Egg-citing Supplemental Material

SUPPLEMENTAL MATERIAL

Egg-citing! Isolation of Protoporphyrin IX from Brown Eggshells and its Detection by

Optical Spectroscopy and Chemiluminescence

Michelle L. Dean, Tyson A. Miller*, and Christian Brückner*

Department of Chemistry, Unit 3060, University of Connecticut, Storrs, CT 06269-3060

* Authors to whom correspondence should be addressed: tyson.miller@uconn.edu, Tel: (860)

486-3052 or c.bruckner@uconn.edu, Tel: (860) 486-2743

Table of Contents of Supplemental Material:

Instructor’s Notes
Chemicals Required S2
Materials Required S4
Safety Precautions S5
Instrumental Parameters S6
Useful Teaching Resources (Poultry, Porphyrins, Eggs, Principles of Fluorescence, S7
Chemiluminescence)
Extensions of the Laboratory Experiments S11
Student Handouts
Pre-laboratory Readings S12
Safety Precautions
S14
Detailed Procedures
Module A (Extraction of Eggshell Pigments) S15
Module B (Identification of the Eggshell Pigments by UV-vis Spectroscopy) S17
Module C (Fluorescence Spectrum of Protoporphyrin) S20
Module D (Chemiluminescence Spectrum of DNPO/H2O2) S22
Module E (Chemiluminescence Reaction in the Presence of the Eggshell Extract) S24
Lab Report Guidelines S25
Post-Laboratory Handout S26
Alternative Procedures
Alternative Preparation of Porphyrin Solution S27
Alternative Procedure for Module E S27

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Instructor’s Notes

Below are explicit experimental procedures that have been performed with a majors and a

non-majors sophomore Organic Laboratory Courses. Four laboratory sections, each composed

of no more than 16 students, working in teams of threes, were able to complete the laboratory in

the allotted time with one UV-Vis and one fluorometer available and mounted on a cart, though

the recording of an excitation-emission spectrum that was omitted because of instrument time

constraints. This experiment was completed following a pre-laboratory lecture that introduced

the basics of UV-Vis and fluorescence spectroscopy.1

Note that the modules do not need to be done all or in the sequential order presented.

Instead, they can be arranged according to the learning objectives set by the instructor.

Materials and Reagents

The brown eggs were store-bought. Washed and dried eggshells from fresh or hard-boiled

eggs are equally fitting for the experiments. All solvents and reagents were reagent-grade

purchased from Fisher-Acros, and were used as received. Absorption spectra were recorded on

a Cary 50 spectrophotometer (Varian), while the chemiluminescence and fluorescence spectra

were recorded on a Cary Eclipse spectrofluorimeter (Varian), all in disposable plastic

(polystyrene or methacrylate) cuvettes suitable for fluorescence spectroscopy.2

1. Please contact the corresponding authors for a copy of the PowerPoint slides that were used.
2. Note that not all plastic cuvettes are resistant to pure ethyl acetate but may be resistant to the ethyl
acetate/methanol mixtures used. We suggest a test to be performed before they are used in the
classroom.
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Chemicals Required

Quantity Module
CAS
Chemical Name Required/ A B* C D E*
Number
Experiment

Ethyl acetate [141-78-6] 30 mL X X

2N Hydrochloric acid [7647-01-0] 50mL X

Methanol, reagent grade [67-56-1] 10 mL X

X
Sodium sulfate, anhydrous [7757-82-6] 1g

Hydrogen peroxide, 30% [7722-84-1] 5 mL X X

3 mM Solution of
bis(2,4dinitrophenyl) oxalate [16536-30-4] 5 mL X X
in ethyl acetate

KOH [1310-58-3] 1 pellet X

* Solution prepared in Module A or any other porphyrin solution in ethyl acetate is

also required, see below.

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Materials Required

Module
Equipment/Glassware/Consumables
A B C D E

Analytical balance, weighing paper or dish X X

Bench top balance, weighing paper or dish X

UV-vis spectrophotometer, w/ cuvettes (4.5 mL)* X

Fluorescence spectrophotometer, w/ cuvettes (4.5 mL)* X X

Ring stand with clamps and rings X

Erlenmeyer flask, 250 mL X

Erlenmeyer flask, 50 mL x 2 X

Filter funnel, small + filter paper X

Cotton X

pH paper X

Separatory funnel w/ stopper, 125 mL X

Graduated cylinder, 50 mL X

Graduated cylinder, 10 mL X X X

Large test tube X

Clean, brown eggshells - 12 g per student (from about 2

X
cooked or fresh eggs)

Pasteur pipettes and bulbs X X X X X

Spatula X X X

* Disposable cuvettes are preferred. However make sure that these cuvettes are compatible
with ethyl acetate/methanol (see also footnote 2 on page S2)

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Safety Precautions:

• This entire extraction procedure should be performed in a fume hood and requires well-

practiced laboratory hygiene, which includes the use of safety goggles and the avoidance

of skin contact with any of the chemicals used.

