Isolation of Biliverdin IXÎ±, as its Dimethyl Ester, from Emu Eggshells

Laboratory Experiment pubs.acs.org/jchemeduc

Isolation of Biliverdin IXα, as its Dimethyl Ester, from Emu Eggshells Steven Halepas, Randy Hamchand, Samuel E. D. Lindeyer, and Christian Brückner* Department of Chemistry, Unit 3060, University of Connecticut, Storrs, Connecticut 06269-3060, United States S Supporting Information *

ABSTRACT: A laboratory experiment is described that extracts the tetrapyrrolic teal-colored biliverdin IXα, as its dimethyl ester, from commercially available emu eggshells. The extraction of ∼10 mg samples of biliverdin is simple and requires two 3 h lab periods: A two-step acid digestion and liquid−liquid extraction, followed by short silica gel flash column chromatography. The deeply colored extract can be characterized by TLC, UV−vis, IR, 1H NMR spectroscopy, ESI+ mass spectrometry, or its diagnostic chemical reduction to the yellow derivative bilirubin. A small-scale variant of this extraction requires only a single 3 h lab period, and the extracts can be analyzed by UV−vis spectroscopy, ESI+ MS, or HPLC. An introduction to the biological origin of biliverdin is provided. This pedagogically flexible project combines natural product isolation with the spectroscopic characterization of a multifunctional biomedically important compound and touches upon many aspects of chemistry and biology. The colorful experiment is appropriate for the introductory organic chemistry laboratory and possesses the prerequisites for a chemistry−biology interlaboratory approach. KEYWORDS: Second-Year Undergraduate, Upper-Division Undergraduate, Laboratory Instruction, Organic Chemistry, Natural Products, NMR Spectroscopy, HPLC, Heterocycles

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INTRODUCTION Grass is green, and blood is red. Thus, few classes of molecules are as conspicuous as the tetrapyrroles, the “pigments of life”.1 The value of the extraction and spectroscopic characterization of natural products in chemical education is well-recognized.2 The key roles of plant pigments (inter alia, the green chlorophyll) in biochemistry and their appealing colors make them privileged molecules in chemical education.3 This should also hold for the animal-based tetrapyrrolic pigments:4 the cyclic porphyrins, their iron complexes (hemes), or their openring degradation products (biliverdins, bilirubins), to name only the most common pigments. These pigments play multiple and crucial roles in human health; thus, their isolation and discussion will find the attention of many students. However, the difficulty of finding readily available and hygienic sources for these compounds inhibits their study in the undergraduate teaching laboratory. Eggs, however, have served as convenient sources for the extraction of natural products. For instance, the isolation of lipids from egg yolks and the extraction of protoporphyrin IX (1) (Scheme 1) from brown chicken eggshells in the teaching laboratory have been described.5,6 Biliverdin IXα (2a) is a linear tetrapyrrole that is the product of the enzymatic oxidative ring cleavage of heme (1Fe) (Scheme 1).7 It is formed during the breakdown of senescent erythrocytes by macrophages and is the compound responsible for the greenish color seen in bruises (before the biliverdin is reduced to the yellow bilirubin (3a) as the bruise is resorbed).7 Biliverdin possesses antimutagenic and antioxidant properties and was shown to be an endogenous tissue protector;8 however, an excess of bilirubin in the body is the hallmark of © XXXX American Chemical Society and Division of Chemical Education, Inc.

hepatic diseases. For example, the characteristic jaundiced appearance is due to the accumulation of bilirubin in the skin and sclera (white of the eye bulb). Biliverdin can be extracted from bile, certain fish bones, frog eggs, or reptile blood, none of which are suitable sources for the teaching laboratory, however (for other sources of biliverdin, see Supporting Information).9 Comparative work of eggshell pigments identified protoporphyrin IX (1) and biliverdin IXα (2a) as the two principle colorants, often found as a mixture (for a note on the nomenclature of porphyrins and biliverdins, see Supporting Information).10 Remarkably, the eggshell pigments of the emu eggshells are almost pure biliverdin IXα (2a). Emus (Dromaius novaehollandiae) are large (18−60 kg, 40−130 lb; up to 2 m, 6.6 ft height) flightless brown birds native to Australia.11 They are related to the better-known and larger African ostrich. The female emu lays a clutch of a dozen or more very large (700 to 900 g, 1.5 to 2.0 lb; 135 mm, 5.3 in long; roughly equivalent to 10−12 chicken eggs), thick-shelled, dark green eggs (Figure 1). Their coloration camouflages the eggs in the shallow ground nests that may be lined with grass and leaves. Emus are farmed for their meat, oil, and leather in their native Australia but also increasingly in the US, Asia, and Europe. Their eggs and eggshells are commercially available from these farms,12 suggesting they can be hygienic sources of biliverdin 2a.13 As an egg is produced in the hen’s reproductive system, the egg is built from the inside out (for a more detailed description Received: June 24, 2017 Revised: August 8, 2017

