Christian Balea, C5-4 TP-FER 19.06.

24

Spectrophotometric study of ferroin

Christian Balea
C5-4

1. Introduction
Coordination complexes occur when a central ion is surrounded by certain molecules, which in this context
are called ligands. Ligands are usually organic molecules that surround the central ion, which is generally
that of a transition metal like iron 1. The complex studied in this lab is ferroin, which is the complex formed
by Fe2+ or Fe3+ ions and the organic molecule ortho-phenanthroline. Ferroin is most stable at a pH between
4 and 52. The maximum absorbance of the complex is at a wavelength of 510 nm2.

Figure 1 Structure of ferroin, the iron-ion is surrounded by a certain number of ortho-phenanthroline-molecules that are
acting as ligands. The complex is characterized by an intense red coloration.

The main characteristic of complexes is their distinct coloration, caused by electron transitions between
orbitals after absorbing light. The electrons give off photons to return from an excited state to their ground
state(1) Benzyl Alcohol. Wikipedia; 2022.
(2) Mode Opératoire NAB.
1
. This fact makes absorption spectrophotometry an ideal analytical method to study coordination
complexes as they absorb light, which can be detected in an absorbance-spectrum.

Spectrophotometry is concerned with the intensity of light that is traveling through an analyte. The
intensity is measured before and after traversal of the absorbing medium, which then allows to measure
how much light is being absorbed by the analyte3. This analytical method allows for the calculation of
concentrations for example, although there are numerous other applications.

During this lab, UV/VIS spectroscopy was used, in which the absorbance of the complex was measured in
the UV and visible light range. The method was used firstly to determine the molar ratio of ion and ligands
in the ferroin complex. The molar extinction factor was also determined, to later allow the determination of
the concentration of an unknown sample of iron sulfate. Lastly, the kinetics of the decomposition of ferroin
in acidic environments was studied and the isobestic point was determined (which is defined as the
wavelength, at which the absorbance of a sample does not change, even when it goes through physical or
chemical changes4).
Christian Balea, C5-4 TP-FER 19.06.24

2. Experimental section
2.1. Materials
The glassware and other equipment used during this lab was the following:

- 100 mL ± 0.1 mL, 50 mL ± 0.1 mL

- 1 mL ± 0.006 mL pipette
- 2 mL ± 0.01 mL pipette
- 3 mL, 4 mL, 5 mL pipettes (error: ± 0.015 mL)
- 10 mL ± 0.02 mL
- 15 mL ± 0.03 mL
- Micropipette, error: ± 0.002 mL

The chemicals used during the whole lab were:

- Ascorbic acid
- Iron(III) chloride hexahydrate ([FeCl3] * 6 H2O)
- 1 M HCL solution
- 0.1 M NaOH solution
- 15 mM ortho-phenanthroline solution (abbreviated as phen in this report)
- Iron(II) sulfate heptahydrate (FeSO4 * 7 H2O)

2.2. Error calculations5

The following equation was used to determine the standard deviation of a series of results:

√
n

∑ (x i−x)2
i=1
S x=
N −1
Equation 1 Standard deviation of a series of results.

2.3. Determination of the stochiometric ratio of phen and iron in ferroin.

Firstly, an ascorbate buffer solution of pH 4.5 was created by weighing 2.4651 g of ascorbic acid and
dissolving it in 1400 mL of deionized water. To this solution, 100 mL of the available 0.1 M NaOH
solution were added. The whole solution was mixed with a stirring magnet and put aside.

With this buffer solution a 5 mM Fe(II) solution was prepared by weighing 0.1408 g of FeSO 4 * 7 H2O and
dissolving it in 100 mL of ascorbate buffer solution. The solution was store in a 100 mL volumetric flask.

