6
transmitted. The transmittance (T) is defined as
the ratio of the transmitted intensity of the light
beam (I
t
) to the initial intensity of the light beam
(I
o
), or T= I
t
/ I
o
. The absorbance (A) is defined as
A=log (1/T). It is found that absorbance is directly
proportional to concentration: A=
ε
b c where A
is the absorbance, c is the concentration (mol/L),
b is the pathlength of the sample (cm), and
ε
is a
constant called the molar absorptivity (L/mol-cm).
This linear relationship between absorbance and
concentration is called Beer’s Law.
The concentration of an unknown sample can be
determined by using Beer’s Law and preparing a
standard curve. A standard curve is prepared by
measuring the absorbance of a series of known
standards at different concentrations. The absor-
bance vs. concentration is then plotted for each
standard (represented by x in Fig. 2). A linear
relationship between the absorbance and con-
centration should then be observed (straight line
in Fig. 2) and a linear standard curve is thus
prepared. It is then possible to determine the
concentration of an unknown sample by measur-
ing its absorbance and comparing it to the stan-
dard curve. The concentration of the unknown
can then be determined as shown by the dotted
line in Fig. 2.
Therefore, if we have a “standard” sample with a
known absorbance and concentration, it is easy
to determine an unknown concentration of the
same substance by measuring its absorbance
and applying Beer’s Law.
I
NTRODUCTION
Fig. 2 Standard Curve
(Beer’s Law)
Fig. 2 Standard Curve
(Beer’s Law)
