AU Biomaterials Design Lab:Protocols/PE Luminescence Spectrophotometer

Description

Luminescence spectroscopy collects information on the way chemicals, that have been electronically excited, decay back to their ground state. Most often spectra include exciting at one specific wavelength and measuring the emission over a range of wavelengths. Luminescence covers several different kinds of excited state decays. We will most often be measuring fluorescence emission (emission from the singlet excited state). For biological studies, fluorescence is often used as a reporter of enzyme activity or presence or a reporter of protein structural properties. The fluorescent amino acid, tryptophan, has a different fluorescence emission in a folded state (when the tryptophan will likely be sequestered inside of the folded protein) than when the protein is unfolded (when the tryptophan will be more solvent exposed). The luminescence spectrophotometer is found in room 207.

Protocol

1. If Necessary
   1. Set up the peltier temperature control device.
   2. Turn on the computer (if not already done)
   3. Open the UVProbe software
   4. Get the computer talking to the instrument by clicking the "Connect Button"
2. Set the measurement "Method"
   1. Click the icon, at the top, that is a yellow circle with a green "M"
   2. Set the wavelength endpoints
      1. Typical for our measurements are: 200 nm to 800 nm
   3. Set the spectrum resolution
      1. Typical for our measurements is 1 nm
   4. Set the acquisition speed/quality
      1. Typical for our measurements is Medium
   5. Set the data collection pathway and the name for the file that will contain every spectrum collected
      1. Change the directory to: C:\Users\Chem Lab\DropBox\CHEM471 2016\UV Vis\Year\Month\Date
      2. Set the filename to something descriptive for all of the samples to be collected
3. Baseline the detector
   1. Option A
      1. Fill the cuvette you will use for the rest of the measurements with the solvent that suspends your analyte
      2. Place the cuvette in the proper holder in the instrument, making sure that light will pass through 2 clear windows
      3. Click "Baseline"
   2. Option B
      1. Don't place a cuvette in the holder at all
         1. Going this route will require you to take a spectrum of your solvent as a blank. You will have to correct all of your subsequent spectra for your solvent's spectrum. This is the best option when there are multiple users on the same instrument during a single day
      2. Click "Baseline"
4. Collect data
   1. Place a sample in a properly cleaned cuvette
   2. Place the cuvette in the proper holder making sure that light will pass through two transparent cuvette windows
   3. Click "Start"
5. Saving data
   1. When the spectrum has been acquired and the instrument has reset itself to its "start" position, you can save your data.
   2. Save data in a format readable by the instrument (.spc files)
      1. From the Menu, select "File > Save As"
      2. Give your file a name that is representative of that particular sample (include descriptors for identity, concentration, or any other important variable)
      3. Click "Save"
   3. Save data in a format readable by analysis software on your computer (.txt files)
      1. From the Menu, select "File > Save As"
      2. Change "Save as type" to "Data Print Table"
      3. Your filename from the previous step will be conserved. Only the file extension will change.
      4. Click Save
   4. Repeat as necessary
6. Shutting down the instrument
   1. Click the "Disconnect" button at the bottom of the screen
   2. Close out of the software (if at the end of the day)
   3. Shut down the computer (if at the end of the day)

1. Open the "Q Blue Wireless Temperature Controller" by clicking its icon on the desktop
2. Set the Temperature
   1. Set the Control Status to "On"
   2. Input your desired temperature
      1. Click "Change" for the Target Temperature and type in the temperature you want for the experiment
      2. For most nanoparticle syntheses, the temperature is 80C
3. Set the stirring
   1. If you need stirring, and have a stir bar in your cuvette, set the stirrer to "On"