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Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
School Name: Nesbru videregående skole
School number: 000706
Candidate name: Monika Nikolaisen
Candidate session number: 000706-019
Research Question:
Investigating the effect of central ion on the absorption and chemiluminescence
of protoporphyrin IX.
IB Subject of Essay: Chemistry
Name of Supervisor: May-Britt Stjerna
Word Count: 3863
Examination session: May 2012
1
Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
ABSTRACT
The research question for this extended essay is as follows: Investigating the effect of central ion on
the absorption and chemiluminescence of protoporphyrin IX. To answer the research question, an
investigation with a four step experimental method was carried out in which zinc and magnesium
central metal ions were introduced to protoporphyrin IX. Firstly, protoporphyrin IX was extracted
from brown eggshells, and secondly, its absorption spectrum was obtained. In the third step,
solutions of Zn2+ and Mg2+ ions were added to separate solutions of protoporphyrin IX to try to
form zinc protoporphyrin IX and magnesium protoporphyrin IX. Their absorption spectra were
obtained and compared to that of protoporphyrin IX and existing sources. Finally, the
chemiluminescent reaction of the substances were performed. The spectra showed that zinc
protoporphyrin IX and magnesium protoporphyrin IX had not been obtained, and there was no
chemiluminescent reaction of the substances. Thus, it was not possible to change the absorption
wavelengths of protoporphyrin IX by introducing central ions of Zn2+ and Mg2+ with the method
used in the experiment. Whether or not the change in structure has an effect on the absorption and
chemiluminescence of protoporphyrin IX is therefore unknown. However, an investigation of
spectroscopic and molecular theory in relation to protoporphyrin IX lead to the conclusion that
forming complex ions from protoporphyrin IX with Zn2+ and Mg2+ central metal ions has an effect
on the absorption and chemiluminescence of protoporphyrin IX.
[Word count: 236]
TABLE OF CONTENTS
Page Number
Introduction......................................................................................................................................4
Background theory...........................................................................................................................4
2
Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
Ultraviolet-visible spectroscopy and fluorescence spectroscopy of protoporphyrin IX....................5
The effect of central ion on the absorption of protoporphyrin IX......................................................6
The effect of central ion on the chemiluminescence of protoporphyrin IX.......................................8
Method.............................................................................................................................................10
Results..............................................................................................................................................12
Observation.......................................................................................................................................14
Conclusion.......................................................................................................................................14
Limitations........................................................................................................................................15
Further development and questions..................................................................................................15
Evaluation........................................................................................................................................16
Appendix..........................................................................................................................................16
Bibliography.....................................................................................................................................17
INTRODUCTION
Protoporphyrin IX, C34H34N4O4, is a highly absorbent and fluorescent substance that emits a deep
red glow in its chemiluminescent reaction. When light passes through the molecule, some of the
light is absorbed as electrons within the molecule gain energy by the light and are excited to a
higher energy level. This is the process of absorption. When the electrons return to their initial
energy level, the absorbed energy is emitted as photons of light, and this is known as fluorescence.
The light has distinctive wavelengths, hence colours, that are determined by the difference between
the energy levels in the molecule. The difference between the energy levels in protoporphyrin IX
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Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
renders the substance red. The chemiluminescence of protoporphyrin IX involves light being
emitted from the substance as a result of a chemical reaction, and this process is connected to the
absorption and fluorescence of the substance.
Research has been done on the absorbent and fluorescent properties of protoporphyrin IX, and there
is one experiment in particular which focuses on explaining UV-vis spectroscopy, fluorescence
spectroscopy and chemiluminescence through the study of protoporphyrin IX.1 Studying these
phenomena, a curious question came to mind; what determines the wavelengths of the light emitted
in the chemiluminescent reaction of protoporphyrin IX, and what can be done to change these
wavelengths? From this, a research question developed: Investigating the effect of central ion on the
absorption and chemiluminescence of protoporphyrin IX. The research question allows for an in
depth study of the chemistry behind protoporphyrin IX's absorbing, fluorescent and
chemiluminescent properties and their connections from an angling that has yet to be approached by
any available literature, that is, investigating the effect on absorption and chemiluminescence after
introducing a central metal ion to the molecule. There are biochemical processes in which zinc
protoporphyrin IX and magnesium protoporphyrin IX are important participants. Zinc is for
example incorporated as the central metal ion in heme in red blood cells when there is a deficiency
of iron or when lead prevents the iron from taking its place as the central metal ion, and the
structure of chlorophyll is derived from magnesium protoporphyrin IX
BACKGROUND THEORY
This experiment will only use UV-vis spectroscopy, which measures absorption wavelengths, and
not fluorescence wavelengths. This is because the school laboratory did not have a fluorescence
spectrometer. Instead, the connections between the absorption and fluorescence of protoporphyrin
IX will be discussed, and an attempt will be made to explain that the fluorescence and
chemiluminescence wavelengths will change as a result of changed absorption wavelengths.
