AU Biomaterials Design Lab:Protocols/PE Luminescence Spectrophotometer: Difference between revisions

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==Protocol==
==Protocol==
# If Necessary
# If Necessary
## The instrument MUST be turned on before the computer. If the computer is already on, you will have to shut it down.
## Set up the [[AU Biomaterials Design Lab:Protocols/Fluorescence Peltier System|peltier temperature control device]].
## Set up the [[AU Biomaterials Design Lab:Protocols/Fluorescence Peltier System|peltier temperature control device]].
## Turn on the computer (if not already done)
## Open the program "FL WinLab"
## Open the UVProbe software
# Measurement setup
## Get the computer talking to the instrument by clicking the "Connect Button"
## Click the spectrum icon on the top toolbar (a rainbow with a spectrum underneath)
# Set the measurement "Method"
## From the "Setup Parameters" tab, choose the type of measurement to preform
## Click the icon, at the top, that is a yellow circle with a green "M"
### Note: For most of what we do, this will be "Emission"
## Set the wavelength endpoints
### Choose a start wavelength
### Typical for our measurements are: 200 nm to 800 nm
#### For tryptophan: 310 nm
## Set the spectrum resolution
### Choose an end wavelength
### Typical for our measurements is 1 nm
#### For tryptophan: 500 nm
## Set the acquisition speed/quality
### Choose an excitation wavelength
### Typical for our measurements is Medium
#### For tryptophan: 290 nm
## Set the data collection pathway and the name for the file that will contain every spectrum collected
### Set the excitation slit width (typical is 10 nm) It is important you note what you use for this value.
### Change the directory to: C:\Users\Chem Lab\DropBox\CHEM471 2016\UV Vis\Year\Month\Date
### Set the emission slit width (typical is 10 nm) It is important you note what you use for this value.
### Set the filename to something descriptive for all of the samples to be collected
### Set the scan speed
# Baseline the detector
#### 100 nm/min is typical for our experiments. It is important you note what you use for this value.
## Option A
### Enter a result filename. (The data are always automatically saved to: C:\flwinlab\data)
### Fill the cuvette you will use for the rest of the measurements with the solvent that suspends your analyte
# Start your measurement
### Place the cuvette in the proper holder in the instrument, making sure that light will pass through 2 clear windows
## Click on the green "Stop Light" icon.
### Click "Baseline"
# Saving your data (This part is convoluted)
## Option B
## The data is automatically saved in a format that is proprietary to the instrumentation software. You need to save it in a format that is readable to you.
### Don't place a cuvette in the holder at all
## In the FLWinLab window, expand the "Graph 1" window, you should see a graph displayed
#### 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
## From the Graph 1 window menu, Choose File > Open from the menu
### Click "Baseline"
## Sort the names by date so your file is easier to find
# Collect data
## Select the file that you want to view and save and click "OK"
## Place a sample in a properly cleaned cuvette
## Click on the name of your file (shown on the bottom of the graph window). This is especially important when there are multiple spectra shown in the same graph.
## Place the cuvette in the proper holder making sure that light will pass through two transparent cuvette windows
## From the Graph 1 window menu, choose File > Save As
## Click "Start"
## Change the file type to: ASCII
# Saving data
## Change the extension of the filename from ".sp" to ".txt"
## When the spectrum has been acquired and the instrument has reset itself to its "start" position, you can save your data.
# Collect more spectra as needed.
## Save data in a format readable by the instrument (.spc files)
# Shut down the instrument
### From the Menu, select "File > Save As"
## Close all FLWinLab windows
### Give your file a name that is representative of that particular sample (include descriptors for identity, concentration, or any other important variable)
## Turn off the instrument
### Click "Save"
## Turn off the computer
## Save data in a format readable by analysis software on your computer (.txt files)
### From the Menu, select "File > Save As"
### Change "Save as type" to "Data Print Table"
### Your filename from the previous step will be conserved. Only the file extension will change.
### Click Save
## Repeat as necessary
# Shutting down the instrument
## Click the "Disconnect" button at the bottom of the screen
## Close out of the software (if at the end of the day)
## Shut down the computer (if at the end of the day)


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


==Notes==
==Notes==

Latest revision as of 10:39, 26 August 2016

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. The instrument MUST be turned on before the computer. If the computer is already on, you will have to shut it down.
    2. Set up the peltier temperature control device.
    3. Open the program "FL WinLab"
  2. Measurement setup
    1. Click the spectrum icon on the top toolbar (a rainbow with a spectrum underneath)
    2. From the "Setup Parameters" tab, choose the type of measurement to preform
      1. Note: For most of what we do, this will be "Emission"
      2. Choose a start wavelength
        1. For tryptophan: 310 nm
      3. Choose an end wavelength
        1. For tryptophan: 500 nm
      4. Choose an excitation wavelength
        1. For tryptophan: 290 nm
      5. Set the excitation slit width (typical is 10 nm) It is important you note what you use for this value.
      6. Set the emission slit width (typical is 10 nm) It is important you note what you use for this value.
      7. Set the scan speed
        1. 100 nm/min is typical for our experiments. It is important you note what you use for this value.
      8. Enter a result filename. (The data are always automatically saved to: C:\flwinlab\data)
  3. Start your measurement
    1. Click on the green "Stop Light" icon.
  4. Saving your data (This part is convoluted)
    1. The data is automatically saved in a format that is proprietary to the instrumentation software. You need to save it in a format that is readable to you.
    2. In the FLWinLab window, expand the "Graph 1" window, you should see a graph displayed
    3. From the Graph 1 window menu, Choose File > Open from the menu
    4. Sort the names by date so your file is easier to find
    5. Select the file that you want to view and save and click "OK"
    6. Click on the name of your file (shown on the bottom of the graph window). This is especially important when there are multiple spectra shown in the same graph.
    7. From the Graph 1 window menu, choose File > Save As
    8. Change the file type to: ASCII
    9. Change the extension of the filename from ".sp" to ".txt"
  5. Collect more spectra as needed.
  6. Shut down the instrument
    1. Close all FLWinLab windows
    2. Turn off the instrument
    3. Turn off the computer


Notes

References

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