1. Turn on the computer. Open the StellarNet software by double clicking on the desktop icon SpectraWiz to open the application.
2. When you first open the software, you should see a screen similar to the one below:
3. Remove the cuvette holder cap. Place your blank cuvette into the cuvette holder and replace the cuvette holder cap.
4. Click on the Scope icon. This will show you the number of counts (photons) the detector receives. You should see a spectrum of counts versus wavelength.
5. Set the integration time so that your spectrum is not saturated but as close to 65,000 counts as possible. Click on the integration time icon 
(and type in a value between 1 and 1000 ms) or use sliding scale 
. In the picture to the right, the spectrum is correctly optimized to be right below the saturation limit (65,536 counts). If a portion of the spectrum is off the screen reduce the integration time.
6. Light Reference. Make sure that your blank cuvette is in the cuvette holder and the cap is on the cuvette holder. Take the light reference by clicking on the yellow light bulb icon 
in the toolbar at the top of the screen.
7. Dark Reference. First, hold the red shutter button for at least three seconds.
You should see the spectrum fall flat everywhere on your graph (A).
Second, while still holding the shutter button, click on the dark light bulb icon 
in the toolbar at the top of the screen. The baseline will drop to zero (B).
Finally, you can release the shutter button. You should see the spectrum return to its original profile.
8. Now that you have taken the dark and light reference, click on the AU (absorbance) icon in the toolbar at the top of the screen to display a spectrum in absorbance units.
9. You should see a nearly flat absorbance spectrum once you switch to absorbance if you have blanked and zeroed the spectrometer correctly.
| Depending on the cuvettes you use, you may see fluctuations below 300 nm. This is simply due to the cuvette absorbing UV radiation at these wavelengths. |
Taking Absorbance Measurements
1. Remove your blank sample from the cuvette holder. Place the most concentrated standard cuvette in the holder and replace the cuvette cap. The instrument will now display absorbance in real time.
2. Look at the spectrum and select the wavelength of maximum absorbance.
3. To find a peak, use the mouse to right click on either of the following icons:
The left arrow will find a peak to the left of the vertical line in the spectra. The right arrow will find a peak to the right of the vertical line in the spectra.
4. You can also right click directly onto the spectrum to find the photon count of a specific wavelength. To find the specific wavelength your cursor is at, look to the bottom of the spectrum. Wave gives the specific wavelength and Val gives the photon count for that particular point in the spectrum.