Because of the relationship between absorption, fluorescence and chemiluminescence, it is
important to discuss fluorescence even though no measurements will be done on this in the
experiment.
Ultraviolet-visible spectroscopy and fluorescence spectroscopy of protoporphyrin IX
Ultraviolet-visible spectroscopy measures the absorption of a substance in the ultraviolet and visible
part of the electromagnetic spectrum. There are different energy levels within a molecule, and the
difference between the energy levels determines the wavelengths of absorption. The greater the
difference, the bigger the energy difference and hence the shorter the wavelength and vice versa. As
each wavelength corresponds to a colour, the difference between the energy levels also determines
the colour of the light emitted. In the protoporphyrin IX molecule there are 22 conjugated πelectrons, and this is what gives the substance its absorbent and fluorescent properties, as well as
determining its wavelengths of absorption and emission. Conjugated π-electrons refer to electrons
that can move freely in a delocalized π-bond resulting from the overlap of p-orbitals of the carbon
atoms.
Fluorescence spectroscopy measures the fluorescence wavelengths of a substance. There is a
1
J. Chem. Educ., 2011, 88 (6), pp 788-792 by Michelle L. Dean, Tyson A. Miller and Christian
4
Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
relationship between the colour, and hence wavelengths of light absorbed by and emitted from a
molecule. When light has been absorbed, the emitted light will take on the complementary colour of
the one which was absorbed. The colour of protoporphyrin IX is red, tending towards orange, hence
the colour of the light absorbed by protoporphyrin IX is blue and cyan, which is confirmed by
looking at its absorption spectrum. Since the light is emitted and absorbed over a range of colours,
protoporphyrin IX also absorbs purple and green wavelengths. Thus there is an interchangeable
relationship between the colour absorbed and the colour emitted by protoporphyrin IX, which
means that if the wavelengths of light absorbed by protoporphyrin IX changes with a central ion,
the wavelengths of emission will change as well, even though these cannot be measured with
fluorescence spectroscopy in this experiment. Following is the absorption and fluorescence spectra
of protoporphyrin IX:
Figure 6:
UV-vis spectrum and fluorescence spectrum of protoporphyrin IX
The x-axis measures the wavelengths absorbed/emitted in nm, and the y-axis
measures the intensity of the absorption/emission. The illustration shows the UV-vis
absorption (solid trace) and fluorescence (dotted trace) spectra of a commercial
sample of protoporphyrin IX in ethyl acetate/MeOH. The most intense absorption
wavelength maximum for protoporphyrin IX is 407 nm, and its most intense
emission wavelength maximum is 635 nm
1
The effect of central ion on the absorption of protoporphyrin IX
Zn2+ and Mg2+ cations react with protoporphyrin IX to form complex ions of zinc protoporphyrin
IX and magnesium protoporphyrin IX with zinc and magnesium as central metal ions. The central
ion may interact with the electrons in the delocalized π-bond in a way which changes the energy
levels of protoporphyrin IX, thus its absorption and fluorescence wavelengths as well. The addition
of a central ion to protoporphyrin IX to form a complex ion is illustrated from figure 1 to figure 2
and 3:
1
Figure 6: Supplemental material to Egg-citing! Isolation of Protoporphyrin IX from Brown Eggshells and its Detection
by Optical Spectroscopy and Chemiluminescence by Michelle L. Dean, Tyson A. Miller and Christian Brückner
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Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
Figure 1: Protoporphyrin IX
Figure 2: Zinc protoporphyrin
IX
Figure 3: Magnesium
protoporphyrin IX
Figure 11, Figure 22, Figure 33.