• Concentrated hydrochloric acid is extremely corrosive with the potential of serious burns.

Extreme caution should be taken when handling this chemical, including the use of gloves

to avoid skin contact. Eye protection!

• Potassium hydroxide is very corrosive and can cause serious burns. Caution should be

taken when handling this chemical, including the use of gloves to avoid skin contact. Eye

protection!

• From the MSDS for bis(2,4-dinitrophenyl)oxalate (DNPO): Irritant. Route of exposure

inhalation. Material is irritating to mucous membranes and upper respiratory tract. May be

harmful by inhalation, ingestion, or skin absorption. Causes eye and skin irritation. Use of

gloves and eye protection!

• From the MSDS for 30% hydrogen peroxide: Strong oxidizer. Contact with other material

may cause fire. Corrosive. Causes burns to skin, eyes, and respiratory tract. Harmful if

swallowed or inhaled. Use of gloves and eye protection!

• When the procedure calls for a dimmed lab, it is important to note that the room does not

need to be totally dark. Before the lab is dimmed, the instructor should make sure that all

students are well prepared to perform the addition of the chemicals in a safe manner!

• All ethyl acetate and MeOH samples must be collected and disposed of appropriately as

flammable organic solvents waste. Some caution should be exercised when disposing

fresh hydrogen peroxide/organic solvent mixtures as they can still evolve oxygen. Do not

close the containers tightly!

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Instrument Parameters

UV-Vis Spectrometer (Cary 50 spectrophotometer, Varian)

Measurement parameters:
Range used to collect the spectrum = 350 to 900 nm
Baseline = ethyl acetate
Scan speed = fast
Overlay = enabled

Fluorimeter- to collect a fluorescence spectrum (Cary Eclipse spectrofluorimeter, Varian)

Measurement Parameters:
Mode = Emission
Excitation wavelength = 407 nm
Range used to collect the spectrum = 615 to 800 nm
Excitation slit = 5 nm*
Emission slit = 5 nm*
Overlay = enabled

* These values were used with a porphyrin extract of Aλ = 407 ~ 1. The porphyrin extract may
need to be further diluted depending on the fluorescence instrument used. We suggest a
dilution to Aλ = 407 in the range of 0.1 to 0.05.

Fluorimeter- to collect a chemiluminescence spectrum

To collect the chemiluminescence spectrum of the DNPO / H2O2 reaction, the following
parameters were used:

Mode = Bio / Chemiluminescence

Gate time = 200 ms
Emission slit = 10 nm
Range used to collect the spectrum = 300 to 700 nm
Averaging time = 0.2 s
Data interval = 1.0 nm

Tip: Using the parameters listed above, a method was defined and saved on the desktop. The
method was opened by the students, thus minimizing set-up time.

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Useful Teaching Resources

Poultry
• Interestingly, the feather colors of a chicken are no predictor for egg color. However, the

color of her earlobes is! If they are light colored, as in the White Leghorn breed, the hen

will likely lay white eggs. If the earlobes are dark (red), the hen will likely produce brown

eggs.

comb
earlobe
wattle

• Information of poultry breeds provided by the Department of Animal Science - Oklahoma

State University: http://www.ansi.okstate.edu/poultry/ (accessed 1/2009)

Eggs

• Interesting fact: Irrespective of their nutritional equality, brown eggs are also frequently

slightly more expensive than white eggs. This may be market-driven where customers

prefer brown eggs but this may also be because the hens that lay brown eggs are

slightly bigger, mature later, or utilize their feed less efficiently than the White Leghorn

(From the Texas A&M University Extension:

http://fcs.tamu.edu/food_and_nutrition/nutrifacts/issue22.pdf (accessed 1/2009)

• Chickscope - A tour through the development of a chicken embryo, provided by the

University of Illinois Urbana-Champaign: http://chickscope.beckman.uiuc.edu/

(accessed 1/2009)

• Eggs: A Virtual Exhibition, provided by the Royal Alberta Museum:

http://www.royalalbertamuseum.ca/vexhibit/eggs/vexhome/oology.htm

(accessed 1/2009)

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Porphyrins

Porphyrins are a naturally occurring class of compounds found ubiquitously in biological

systems. They are responsible for a number of vital roles. For instance, hemes – protoporphyrin

IX iron complexes – are the prosthetic group in hemoglobin, thus acting as carriers of oxygen.

