Conversion of absorbance to nmol p-nitrophenol/ml?

Question

I have no idea how convert my absorbance to nmol p-nitrophenol/ml, i have my values for absorbance but how do i get it in nmol X/ml? bearing in mind the value for absorbance was 405, and i used 10mM (ml) of p-PNP

also how do obtain an initial rate of reaction? is that the same of finding the gradient on a graph

Answer 1

It sounds like an enzymatic assay for phosphatases. I did that myself a long time ago.
If I remember correctly 405 should be the wavelength at which you are measuring the absorbance, unless you mean that the value was 0.405

You need to make a standard curve: measure the absorbance for different samples of p-nitrophenol of known concentration (you prepare them yourself) and then do the plot absorbance vs concentration. You find the equation for the curve by linear regression and then you can use it to find out the concentration of p-nitrophenol in your samples. Be sure to have alkaline solutions in order to have practically all of the p-nitrophenyl molecules in the p-nitophenoxy form as the anionic form is yellow and the protonated colorless.

The exact math depend on the specifics of your experimental set-up.

As for the second question it is not very clear what you mean.

Answer 2

are you asking about beers law A= ebc, if you have absorbance A, path length b, extinction coeficient e, then u find concentration in mol/L then convert to nano mol per mililiter.