Beer-Lambert equation

nobahar

1. The problem statement, all variables and given/known data
I need to use the Lambert-Beer equation to plot optical density of a lactate solution.


2. Relevant equations
[tex]A=\varepsilon\ell C[/tex]
(The path length is 1 cm)

3. The attempt at a solution
This is one of my four samples (if you can show me where I'm going wrong with this then I can apply the correction to the others):
2.22 [tex]\mu[/tex]mol in 1.5ml, therefore there is 0.296 [tex]\mu[/tex]mol in 0.2ml
This info seems to match to the example graph I have.
But using the formula above, where [tex]\varepsilon[/tex]=6.22 for micromol/ml, I get:
6.22 x (0.296/0.2) micromol/ml = 9.2056
This doesn't match up with the graph I have! Any help appreciated! Please!

Ygggdrasil

Beer's law is accurate only for values of A from about 0.1 to 1.0. Experimentally, it is difficult to accurately measure absorption values outside this range.

nobahar

That's the main problem, the results from the example graph are at a maximum of 0.4 for the absorption at 340nm (y-axis) when plotted against amount in micromoles up to 0.4 on the x-axis.
I can get a fairly close approximation (the graph itself is supposed to be an estimate) if I use the following formula:
(amount in micromoles x amount in ml) x 6.22. But this equation makes no sense; or at least I can't see the reasoning behind it.

Ygggdrasil

The slope of the best fit line to the graph should give you ε.