User:Anneliese Faustino/Notebook/671 Nanoparticles/2016/08/31

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UV-Vis Instrument Protocol

This protocol was followed for all UV-Vis measurements and was taken from here.

  1. If Necessary
    1. Turn on the instrument (if not already done)
    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"

Methods

Stock solutions were prepared by Dr. Hartings on 160830 as such:

stock solutions

  1. The NaOH and HCl samples were made exactly as designed.
  2. AuCl3
    1. mass = 10.13 mg
    2. 10.0 mL volumetric flask
    3. concentration = 3.34 mM
  3. Fructose
    1. mass = 23.48 mg
    2. 10.0 mL volumetric flask
    3. concentration = 13.0 mM
  4. Bovine Serum Albumin
    1. mass = 22.72 mg
    2. 10.0 mL volumetric flask
    3. concentration = 34.0 mM
0 mM Fructose
pH μL of 1 mM HClstock μL of 1 mM NaOHstock μL of 1 M NaOHstock μL of AuCl3stock μL of Fructosestock μL of BSAstock μL of Water
4 500 0 0 374 0 460 3666
5 50 0 0 374 0 460 4116
6 5 0 0 374 0 460 4161
7 0 0 0 374 0 460 4166
8 0 5 0 374 0 460 4161
9 0 50 0 374 0 460 4116
10 0 500 0 374 0 460 3666
11 0 0 5 374 0 460 4161
12 0 0 50 374 0 460 4116
0.0625 mM Fructose
pH μL of 1 mM HClstock μL of 1 mM NaOHstock μL of 1 M NaOHstock μL of AuCl3stock μL of Fructosestock μL of BSAstock μL of Water
4 500 0 0 374 24 460 3642
5 50 0 0 374 24 460 4092
6 5 0 0 374 24 460 4137
7 0 0 0 374 24 460 4142
8 0 5 0 374 24 460 4137
9 0 50 0 374 24 460 4092
10 0 500 0 374 24 460 3642
11 0 0 5 374 24 460 4137
12 0 0 50 374 24 460 4092

AuNP synthesis results


pH 4 through 12 (left to right), 0.25 mM Au, 3.1 μM BSA, 0 mM fructose

20160831 mrh BSAAuNP.JPG

pH 4 through 12 (left to right), 0.25 mM Au, 3.1 μM BSA, 0.0625 mM fructose

20160831 mrh BSAFructoseAuNP.JPG

It should be noted that 0.0625 mM fructose, pH 7 formed fibers, not nanoparticles, and this sample will be redone at a later date.

The cuvette was washed with deionized water, a sodium dodecyl sulfate solution, 0.1M HCl, and again with deionized water between each sample analysis.

Data

Analyzed raw data can be found in the Excel spreadsheet File:Anneliese Faustino Analyzed Data 160831.xlsx.

The UV-Vis analysis of the Au NPs with [Fructose]= 0mM and 0.0625 mM is shown in the four graphs below. Drift was found to have occurred during the course of this three hour experiment. To correct for this, the respective absorbances at 800 nm were subtracted from each spectrum. Both the baseline corrected and non-baseline corrected spectra are shown. Samples with pH 11 and 12 were visibly clear (unlike the other samples, which were visibly violet) and had flat spectra with no maximums unlike the rest of the samples (pH 4-9), which were found to have maximums around 530 nm. No trends based on pH, absorbance, or [Fructose] were visible from the graphs.

Uv gold 0mM BC.PNG Uv gold 0mM NC.PNG

Uv gold 0.0625mM BC.PNG Uv gold 0.0625 NC.PNG


530 nm was found the be the maximum for the majority of the spectra. The A(530) of the Au NPs with [Fructose]= 0mM and 0.0625 mM is shown in the four graphs below. Both the baseline corrected and non-baseline corrected spectra are shown. Samples with pH 11 and 12 were not found to have peaks at 530 nm. No trends based on pH, absorbance, or [Fructose] were visible from the graphs.

A530 gold 0mM BC.PNG A530 gold 0mM NC.PNG

A530 gold 0.0625mM BC.PNG A530 gold 0.0625mM NC.PNG


It should be noted that pH 7 is not included in the graphs of 0.0625 mM Fructose AuNP, because it was not run (it was prepared as fibers, not NPs); this sample will be run and added to the graph on a later date.