Porphyrins are aromatic, tetrapyrrolic macrocycles incorporating 22 conjugated π-electrons.

This large π-system is the origin of their deep color, i.e., their strong absorbance in the visible

range.3,4 The UV-vis spectra of porphyrins are due to strongly allowed π-π* transitions and have

been discussed in this Journal.5,6,7,8,9,10 Porphyrins are also superbly fluorescent. Both features

make them ideal study objects for UV-vis and fluorescence spectroscopy,11 and make them

ideal fluors in chemiluminescence reactions.

3 5 7
4 6
2 8
1 NH N 9
21 22
20 10
24 23
19 N HN 11
18 12
16
17 15 14 13 13

Porphin

The fundamental system is the fully unsaturated, cyclic tetrapyrrolic pigment porphin. Once

substituted, it is named porphyrin. Positions 2,3,7,8,12,13,17, and 18 are referred to as the ß-

positions; positions 5,10,15, and 20 as the meso-positions. All substituted porphyrins can be

named systematically with this system, but for convenience, the naturally occurring porphyrins

are named according to the trivial Fischer nomenclature, shown in the Table below.

3. Milgrom, L. R. The Colours of Life: An Introduction to the Chemistry of Porphyrins and Related
Compounds. Oxford University Press: Oxford, 1997.
4. Kadish, K. M.; Smith, K. M.; Guilard, R., Eds., The Porphyrin Handbook. Academic Press: San Diego,
2000 (Vol. 1-10), 2003 (Vol. 11-20).
5. Brisbin, D. A.; Asgill, J. O. J. Chem. Educ. 1974, 51, 211-213.
6. Arnold, D. P. J. Chem. Educ. 1988, 65, 1111-1112.
7. Marsh, D. F.; Mink, L. M. J. Chem. Educ. 1996, 73, 1188-1190.
8. Falvo, R.; Mink, L. M.; Marsh, D. F. J. Chem. Educ. 1999, 76, 237-239.
9. Basu, P. J. Chem. Educ. 2001, 78, 666-669.
10. Brückner, C. J. Chem. Educ. 2004, 81, 1665-1669.
11. Schuster, G., Acc. Chem. Res. 1979, 12, 366-373.
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Fischer nomenclature for selected naturally occurring porphyrins

Trivial Name Substituentsa and Locants

2 3 7 8 12 13 17 18
Deuteroporphyrin Me H Me H Me PH Me PH
Hematoporphyrin Me Et(OH) Me Et(OH) Me PH Me PH
Mesoporphyrin Me Et Me Et Me PH Me PH
Protoporphyrin IXb Me Vn Me Vn Me PH Me PH
Uroporphyrin A H P H A H PH AH PH AH PH
Coproporphyrin Me AH Me AH Me AH Me AH

a Et(OH) = -CH(OH)CH , Vn = -CHCH , AH = -CH COOH, PH = -CH CH COOH;

3 2 2 2 2
b The roman numerals refer to Fischer’s type nomenclature for naming all the possible regioisomers of
one set of substituents. For papers on the enumeration of permutational isomers in porphyrins, see:
(a) Tapscott, R. E.; Marcovich, D. J. Chem. Ed. 1978, 55, 446-447. (b) Pilgrim, R. L. C. J. Chem. Ed.
1974, 51, 316-318.

Principles of UV-Vis and Fluorescence Spectroscopy:

• Visible and Ultraviolet Spectroscopy, provided by William Reusch of Michigan State

University: http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm

(accessed 1/2009)

• Fluorescence Excitation and Emission Fundamentals, site provided by the Olympus

Corporation: http://www.olympusconfocal.com/theory/fluoroexciteemit.html (accessed

1/2009)

• Fluorescence Tutorials, site provided by Invitrogen:

http://www.invitrogen.com/site/us/en/home/support/Tutorials.html (accessed 1/2009)

Chemiluminescence

Chemiluminescence describes any process in which some or all of the energy of a

chemical reaction is released in the form of light. Generally, a high-energy compound is

generated by chemical means that, upon further reaction, forms an excited state intermediate

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that relaxes to its ground state by giving off light. Peroxyoxalates, generated from oxalate esters

and hydrogen peroxide, have long been known to fragment and undergo such a

chemiluminescence reaction,12,13,14 emitting light in the UV-region, and have been described in

this Journal.15,16,17 The addition of a suitable fluor to the UV-emitting chemiluminescence makes

the emission visible. Chemiluminescence reactions in the presence of a fluorophore are the

underlying working principle of glow sticks or firefly emissions, and were also discussed in this

Journal.18

Further reading:

• Chemiluminescence of oxalate esters, provided by the University of Leeds, UK:

http://www.chem.leeds.ac.uk/delights/texts/VV_exp_26.htm (accessed 1/2009)

