In the Classroom edited by
JCE DigiDemos: Tested Demonstrations
Ed Vitz
The Fluorescence of Lignum nephriticum: A Flash Back to the Past and a Simple Demonstration of Natural Substance Fluorescence submitted by:
Mark Muyskens Department of Chemistry and Biochemistry, Calvin College, Grand Rapids, MI 49546;
[email protected] checked by:
Ed Vitz Department of Chemistry, Kutztown University, Kutztown, PA 19530
This article describes a simple and visually striking demonstration of fluorescence from the aqueous extract of the tropical hardwood Pterocarpus indicus, also known as narra. Its simplicity and dramatic pH dependence make this demonstration particularly attractive as an example of a natural substance that is strongly fluorescent. The phenomenon also has historical significance because it represents the first recorded observation of fluorescence, noted over 400 years ago when the wood was known as Lignum nephriticum. In addition to the demonstrations described in this article, a companion JCE Classroom Activity gives the details for a hands-on activity based on narra extract fluorescence (1). Fluorescence occurs when a substance absorbs light in one wavelength region and at the same time emits light in a different region of the spectrum, normally of longer wavelength. Fluorescent substances are encountered on a daily basis in modern society, where fluorescence is used to capture our attention: traffic-safety cones, school-crossing signs, store price labels, and retail packaging. Here a fluorescent pigment absorbs ultraviolet and visible light from the sky or room lights and emits visible light in a different wavelength region from that absorbed. We notice the object in part because it has a luminosity that is enhanced relative to the nonfluorescent objects around it. The characteristic shift to longer wavelength from absorbed light to emitted light is known as the Stokes shift. The topic of fluorescence engages not just everyday uses, but also many important scientific applications. Three significant examples are (i) green fluorescent protein, which is used as an important biochemical tool for research involving gene expression (2); (ii) automated DNA sequencing based on fluorescence, which has significantly accelerated the human genome project; and (iii) enzyme-linked immunoassay (ELISA) with fluorescence detection, which plays a role in detection of mad cow disease. Therefore, even brief introductions to the topic of fluorescence can tie into exciting developments in research. While most of the fluorescent objects placed in our daily path contain synthetic fluorescent pigments, there are good examples of natural substances that are fluorescent. A recent article and activity published in this Journal (3), which focused on fluorescence and other kinds of luminescence, included the fluorescence of chlorophyll, quinine (found in tonic water), and specimens of minerals. Another good exwww.JCE.DivCHED.org
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Kutztown University Kutztown, PA 19530
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ample is riboflavin (vitamin B2) in aqueous solution, which gives bright yellow fluorescence under a black light. While the observation of chlorophyll fluorescence usually involves extraction by a flammable solvent, this demonstration involves aqueous extraction of narra wood. The place of fluorescence in a college chemistry curriculum is most likely in analytical and physical chemistry. However, this demonstration can play a role in any part of the curriculum that may touch on fluorescence. It can tie into demonstrations of the pH dependence of solutions such as indicators or serve as an entry in discussions of the interaction of light, color, and molecules. Fluorescence in general plays such an important role in science and society that it deserves more coverage in the high school and college chemistry curriculum. Demonstration and Discussion Pterocarpus indicus is a large, rainforest tree from Southeast Asia, the Philippines, and Malaysia that produces a choice hardwood used for furniture, cabinetry, and carving. It is known by a variety of common names besides narra including angsana, Malay padauk, and Amboyna burl. The wood can be purchased from a number of companies in the United States that supply exotic wood species to wood workers. A small piece of wood about 24 in. × 2 in. × 2 in. will provide enough shavings for many demonstrations and experiments, and pieces of about this size can be purchased for under $15 including shipping costs.1 Not all wood-supply businesses are careful to include the species name in their product description. For example, a wood simply called padauk turned out to be Pterocarpus soyaxii (African padauk) from Cameroon, a closely related species that produces a significantly weaker fluorescence in water. However, wood samples with the common names listed above, particularly samples that were listed specifically as Pterocarpus indicus, give good results. Eysenhardtia polystachya is another wood species tied historically to narra with similar fluorescence properties. It is a shrub or small tree native to Mexico and Southwest United States, also known as Mexican kidneywood. This wood apparently has much less commercial value because a Web search on this species yielded botanical information but no commercial sources. Observing the fluorescence from an aqueous infusion of narra is straightforward and produces eye-catching results.