From this Fe(II) solution, 7 different solutions were prepared by adding different volumes of the 15 mM
phen solution and diluting them all to 100 mL with the previously prepared buffer solution. The
compositions of the solutions are as follows:

Table 1 preparation of the solution to determine the molar ratio of the complex between iron and phen.
# of solution V(Fe(II)) [mL] V(phen) [mL] Vtot
1 1.5 0 100
2 1.5 0.5 100
3 1.5 1 100
4 1.5 1.5 100
5 1.5 2 100
6 1.5 2.5 100
Christian Balea, C5-4 TP-FER 19.06.24

7 1.5 3 100

After mixing the solutions, they were left for 15 minutes before being analyzed, to ensure that all the
complexation reactions had been completed. After 15 minutes, the absorbance of the solutions was
analyzed at 510 nm. A blank was also recorded, using the pure buffer solutions, to ensure more accurate
results.

2.4. Determination of the molar extinction coefficient ε of ferroin.

Firstly, 100 mL of a stock solution were prepared by mixing 15 mL of the 5 mM Fe(II) solution and 15 mL
of the 15 mM phen solution and diluting the solution to 100 mL with the buffer solution. From this stock
solution, 5 solutions were prepared with increasing concentrations according to the following table:

Table 2 Composition of the standards for the determination of the molar extinction coefficient of ferroin.
# of solution Vstock [mL] Vtot [mL]
1 1 50
2 2 50
3 3 50
4 4 50
5 5 50

The absorbance of these standard solutions was measured at 510 nm.

2.5. Determination of the concentration of ferroin of an unknown sample.

The unknown sample consisted of an unknown mass of iron sulfate. To analyze this sample, it was
dissolved in 100 mL of the ascorbate buffer solution and stored in a volumetric flask. Two solutions were
prepared from this solution by taking 1 mL of this solution and mixing it with 2 mL of the phen solution.
The two solutions diluted to 100 mL with ascorbate buffer. Finally, the absorbance of the 2 solutions was
measured at 510 nm.

2.6. Kinetic study of the decomposition of ferroin in an acidic environment.

Firstly, 100 mL of 5 mM aqueous Fe(II) solution were prepared by weighing 0.1395 g of FeSO 4 * 7 H2O
and dissolving it in 100 mL of deionized water. Then, 1.5 mL of the Fe(II) solution were mixed with 1.5
mL of phen solution to form ferroin. Again, the solutions were left for 15 min to ensure that the
complexation had completed. After 15 minutes, 10 mL of 1 M HCl solution were added to the complex
and a sample was taken to measure its absorbance at 510 nm. After this, every 30 minutes a sample was
taken from both solutions and its absorbance was measured at 510 nm. One solution was kept at room
temperature, while another was kept in a 30°C water bath.

2.7. Visualization of an isobestic point.

To begin, 100 mL of a 5 mM FeCl3 solution was prepared by weighing 0.136 g of iron chloride and
dissolving it in 100 mL of deionized water. A 10 mM aqueous solution of ascorbic acid was prepared by
dissolving 0.1764 g of acid in 100 mL of deionized water. 10 mL of this solution were taken and diluted 10
times in a 50 mL volumetric flask, resulting in the desire 10 mM concentration.

From the Fe(III) solution and the 15 mM phen solution, a solution was made, which will be called complex
solution, by mixing 10 mL of Fe(III) solution and 10 mL of phen solution and diluting it to 100 mL with
deionized water. From this solution, 5 other solutions were made by adding different volumes of ascorbic
acid solution and diluting them to 100 mL with deionized water according to this table:
Christian Balea, C5-4 TP-FER 19.06.24

Table 3 Preparation of the solutions for the visualization of an isobestic point.

# of solution Vcomplex [mL] Vascorbic acid [mL] Vtot [mL]
1 4 0 100
2 4 1 100
3 4 2 100
4 4 3 100
5 4 4 100

The absorbance of these solutions was measured from wavelengths starting at 800 nm until 300 nm and the
entire absorbance spectrum was measured for this experiment.

3. Results and discussion

3.1. Determination of the molar ratio between iron and phen in ferroin.
The absorbance values of each solution at 510 nm are compiled in the following graph:

Table 4 Absorbance at 510 nm of the 7 solutions.

