Posted by **~christina~** on Friday, October 10, 2008 at 6:59pm.

If I have a graph of concentration in Absorbance at 400nm vs [micromolar]of para-nitrophenol

The slope would be the extinction coefficient. (y=mx) if forced through 0

The problem is what is y?

I know y is the absorbance but it's given on a paper i have as,

**y= Absorbance at lmax**
**Can someone explain what lmax means?**
I need to find the concentration of an unknown from this standard curve and I think I find it by first finding the epsilon from the graph, by using..

**1.** y=mx then:

m= # microM^-1 cm^-1

(not sure if it needs to be in M^-1 or how to convert it to that if I need it to be)

The m is the epsilon.

**The problem is that the graph's slope is 0.003653 is molar absorption coefficient supposed to be that small?**
**2.** then I take that and use it to find the concentration of the unknown solution, from beer's law, since I have the absorption of it.

**Another issue is that the sample I did measure, was of a solution that I diluted in a ration of 1ml sample to 3ml pH10 buffer. The standard curve sample of 0.1M Paranitrophenol was also diluted with buffer prior to being analyzed, with same ratio of dilution.**
First, finding the concentration of the diluted unknown is okay, as long as the absorption coefficient is right...by using A= ebc

A= known

e= calculated from graph above

c= concentration (can be found)

b= 1cm

**But how am I to find the original concentration of the unknown stock paranitrophenol if I did not analyze it and was not told to....instruction ssay to, "calculate the concentration of the diluted unknown Paranitrophenol and the original unknown stock paranitrophenol using the standard curve generated"**
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