SCL9.

UV-vis spectroscopy
The purpose of this experiment was to use ultraviolet-visible spectroscopy to identify the spectrums which
belonged to the different known concentrations of rhodamine B solution, and then use this information to
find the extinction coefficient of rhodamine B and thus the concentration of an unknown solution.

Ultraviolet-visible spectroscopy (UV-vis) is a technique which uses radiation to promote bonding electrons
into higher energy levels. Based on the energy level, when an electron is promoted, the radiation absorbed
by that electron will vary at different wavelengths. This can be used in UV-vis spectroscopy to derive a
spectrum for a solution which can then be used to identify specific properties, such as concentration for
this experiment (Convention, 2018).
Under certain conditions, we can derive 𝐴 = 𝜖𝑐𝑑 from the Beer-Lambert law, where A is the absorbance at
a wavelength, ϵ is the molar absorptivity constant, c is the concentration and d is the path length through
the solution (Zhang, 2018). The law states that the absorbance is proportional to the concentration,
therefore the linear relationship can be used to determine molar absorptivity. Furthermore, the molar
absorptivity can be used to determine the concentration of an unknown solution.

Higher concentration solutions will have more bonding electrons to absorb the radiation, therefore more
radiation can be absorbed overall. This means the height of the peaks at a given wavelength can be used to
identify the concentrations of the solutions.

Procedure

• A 100cm³ stock solution of rhodamine B with concentration 62.6µmol/dm3 was prepared. This was
achieved by measuring 0.003g of rhodamine B powder, transferring it to a 25cm³ graduated
cylinder, washing off any remaining powder and then making it up to 25cm³ using deionised water.
The solution was transferred to a separate beaker, and then the graduated cylinder was made up
to 25cm³ and transferred to the beaker three further times, to make up the 100cm³ stock solution.
• Using a graduated pipette with the stock solution and deionised water, 20cm³ of each of the
diluted solutions (5.00µmol/dm³, 1.25µmol/dm³, 0.31µmol/dm³, 0.078µmol/dm³ and
0.019µmol/dm³) were prepared in the graduated cylinder and transferred to separate labelled
containers.
• The solutions in the containers were transferred to separate cuvettes, along with a sample of
deionised water for use as reference. The cuvettes were placed in the spectrometer for analysis,
starting with the deionised water, followed by the solution with the next lowest concentration. The
spectra produced were saved.
• A solution with an unknown concentration which was pre-prepared was also placed in the
spectrometer, so that the concentration can be determined from the spectrum produced. The
cuvettes were then placed in the spectrometer two more times for reliable results.

The spectrometer recorded spectra between a wavelength of 300-700nm, which means that the range of
the cuvettes had to be within that region for the results to be valid. Therefore, 200-800nm cuvettes were
used. The mass scale was not accurate enough for the original stock solution with a concentration of
66µmol/dm³, thus the mass required was rounded and the actual concentration of solution was calculated.
The uncertainty of the mass scale was ±0.0005g, and ±0.1cm³ for the 25cm³ graduated cylinder and
±0.0002cm³ for the graduated pipette. Using this, the maximum and minimum concentrations can be
calculated, thus, the error for each concentration is:

• Stock: 62.6 ± 10.7µmol/dm3

• 5.00 ± 0.84µmol/dm3
• 1.25 ± 0.21µmol/dm3
• 0.31 ± 0.052µmol/dm3
• 0.078 ± 0.014µmol/dm3
• 0.019 ± 0.0040µmol/dm3

Zamir Sarvari 180410101

 SCL9. UV-vis spectroscopy

The UV-vis spectroscopy spectrum of the different concentrations

of rhodamine solution
0.7 5±0.84, 554

0.6 1.25±0.21, 554

0.5 Unknown, 555

Absorbance

0.4 0.31±0.052, 555

0.3 0.078±0.014, 555

0.2
0.019±0.0040, 554
0.1

0
300 350 400 450 500 550 600 650 700
Wavelength (𝜆) / nm

5 1.25 0.31 0.078 0.019 Unknown

Knowing the greater peaks belong to solutions of greater concentration, each spectrum can be associated.
The peak wavelength for each concentration is labelled, thus it can be seen that the peak lies at 554-
555nm. The absorbance values from these wavelengths will be averaged and used to find the molar
absorptivity. The spectrum for the unknown solution can be seen between the spectra for 1.25µmol/dm³
and 0.31µmol/dm³, therefore we already know the concentration must lie between those values.

Absorbance
Concentration / µmol/dm³
Wavelength (L) / nm 5 1.25 0.31 0.078 0.019 Unknown
554 0.648 0.154 0.036 0.01 0.003 0.059
555 0.648 0.153 0.036 0.011 0.003 0.059
avg 0.648 0.1535 0.036 0.0105 0.003 0.059
520 0.228 0.055 0.014 0.004 0.001 0.021

A graph of absorbance against concentration to determine the

molar absorptivity
0.7

0.6

0.5
y = 0.1298x - 0.0026
0.4
Absrobance

0.3 554-555
520
0.2
y = 0.0456x - 0.0003
0.1

0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
-0.1
Concentration / µmol/dm³

Zamir Sarvari 180410101

 SCL9. UV-vis spectroscopy

It can be seen from the graph that the A/c value at 554-555nm is 0.1298dm³/µmol (129811.29dm³/mol)
and 0.0456dm³/µmol (45575.89dm³/mol) at 520nm. Using this, the molar absorptivity can be identified by
dividing by the path length, which in this case is 1.25cm. Therefore, the molar absorptivity is
103849.03dm³/mol/cm between 554-555nm, and 36460.72dm³/mol/cm at 520nm.

To find the concentration of the unknown sample, this molar absorptivity value must be substituted into
𝐴
the Beer-Lambert law. Rearranging the Beer-Lambert law to give concentration: 𝑐 = 𝜖𝑑, and substituting
the absorbance value given in the table, along with the molar absorptivity and path length, gives a
concentration of 0.45µmol/dm³, which fits within our range specified from before.

Percentage error:
106000 − 103849.03
( ) 𝑥100 = 2.02%
106000
(Prahl, 1998)

Conclusion

To conclude, our molar absorptivity constant value is very accurate, however it cannot be certain whether
this is due to random error. The conclusion is rather strong, since many of the errors are accounted for,
however there were many systematic errors out of our control during the experiment. The graph shows
that as the concentration increases, the intensity of the peak also increases.
The procedure had multiple errors, such as reading the apparatus using the eye, which could lead to many
potential human errors. The mass scale did not have enough accuracy for an accurate reading and it was
sensitive, and the amount of powder was very small, therefore making it difficult to get an accurate value.
It also was very difficult to maintain a stable value. This may be due to the ventilation in the room at the
time. To avoid this next time, the air conditioning must be kept off during the experiment. Although repeats
were done for the spectrometer were done, the whole experiment was only done once, which means that
our results may not be reliable.

References
Convention, T. U. S. P., 2018. Qualification of UV-VIS Spectrophotometers. Food Chemical Codex, p. 39.

Prahl, S., 1998. Rhodamine B. [Online]

Available at: https://omlc.org/spectra/PhotochemCAD/html/009.html
[Accessed 25 November 2018].

Zhang, H., 2018. SCL8-11. Nano-engineered Composites with Multi-functionalities, p. 6.

The United States Pharmacopeial Convention. (2018). Food Chemicals Codex (11th Edition) -
Qualification of UV-VIS Spectrophotometers. (pp. 39). The United States Pharmacopeial Convention.

Zamir Sarvari 180410101