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DOI: 10.1021/acs.jchemed.7b00449 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

eggshell dyes are exclusively found embedded in the cuticle, a thin protein matrix layer on the outside of the eggshell.10 The cuticle is generated by dedicated glands that also produce the pigments in the final hours before the eggs are laid. The property of calcium carbonate and cuticle to dissolve under strongly acidic conditions, thus releasing the acid-stable biliverdin, enables the extraction of the pigment.10,13 The pleasing deep coloration of biliverdin allows for naked-eye TLC and column chromatography isolation of this natural product while its 1H NMR spectroscopic characterization exemplifies the analysis of a complex natural product with multiple diagnostic features. Moreover, the NaBH4 reduction of the biliverdin dimethyl ester rapidly generates the brown bilirubin dimethyl ester. Biliverdin and bilirubin (and porphyrins) will expose the students to two important classes of tetrapyrroles and serve as an introduction to the origin of color in πconjugated molecules.

Scheme 1. Biological Formation of Biliverdin IXα by Oxidation (and Demetallation) of Heme (1Fe), the Reduction of Biliverdin (2a) to Bilirubin (3a), and in Vitro Esterification of 2a and Reduction of 2b As Described in This Experiment

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EXPERIMENT This experiment is a straightforward isolation, chromatographic purification, and 1H NMR, IR, and UV−vis spectroscopic analyses of biliverdin IXα, as its dimethyl ester 2b, from commercially available emu eggshells. Students may work individually or in pairs and complete the experiment within two 3 h laboratory periods. Dark green emu eggshells (approx 30− 50 g) are broken into large pieces and briefly (∼20 min) submerged in 3 N aq HCl at ambient temperature. This treatment loosens the thick, white membrane from the inner face of the eggshell, allowing for manual separation from the rinsed shells; the removal of this membrane greatly facilitates the extraction of the biliverdin dimethyl ester. The green eggshells are ground to a powder in a commercial blade coffee grinder, added to a 2 M methanolic H2SO4 solution, and heated to gentle reflux for 1 h. After neutralization, crude 2b is isolated by extraction with ethyl acetate from the MeOH/aqueous solution and purified on a silica gel flash chromatography column with ethyl acetate as the eluent. After workup, pure 2b is obtained as a dark blue-green material and is characterized by TLC, UV−vis, IR and 1H NMR spectroscopy, and ESI+ MS. In addition, a more rapid small-scale extraction of only 50− 200 mg of eggshells (emus, chicken, quail, or other colored bird eggshells) is also described (Supporting Information). This procedure provides enough of the primary pigments (depending on the eggshells chosen, biliverdin and/or protoporphyrin IX, as their dimethyl esters) for their characterization using UV−vis spectroscopy, ESI+ MS, TLC, or normal-phase HPLC with UV-detection. Reduction of 2b to bilirubin dimethyl ester (3b) (Scheme 1) can be accomplished by addition of NaBH4 to a solution of 2b in ethyl acetate containing methanol; the blue-green color of 2b converts to the yellow color of 3b within minutes, after which the reaction should be quenched with acetic acid. Bilirubin derivative 3b can be characterized by UV−vis spectroscopy and ESI+ MS.

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HAZARDS Suitable personal protection equipment (splash goggles, lab coat, gloves) must be worn when working with strong mineral acids (aqueous hydrochloric acid, HCl; sulfuric acid, H2SO4). Proper precautions must be taken when working with flammable solvents (ethyl acetate, methanol, hexanes): fume hood and absence of flames or other ignition sources. The use

Figure 1. Emu eggshell purchased at Floek’s Country Ranch, Tucumcari, NM.

of ovogenesis, see Supporting Information). Porous layers of calcite, a white, crystalline form of calcium carbonate (CaCO3), mixed with structural proteins, form the eggshell structure. The B




The first acid digestion of coarse emu eggshell fragments using 3 N HCl at ambient temperature dissolves some eggshell calcium carbonate according to the equation

of a well-ventilated fume hood is recommended for the extraction procedures under reflux conditions. Silica gel is an inhalation hazard; the generation of dusts should be avoided. Sodium borohydride (NaBH4) is flammable, particularly in contact with protic solvents. It is hazardous (irritant, strongly corrosive) in the case of skin or eye contact. Seek immediate medical attention upon ingestion or inhalation. Deuterated chloroform is a volatile irritant suspected to be a carcinogen. n‑Hexane is a neurotoxin. Biliverdin DME (2b) does not carry any significant safety threats. After handling the dry eggshells, hands should be carefully washed with soap. Provide liquid and solid waste containers for the proper disposal of all materials.