Zinc protoporphyrin IX and magnesium protoporphyrin IX are complex ions. A complex ion is an
ion which has a central metal ion bonded to surrounding atoms known as ligands. When cations of
Zn2+ or Mg2+ react with protoporphyrin IX, they accept pairs of electrons from nitrogen atoms
which were previously bonded to a hydrogen atom. This way, the nitrogen atoms act as ligands. The
bonds between the central metal ion and the nitrogen atoms is a dative covalent bond, meaning that
both of the shared electrons come from the nitrogen atoms. This electron sharing is similar for all
complex ions, varying with the central metal ion and the number of surrounding atoms, otherwise
known as the coordination number. Metals that can behave as the central metal ion in complex ions
are those with low energy unfilled d- and p-orbitals that can accept lone pairs of electrons from
ligands. Magnesium has unfilled 3p orbitals, and can therefore act as the central ion of a complex
ion. Zinc has an unfilled 4p orbital that can bond to ligands.
One can determine whether or not the difference in energy levels in the protoporphyrin IX molecule
has increased or decreased as a result of the central ion by looking at its absorption spectrum. The
bigger the difference between the energy level to which an electron is prompted from its initial state,
the more energy the emitted photon of light has when the electron returns to its initial state. The
farther to the right in the spectrum the wavelengths are, the greater the wavelengths are, thus they
also carry less energy. This means that, if the wavelengths have decreased with a central ion, the
difference between the energy levels between which the electron has been excited has increased. If
the wavelengths have increased, the difference between the energy levels has decreased.
The most intense maximum absorption wavelength of protoporphyrin IX is 407 nm. A source says
that the most intense maximum absorption wavelength of zinc protoporphyrin IX is 426 nm4. If this
is true, there will be a bathochromic shift, a shift to longer wavelengths, for zinc protoporphyrin IX.
This means that the difference between the energy levels in the protoporphyrin IX molecule
1
Figure 1: SUPPLEMENTAL MATERIAL to Egg-citing! Isolation of Protoporphyrin IX from Brown Eggshells and its
Detection by Optical Spectroscopy and Chemiluminescence by Michelle L. Dean, Tyson A. Miller and Christian
Brückner
2
Figure 2: http://en.wikipedia.org/wiki/File:Zinc_protoporphyrin.svg
3
Figure 3: Figure 3 was made from Figure 2 in Adobe Photoshop CS4
4
http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=282820|SIAL&N5=SEARCH_CONCAT_PNO|
BRAND_KEY&F=SPEC
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Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
between which the electrons are excited decreases. No sources were found which mention the
maximum absorption wavelength of magnesium protoporphyrin IX, hence the nature of its
absorption shift and its effect on the difference between the energy levels within the protoporphyrin
IX molecule may be determined in the experiment. The study of the absorption shifts of the
complex ions in relation to energy levels is important as it demonstrates whether or not the
difference between the energy levels in the protoporphyrin IX molecule has decreased or increased
as a result of the central ion, thus illustrating an effect of central ion on the absorption of
protoporphyrin IX.
The effect of central ion on the chemiluminescence of protoporphyrin IX
When studying the colour of the light emitted in the chemiluminescent reaction of protoporphyrin
IX, its connection to absorption and fluorescence is important. Just as there is a connection between
the absorption and fluorescence of a substance, there is a connection between the fluorescence and
the chemiluminescence of a substance. The fluorescence of protoporphyrin IX is thus imperative for
the study and explanation of the chemiluminescence of protoporphyrin IX. In the fluorescence of a
substance, light is emitted after a gain of energy through absorption, whilst in the
chemiluminescence of a substance, light is emitted after a gain of energy through a chemical
reaction. The difference in the energy levels between which electrons are excited in the
protoporphyrin IX molecule is the same in both its fluorescence and chemiluminescent reaction, and
the emitted light has the same colour. Thus if one changes the fluorescence of protoporphyrin IX, its
chemiluminescence will change accordingly. By changing the absorption wavelengths of
protoporphyrin IX with a central ion, thus its fluorescence wavelengths, the colour of light emitted
in its chemiluminescent reaction will change as well.
