Page 45)

Sample Experiments
Palm-SPEC Operating Instructions 42
Absorbance Experiments
Absorbance spectra are a measure of how much light a sample absorbs. For most samples, absorbance is
linearly related to the concentration of the substance. OOIPS2000 calculates absorbance (A
λ
) using the following
equation…
S λλλλ - D λλλλ
A
λλλλ
= - log
10
(
R λλλλ - D λλλλ
)
…where S λ is the sample intensity at wavelength
λ
, D λ is the dark intensity at wavelength
λ
, R λ is the reference
intensity at wavelength
λ
.
Absorbance is also proportional to the concentration of the substance interacting with the light (this is known as
Beer’s Law). Common absorption applications include the quantification of chemical concentrations in aqueous or
gaseous samples.
Follow the steps below to take an absorbance measurement using OOIPS2000:
1. Place OOIPS2000 in scope mode by clicking the scope mode icon on the toolbar or selecting Spectra |
View Mode | Scope from the menu bar.
2. Ensure that the entire signal is on scale. The intensity of the reference signal should peak at about 3500
counts. If necessary, adjust the integration time until the intensity is approximately 3500 counts.
3. Place a sample of the solvent into a cuvette, place the cuvette into the sampling chamber, and take a
reference spectrum. You must take a reference spectrum before measuring absorbance.
Note: Do not put the sample itself in the path when taking a reference spectrum, only the solvent.
Select Spectra | Store Reference Only from the menu to store the reference. This command merely
stores a reference spectrum in memory. You must select File | Save | Reference from the menu bar to
permanently save the spectrum to disk.
4. Take a dark sample by selecting Spectra | Store Dark Only from the menu. OOIPS2000 turns the light
source off, takes a dark sample, and turns the light source back on. This command merely stores a dark
spectrum in memory. You must select File | Save | Dark from the menu to permanently save the
spectrum to disk.
You must take a dark spectrum before measuring absorbance.
5. Put the sample in the cuvette holder and take an absorbance measurement by clicking on the
Absorbance Mode icon on the toolbar or selecting Spectra | View Mode | Absorbance from the menu.
To permanently save the spectrum to disk, click the Save icon on the toolbar or select File | Save |
Processed from the menu bar.
Note: If you change any sampling variable (integration time, averaging, smoothing, etc.), you must store
a new dark and reference spectrum.