# User:Melissa Novy/Notebook/CHEM-571/2012/11/14

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 Revision as of 16:46, 14 November 2012 (view source)← Previous diff Revision as of 14:38, 15 November 2012 (view source) (→Objectives)Next diff → Line 20: Line 20: ** Analyze the concentration of HRP stock solution with UV-vis. ** Analyze the concentration of HRP stock solution with UV-vis. ** The molar absorptivity coefficient of HRP at 403 nm is 1.02×105 M-1 cm-1. ** The molar absorptivity coefficient of HRP at 403 nm is 1.02×105 M-1 cm-1. - * Dialyze fractions of ADA made on x, x, and x. + * Dialyze fractions of ADA made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/03|2012/10/03]] and [[User:Melissa_Novy/Notebook/CHEM-571/2012/11/06|2012/11/06]]. ==Au-ADA Solutions== ==Au-ADA Solutions==

## Revision as of 14:38, 15 November 2012

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## Objectives

• Make Au-ADA solutions at mole ratios of Au:ADA from 60 to 150.
• Make Au-HRP solutions at mole ratios of Au:HRP from 60 to 150.
• Analyze the concentration of HRP stock solution with UV-vis.
• The molar absorptivity coefficient of HRP at 403 nm is 1.02×105 M-1 cm-1.

• Ten solutions at Au-ADA mole ratios of 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 were made.
• Please refer to Dhea Patel's entry for theoretical and actual calculations of stock solutions, and volumes of stock solutions and deionized H2O added to each solution.
• Note that ADA made on 2012/10/03 was used to make the solutions. The solution of HAuCl4 was made on 2012/09/05.

## Au-HRP Solutions

• Ten solutions at Au-HRP mole ratios of 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 were made.
• Please refer to Dhea Patel's entry for theoretical and actual calculations of stock solutions, and volumes of stock solutions and deionized H2O added to each solution.
• Note that 130, 140, 150 Au-HRP solutions were recalculated to account for a new stock solution at different concentration of HRP.
• The concentration of the new HRP stock solution was calculated from its absorbance at 403 nm and its molar absorptivity coefficient.
• Its concentration was calculated to be 28.5 μM.
```  Abs.: 2.1
Path length (b): 1 cm
Molar absorptivity coefficient (ε) = 1.02×105 M-1 cm-1
Concentration (c) = 2.1/(1 cm)(1.02×105 M-1 cm-1) = 28.5 μM
```

• Frac 1,3 of ADA, made on x, was used. Its concentration was 10.35 μM, as determined by Bradford assay.
• It was diluted to 1 ng/μL, then to 0.1 ng/μL using phosphate buffer, prepared on x.
```  Initial concentration of ADA: 10.35 μM in 1 mL of solution
(10.35×10-6)(0.001 mL) = 1.035×10-8 mol ADA
(1.035×10-8 mol)(42000 g/mol) = 0.0004347 g ADA/mL
(0.0004347 g ADA/mL)(1 mL/1000 μL)(109 ng/1 g) = 434.7 ng/μL
```
```  (434.7 ng/μL)(V1) = (1 ng/μL)(1000 μL)
V1 = 2.3 μL
(1 ng/μL)(V1) = (0.1 ng/μL)(1000 μL)
V1 = 100 μL
```