Ab Ovo – Fluorescence of a Porphyrin Solution Isolated from an Eggshell
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The following article was originally published in the journal for educators Chemia w Szkole (eng. Chemistry in School) (6/2014):
The phrase ab ovo comes from Latin and literally means "from the egg," which many consider a shortened form of the Latin proverb ab ovo ad mala ("from the egg to the apple"), referring to a Roman feast that traditionally began with the consumption of eggs. This phrase is commonly used to mean "from the beginning" [3]. But what does a hen's egg have to do with chemistry?
It turns out that eggshells, especially those of brown eggs, contain a significant amount of compounds from the porphyrin group. From a chemical standpoint, they are derivatives of porphine, whose molecule consists of four five-membered heterocyclic pyrrole rings, interconnected by methine bridges (Fig. 1) [1].
The porphyrin skeleton is present in the structure of many compounds that play essential biological roles. It appears in the heme molecule (as an iron complex), which serves as a prosthetic group in hemoglobin, myoglobin, and cytochromes, as well as in chlorophyll molecules (as a magnesium complex).
Porphyrin derivatives, under appropriate conditions, often exhibit strong fluorescence. In this experiment, I will describe how to isolate these compounds from a commonly available material—hen eggshells—using simple methods.
Experiment
A significant advantage of this experiment is that it can be conducted using easily accessible and relatively low-risk substances [2]. The primary raw material is eggshells, especially those with a brown color (Photo 1). White eggshells are unsuitable because they contain significantly fewer of the chemical compounds of interest.
After breaking the egg, the shell should be washed in warm water to remove any remaining egg white and then dried.
To isolate the porphyrins contained in the shell, we need:
- Hydrochloric acid HCl (Cp = 15%) or acetic acid CH3COOH (as vinegar, Cp = 10%),
- Ethyl acetate CH3COOC2H5.
The best results are obtained using hydrochloric acid HCl; however, it can be replaced with vinegar, which is a solution of acetic acid CH3COOH at a concentration of up to 10%. Ethyl acetate should ideally be chemically pure, though it is also an ingredient in many nail polish removers, making it an interesting avenue for further experimentation.
Preparing the experiment is straightforward. The cleaned eggshell should be crushed into small fragments, which are then covered with 2 cm3 (0.07 fl oz) of hydrochloric acid HCl or a similar amount of acetic acid CH3COOH.
A reaction between the acid and the calcium carbonate CaCO3 in the shell immediately begins, following the equation:
This reaction produces large amounts of gaseous carbon dioxide CO2, causing the mixture to foam. During the dissolution of calcium carbonate CaCO3, the previously trapped porphyrins are released—allowing for their extraction. To the foaming mixture, 5-7 cm3 (0.17-0.24 fl oz) of ethyl acetate CH3COOC2H5 is added, the mixture is shaken, and left to stand (Photo 2). When using hydrochloric acid, this process takes about fifteen minutes, whereas with acetic acid, it may require several hours.
After the specified time, the mixture is shaken once again, solid residues are filtered out, and the liquid is poured into a graduated cylinder. Phase separation occurs quickly: the lower aqueous phase and the upper organic solvent phase containing the extracted porphyrins (Photo 3A). Neither layer shows visible coloring.
The appearance, however, changes dramatically when the sample is illuminated with ultraviolet light (wavelength λ=366 nm). The upper (organic) phase, containing dissolved porphyrins, exhibits a distinctly bright red fluorescence (Photo 3B). Notably, the lower aqueous phase shows no visible fluorescence.
Explanation
A hen's eggshell contains various porphyrin derivatives, including protoporphyrin IX, coproporphyrin, uroporphyrin, and others. In brown eggshells, protoporphyrin IX, also known as ooporphyrin (Fig. 2), is the predominant compound, imparting its characteristic color [5].
Protoporphyrin IX is one of the precursors of the biologically significant compounds previously mentioned (heme, chlorophylls, cytochromes). It is not surprising that the sequence of chemical processes leading to its biosynthesis is highly conserved throughout the evolution of life on Earth—there are no significant differences even between such evolutionarily distinct organisms as bacteria and mammals [4].
A solution of protoporphyrin IX in an organic solvent, like other porphyrin derivatives (e.g., chlorophyll), exhibits strong red fluorescence when exposed to ultraviolet light.
This phenomenon is explained by the Stokes shift, which refers to the shift of the emission spectrum maximum relative to the absorption spectrum maximum for a given excited state. In a Stokes shift, the emission peak occurs at a longer wavelength (λ) than the absorption peak (Fig. 3).
This can be easily explained using a simplified Jablonski diagram (Fig. 4).
A molecule in its ground state, which is the lowest energy state, can absorb a specific amount of energy (in this case, ultraviolet radiation), allowing it to transition to an excited state with higher energy. However, part of this energy is rapidly dissipated through non-radiative transitions, such as vibrational relaxation, leading the molecule to relax to a lower excited state. This situation is inherently unstable, so the molecule quickly returns to its ground state, releasing the remaining energy as radiation. Since the emitted radiation has lower energy than the absorbed radiation, this results in an increase in wavelength (λ).
References
- [1] Bojarski J., Chemia organiczna, Wydawnictwo Uniwersytetu Jagiellońskiego, Kraków 1999.
- [2] Brandl H., Weiß D., Experimente mit Pflanzeninhaltsstoffen - Fluoreszenzfarbstoffe in der Natur, Chemie in unserer Zeit, 2013.
- [3] Gloger Z., Encyklopedia staropolska, Warszawa 1900-1903.
- [4] Layer G., Reichelt J., Jahn D., Structure and function of enzymes in heme biosynthesis, Protein Science, 2010.
- [5] With T. K., Porphyrins in egg shells, The Biochemical Journal, 1973.
All photographs and illustrations were created by the author.
Marek Ples