• Chemiluminescence of luminal - with an explanation, mechanism and experimental

section, provided by the University of Bristol, UK:

http://www.chm.bris.ac.uk/webprojects2002/fleming/index.htm (accessed 1/2009)

12. Schuster, G., Acc. Chem. Res. 1979, 12, 366-373.

13. Stevani, C. V.; Silva, S. M.; Baader, W. J. Eur. J. Org. Chem. 2000, 4037-4046.
14. Lee, J. H.; Rock, J. C.; Schlautman, M. A.; Carraway, E. R. J. Chem. Soc., Perkin Trans. 2. 2002,
1653-1657.
15. Kuntzleman, T. S.; Comfort, A. E.; Baldwin, B. W. J. Chem. Educ. 2009, 86, 64-67.
16. Mohan, A. G.; Turro, N. J. J. Chem. Educ. 1974, 51, 528-529.
17. McCapra, F. Methods Enzymol. 2000, 305, 633-659.
18. Roser, C. E.; McCluckey, C. L. J. Chem. Educ. 1999, 76, 1514-1515.
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Extensions of the Laboratory Experiment

1. Brightness of a Fluor

This experiment may also serve as an illustration of what is referred to as the brightness

of a fluorophore, which is computed by multiplying the extinction coefficient by the

fluorescence quantum yield (ε x Φ). Depending on class-appropiate discussion, this issue

may be addressed to varying depth and correlated to the very high sensitivity tests for blood

used in forensics.19

2. Discussion of Energy Transfer Processes

As an extension to this experiment the energy transfer processes as they relate to

photosynthesis or fluorescence-resonance energy transfer (FRET) can be introduced

together with work on green fluorescent protein that was recognized by the 2008 Nobel

Prize in Chemistry.20

3. Separation of Egg Shell Pigments by TLC

An extraction with concomitant esterification of the propionic acid functionalities of the

porphyrins using H2SO4/MeOH has been described, allowing their analysis by TLC.21

19. Chu, E. J.; Chu, T. C. J. Chem. Educ. 1953, 30, 178-179.

20. Nobel Prize in Chemistry 2008. http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/
(accessed July 2009).
21. With, T. K. Biochem. J. 1973, 137, 597-598.
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Student Handouts - Pre-Laboratory Reading and Laboratory Procedure

The following handout is given to the students a week before they will perform this lab.

Egg-Citing
Extraction and Detection of Porphyrins by Chemiluminescence

Have you ever considered what gives brown eggs their color? The 1930 Nobel laureate
Hans Fischer can be credited for initially recognizing that the brown spots on the shells of
seagull eggs were composed of porphyrins. Initially called ooporphyrin, the principal component
was eventually identified by him as protoporphyrin IX.

NH N

N HN

HO2C CO2H

Protoporphyrin IX (PPIX)
C34H34N4O4
MW: 562.7

The goal of this laboratory experiment is to expose you to an organic extraction method
used to extract protoporphyrin IX (PPIX) from brown eggshells. Following the extraction, the
main component of the solution will be identified and quantified using UV-Vis spectroscopy.
Porphyrins are highly fluorescent compounds, and therefore a fluorescence spectrum of the
extract can be obtained. The fluorimeter will also be used to measure the light generated by a
chemiluminescence reaction of bis(2,4-dinitrophenyl)oxalate (DNPO) with hydrogen peroxide
(H2O2). Lastly you will see how porphyrins, such as PPIX, can be detected using an eye-
catching chemiluminescence reaction. With the collected spectra and observations made during
the laboratory, you will be able to determine the photophysical role that the porphyrin plays in
the chemiluminescence reaction to produce the observed glow.

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Recommended Readings:

You will be introduced to new spectroscopic methods that are used to measure the chemical
and photophysical phenomena observed in this lab. Since light absorption and emission (as
measured by UV-Vis absorption and fluorescence emission) by organic molecules may have
been only briefly mentioned during your Organic Chemistry course, it will be helpful to view the
following links below. These websites along with the pre-laboratory lecture will provide you will
ample material to understand the spectra that will be collect and allow you to draw conclusions.

UV-Vis Spectroscopy
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm

Fluorescence spectroscopy
http://www.invitrogen.com/site/us/en/home/support/Tutorials.html

Begin with tutorial 1 “Get a basic understanding of fluorescence” slides 1-11 and then proceed
to tutorial 2 “Learn how to interpret excitation and emission spectra”. The other tutorials are not
relevant to this class.

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Safety Precautions:

1. Eye protection must be worn at all times during lab, even when analyzing your sample
using the instruments.

2. Perform Module A: “Extraction of Eggshell Pigments” in the fume hood.

3. Hydrochloric acid (HCl) is corrosive and has the potential to cause serious chemical
burns. Use caution and gloves when handling this chemical.