CaCO3(s) + 2HCl(aq) → CaCl 2(aq) + CO2 (g) + H 2O

This allows the mechanical separation of a tough inner membrane from the eggshell, without apparent digestion of the dye-bearing cuticle layer. This facilitates the subsequent digestion step using H2SO4/MeOH at reflux temperature. This acid/solvent combination releases biliverdin (2a) from its proteinaceous matrix, esterifies it to its dimethyl ester (2b) (Scheme 1), and converts the eggshell calcium carbonate (CaCO3) to calcium sulfate (CaSO4), according to

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STUDENT ACTIVITY AND DISCUSSION The isolation process is a modern adaptation of a classic procedure and involves a two-step acid digestion.13 The first step (using 3 N HCl) only etches the eggshells and allows the manual removal of an inner membrane, facilitating the second step, a complete acid digestion of the eggshells (using 2.0 M H2SO4 in MeOH) in a standard reflux setup. This step concomitantly esterifies biliverdin diacid 2a to form the dimethyl ester 2b (Scheme 1). Both acid-digestion steps are time-insensitive and can be interrupted at any given time with no visible degradation of the biliverdin. The esterification facilitates isolation (extraction from an aqueous to an organic phase) and purification (by short silica gel flash column chromatography) of biliverdin 2b. Depending on the target audience and available instrumentation, the extract can be analyzed by TLC; UV−vis, IR, and 1H NMR spectroscopy; and ESI+ mass spectrometry. In a biomimetic fashion, the teal biliverdin 2b can be readily reduced with NaBH4 to greenyellow bilirubin 3b (Figure 2).14

CaCO3(s) + H 2SO4 (aq) → CaSO4 (aq) + CO2 (g) + H 2O

The resulting off-white chalky filter cake (mainly composed of CaSO4 and undigested eggshell CaCO3) and the dark bluegreen filtrate attest to the efficiency of the eggshell pigment extraction. The methanolic filtrate is diluted with water, and the biliverdin dimethyl ester (2b) is extracted into an organic phase (ethyl acetate). Optional silica gel flash chromatography (100% ethyl acetate) yields biliverdin IXα dimethyl ester (2b) of high purity. Typically, the yields varied with the degree to which the biliverdin was released from the eggshell in the sulfuric acid digest, the skill of the student, and the coloration of the eggshell (that ranged from light teal to nearly black-blue-green). We found that in one class (n = 14) our students extracted from 50 g eggshells (corresponding to one-third to one-half of an eggshell) between traces and 20 mg of pure, crystallized biliverdin DME (2b). In most cases, the yield of 2b was sufficient to allow an acquisition of a high-quality 1H NMR spectrum using 16−64 scans. If only samples for UV−vis spectroscopy, mass spectrometry, and HPLC are desired, the extraction procedure can be scaled down by a factor of 100, or more, and the membrane removal and chromatography steps can be omitted. TLC confirmed the purity of the extract (see Supporting Information); the presence of ester- and lactam-type carbonyl groups was identified by students in an IR spectrum of 2b (see Supporting Information). The diagnostic spectroscopic data of the purified teal extract identified it as biliverdin dimethyl ester (2b): the broad, two-band UV−vis spectrum (Figure 2) is characteristic for open-chain tetrapyrrolic pigments in general,14 and conforms to the published spectrum of biliverdin dimethyl ester (2b).15 It is distinctly different from the UV−vis spectrum of the macrocycle-aromatic protoporphyrin IX (see Supporting Information), highlighting the effect of macrocyclization on the electronic properties of molecules. The 1H NMR spectrum of 2b was well-resolved and showed all carbon-bound hydrogens; the NH hydrogens are, depending on the amount of moisture present, broadened and sometimes not visible (Figure 3). The number of signals indicated the absence of any symmetry in the molecule, and functional groups could be identified on the basis of their diagnostic peak positions and peak patterns. A more detailed discussion of their assignment is provided in the Supporting Information. Biliverdin formed by the natural degradation of heme is, in nature, further metabolized by enzymatic reduction to the yellow compound bilirubin (3a) (Scheme 1).7 This reduction can also be performed chemically on the biliverdin dimethyl

Figure 2. UV−vis spectrum of purified biliverdin dimethyl ester 2b (in EtOAc) and crude bilirubin 3b (made by addition of NaBH4/MeOH to 2b in the cuvette); representative student data.

