Appendix 1

Operation of the Ocean Optics Red Tide UV/Vis Spectrometers using the SpectraSuite Software:

  1. Instrument Set-Up
    1. Log onto the computer using your username and password. 
    2. Connect the AC power supply to the UV/VIS source and plug it into an outlet.  (The USB cable must NOT be attached at this point.)
    3. Connect the spectrometer to the PC using the USB cable.  You should hear the fan on the spectrometer begin to run.
    4. Launch the software by selecting:
    5. Start→Programs→OceanOptics→SpectraSuite→SpectraSuite
    6. Click the Strobe/Lamp Enable box to turn on the light sources.  (Ideally the lamp should warm up for 20-30 minutes before taking measurements.)  You should see a screen similar to this:
    7. Configure the software through the following steps:
      1. The intensity of the two lamps (UV and visible) must be balanced.  Right-click on the spectrometer picture in the upper left of the screen.  Select Spectrometer Features and then the UV-Vis tab.  The window shown below should appear.  Balance the output by changing the Visible Lamp Intensity until the main plateaus in the left and right sides of the lamp output are at approximately the same count level.
      2. We want to maximize the detector signal without overranging the detector signal.  Increase the Integration Time (the time the detector collects data) to increase the detector signal.  Ideally the red intensity curve will rise to near the maximum count level (typically ~4000 counts), but not be completely flat anywhere.  If it is flat on top anywhere, reduce the integration time until this no longer happens.  This curve is the intensity output of the light sources at each wavelength.
      3. Decrease the amount of noise in your spectrum by adjusting the Scans to Average and the Boxcar width settings.  The spectrum below was acquired by averaging 5 scans with a boxcar width of 10 and an integration time of 182 ms.
  2. Take Dark and Light Reference Spectra.
    1. Obtain the dark spectrum by first turning off the light sources (uncheck Strobe/Lamp Enable).  Wait for the detector signal to drop to zero, then click the Store Dark Spectrum button on the toolbar.
    2. Recheck the Strobe/Lamp Enable button and wait for the lamps to return to their full intensity.  Fill a clean cuvette approximately 2/3-full with your reference solution, wipe the sides of the cuvette with a methanol-soaked Kimwipe, and then place the cuvette in the instrument.  Make sure the cuvette goes all the way to the bottom.  (You should never touch the cuvette on the sides where the light shines through.)  Obtain a reference spectrum by clicking the Store Reference Spectrum button on the toolbar.
    3. Note:  If you change any of the settings from above (integration time, scans to average, boxcar width), you must repeat steps 1 and 2.
  3. Data Collection
    1. Change the display from Scope mode to Absorbance mode by clicking the absorbance button:
    2. Replace the reference solution with your sample solution, and you will see the absorption spectrum of the sample appear.  The instrument will continuously average the spectrum and update the display.
    3. You may use the scale-adjustment tools above the graph to zoom in on the region of interest.  This affects only the display of the data, not the data itself.
    4. If the cursor is not already on, click inside the spectrum box to turn it on.   Move the cursor by clicking the desired point on the spectrum, or by using the Wavelength box at the bottom of the screen.  Notice that the absorbance at the cursor wavelength is also displayed at the bottom of the screen.
    5. Save your data by clicking on the Save icon in the tool bar:

      Do NOT use the File →Save→Save Spectrum sequence
      from the menu bar.  This turns on the continuous saving function,
      and you do not want that. 

      A dialog box will open like the one below.  Select Processed Spectrum and Tab Delimited.   Browse to the location where you want to save your data and enter a filename.  Click Save twice.
    6. Replace the sample in the cuvette with your next sample to be measured, and repeat steps 3-5.
  4. Instrument Shut-Down (You must disconnect the spectrometer from the PC in the following order.)
    1. Close down the spectrometer software.
    2. Disconnect the AC power cable.
    3. Disconnect the USB cable.
  5. Accessing Your Data
    1. From Excel, click the Excel icon in the upper left-hand corner of the screen and select Open.
    2. For Files of Type be sure to select All Files.  Find your data file, select it, and click Open.
    3. When the Text Import Wizard appears, simply click Finish.  Your file will load into a spreadsheet.  After listing some information about your experiment, you’ll notice two columns of numbers.  The column on the left is wavelengths, and the column on the right is absorbances.