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Spectrophotometry
Spectrophotometer CCD 2
Ref : 701 606
We note that the absorption value is negative for a wavelength less than 420 nm.
This is due to the fact that, in this area, the signal amplitude is weak and close to zero when
using a polystyrene vessel. Furthermore, the light source was able to evolve between the
calibration and the acquisition.
In order to eliminate these negative values, the light source must be lit for 20 minutes before
calibration and measurements.
Note: this does not disturb the measurement for
2.2.3 Beer-Lambert
Beer-Lambert law is often used to determine the concentration of an unknown solution.
For each experiment in this tab, it is necessary to keep the light source lit for 20
minutes before any acquisition. In fact, you will need a stabilized source in order to
compare the absorptions, at a given wavelength and for different concentrations. The
experiment may take several minutes. THE SOURCE MUST BE STABLE.
Reminders:
Beer-Lambert law allows highlighting the proportionality between the absorption of a
solution for a chosen wavelength and the concentration of this solution.
This law is specified by the following relation:
With:
l: the optical path length in cm
c: the concentration in mol.L-
Application limit:
Beer-Lambert law is valid for diluted solutions. When the concentration of solutions to be
measured is very high, the properties of the molecules evolve.
Furthermore, the spectrophotometer CCD2 is calibrated to allow absorption measurements
between 0 and 2.5 Abs.
Experiment:
The first step consists in calibrating the device. For this, it is recommended to keep the
integration time automatic adjustment.
Place your vessel filled with solvent to measure the reference spectrum. Make sure
that no filter is placed in the vessel carrier.
Note: the device also measures the black to avoid saving the optical sound that could
disturb the measurement.
Choose an operating wavelength. It can be chosen arbitrarily or depending on an
absorption experiment that would have allowed to determine the wavelength for which the
solution absorption is at a maximum.
After that, choose the name of the variable to be placed as abscissa and its unit. In general,
the variable is the concentration and its unit is mol.L
variable (temperature for example) or another unit such as mmol.L
Alternately place in the vessel carrier the tanks with solutions of known concentration.
Enter, for each solution, the value of the known variable X.
When all the absorption measurements of your known solutions are conducted, plot the
calibration curve.
Place the tank containing a solution of unknown concentration and determine its
absorption. Deduce its concentration.
Note: clicking on « measure » displays the absorption and concentration in one click!
ENGLISH
the molar extinction coefficient (in m
1
.
max
3
.mol
-1
.cm
-1
)
-1
. However, you can choose another
21
-1
.
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