This process requiring two 3 h laboratory sessions can be accelerated to a single 3 h session if the amount of eggshells is significantly reduced and the crude extract analyzed by UV−vis spectroscopy, ESI+ mass spectrometry, and HPLC. The advantage of the latter procedure is that any eggshell can be analyzed and the presence and ratio of biliverdin and protoporphyrin in the eggshells determined. Overall, these experiments exemplify the isolation and chemical modification of a natural product using standard methodologies and the spectroscopic analysis of a complex, multifunctional biologically relevant molecule with multiple diagnostic features. C




Figure 3. 1H NMR spectrum (CDCl3, 400 MHz) of 12 mg of purified recrystallized biliverdin dimethyl ester 2b. Student samples may be pooled should the yields per individual student be suspected to be too low for the recording of a well-resolved spectrum.

The spectroscopic analyses of biliverdin dimethyl ester provided an exercise in the spectroscopic characterization of a fairly complex molecule: The interpretation of its IR spectrum, particularly with respect to the presence of carbonyl functionalities, was readily understood by the audiences. The UV−vis spectra of the extract and its reduction product were compared to literature-published spectra of biliverdin, bilirubin, and protoporphyrin, to verify its identity (see also Supporting Information).6,7,15 The spectra were interpreted by comparison of the degrees of conjugation present in the corresponding molecules. Most second-year undergraduates did not have the knowledge base to truly comprehend the photophysical origins of the UV−vis bands observed, but all audiences benefited from the qualitative discussion of π-conjugation pathways and associated colors. The participants of our workshop were treated to a more comprehensive discussion of the photophysical aspects of the tetrapyrroles.6 This experiment introduces our research students to some crucial techniques used in their synthesis-based research. The ESI+ mass spectrum was essentially self-explanatory for second-year or upperdivision undergraduate organic students once they understood the principle of ionization operating in ESI+ MS. Discussion of the acid-catalyzed esterification mechanism illustrated the dual role of sulfuric acid in the extraction. Interpretation of the 1H NMR spectrum was the most challenging, but after some guidance, the students recalled much of what they had learned in their lecture courses. Once the final peak assignments were made and all hydrogens were assigned, a deep sense of accomplishment was felt. The small-scale extraction protocols and HPLC analysis of the extracts allow for the screening of a variety of eggshells and a (semi)quantitative determination of the presence of the two major pigments, allowing a study of the pigments in a variety of brown (hen, quail) and green (Araucana fowl, emu) commercially available eggshells.10,12 The isolation procedure reaffirmed acid−base and precipitation concepts that students learned in general chemistry. In general, we found that life and natural science majors were particularly taken with chemistry experiments that touched upon biological problems, making eggs wonderful teaching tools.6,16 Since the majority of students taking advanced laboratory chemistry classes are not chemistry majors, creating this link to their core interests and other courses presented an

ester (2b) (Scheme 1). Addition of a pinch of NaBH4 to a cuvette of a solution of 2b (in EtOAc with a drop of MeOH) accomplishes this reduction (Figure 2). The drastically altered optical spectrum of the resulting bilirubin dimethyl ester (3b) is rationalized by the reduction of the central carbon, leading to interruption of the tetrapyrrolic π-conjugation by formation of two conjugated dipyrrinone units that are isolated from one another, reiterating the effect conjugation has on the electronic properties of the tetrapyrroles. ESI+ (30 V cone voltage, 100% acetonitrile) is an excellent ionization method for a mass spectrometric analysis of the biliverdin (and bilirubin). In both cases, the monoprotonated parent molecule was observed (see Supporting Information for representative student spectra of 2b and 3b). HPLC analysis of the eggshell extracts using a gradient elution protocol (hexanes and ethyl acetate) on a normal-phase column with detection wavelength set at 400 nm provides good separation and detection of the tetrapyrrolic compounds (see Supporting Information for details). Representative HPLC chromatograms of eggshells containing either biliverdin or protoporphyrin, extracted as their dimethyl esters, are presented in the Supporting Information.