# of solution Absorbance [AU]
1 0.0069
2 0.2852
3 0.5653
4 0.8448
5 0.9565
6 0.9765
7 0.97

n( phen)
From this data, the absorbance of the solutions was plotted as a function of the molar ratio :
n¿¿

Absorbance as a function of molar ratio

1.2

1
Absorbance [AU]

0.8

0.6

0.4

0.2

0
0 1 2 3 4 5 6 7
molar ratio [-]

Graph 1 Absorbance as a function of molar ratio. The orange points represent an excess of phen that can no longer react to
form ferroin, and the blue points represent the moment at which the phen is being consumed to produce ferroin.
Christian Balea, C5-4 TP-FER 19.06.24

It is known that ferroin is formed, following this reaction2:

Reaction 1 Formation of ferroin in aqueous solution. The unknown stochiometric coefficient n will be determined from the
experimental data of this experiment.

Because during the experiment, the concentration of Fe 2+ was kept constant, at a certain volume of phen
solution added, no more ferroin will be formed, as there will no longer be any Fe 2+ in solution that could
react. This means, that from a certain molar ratio, the absorbance of the ferroin solution will remain
constant as the concentration of ferroin will also remain constant. This can be seen in graph 1, from a molar
ratio of 4, the absorbance no longer increases, indicating that no more ferroin is produced. When plotting
the linear trendlines of the orange series and the blue series, the intersection has an x-value of around 3.3.
Because stochiometric coefficients are integers, it can be concluded that n is equal to 3. By comparing this
result with the chemical formula of ferroin, n is in fact equal to 3, implying that the exact chemical formula
of ferroin is [Fe (phen)3]2+.6

3.2. Determination of the molar extinction coefficient of ferroin.

The absorbances of the 5 standard solutions at 510 nm are compiled in the following table:

Table 5 Absorbances of the 5 standard solutions.

# of standard Absorbance [AU]
1 0.1555
2 0.3132
3 0.4779
4 0.6422
5 0.8055

By calculating the concentration of the 5 standards, the absorbance was plotted as a function of
concentration:

Absorbance as a function of concentra-

0.9
tion
0.8
f(x) = 1.61494285714286 x − 0.00468571428571435
0.7 R² = 0.999865271276185
Absorbance [AU]

0.6
0.5
0.4
0.3
0.2
0.1
0
0 0.1 0.2 0.3 0.4 0.5 0.6
Concentration
absorbance [mM]
Linear (absorbance)
Graph 2 Calibration curve for the determination of the molar extinction coefficient of ferroin.
Christian Balea, C5-4 TP-FER 19.06.24

The Beer-Lambert law offers a mathematical relationship between the absorption and concentration of a
compound2:

A=ε ∙l ∙ c ¿
Equation 2 The Beer-Lambert law. A is the absorbance of the sample, ε is the molar extinction coefficient which depends on
the compound that is being analyzed and l is the thickness of the traversed medium. During all experiments, l had a value of
1 cm.

Because the thickness of the cuvette was 1 cm, the molar extinction coefficient is simply the slope of the
linear trendline divided by the thickness of the cuvette. It follows, that the molar extinction coefficient is
equal to 1614.9 L*mol-1*cm-1 because absorption has no units. The R 2 unit of the linear trendline is very
close to 1, implying that the Beer-Lambert law is being closely followed by ferroin and that this calibration
curve can be confidently used to calculate the concentration of the unknown sample provided.

3.3. Determination of the ferroin concentration of an unknown sample

The absorbance of the two sample solutions was measured and the values were inserted into the calibration
curve:

Absorbance of the samples

1
0.8
Absorbance [AU]

0.6
0.4
0.2

0
0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55
Concentration [mM]

calibration curve Linear (calibration curve)

Sample 1 Sample 2
Graph 3 Absorbance of the samples inserted into linear trendline.

Using the equation of the calibration curve, the ferroin-concentrations of the two samples was calculated,
and from these values, the mass of iron sulfate was calculated. The results are compiled in the following
table:

c([Fe phen3]2+) [M] c(Fe2+) [M] n(Fe2+) [mol] m(FeSO4) [g]

Sample 1 0.000456 0.045600 0.004560 1.268
Sample 2 0.000459 0.045900 0.004590 1.276
Average 1.272
standard deviation 0.006
Table 6 Results of the determination of the mass of an unknown sample.