4. From the MSDS for bis-(2,4-dinitrophenyl)oxalate (DNPO): irritant. Material is irritating to

mucous membranes and upper respiratory tract. May be harmful by inhalation, ingestion,
or skin absorption. Causes eye and skin irritation. Gloves may be worn when handling the
solid DNPO or prepared solution of DNPO to avoid skin contact.

5. From the MSDS for 30% hydrogen peroxide: Strong oxidizer. Contact with other material
may cause fire. Corrosive. Causes burns to skin, eyes, and respiratory tract. Harmful if
swallowed or inhaled. Gloves should be worn when handling this compound.

6. When you are done collecting your spectra, empty your cuvettes into the provided organic
waste containers.

7. When the procedure calls for a dimmed room, assure that your classmates are prepared
for the absence of lights. Also, be prepared to perform the addition of the chemicals in the
near-dark in a safe manner.

8. Be sure to separate and dispose all your wastes accordingly into the labeled waste
receptacles.

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PROCEDURES
Module A: Extraction of Eggshell Pigments

1. Weigh the dried eggshells from two medium sized brown eggs (about 12 g).

2. Compare the color of your eggs to that of the color of the eggs from two other groups in
your lab section.

3. Add 50 mL of 2 M hydrochloric acid into a 250 mL Erlenmeyer flask.

4. Then add 25 mL of ethyl acetate, followed by the dried eggshells which are broken only
small enough to fit into the flask. Leave them as coarse as possible to prevent excessive
foaming. As the hydrochloric acid etches / dissolves the eggshells, carbon dioxide evolves,
so be sure not to stopper the flask. The porphyrins are extracted into the organic phase,
indicated by the successive (light pink) coloration of the organic phase.

3. Allow this mixture to sit (do not stir but you may swirl the flask) for 15 to 20 min, or until the
bubbling has subsided. The bubbling may not completely stop, but the eggshells should
appear nearly colorless. At this time the solution should be nearly neutral. You may check
this using a piece of pH paper. Dip a piece of pH paper (about one inch in length) into the
aqueous portion of the extract solution, and compare the color to the chart on the pH
paper roll to determine the pH.

4. Break up the emulsion and foam that has formed in the Erlenmeyer flask. Thus, add about
5 mL portions of methanol until the organic phase cleanly separates from the aqueous and
solid phases.

5. Plug a funnel with a piece of cotton and place it over a 100 mL Erlenmeyer flask as shown
in Figure 1 below. Pour your mixture with the eggshells into the funnel. Once the liquids
drained, rinse the shells twice with about 5 mL of ethyl acetate.

cotton plug

Figure 1. Filtration set-up

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6. Transfer the filtrate into a separatory funnel. Add about 15 mL of water. Wash the organic
phase by stoppering the funnel, and shaking it gently to mix the two layers. Be sure to vent
the separatory funnel frequently.

7. Place the separatory funnel in the ring stand and allow the organic and aqueous phases to
separate.

8. Collect the aqueous waste layer in a beaker.

9. Wash the organic phase two more times by repeating the addition of water, washing, and
draining steps.

10. Collect the colored (and turbid) organic phase in a clean 125 mL Erlenmeyer flask. Add
about the drying agent sodium sulfate (about a spoon full) to the organic phase to dry the
solution. Swirl the flask gently. The organic phase should become clear.

11. Place a filter, fitted with filter paper, atop a clean and dry 125 mL Erlenmeyer flask and
pour the dried organic phase through the filter.

12. Rinse the filter with 5 mL of methanol.

13. Record the final volume of the dried porphyrin extract using a graduate cylinder.

In your notebook:

A.1. Describe the eggshells that were used in this Module (color, mass, etc.). How did
the color or your eggshells compare with those of two other groups?

A.2. Record the final volume of the eggshell extract.

A.3. Are the eggshells remaining in the funnel entirely colorless?

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Module B: Identification of the Eggshell Pigments by UV-vis Spectroscopy

1. Place a cuvette containing ethyl acetate into the cell holder of the UV-Vis spectrometer
and close the door.

2. Double-click on the icon that is found on the desktop titled “Egg-citing-UV-Vis Method.”

3. Press the “Start” icon, as shown in Figure 2, to collect the absorption spectra from 350 to
900 nm.

Click here to save Click on this icon to

your data. In the To collect your UV-Vis close the program
drop-down menu spectrum click on this when you have
choose “save data.” icon. finished this module.