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EDUCATIONAL ASPECTS This experiment is suitable for second-year or upper-division undergraduate organic chemistry laboratory classes in which a natural product isolation and characterization project that utilizes a number of standard laboratory techniques (reflux setup, extraction, TLC, column chromatography, use of rotary evaporator, product precipitation, handling of very small quantities), spectroscopic methods (UV−vis, IR, MS, 1H NMR), and separation methods (TLC, HPLC) is desirable. Also, the experiment, due to the intense colors involved, is satisfying even in the absence of some, or all, of the instrumentation needed for the spectroscopic characterization/HPLC separation of the products. The experiment was tested on a variety of audiences (undergraduate students newly joining our research group and second-year undergraduate organic laboratory classes), and it was made the centerpiece of a 3 day high school teacher training workshop. The experiments were performed primarily by individuals but are also suitable for pairs of students. D




(3) Dias, A. M.; La Salete Ferreira, M. Supermarket Column Chromatography of Leaf Pigments” Revisited: Simple and Ecofriendly Separation of Plant Carotenoids, Chlorophylls, and Flavonoids from Green and Red Leaves. J. Chem. Educ. 2015, 92 (1), 189−192. (4) This plant/animal distinction is oversimplified: While the dominant tetrapyrroles in animals are indeed porphyrins, some chlorins may also be found (in nonphotosynthetic capacities). Inversely, the metabolic and photosynthetic apparati of plants also contain hemes. (5) (a) Stanish, I.; Zabetakis, D.; Singh, A. Efficient Separation of Yolk from White in Boiled Chicken Eggs Leads to Convenient Extraction of Biologically Viable Phosphatidylcholine Lipids. J. Chem. Educ. 2002, 79 (4), 481−483. (b) Bendinskas, K.; Weber, B.; Nsouli, T.; Nguyen, H. V.; Joyce, C.; Niri, V.; Jaskolla, T. W. A Teaching Laboratory for Comprehensive Lipid Characterization from Food Samples. J. Chem. Educ. 2014, 91 (10), 1697−1701. (6) Dean, M. L.; Miller, T. A.; Brückner, C. Egg-Citing! Isolation of Protoporphyrin IX from Brown Eggshells and Its Detection by Optical Spectroscopy and Chemiluminescence. J. Chem. Educ. 2011, 88 (6), 788−792. (7) (a) Frydman, R. B.; Frydman, B. Heme Catabolism: A New Look at Substrates and Enzymes. Acc. Chem. Res. 1987, 20 (7), 250−256. (b) McDonagh, A. F. Turning Green to Gold. Nat. Struct. Biol. 2001, 8 (3), 198−200. (8) (a) Bulmer, A. C.; Ried, K.; Blanchfield, J. T.; Wagner, K. H. The Anti-Mutagenic Properties of Bile Pigments. Mutat. Res., Rev. Mutat. Res. 2008, 658 (1−2), 28−41. (b) Bois-Choussy, M.; Barbier, M. Photo-Oxidation and Photoprotection in the IXγ Bile Pigment Series: A Comparison of the Photoprotective Roles of Pterobilin, Phorcabilin and Sarpedobilin in vitro. Tetrahedron 1983, 39 (11), 1915−1918. (d) Nakagami, T.; Toyomura, K.; Kinoshita, T.; Morisawa, S. A Beneficial Role of Bile Pigments as an Endogenous Tissue Protector: Anti-Complement Effects of Biliverdin and Conjugated Bilirubin. Biochim. Biophys. Acta, Gen. Subj. 1993, 1158 (2), 189−193. (e) Dorazio, S. J.; Halepas, S.; Bruhn, T.; Fleming, K. M.; Zeller, M.; Brückner, C. Singlet Oxygen Oxidation Products of Biliverdin IXα Dimethyl Ester. Bioorg. Med. Chem. 2015, 23 (24), 7671−7675. (9) Fang, L.-S.; Bada, J. L. A comparative study of the occurrence, extent of conjugation, and excretion of the bile pigment biliverdin in marine fish. Marine Biol. Lett. 1983, 4, 341−348. (10) Kennedy, G. Y.; Vevers, H. G. A Survey of Eggshell Pigments. Comp. Biochem. Physiol. 1976, 55B, 117−123. (11) Wikipedia entry for “Emu”. http://en.wikipedia.org/wiki/Emu (accessed Aug 2017). (12) Eggshells of the American robin (Turdus migratorius) and many other thrushes are light blue (robin’s egg blue) because of their high biliverdin contents. However, it should be made abundantly clear to the students that collecting and possessing wild birds’ eggs is illegal in the US, the EU, and many other countries. (13) Tixier, R. Contribution a L’étude De L’ester Méthylique De La Biliverdine Des Coquille D’oeufs D’emeu. Bull. Soc. Chim. Biol. 1945, 27, 627−631. (14) Falk, H.: The Chemistry of Linear Oligopyrroles and Bile Pigments; Springer Verlag: NY, 1989. (15) Margulies, L.; Stockburger, M. Spectroscopic Studies on Model Compounds of the Phytochrome Chromophore. Protonation and Deprotonation of Biliverdin Dimethyl Ester. J. Am. Chem. Soc. 1979, 101 (3), 743−744. (16) (a) Cortel, A. Equilibrium with Fried Eggs of PbI2 and KPbI3. J. Chem. Educ. 1997, 74 (3), 297. (b) Eagle, C. T.; Dearman, B. M.; Goodman, A. B. Chemistry for Breakfast: Approaching Kinetics and Uncovering Everyday Chemistry by Cooking Eggs. Chem. Educ. 2003, 8 (2), 122−124. (c) Choi, M. M. F.; Wong, P. S. Using a Datalogger To Determine First-Order Kinetics and Calcium Carbonate in Eggshells. J. Chem. Educ. 2004, 81 (6), 859−861. (d) Mebane, R. C.; Rybolt, T. R. Chemistry in the Dyeing of Eggs. J. Chem. Educ. 1987, 64 (4), 291−293. (e) Newton, T. A. Measurement of Eggs: A General Chemistry Experiment. J. Chem. Educ. 1990, 67 (7), 604−605.