In the chemiluminescent reaction of protoporphyrin IX, TCPO (bis(2,4,6-trichlorophenyl)
ethanedioate) reacts with hydrogen peroxide to form phenol and cyclic peroxide. Protoporphyrin IX
is added to the reaction and the molecules are exited by the cyclic peroxide. The exited molecules
return to their ground state and photons of light are released. By replacing protoporphyrin IX with
zinc protoporphyrin IX and magnesium protoporphyrin IX, the object is to investigate if there is a
change in the colour of the emitted light as a result of changed absorption and fluorescence
wavelengths. The chemiluminescent reaction of protoporphyrin IX is illustrated by a process of
three steps:
1. TCPO (Bis(2,4,6-trichlorophenyl) ethanedioate) reacts with hydrogen peroxide to form
phenol and cyclic peroxide (1,2-dioxethane-3,4-dione)
Bis(2,4,6-trichlorophenyl) ethanedioate + hydrogen peroxide =>
2 phenol + cyclic peroxide
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Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
Figure 4: Formation of phenol and cyclic peroxide from TCPO and hydrogen peroxide
The reaction is initiated in order to form cyclic peroxide, which is needed in the next step to excite
the protoporphyrin IX molecule
1
2. Cyclic peroxide reacts with protoporphyrin IX to form CO2 and an excited protoporphyrin
IX molecule
Cyclic peroxide + protoporphyrin IX => 2CO2 + protoporphyrin IX*
Figure 5: Cyclic peroxide reacts with protoporphyrin IX to form an excited protoporphyrin IX
molecule and 2 CO2 molecules
The cyclic peroxide molecule is unstable, and decomposes to CO2. The excess energy excites the
protoporphyrin IX molecule, meaning that electrons within the molecule are excited from a lower
energy level to a higher energy level. The excited molecule is represented by *
2
3. The emission of a photon from the excited protoporphyrin IX molecule
Protoporphyrin IX* => Protoporphyrin IX + light
When the electron that has been excited to a higher energy level returns to its ground state, a photon
of light is emitted. The sum of all photons emitted from the protoporphyrin IX molecules is seen as
1
Figure 4 was made using Figure 1, ChemSketch and Adobe Photoshop CS4
Figure 5 was made using Figure 1, ChemSketch and Adobe Photoshop CS4
2
8
Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
a deep, red glow.
Zn2+ and Mg2+ ions are added to two different solutions of protoporphyrin IX and the
chemiluminescent reaction of protoporphyrin IX is repeated, replacing step two of the reaction with
1: Zinc protoporphyrin IX and 2: Magnesium protoporphyrin IX. This gives step 3:
1: Zinc protoporphyrin IX* => Zinc protoporphyrin IX + light
2: Magnesium protoporphyrin IX* => Magnesium protoporphyrin IX + light
Although the wavelengths of light emitted may change with the central ion, this may not be spotted
with the unaided eye. This is because the absorption wavelengths of protoporphyrin IX only change
slightly (19 nano meters with the zinc ligand), and since the fluorescence wavelengths of zinc
protoporphyrin IX correspond to a colour of red, as with the protoporphyrin IX molecule, a change
in the colour emitted in the chemiluminescence reaction is unlikely to be seen, thus making the
absorption spectra of the new solutions the key to determine changes in chemiluminescence.
METHOD1
Step 1: Extraction of protoporphyrin IX
As a safety precaution, this module was performed in a fume hood.
1) About 12 g of brown eggshells were weighed out and dried.
2) 50 mL of 2 M hydrochloric acid were added to a 250 mL Erlenmeyer flask.
3) 25 mL of ethyl acetate were added into the Erlenmeyer flask, followed by the eggshells.
4) The mixture sat for 15-20 minutes. The pH of the solution was checked to confirm that it was
close to neutral.
5)The organic phase was filtered, washed and dried with anhydrous sodium sulphate. 2x5 mL of
methanol was added.
6) The final volume of the porphyrin extract was recorded.