Figure 2. Interface to control the UV-Vis spectrometer

4. In the box that appears name the spectrum as follows: UV-followed by group members
initials (Example for John Doe and Mike Smith= UV-JDMS. Press “OK” once you have
entered a name.

5. If the Soret band, the most intense peak, exceeds an absorbance of 2 (a value close to the
detector limit), return the porphyrin extract to the remaining stock eggshell extract solution.
Dilute this by adding 25 mL portions of methanol to the stock solution. After each addition
measure the absorption spectrum by following steps 1-3. If the absorbance is still above 2,
repeat this step. Be sure to record the new volume of the stock solution after the final
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dilution step.

6. Sketch, or print, the spectrum in your notebook, being sure to record the wavelength that
each peak appears at. Also, record the relative absorbance of the peaks that are
observed.

7. Save your file. To do so click on “File” at the top of the screen and choose “Save data.”
Save it to the “Eggciting Fall 2008” folder on the desktop. Name the file in the same way
that you did your spectrum.

8. Close this program by clicking on the X in the top right corner.

9. Dispose of the solution in the cuvette in the designated organic waste containers. Then
rinse the cuvette with a small amount of methanol. DO NOT USE ACETONE OR WATER.

In your notebook

B.1. Write down the file name assigned to your UV-Vis spectrum.

B.2. Sketch the collected UV-Vis spectrum. Be sure to label the wavelength of each peak
that appears and the relative absorption.

B.3. Describe how your UV-vis spectrum compares to the reference spectrum shown
below.

B.4. Using the spectra collected, calculate the concentration of protoporphyrin IX using
the Beer-Lambert Law.

A=εcl

A = absorbance as measured in the UV-Vis (unitless); ε molar extinction coefficient

(L⋅mol-1⋅cm-1); c = porphyrin concentration (mol⋅L-1); l = path length of cuvette (cm) In
the case of the cuvettes that you will use in this experiment, the length will be 1 cm.
The information required to complete this calculation is given below.

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Spectroscopic data for protoporphyrin IX22

UV-vis absorption Fluorescence Fluorescence

spectrum, spectrum quantum yield

λmax / nm (ε/M-1cm-1) λmax-em / nm (rel. I) φF (%)

(in CHCl3)* (in MeOH)* (in MeOH)*
407 (171,000), 635 (1.0) 15.5
505 (14,150), 698 (0.2)
541 (11,600),
575 (7,440),
630 (5,380)
* Solvatochromic effects for the porphyrins are below ± 5 nm.

Figure 3. UV-vis absorption (solid trace) and fluorescence (dotted trace) spectra of a
commercial sample of protoporphyrin IX in ethyl acetate/MeOH.

B.5. Using the final volume of your extracted solution and its concentration, determine the
number of moles of protoporphyrin IX that were extracted from the brown eggshells.

B.6. How many moles of protoporphyrin IX were extracted per gram of eggshell?
Compare this number with two groups that you compared your dry eggs with in
Module A? How does your value compare? Explain your observation.

22. Data from: (a) Fuhrhop, J.-H.; Smith, K. M. Laboratory Methods in Porphyrin and Metalloporphyrin
Research, Elsevier: New York, 1975. (b) Lozovaya, G. I.; Masinovsky, Z.; Sivash, A. A. Origins of Life and
Evolution of the Biosphere 1990, 20, 321-330.
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Module C: Fluorescence Spectrum of Protoporphyrin IX*

1. Place 1 mL of the eggshell extract into a clean and dry cuvette. Then add 1.5 mL of ethyl
acetate.

2. Double-click on the icon found on the desktop titled “Egg-citing- Fluorescence Method”

3. The screen that will appear will look very similar to the application used to collect your UV-
Vis spectrum. Click on the “Start” icon at the top of the screen, as shown in Figure 4, to
collect your spectrum.

Click on this icon to

collect your spectra.

Once you have collected your

spectra, click on this icon to enlarge
the spectra to fit the screen.

Figure 4. Interface to control the fluorimeter

4. Sketch, or print, the spectrum in your notebook, being sure to record the wavelength that
each peak appears at. Also, record the fluorescence intensity of the peaks that are
observed.

5. Save your file. To do so click on “File” at the top of the screen and choose “Save data.”
Save it to the “Eggciting Fall 2008” folder on the desktop. Name it as fluor-group member
initials. (Example for John Doe and Mike Smith= fluor-JDMS)

6. Click on the “Setup…” icon on the left of the screen. Change the excitation wavelength to
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480 nm. Then click “Ok” at the bottom of the Setup window to save the changes.

7. Click on the “Start” icon at the top of the screen. Once the spectrum has been collected,
record the relative intensity of the peak that occurs at 635 nm.

8. Repeat steps 6 and 7 until in 20 nm increments until an excitation wavelength of 620 nm

has been reached.

