# User:Melissa Novy/Notebook/CHEM-571/2012/09/18

(Difference between revisions)
 Revision as of 20:44, 18 September 2012 (view source)← Previous diff Revision as of 22:36, 18 September 2012 (view source)Next diff → Line 14: Line 14: |colspan="2"| |colspan="2"| - =Objectives== + ==Objectives== * Make stock solutions of sodium phosphate, 4-aminoantipyrine, H2O2, and horseradish peroxidase. * Make stock solutions of sodium phosphate, 4-aminoantipyrine, H2O2, and horseradish peroxidase. * Perform an assay on the enzymatic rate of horseradish peroxidase. * Perform an assay on the enzymatic rate of horseradish peroxidase. + ** Vary the concentration of H2O2, while keeping the concentration of AAP constant, then vary the concentration of APP while keeping the concentration of H2O2 constant. ==0.2 M Sodium Phosphate Buffer in H2O== ==0.2 M Sodium Phosphate Buffer in H2O==

## Revision as of 22:36, 18 September 2012

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## Objectives

• Make stock solutions of sodium phosphate, 4-aminoantipyrine, H2O2, and horseradish peroxidase.
• Perform an assay on the enzymatic rate of horseradish peroxidase.
• Vary the concentration of H2O2, while keeping the concentration of AAP constant, then vary the concentration of APP while keeping the concentration of H2O2 constant.

## 0.2 M Sodium Phosphate Buffer in H2O

• Calculations:
• 250 mL of 0.2 M sodium phosphate stock solution requires the following:
MW sodium phosphate: 163.94 g/mol
Mass sodium phosphate:
250 mL × (10-3 L/mL) × 0.2 M × (10-3 mol/mmol) × 163.94 g/mol = 13.4035 g sodium phosphate
Actual mass sodium phosphate: 13.4031 g
Actual concentration:
13.4031 g × (1 mol/163.94 g) × (1/0.25 L) = 0.327 M in H2O
• 13.4031 g of sodium phosphate was dissolved in about 200 mL of H2O.
• The pH was adjusted to 7.00 by adding 20 mL of 1 N HCl dropwise.
• The solution was then diluted to a final volume of 250 mL with additional H2O.

## Spectrophotometric Analysis of Horseradish Peroxidase Enzyme Activity Rate at 510 nm

• Final concentrations of stock solutions in total volume of 1.5 mL:
 Stock Solution Initial Concentration, M1 [M] Volume Added V1 [mL] Final Concentration, M2 [M] H2O2 0.000034 0.75 0.000017 AAP 0.0025 0.7 0.001166667 Phenol 0.018 0.01 0.00012
• The concentration of H2O2 was varied by serial dilution as follows, while the concentrations of all other solutions were kept constant:
• 8.5 μM, 4.25 μM, 2.125 μM, 1.0625 μM.