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monochromatic
CHEM 305
b
P0 is the incident radiant power or intensity
P is the radient intensity that remains
b is the path length
Physical principle
Recall the potential energy function that we had for a diatomic molecule.
CHEM 305
T = I / I0
%T = 100 T
Absorbance
A = log10 I0 / I
A = log10 1 / T
A = log10 100 / %T
A = 2 - log10 %T
CHEM 305
Beer-Lambert Law
The Beer-Lambert law relates the absorbance to the concentration:
A=bc
where A is absorbance (no units, since A = log10 P0 / P ), is the molar
absorbtivity or extinction coefficient with units of L mol-1 cm-1, b is the path
length of the sample i.e. the path length of the cuvette in which the sample
is contained (in cm) and finally, c is the concentration of the compound in
solution, expressed in mol L-1.
Of course, we can also express transmittance in terms of concentration, but
the Beer-Lambert law is more useful because the relationship between
absorbance and concentration is linear.
CHEM 305
CHEM 305
http://www.chem.ufl.edu/~itl/3417_s98/spectroscopy/beerslaw.htm
and
Therefore, we have
log (I0/I) =
NA c b
2.303*1000
A=bc
or
with
NA
2.303*1000
This shows how the extinction coefficient is related to the cross-sectional areas of
absorption. E.g.
Absorption atoms
molecules
9 kDa protein
CHEM 305
10-12 cm2
10-16 cm2
10-17 cm2
3*108 M-1cm-1
3*104 M-1cm- 1
7*103 M-1cm- 1
if c is too
high, dilute!
CHEM 305
2 HPLC Trace
set absorbance wavelength = 280 nm
time
CHEM 305
3 Changes in configuration
CHEM 305