MATERIALS 1. Standard PNP solution (0.1mM) 2. pH10.0 buffer 3. Unknown PNP solution ( A or B ) METHOD 1. Dilute the standard PNP solution appropriately with pH10.0 buffer ( 4mL final volume) to make 5known standards containing 10M,20M,30M,50M and 75MPNP. 2. Measure the Absorbance at the wavelength where PNP absorbs maximally (Amax, which you determined in Experiment 3-2). 3. Dilute 1mL of one of the unknowns with 3mL of buffer, and measure at the same wavelength. RESULTS AND CALCULATIONS - Plot the known concentrations of PNP vs. Amax for the data obtained with the standards. This plot is known as a standard curve. - Read the concentration of the unknown from the standard curve. QUESTIONS 3-4 Why would you want to use the wavelength of maximum absorption for this determination? 3-5 Calculate the amount of PNP in nmol present in each of the above solutions in the 4mL of the diluted PNP solutions 3-6 Calculate the concentration of the unknow PNP solution