User:Anneliese Faustino/Notebook/671 Nanoparticles/2016/09/06

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Sample Preparation

BSA Ocean Optics Final Sample Solution Concentrations:

  1. 3 mL total
  2. 0.25 mM Au
  3. 3.125 μM BSA
  4. HCl or NaOH appropriate for your pH

Stock solution Concentrations:

  1. 5.08 mM AuCl3
  2. 34 mM BSA
  3. 1 mM HCl
  4. DI HOH

Solution Transfer Volumes:

  1. 2278 uL HOH
  2. 147 uL AuCl3
  3. 275 uL BSA
  4. 300 uL HCl

Methods

  1. After following the Ocean Optics protocol below to set up the instrument, we analyzed the prepared sample.
  2. A scan was taken every three minutes and transferred to Dropbox.

Protocol for Ocean Optics

  1. Extra Steps
    1. Do you need temperature control or need stirring?
      1. See the protocol for controlling the cuvette controller Quantum Northwest Cuvette Holder
  2. Turn on the jaz spectrophotometer (red on-off button)
  3. Turn on the Lamps (Ocean Optics DH-2000)
    1. Turn on the switch in the back of the controller box
    2. Press the blue "Deuterium" button and wait for the light to stay on
    3. Press the red "Halogen" button
    4. The lamps need 15 minutes to warm up before you are ready to proceed to the next step.
  4. Starting the software
    1. Open the OceanView software.
      1. Click on the "Create New Spectroscopy Application" icon (looks like a bunch of shark fins stacked on one another)
        1. Click on "Spectroscopy"
        2. Select JAZA1666 and click "Next"
        3. Select "Absorbance" and click "Next"
        4. Set Acquisition Parameters
          1. Set "Integration Time" to 1 ms
          2. Set "Scans to Average" to 100
          3. Click "Next"
        5. Store Reference Spectrum
          1. Make sure that the toggle on the light source is switched to "Open"
          2. Click on the light bulb icon
            1. A spectrum should appear in the middle display
          3. Click "Next"
        6. Store Background Spectrum
          1. Make sure that the toggle on the light source is switched to "Closed"
          2. Click on the light bulb icon
            1. A spectrum should appear in the middle display
          3. Click "Finish"
        7. Make sure that the toggle on the light source is switched to "On"
  5. Set up your data acquisition schedule
    1. Click on the "Save graph to file/files" icon (the floppy disc icon on the lower icon panel)
    2. Click "Yes" for "You must configure save parameters first. Do you want to configure now?"
    3. Set Save Options
      1. Choose the "Between saved scans, wait at least" option
      2. Set the time to whatever the experiment requires
        1. For most nanoparticle syntheses, this will be 2 minutes
      3. Choose the "Stop after this amount of time" option
        1. For most nanoparticle syntheses, this will be 3 hours
    4. Set File Options
      1. Save to Directory: C:\Users\Lab Admin\DropBox\CHEM471 2016\Ocean Optics\Year\Month\Date
      2. Select Open
      3. Set Basename to something that will be descriptive of your data for example Lysozyme_AuNP_Ratio45_80C
      4. Click "Apply"
      5. Click "Exit"
  6. Start data collection
    1. After you have your sample, cuvette, and cuvette holder properly prepared perform the following:
      1. Click the "Save graph to file/files" icon (the floppy disc icon on the lower icon panel)
      2. Data files should be showing up in your selected folder. If it isn't, you have done something wrong.
  7. Shutting down
    1. When your data collection is finished, perform the following steps
      1. Shut down the OceanView software
      2. Turn off the jaz spectrophotometer
      3. Turn off the lamps (main switch in the back)
      4. Shut down the Quantum Northwest Cuvette Holder if necessary
  8. Analyzing Data
    1. Open data compilation folder.
    2. Open copy command.txt document.
    3. In data compilation folder shift right click and choose open command window here.
    4. Copy the first line from the copy command.txt document and paste into the command window. Hit enter.
    5. It will prompt “location?” Copy the location of the saved scans, paste it into the command window, and hit enter.
    6. Then copy the second line from the copy command.txt document, paste into the command window, and hit enter.
    7. It will prompt “number of files.” Enter the number of files that it has copied which will be immediately above the prompt.
    8. It will prompt “number of lines per file.” Enter 2048.
    9. It will prompt “number of lines before data.” Enter 17.
    10. It will prompt “Are you sure (Y/N)?” Enter Y.
    11. In the data compilation there will a document titled out.txt. Open out.txt and save as an excel file in the folder with the saved scans and rename to match the folder.
    12. Delete out.txt if still saved in data compilation folder.

Data Analysis

Data were imported and compiled into an Excel file. From here, the maximum absorbance and maximum wavelength over time were analyzed. The resulting data are shown in the two graphs below. Image:Max abs over time.PNG Image:Max wavelength over time.PNG

The maximum absorbance over time graphs shows the expected increase in absorbance over time as the nanoparticles form.

The maximum wavelength over time is unchanged until 3000-3120 seconds at which point there is an abrupt increase from 453.4nm to 536.54 nm.

The absorbance at 535.15 nm was taken and analyzed over time. Image:A353 over time.PNG

From this graph, the gradual increase of absorbance over time is visible.


The raw data file can be found in this Excel worksheet: Image:BSA AuNP pH4.xlsx.


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