Step 2: Identification of protoporphyrin IX by UV-vis spectroscopy
Before the procedure began, the UV-vis spectrometer was connected to the computer. The computer
1
The experimental method used in this extended essay is an adaptation of the experimental method in the laboratory
experiment Egg-Citing! Isolation of Protoporphyrin IX from Brown Eggshells and Its Detection by Optical
Spectroscopy and Chemiluminescence (see bibliography for source). For detailed information on the chemicals and
materials used in the method, see apparatus list in appendix. The adaptations to the original method are also stated in
the appendix.
9
Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
programme Quantum Spectroscopy Software was used to obtain the spectrum.
1) A black block was placed into the cell holder of the UV-vis spectrometer to determine the dark
spectrum, after which a cuvette of ethyl acetate was placed into the cell holder to determine the
reference spectrum.
2) A cuvette of the porphyrin extract was placed into the cell holder of the UV-vis spectrometer, and
its absorption spectrum (in the range 350-900 nm) was obtained.
Step 3: Addition of Zn2+ and Mg2+ ions to protoporphyrin IX to form zinc protoporphyrin IX
and magnesium protoporphyrin IX and identification of the solutions by UV-vis spectroscopy
1) 3 grams of of magnesium nitrate was dissolved in 100 ml of ethyl acetate. The excess magnesium
nitrate was filtrated out of the solution. 2 mL of this solution was then added to 2 mL of the
porphyrin extract.
The same procedure was repeated by replacing magnesium nitrate with zinc nitrate. After the
cations had been added to the protoporphyrin IX extract, the UV-vis spectra of the solutions were
obtained. This was done in the same manner as that in Step 2.
Step 4: Chemiluminescence reaction of protoporphyrin IX
1) 5 mL of the protoporphyrin IX extract was poured into a large test tube and a pellet of KOH was
added to increase the intensity of the reaction.
2) 1 mL of 30% hydrogen peroxide was added to the solution
3) The room was darkened in order to observe the chemiluminescent reaction
4) 2 mL of a 7 mM TCPO solution was added. The TCPO solution was made by adding 10 mL of
ethyl acetate into a 25 mL Erlenmeyer flask, and then adding 0,030g of TCPO.
Step 4 was repeated by replacing the protoporphyrin IX solution with 1: the zinc protoporphyrin IX
solution and 2: the magnesium protoporphyrin IX solution.
RESULTS
Step 1
The protoporphyrin IX was successfully extracted from the eggshells. The step was preformed
several times, and the spectra were equal except for in their intensities, which could have been
caused by variations in pigment contents of the eggs, as some were browner than others.
10
Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
Step 2
The UV-vis spectra of protoporphyrin IX was obtained. To have a fixed point of comparison, the
same protoporphyrin IX solution was used to react with zinc and magnesium cations as well as in
the chemiluminescent reaction. Following is the spectrum of the solution:
Figure 7: UV-vis absorption spectrum of protoporphyrin IX
The y-axis measures intensity of absorption and the x-axis measures wavelengths in nano
meters. The graph corresponds well to Figure 6; the spectrum of the commercial sample.
Coordinates of the maximum absorption wavelengths:
(404, 1,29), (500, 0,41), (533, 0,23), (571, 0,14)
Step 3
The colour of the protoporphyrin IX solution turned yellow, nearly colourless, when the
protoporphyrin IX solutions were reacted with the cations to form complex ions, and a precipitate
was formed for the addition of both Zn2+ and Mg2+ to protoporphyrin IX. The spectra of the
solutions show slight shifts to the left compared to the spectrum of protoporphyrin IX:
Figure 8: Protoporphyrin IX solution + Zn2+
The y-axis measures intensity of absorption and the x-axis measures wavelengths in nano
meters.
11
Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
Figure 9: Protoporphyrin IX solution + Mg2+
The y-axis measures intensity of absorption and the x-axis measures wavelengths in nano
meters.
1
Absorption wavelength maximums of the different solutions:
Table 10: Absorption wavelength maximums and their respective intensities for
the different solutions
Step 4
The protoporphyrin IX solution emitted a red glow in its chemiluminescent reaction, which lasted
for a couple of seconds. Since there had been no signs to indicate that zinc protoporphyrin IX and
magnesium protoporphyrin IX had formed, the significance of testing whether or not they emitted
light in a chemiluminescent reaction was little. Nevertheless, it was tried, and the assumptions were
confirmed as no glow was observed in the reaction.
