Ocean Optics CHEM2000 - Manual (Page 28)

3: Experiment Tutorial
To discover the unknown concentration of a substance in a solution, you must first take spectral scans of a
series of solutions with different known concentrations of the same substance. You begin this process by
taking an absorbance spectrum of the solution with the highest known concentration.
► Procedure
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 solution with the highest known concentration 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. To save the spectrum, select File | Save
Spectral Values from the menu.
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. To save the spectrum, select File | Save Spectral Values from the menu.
3. Take the solution with the highest known concentration and put it in the cuvette holder. Make
sure nothing is blocking the light going to your sample. Then select Absorbance under Mode of
Operation. Click on the Scan button in the display area to take a scan. Make sure Single is
selected. To save the spectrum, select File | Save Spectral Values from the menu.
4. Now select the wavelength for monitoring the concentration of your solutions by choosing
Spectrometer | Select Concentration Wavelength from the menu. Move the cursor to the
highest absorbance peak of the spectrum of the solution with the highest known concentration and
choose Select.
5. Remove the solution with the highest known concentration. Select Spectrometer | Calculate a
Calibration Curve from the menu. The Calculate Calibration Curve dialog box opens. Now you
will begin taking scans of the rest of your series of standard solutions with known concentrations,
from the lowest known concentration to highest, all while working in this dialog box.
6. If you wish, you can take a new reference and a new dark scan for each solution by choosing
Scan | Dark and Scan | Reference from the menu of this dialog box. However, in this case, it is
not necessary. If no reference or dark scan is taken at this point, the software will use the
reference and dark scans taken in Steps 1 and 2 to calculate absorbance.
7. Take the solution with the lowest known concentration and put it in the cuvette holder. Enter the
known concentration of the standard solution in the chart in the Concentration column, next to
Solution #1.
22 PRELIMINARY DRAFT 105-00000-CHM-02-0405