User:Melissa Novy/Notebook/CHEM-571/2012/09/19

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(Objectives)
Current revision (11:26, 9 October 2012) (view source)
(Notes)
 
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   '''Mass luminol''':
   '''Mass luminol''':
         10 mL × (10<sup>-3</sup> L/mL) × (2.5 × 10<sup>-3</sup> M) × (10<sup>-3</sup> mol/mmol) × 177.16 g/mol = 0.00443 g luminol
         10 mL × (10<sup>-3</sup> L/mL) × (2.5 × 10<sup>-3</sup> M) × (10<sup>-3</sup> mol/mmol) × 177.16 g/mol = 0.00443 g luminol
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   '''Actual mass sodium phosphate''': 0.0044 g
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   '''Actual mass luminol: 0.0044 g'''
   '''Actual concentration''':  
   '''Actual concentration''':  
         0.0044 g × (1 mol/177.16 g) × (1/0.010 L) = 2.5 × 10<sup>-3</sup> M in H<sub>2</sub>O
         0.0044 g × (1 mol/177.16 g) × (1/0.010 L) = 2.5 × 10<sup>-3</sup> M in H<sub>2</sub>O
* The concentration of the luminol stock solution was varied by serial dilution as follows:
* The concentration of the luminol stock solution was varied by serial dilution as follows:
-
** 1.25 mM, 625 μM, 312.5 μM, and 156.25 μM.  
+
** 1.25 mM, 625 μM, 312.5 μM, and 156.25 μM.
 +
** 10 mL solutions of each concentration were made.
* Notes:
* Notes:
-
** Luminol did not dissolve completely in H<sub>2</sub>O, so Na<sub>2</sub>CO<sub>3</sub> was added in a ratio of ? g for every 500 mL of solution to aid its dissolution.
+
** Luminol did not dissolve completely in H<sub>2</sub>O, so Na<sub>2</sub>CO<sub>3</sub> was added in a ratio of 4 g for every 500 mL of solution to aid its dissolution.
*** 0.08 g of Na<sub>2</sub>CO<sub>3</sub> was added to the luminol solution.  
*** 0.08 g of Na<sub>2</sub>CO<sub>3</sub> was added to the luminol solution.  
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** After the luminol dissolved, 0.48 g Na<sub>2</sub>HCO<sub>3</sub> was added to adjust the pH of the solution to about 7.
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** After the luminol dissolved, 0.48 g NaHCO<sub>3</sub> was added to adjust the pH of the solution to about 7.
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*** ? g of Na<sub>2</sub>HCO<sub>3</sub> was added to the luminol solution.
+
*** 0.48 g of NaHCO<sub>3</sub> was added to the luminol solution.
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==Notes==
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* Please refer to the [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/25|entry on 2012/09/25]] for HRP-luminol fluorescence spectra and data analysis.

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Objectives

  • Complete horseradish peroxidase enzymatic activity assay from 2012/09/18.
  • Make 2.5 mM luminol (MW 177.16) stock solution for horseradish peroxidase chemiluminescence assay.
  • Using a fluorimeter, collect emission spectra at 425 nm for horseradish peroxidase chemiluminescence assay.

Luminol Stock Solution in H2O and Carbonate Buffer

  • Calculations:
    • 10 mL of 2.5 mM luminol stock solution requires the following:
  MW luminol: 177.16 g/mol
  Mass luminol:
        10 mL × (10-3 L/mL) × (2.5 × 10-3 M) × (10-3 mol/mmol) × 177.16 g/mol = 0.00443 g luminol
  Actual mass luminol: 0.0044 g
  Actual concentration: 
        0.0044 g × (1 mol/177.16 g) × (1/0.010 L) = 2.5 × 10-3 M in H2O
  • The concentration of the luminol stock solution was varied by serial dilution as follows:
    • 1.25 mM, 625 μM, 312.5 μM, and 156.25 μM.
    • 10 mL solutions of each concentration were made.
  • Notes:
    • Luminol did not dissolve completely in H2O, so Na2CO3 was added in a ratio of 4 g for every 500 mL of solution to aid its dissolution.
      • 0.08 g of Na2CO3 was added to the luminol solution.
    • After the luminol dissolved, 0.48 g NaHCO3 was added to adjust the pH of the solution to about 7.
      • 0.48 g of NaHCO3 was added to the luminol solution.

Notes

  • Please refer to the entry on 2012/09/25 for HRP-luminol fluorescence spectra and data analysis.



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