31
Concentration Experiments
The absorbance of a solution is related to the concentration of the species within it. The relationship, known
as Beer’s Law, is:
A
λ
=
ε
λ
c
l
where
A
is the absorbance at wavelength
λ
,
ε
is the extinction coefficient of the absorbing species
at wavelength
λ
, c is the concentration and
l
is the optical pathlength.
Concentration is the amount of a specified substance in a solution. Graphs of absorbance vs. concentration
are known as Beer’s Law plots. These are prepared by measuring the light absorbed by a series of solutions
with different
known
concentrations. The length of the sample -- such as the path length of our cuvette
holder -- and the wavelength chosen for monitoring the amount of light absorbed are constants. A linear
plot from taking scans of these standard solutions with known concentrations is then obtained. The plot is
then used to determine the
unknown
concentrations of substances in solutions.
In order to discover the unknown concentration of a substance in a solution, you must first take spectral
scans of a series of solutions with different known concentrations of the same substance. You begin this
process by taking an absorbance spectrum of the solution with the highest known concentration.
1.
Select
Scope
under
Mode of Operation
in the software display area. Make sure the signal is on scale.
Adjust acquisition parameters so that the peak intensity of the reference signal is about 3500 counts.
Take a reference spectrum by first making sure nothing is blocking the light path going to your
spectrometer. The solution with the highest known concentration you want to measure must be absent
while taking a reference spectrum. Take the reference reading by clicking the
Reference
button in the
software display area. To save the spectrum, select
File | Save Spectral Values
from the menu.
2.
While still in
Scope Mode
, take a dark spectrum by first completely blocking the light path going to
your spectrometer. (If possible, do not turn off the light source. If you must turn off your light source
to store a dark spectrum, make sure to allow enough time for the lamp to warm up before continuing
your experiment.) Take the dark reading by clicking the
Dark
button in the software display area. To
save the spectrum, select
File | Save Spectral Values
from the menu.
3.
Take the solution with the highest known concentration and put it in the cuvette holder. Make sure
nothing is blocking the light going to your sample. Then select
Absorbance
under
Mode of
Operation
. Click on the
Scan
button in the display area to take a scan. Make sure
Single
is selected.
To save the spectrum, select
File | Save Spectral Values
from the menu.
4.
Now select the wavelength for monitoring the concentration of your solutions by choosing
Spectrometer
| Select Concentration Wavelength
from the menu. Move the cursor to the highest absorbance peak of
the spectrum of the solution with the highest known concentration and choose
Select
.
5.
Remove the solution with the highest known concentration. Select
Spectrometer | Calculate a
Calibration Curve
from the menu. The Calculate Calibration Curve dialog box opens. Now you will
begin taking scans of the rest of your series of standard solutions with known concentrations, from the
lowest known concentration to highest, all while working in this dialog box.
6.
If you wish, you can take a new reference and a new dark scan for each solution by choosing
Scan |
Dark
and
Scan | Reference
from the menu of this dialog box. However, in this case, it is not
necessary. If no reference or dark scan is taken at this point, the software will use the reference and
dark scans taken in Steps 1 and 2 to calculate absorbance.
7.
Take the solution with the lowest known concentration and put it in the cuvette holder. Enter the known
concentration of the standard solution in the chart in the
Concentration
column, next to
Solution #1
.