Due to the small standard deviation of these results, it is most probable that the result is correct. The two
absorbances of the samples are on the linear calibration curve, meaning that the ferroin concentrations have
been correctly calculated and obey the Beer-Lambert law. The only source of error is a mistake when
calculating the mass of iron sulfate.
Christian Balea, C5-4 TP-FER 19.06.24

3.4. Kinetic study of the dissociation of ferroin

It is known that the dissociation of ferroin in acidic media is of order 1, implying that the rate law can be
expressed in the following manner2:

v=k ∙ c ¿
Equation 3 Rate law of the dissociation of ferroin in an acidic environment. The rate constant k is determined experimentally
and depends on temperature.

With the measured absorbances at 30-minute time intervals, the concentration of ferroin was plotted as a
function of time:

Ferroin concentration as a function of

0.500 time
0.450
0.400
c([Fe phen3]2+ [mM]

f(x) = − 0.00277024377154416
0.000903048692385493xx++0.414948294012013
0.411381509691002
0.350 0.983213578705955
R² = 0.999257263903683
0.300
0.250
0.200
0.150
0.100
0.050
0.000
0 20 40 60 80 100 120 140

room temperature Time [min] Linear (room temperature)

30 degrees Linear (30 degrees )
Graph 4 Concentration of ferroin as a function of time. The rate constant k is the slope of the linear trendline.

According to these two graphs, the rate constant of a chemical reaction is very dependent on temperature as the
rate constant at 30°C is more than 3 times larger than the one at room temperature. To conclude these
experiments, the rate constants of the dissociation of ferroin in acidic media are -0.0009 min -1 and -0.0028 min-1
at room temperature and 30°C respectively.

3.5. Visualization of an isobestic point

To visualize an isobestic point of this iron-complex, the entire absorbance spectrum for wavelengths
between 300 and 800 nm was plotted:

Graph 5 Absorbance spectrum of ferroin. The isobestic point is the intersection of the spectra of all the
five samples.
Christian Balea, C5-4 TP-FER 19.06.24

The isobestic point is defined as a wavelength, at which the absorbance of a compound does not change,
even if it undergoes chemical or physical changes. Thus, the isobestic point of ferroin can be detected as
being the unique intersection point of all the spectra of the samples, because it is the only absorbance value
that is constant, despite the ferroin changing concentrations and thus, undergoing a physical change. It
seems to be situated around 370-380 nm.

4. Conclusion
During this lab, ferroin was studied intensely using spectrophotometry. The stochiometric ration between
phen and iron was measured to exactly determine the molecular formula of ferroin. The molar extinction
coefficient of ferroin was determined, to enable calculations of concentrations of unknown ferroin samples
using the Beer-Lambert-law. The decomposition in acidic media of ferroin was also studied from a kinetic
point of view to better understand the effect of temperature on this reaction and its kinetics in general.
Finally, an isobestic point was visualized using ferroin at different concentrations.

The results of this lab can be further expanded upon to, for example, determine the activation energy of the
decomposition of ferroin, by making more experiments as the ones described in chapter 2.5. at more
temperatures, to exactly study the temperature dependence of the rate constant. The knowledge of the
activation energy could help the development of catalysts for this reaction. The spectrometric
measurements performed can now help determine the concentration of ferroin in many samples and could
also be used to further study the kinetics of ferroin, or maybe, even study the chemical properties of
ferroin, like its behavior at different pH ranges or its behavior when reacting with other chemicals. The
isobestic point of ferroin could be used to study reaction rates of reactions involving it, as the constant
absorbance creates a reference point, that could prove itself to be very useful when performing kinetic
studies. Because of the low standard deviations of the results and the R 2 values that are mostly very close
to 1, imply that the results presented in this report are trustworthy and most probably correct.
Christian Balea, C5-4 TP-FER 19.06.24

5. Bibliography
(1) Coordination Complex. Wikipedia; 2022.
(2) Mode opératoire FER, Dr MER Anne-Sophie Chauvin, Dr Julien Andres, EPFL 2022
(3) Spectrophotometry. Wikipedia; 2022.
(4) Isosbestic Point. Wikipedia; 2022.
(5) Analyse Quantitative, Dr. Christophe Roussel, EPFL 2022
(6) Ferroin. Wikipedia; 2022.