User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/11/27

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Objective

  • to analyze the AuHRP and AuADA solutions made on November 14th using both UV-vis and AAS.
  • to prepare AuADA solutions using dialyzed ADA (the ADA was dialyzed from November 14th until November 27th).
  • to resuspend AuHRP fibers in Tris Buffer

Description

Analyzing AuHRP and AuADA Mixtures

  • UV-vis
    • 3mL of each sample were pipetted using a Pipetman into a quartz cuvette and placed in the UV-vis.
    • The data was collected, corrected for water, and plotted. Please see below under "Data" for the graph.
  • AAS
    • HCl was used as a blank and the AAS was auto-corrected for water.
    • 8 standards were used: 5ppm Au, 10ppm Au, 15pp Au, 20ppm Au, 25ppm Au, 30ppm Au, 35ppm Au, and 40ppm Au.
    • Each sample of AuHRP and AuADA was run and the measured concentration was collected and plotted.

"Preparing AuADA solutions"

  • Please refer to Kay's entry for details on removing the ADA from the snake-skin dialysis tubing and preparing the AuADA solutions.
    • Note that in preparing the AuADA solutions, the only difference in preparation from that of November 14th was the use of dialyzed ADA, rather than the purified ADA solution.

"Resuspension of Protein Fibers in Tris Buffer"

  • Please refer to Kay's entry for details on fiber resuspension, including Tris buffer volumes and concentrations.
    • Tris buffer was added to 130, 140, and 150 molar ratio Au:HRP fibers and allowed to sit over night.

Data/Observations

UV-VIS

Image:UV-Vis_Spectra_of_AuADA_11142012.JPG

  • The spectra show a consistent peak at 520 nm, indicating the presence of AuNPs.
  • There was a direct relationship between the absorbance and the mole ratio of Au:HRP. As the mole ratio increased, the absorbance increased.
  • The 130 AuHRP solution contained 1 mL less of H2O due to an error during preparation of the solution, which may account for the similarity in absorbance peake for 130 and 140 mole ratio Au:HRP solutions.

Image:Absorbance_at_525nm_versus_60_to_150_Mole_Ratio_of_AuHRP_samples.jpg

  • This graph plots the absorbance values at 525nm (where the peak was detected) against the mole ratios of the AuHRP solutions.
  • This graph better displays the direct relationship between peak absorbance and mole ratio.

Image:UV-Vis_Spectra_of_AuADA.JPG

  • No peaks were formed in this spectra, indicating the absence of AuNPs.
  • The solutions themselves were colorless, so these results were expected.
  • The ADA used in these solutions was pre-dialysis, so the ADA was in elution buffer containing high levels of imidazole, which may have interfered with the AuNP formation.

AAS

Standard HCl sample Concentration of HCl [ppm] Absorbance(Abs.)
150.0934
280.144
3100.1751
4150.2543
5200.3375
6250.4134
7300.4774
8400.6225
  • This table shows the absorbance values of each of the standard used for AAS.
Au/HRP samples Absorbance(Abs.) Concentration[ppm]
600.08273.8088
700.10425.2282
800.11025.6243
900.14287.7765
1000.15848.8064
1100.1669.3081
1200.190110.8992
1300.275416.5305
1400.214412.5034
1500.247314.6754
  • This table lists the absorbance of each AuHRP sample and its Au concentration in ppm.

  • This graph plots the concentration of gold, in ppm, in the AuHRP solutions against the Au:HRP mole ratios. This confirms the results from the UV-vis that showed the direct relationship between the concentration of gold and the mole ratio of Au:HRP in the solutions. However, because the concentration of gold is being measured in ppm and the concentration of gold was the variable when making the solutions, the %difference concentration needs to be plotted against the mole ratio.


Au/ADA samples Absorbance(Abs.) Concentration[ppm]
600.04171.1021
700.05712.1188
800.06052.3432
900.07613.3731
1000.10415.2261
1100.08283.8154
1200.08543.9871
1300.1025.083
1400.1125.7431
1500.1166.0072
  • This table lists the absorbance of each AuADA sample and its Au concentration in ppm.

  • This graph plots the concentration of gold, in ppm, in the AuADA solutions against the Au:ADA mole ratios.
  • There is an outlier peak at 100 Au:ADA, with a concentration of 5.2261ppm.



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