Observation
There seems to be a similar pattern in the change of wavelengths for protoporphyrin IX + Zn2+ and
protoporphyrin + Mg2+, as they both shifted slightly to the left, except for that the wavelengths of
protoporphyrin IX + Zn2+ shifted a bit more to the left than protoporphyrin IX + Mg2+. The fact that
the shifts are the same is not the curious change, seeing as the absorption wavelengths for
magnesium protoporphyrin IX are unknown, and therefore might very well be similar to that of zinc
protoporphyrin IX. What is worth noting is that the absorption wavelength maximum of the
protoporphyrin IX + Zn2+ solution is not in the vicinity of what zinc protoporphyrin is sourced to be,
426 nm. It has in fact shifted the opposite way, which questions the results after adding Zn2+ to
protoporphyrin IX. Since the spectrum of protoporphyrin IX + Zn2+ is not close to 426 nm and the
1
The spectra in Figure 7, 8 and 9 were obtained from the computer programme Quantum Spectroscopy Software
12
Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
same change occurred for protoporphyrin IX + Mg2+, it is likely that magnesium protoporphyrin IX
was not formed either.
The results from the experiment indicate that it is not possible, with the method used in the
experiment, to form zinc protoporphyrin IX and magnesium protoporphyrin IX by adding Zn2+ and
Mg2+ cations to protoporphyrin IX. Thus it is not possible to prove the effects of central ion on the
absorption and chemiluminescence of protoporphyrin. The absorption wavelength maximums of
magnesium protoporphyrin IX are therefore still unknown, as well as the nature of its absorption
shift as compared to protoporphyrin IX. Furthermore, the absorption wavelengths of zinc
protoporphyrin IX could not be confirmed.
CONCLUSION
As stated in the results, in the experiment it was not possible to form zinc protoporphyrin IX or
magnesium protoporphyrin IX by adding solutions of these ions to protoporphyrin IX solutions.
This indicates that perhaps another method is required to form the solutions. Unfortunately, this also
leaves one in the dark about the effect of central ion on the absorption wavelengths and
chemiluminescene of protoporphyrin IX. The theory and evidence presented does, however, suggest
that there may be an effect of Zn2+ and Mg2+ central metal ions on the absorption and
chemiluminescence of protoporphyrin IX. It is likely that the central ion interacts with the
delocalized π-bond, and as a result changes the absorption and chemiluminescence wavelengths of
protoporphyrin IX. Zinc protoporphyrin is said to have different absorption wavelengths than
protoporphyrin IX, which is most likely due to the one thing that distinguishes it from
protoporhyrin IX; the Zn2+ central ion. For zinc protoporphyrin IX, the distance between the energy
levels between which the electrons are excited decrease and the absorption wavelengths increase.
As a result, there is a change in fluorescence wavelengths related to the complementary colour of
the absorption, thus chemiluminescence wavelengths will change as well. For the magnesium
central ion, this effect remains unknown, although an effect of similar nature is likely due to the
similar changes in structure for zinc and magnesium central ions. Thus, the formation of zinc
protoporphyrin IX and magnesium protoporphyrin IX solutions from protoporphyrin IX is likely to
change its absorption and chemiluminescence wavelengths even though it could not be
experimentally demonstrated in this essay.
Limitations
The lack of a fluorimeter to measure the fluorescence and chemiluminescence spectra of
protoporphyrin IX restricted the confirmation of the new protoporphyrin solutions through changes
in their fluorescence wavelengths and the knowledge of their direct effect on the
chemiluminescence of protoporphyrin IX. Although a fluorimeter was not available, spectroscopic
theory leads one to the deduction that if there has been a change in absorption wavelengths, this
would have led to a change in fluorescence wavelengths and thus chemiluminescence as well. This
is relevant as the experiment attempted to change the wavelengths of light emitted in the
chemiluminescent reaction of protoporphyrin IX, which is dependent on the fluorescence of
protoporphyrin IX.