educational benefit. While avian eggs were familiar to all students, the students were unfamiliar with emu eggshells. We found that their sheer size and coloration held deep fascination. This fascination was leveraged to get students to think about topics the instructor liked to emphasize, be it the structure and function of tetrapyrrolic naturally occurring pigments (our particular specialty), or the chemistry of bruises. When the study is extended beyond emu eggshells, a discussion of the many roles attributed to eggshell coloration is an obvious extension of this experiment (see Supporting Information).10 This experiment confirms that eggs are a wonderful and versatile object for an interlaboratory approach to study chemistry and biology. Additional teaching material in support of this endeavor is available in the Supporting Information.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00449. Illustrated detailed instructions of the extraction procedure, instrument and materials lists, a reproduction of all spectra, and resources on eggs, ovogenesis, biliverdin, and the nomenclature of tetrapyrrolic pigments (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Randy Hamchand: 0000-0002-9078-7550 Christian Brückner: 0000-0002-1560-7345 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the many students and the members of the high school teacher “Eggciting” workshop that tested the experiments. The work was supported by the NSF through Grants CHE-1058846, CHE-1465133 (to C.B.), and HRD-1400382 (fellowship to R.H.).

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REFERENCES

(1) Milgrom, L. R. The Colours of Life: An Introduction to the Chemistry of Porphyrins and Related Compounds; Oxford University Press: New York, 1997. (2) For recent examples, see: (a) Purcell, S. C.; Pande, P.; Lin, Y.; Rivera, E. J.; Paw U, L.; Smallwood, L. M.; Kerstiens, G. A.; Armstrong, L. B.; Robak, M. T.; Baranger, A. M.; Douskey, M. C. Extraction and Antibacterial Properties of Thyme Leaf Extracts: Authentic Practice of Green Chemistry. J. Chem. Educ. 2016, 93 (8), 1422−1427. (b) McLain, K. A.; Miller, K. A.; Collins, W. R. Introducing Organic Chemistry Students to Natural Product Isolation Using Steam Distillation and Liquid Phase Extraction of Thymol, Camphor, and Citral, Monoterpenes Sharing a Unified Biosynthetic Precursor. J. Chem. Educ. 2015, 92 (7), 1226−1228. (c) Garber, K. C. A.; Odendaal, A. Y.; Carlson, E. E. Plant Pigment Identification: A Classroom and Outreach Activity. J. Chem. Educ. 2013, 90 (6), 755− 759. (d) Chrea, B.; O’Connell, J. A.; Silkstone-Carter, O.; O’Brien, J.; Walsh, J. J. Nature’s Antidepressant for Mild to Moderate Depression: Isolation and Spectral Characterization of Hyperforin from a Standardized Extract of St. John’s Wort (Hypericum perforatum). J. Chem. Educ. 2014, 91 (3), 440−442. E


Isolation of Biliverdin IXÎ±, as its Dimethyl Ester, from Emu Eggshells

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