9. Close the program by clicking on the X in the top right corner, as shown in Figure 4.

10. Return your extracted porphyrin solution in your cuvette to your stock solution. Then rinse
the cuvette with a small amount of methanol. DO NOT USE ACETONE OR WATER.

In your notebook:

C.1. Sketch the spectra collected. Be sure to label the wavelength of each peak that
appears and the fluorescence intensity.

C.2. Construct a graph of the excitation wavelength vs. the relative intensity.

C.3. Describe how changing the excitation wavelength affects the emission spectrum and
how this graph compares to the UV-Vis spectrum that was previously collected.

* Steps 6 through 8, the acquisition of an excitation-emission spectrum, and associated questions C.2
and C.3 were not tested with a large group of students due to time and instrument availability.

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Module D: Chemiluminescence Spectrum of DNPO/H2O2

1. Prepare a 7 mM solution of DNPO in ethyl acetate. To do this, place 10 mL of ethyl

acetate into a 25 mL Erlenmeyer flask, then add 0.030 g of DNPO. Swirl the flask gently
until all the DNPO has dissolved.

2. Place 2 mL of the 7mM DNPO solution prepared in the previous step, into a cuvette.

3. Double-click on the “Egg-citing- Chemi Method” icon found on the desktop.

4. To collect your data click on “Start” at the top of the screen. This will bring up a window
that counts down until it starts acquiring data, as shown in Figure 5. At this time add
about 1 mL of 30% hydrogen peroxide using a glass pipette to the cuvette and mix using
the pipette. Caution - 30% H2O2 is a strong oxidant and bleaches skin and clothing.
Once mixed quickly but carefully place the cuvette into the holder of the fluorimeter,
close the sample compartment door of the fluorimeter, and start the data acquisition by
clicking “OK” in the countdown window, as depicted in Figure 5.

Click here once you have

mixed the DNPO and H2O2,
placed the cuvette in the
fluorimeter, and closed the
door.

Figure 5. Appearance of the interface when data acquistion is initiated to collect a

Chemiluminescence spectrum

5. When the data acquistion is complete, enlarge the spectrum by clicking on the arrow
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icon, as shown in Figure 5.

6. Sketch, or print, the spectrum in your notebook, being sure to record the wavelength that
each peak appears at. Also, record the relative absorbance of the peaks that are
observed.

7. Save your file. To do so click on “File” at the top of the screen and choose “Save data.”
Save it to the “Eggciting Fall 2008” folder on the desktop. Name it as chemi-group
member initials. (Example for John Doe and Mike Smith= chemi-JDMS)

8. Close the application by clicking on the X in the top right hand corner.

9. Discard the solution in the cuvette to the appropriate waste container. Then rinse the
cuvette with a small amount of methanol. DO NOT USE ACETONE OR WATER.

In your notebook:

D.1. Sketch the spectra collected. Be sure to record the wavelength of each peak that
appears and the fluorescence intensity.

D.2. What is the range of emission that this chemical reaction produces? Does this
have any relationship to the porphyrin spectrum? If so, describe the relationship

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Module E: Chemiluminescence Reaction in the Presence of the Eggshell Extract

Please read the entire procedure for this module before setting up this reaction.

If a 7 mM solution of DNPO in ethyl acetate has not already been prepared, do so at this time.
To prepare this solution follow the procedure in Step 1 of Module D.

Part A: Chemiluminescence Measured in the Fluorimeter*

1. Repeat the procedure as described for Module D, replacing the 2 mL of 7 mM DNPO in

step 2 with 1 mL of 7 mM DNPO in ethyl acetate and 1 mL of the eggshell extract that
was prepared in Module A. Use the method titled “Egg-citing- Finale Method” on the
desktop to collect your data.

Note: Proceed onto Part B only once all of your classmates have completed Part A and you are
instructed to do so. You instructor will darken the room within 5 minutes of the completion of
Steps 1-3, since the solution prepared is not stable.

Part B: Chemiluminescence as Seen with the Naked Eye

1. In a large test tube place 5 mL of the extracted porphyrin solution prepared in Module A.

2. Add 3 mL of 30% hydrogen peroxide to the solution.

3. Wait for your instructor to darken the room and to give you permission to proceed with
your experiment.

4. Add 2 ml of the 7 mM solution of DNPO, prepared in Module D to the solution. Swirl the
test tube and observe the reaction.

5. Wait for the light to come back on before proceeding with the rest of the lab.

This procedure may be repeated a second time. However, this time a pellet of KOH following
step 1 to intensify the reaction.

In your notebook:

E.1. Record your observation of the reaction following the addition of DNPO to the test
tube.