Access to commercial solutions of zinc protoporphyrin IX and magnesium protoporphyrin IX
would have provided information about the absorption wavelengths of the substances, and the
differences of these as compared to that of protoporphyrin IX. Furthermore, the commercial
13
Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
solutions could have been used in the chemiluminescence reaction to reveal if the colour of the
emitted light changes, if the solutions would emit light in a chemiluminescent reaction at all.
Unfortunately, these substances are too expensive for one time use.
Further development and questions
The research question is essentially left unanswered, so an obvious direction for the development of
the essay is to further investigate how to form zinc protoporphyrin IX and magnesium
protoporphyrin IX from protoporphyrin IX. This way, an empirical, as opposed to theoretical,
investigation of the effect of central ion on the absorption and chemiluminescence of
protoporphyrin IX would be possible. However, if no new method to form the substances was found,
commercial ones would have to be bought. Another extension of the experiment is to investigate the
effect of other central metal ions on the absorption of protoporphyrin IX. Some cations may give
greater changes in absorption wavelengths, such as Fe2+ or Fe3+, which form coloured complex ions
due to their partially filled d-orbitals. That way, the colour of the light emitted in the
chemiluminescent reaction of protoporphyrin IX might change enough to be spotted with the naked
eye. There are porphyrins and porphines with similar structures to zinc protoporphyrin IX and
magnesium protoporphyrin IX that have different central ions and higher absorption maximums1.
The central ions of these solutions might have been reacted with protoporphyrin IX to see if greater
absorption wavelength changes occur than those for zinc protoporphyrin IX and magnesium
protoporphyrin IX. Obtaining a shift of absorption wavelengths which extends outside the colour
range of the absorption wavelengths of protoporphyrin IX is more likely to cause a visible change in
the colour of the light emitted in its chemiluminescent reaction than absorption changes which only
vary in shades of the same colour (which is the case for Zn2+, that for Mg2+ being unknown).
Another question is whether or not there is an effect of the cation charge on the absorption
wavelengths and chemiluminescence of protoporphyrin IX. Different positive charges on cations
may interact differently with the negatively charged delocalized π-electrons in the molecule. It may
be possible that ions of charge 2+ are the only ones that can act as central ions in protoporphyrin IX.
Additionally, more research could be done to discover in greater depth which changes occur with
the energy levels, bonds and electrons within the molecule when central ions are introduced. These
are all question which could have been addressed given a bigger word limit.
EVALUATION
Several factors may have affected the results of the experiment. The extraction process of
protoporphyrin had to be carried out each time a new solution was needed, and this limited the
amount of solution available to form zinc protoporphyrin IX and magnesium protoporphyrin IX. A
greater amount of the solutions, and thus the ability to do more trials, would have given more
accurate results and allowed for more experimentation with the addition of ions. More solutions
would also have made it possible to obtain several absorption spectra for the solutions and drawing
an average. This would have given more accurate absorption wavelength maximums, as well as
giving more precise information with which to interpret the absorption shifts. Perhaps this would
have shown that the absorption shifts after adding Zn2+ and Mg2+ were the same, or perhaps it
would have confirmed the results in step 3, and shown that the shifts were in fact not the same,
implying that the ions in some way must have affected the spectra. Other sources of error or
inaccuracy include TCPO being used instead of DCPO in the chemiluminescent reaction of
1
http://www.sigmaaldrich.com/materials-science/material-science-products.html?TablePage=19353708
14
Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
protoporphyrin IX. If TCPO reacts in a different way than DCPO, this might have had an effect on
the results of the chemiluminescent reaction. Additionally, the usage of magnesium sulphate as a
drying agent instead of sodium sulphate might have changed the outcome of the experiment.
However, the absorption spectra of the purified protoporphyrin IX corresponded to the absorption
spectra of a commercial sample of protoporphyrin IX, indicating that it is correct. Additionally, after
adding Mg2+ ions to the protoporphyrin IX solution the spectra changed, therefore it is probable that
there was no change in the spectra when it was dried with anhydrous magnesium sulphate.