E.2. Using the spectra collected from the previous modules explain the role that the
porphyrin plays in the DNPO/H2O2 reaction to produce the glow observed. It will be
helpful think about what each spectrum is measuring on a microscopic level.

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Lab Report

This lab report will not be a formal write up. Please answer the questions that are listed
below. Be sure to show all of your work for any calculations that you perform. Each member of
the group must submit their own answers.

1. In your own words, describe the purpose of the Egg-citing! laboratory experiment.

2. Sketch the UV-vis spectrum of the eggshell extract.

3. Calculate the concentration of the protoporphyrin IX in the extracted solution.

4. Based on the total volume of the extracted solution, how many moles of protoporphyrin
IX were extracted?

5. How many moles of protoporphyrin IX were extracted per gram of eggshell? How did
this value compare with the values obtained by two other groups? Explain and
rationalize your observation.

6. Sketch the fluorescence spectrum of the eggshell extract.

7. Sketch the chemiluminescence spectrum of the DNPO/H2O2 reaction.

8. At what wavelength of light did the glow that was observed in Module E occur? What
reactant is responsible for the glow observed?

9. Provide an explanation of how the Protoporphyrin IX interacts with the DNPO/H2O2

reaction. Please use the spectra that were collected during this experiment to support
your answer.

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Student Handout- Post-Laboratory

This is given to the students after they have completed their lab report, but before they have
taken the post-laboratory quiz.

Egg-Citing!
Extraction and Detection of Porphyrins by Chemiluminescence

Post-Laboratory
Were you able to figure out how the three spectra that were collected (absorption and
fluorescence of the porphyrin extract and the chemiluminescence spectrum of the DNPO/H2O2
reaction) fit together to explain the bright red glow observed in Module E?

It will be helpful to consider what process each spectra represents. How does protoporphyrin IX
interact with the light produced by the chemical reaction shown below?

O O NO2 H2O2, HO
O O cat. base
*
O O
2 O2N +
O2N NO2 NO2 OO
NO2
DNPO

2 CO2 + light

If all this still seems unclear visit the following website:

http://www.chem.leeds.ac.uk/delights/texts/VV_exp_26.htm

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Alternative Procedures

Alternative Preparation of Porphyrin Solutions:

A porphyrin solution suitable for Modules B-E can be prepared by dissolution of several

commercially available or readily synthesized porphyrins:

• Protoporphyrin IX ([553-12-8], Aldrich P8293, 1 g $56.60)23

• Hematoporphyrin ([14459-29-1], Aldrich H5518 (50% purity – the bulk of the 50%

‘impurity’ are other porphyrins such as hydroxyethyl-vinyl-deuteroporphyrin and

protoporphyrin and are not detrimental to the experiments), 1 g $18.00)23

• meso-Tetraphenylporphyrin ([917-23-7], Aldrich 247367, 250 mg $59.20).23, 24

Porphyrin (~5 mg) is placed into the Erlenmeyer flask, dissolved in ethyl acetate (25 mL)

and glacial acetic acid (2-3 drops) is mixed into this solution. The solution can be kept in the

dark (flasks should be wrapped with aluminum foil) in a stoppered flask for at least two weeks.

Chemiluminescence Demonstration (Variation of Module E - Chemiluminescence in

the Presence of the Brown Eggshell Extract):25

While the demonstration can be performed in many different vessels, a large or extra-large

test tube is most suitable. 20 Parts of the solution prepared in Module A (either the ethyl

acetate/MeOH brown eggshell extract or a ethyl acetate solution of a commercial porphyrin) are

placed into the test tube and 2 parts 30% H2O2 solution and one or two KOH pellets are added.

The biphasic mixture is briefly swirled. Then 1 part of a DNPO solution (0.3 mM in ethyl acetate)

is added (by pipette/syringe) and the mixture stirred or swirled. An intense red glow is

instantaneously observed that is best visible under dimmed light conditions.

23. All prices based on Aldrich 2008 Catalogue.

24. This porphyrin is also readily synthesized: (a) Marsh, D. F.; Mink, L. M.; J. Chem. Ed. 1996, 73, 1188-
1190. (b) Falvo, R. E.; Mink, L. M.; Marsh, D. F. J. Chem. Ed. 1999, 76, 237-238.
25. Adapted from: Brandl, H.; Albrecht, S. Prax. Naturwiss., Chem. 1990, 39(3), 17-24 (CAN 113:39463).
Brandl, H. Prax. Naturwiss., Chemie in der Schule, 2002, 51(3), 18-22 (CAN 137:93337). A variation
of this chemiluminescence experiment is also found in: Chemical Curiosities, Roesky, H. W.; Möckel,
K., VCH: Weinheim, 1996, 208-209.
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