APPENDIX
Adaptations to the original experimental method
The method originally consisted of five modules. In this method, Module C (Fluorescence
Spectrum of Protoporphyrin) and Module D (Chemiluminescence Spectrum of TCPO/H2O2) were
omitted because of the lack of a fluorimeter. Step 3 was added to account for the change of the
formation of zinc protoporphyrin IX and magnesium protoporphyrin IX, in which the absorption
spectra of the solutions were obtained as well. The school laboratory did not have DNPO, neither
was it possible to order this substance, therefore TCPO (bis(2,4,6-trichlorophenyl) ethanedioate)
was ordered and used as a replacement instead. Because the school laboratory did not have sodium
sulphate, magnesium sulphate was used as a drying agent instead.
Apparatus
Chemicals
Ethyl acetate, 30 mL
2 moles*dm-3 Hydrochloric acid, 50mL
Methanol, reagent grade, 10 mL
Sodium sulphate, anhydrous, 1 g
Hydrogen peroxide, 30%, 5 mL
3 mmoles*dm-3 Solution of bis(2,4,6-trichlorophenyl) ethanedioate (replacement for
bis(2,4dinitrophenyl) oxalate in the original experimental method) in ethyl acetate, 5 mL
Solid KOH, 1 pellet
Magnesium nitrate
Zinc nitrate
Materials Required (equipment/glassware/consumables)
Analytical balance, weighing paper or dish, accuracy +/- 0,01 grams
Bench top balance, weighing paper or dish
UV-vis spectrophotometer Amadeus, w/cuvettes (4.5 mL)
Ring stand with clamps and rings
Erlenmeyer flask, 250 mL
2 Erlenmeyer flask, 50 mL
Filter funnel, small + filter paper
Cotton
pH paper
Separatory funnel w/ stopper, 125 mL
Graduated cylinder, 50 mL
Graduated cylinder, 10 mL
Large test tube
Clean, brown eggshells – about 12 g (from about 2 cooked or fresh eggs)
15
Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
Pasteur pipettes and bulbs
Spatula
BIBLIOGRAPHY
Egg-Citing! Isolation of Protoporphyrin IX from Brown Eggshells and
Its Detection by Optical Spectroscopy and Chemiluminescence
J. Chem. Educ., 2011, 88 (6), pp 788-792 by Michelle L. Dean, Tyson A. Miller and Christian
Bruckner
Date of last consultation: 13th of August 2011
SUPPLEMENTAL MATERIAL to Egg-citing! Isolation of Protoporphyrin IX from Brown
Eggshells and its Detection by Optical Spectroscopy and Chemiluminescence
By Michelle L. Dean, Tyson A. Miller and Christian Brückner
Date of last consultation: 13th of August 2011
Turning on the Light: Lessons from Luminescence
Vol. 82 No. 1 January 2005, Journal of Chemical Education
Date of last consultation: 14th of August 2011
Porphyrines
http://www.sigmaaldrich.com/materials-science/material-scienceproducts.html?TablePage=19353708
Date of last consultation: 13th of August 2011
Zinc protoporphyrin IX
http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=282820|SIAL&N5=SEARC
H_CONCAT_PNO|BRAND_KEY&F=SPEC
Date of last consultation: 5th of September 2011
UV-Visible Spectroscopy
http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/UV-Vis/uvspec.htm#uv1
Date of last consultation: 12th of August 2011
Visible and Ultraviolet Spectroscopy
http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/UV-Vis/spectrum.htm 05081
Date of last consultation: 12th of August 2011
What is chemiluminescence?
http://www.scienceinschool.org/2011/issue19/chemiluminescence
Date of last consultation: 13th of August 2011
16
Investigating the effect of central ion on the absorption and chemiluminescence of protoporphyrin IX.
File:zinc protoporphyrin IX.svg
http://en.wikipedia.org/wiki/File:Zinc_protoporphyrin.svg
Date of last consultation: 16th of October 2011
Conducting Polymers Conjugated Double bonds
http://www.chm.bris.ac.uk/webprojects2001/parrott/pages/conjugated_double_bonds.htm
Date of last consultation: 5th of August 2011
Chemistry by John Green and Sadru Damji, 3rd edition first published in 2007 by IBID Press,
Victoria.
Date of last consultation: 17th of October 2011
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