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Adequate shavings of the yellowish-red, fragrant hardwood can be obtained with a knife. However, it is easier to generate a large quantity of shavings by using a wood plane. A single shaving (∼0.1 g) placed in 50 mL of room temperature tap water for a few minutes is sufficient to produce a solution that exhibits blue fluorescence.2 The aqueous solution is a yellow–amber color. The shavings can be left in the solution, or removed by decanting or filtering. The blue tinge from fluorescence is more easily seen in a concentrated solution (3 grams of shavings placed in 200 mL of room temperature water with a drop of 1 M NaOH for at least an hour). The blue fluorescence is easily observed by eye at the surface of the solution from at least four different excitation sources: sunlight, fluorescent room light, a long-wave ultraviolet lamp (black light), and an ultraviolet light-emitting diode3 (UV LED; maximum wavelength, ∼400 nm). While fluorescence in general is more easily observed in a darkened room, narra extract fluorescence is bright enough to be viewed satisfactorily in normal room light when excited using a UV LED. Pouring the amber solution in direct sunlight while viewing the flowing liquid against a dark background can produce the spectacular effect of a blue stream. Although the fluorescent substance in the extract is susceptible to degradation in sunlight, a glass vessel of the liquid sitting in a sunny location provides a great show for several weeks and makes a wonderful discussion starter on the topic of fluorescence. A solution kept out of direct sunlight at room temperature will remain fluorescent for months. The fluorescence is strongly pH dependent with the transition occurring from a pH of 6 (non-fluorescent) to 8 (fluorescent). A pH series of solutions in room light and under black light illumination are shown in Figure 1. The visible absorption spectrum of the narra solution changes with pH as well, the solution becoming darker yellow in color as it becomes more fluorescent. For a spectacular show, illuminate
Figure 1. pH dependence of the aqueous extract of narra. Standard 1-cm cuvettes are labeled with the pH. The top series (A) shows the room light appearance with a transition from nearly colorless to yellow occurring between pH 6 and 8. The bottom series (B) shows the same set of cuvettes under black light ultraviolet illumination with a transition from non-fluorescence to blue fluorescence (maximum emission at 465 nm) occurring from pH 6 to 8 and a second transition from fluorescent to non-fluorescent from pH 11 to 13.
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a sample of the non-fluorescent solution (pH below 6) with a black light in a darkened room and add a small quantity of base to the solution (a single drop of 0.1 M NaOH is usually sufficient); the solution becomes brightly fluorescent. Figure 1 also shows that there is a second transition from fluorescent to non-fluorescent at high pH, between 11 and 13. Thus, it is possible with a strong base to raise the pH sufficiently to achieve a non-fluorescent result. The watersoluble constituents of the wood will lower the pH of deionized water enough that it normally requires raising the pH to observe strong fluorescence. On the other hand, tap water may have enough natural buffer capacity to moderate the pH change and may not require any pH adjustment for good fluorescence. Carrying out side-by-side extractions with deionized water and tap water can be an entry point to discussing the difference between these two types of water; tap water contains dissolved solids that buffer the effect of adding narra shavings. The aqueous extract from African padauk exhibits a similar, pH dependent, blue fluorescence, but it has a much weaker intensity than narra for the same quantity of wood. Use of this demonstration in a small lecture forum (circa 50 students) can be done in more than one way. Use of a concentrated solution in its fluorescent form (see above) on an overhead projector will show the visible color in the projected image, and the blue fluorescence excited by the projector lamp is visible at the bottom of the beaker. Lowering the pH will show the loss of fluorescence and the change of color. In a more hands-on approach, the fluorescent and nonfluorescent narra solution can be placed in labeled cuvettes and passed among the students along with a UV LED keychain flashlight3 for excitation. The two samples are easily distinguished by the bright blue fluorescence in the sample with elevated pH. The Stokes shift, one of the key features of fluorescence, can be demonstrated quite easily using a yellow filter. The Roscolene Medium Lemon yellow filter #8064 is an ideal filter because its absorbance spectrum coincides very well with the absorbance spectrum of the fluorescent component of narra. Placing the filter between the excitation source and the solution blocks the fluorescence because the filter blocks the wavelengths needed for fluorescence. When the filter is placed between the solution and the observer, the fluorescence is still visible because the filter passes light emitted as fluorescence. Figure 2 shows the four spectra involved in this demonstration: (A) the UV emission spectrum of the black light, (B) the fluorescence excitation spectrum, which is the fluorescence emission as it depends on excitation wavelength and is directly related to where fluorescent components of narra absorb,5 (C) the fluorescence emission spectrum, and (D) the transmission spectrum of the yellow filter. The overlap between curves A and B indicates where the black light is absorbed by the fluorescent component of the narra solution. The yellow filter absorbs the black light emission, but passes a portion of the narra solution emission. Based on the spectra, it appears that the filter absorbs a significant quantity of the fluorescent emission, however to the human eye the fluorescence appears to be undiminished. A careful observer will note a subtle shift in the color emitted from blue to green, because the filter removes a portion of the blue light from the emission. It is clear from these simple observations
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that the light emission is in a different wavelength region from the light absorbed, and because blue light has longer wavelengths than violet light it illustrates the Stokes shift. Historical Background According to Partington (4), the first recorded observation of fluorescence dates to the year 1565.6 The writings of the Spanish physician and botanist Nicolás Monardes describe a wood from New Spain called Lignum nephriticum that was used as a medical treatment for liver and kidney ailments. Monardes also mentions that water in contact with the wood assumes an unusual blue color. Lignum nephriticum was well known in 16th and 17th century Europe owing to cups or chalices made of this exotic wood that likewise exhibited a curious blue color at the surface of water placed in the cup. Robert Boyle in 1664 reported that the blue tinge was dependent on pH (4). Sir Isaac Newton in 1672 included his observations of the phenomenon in his efforts to formulate his theory of light and color (5). However, the botanical origin of Lignum nephriticum was lost by the mid 18th century. Safford’s work in 1915 reestablished the origins of Lignum nephriticum (7). There are two different woods that became confused as one, both giving similar fluorescent results in water. The two woods are narra and Mexican kidneywood mentioned earlier in this article. Safford is convinced that Robert Boyle’s observations were based on Mexican kidneywood, whereas the once-famous cups came from
Figure 2. Spectra related to narra extract fluorescence. (A) The black light emission spectrum, gray line (48 in. black light fluorescent tube, Phillips F40T12/BLB); (B) the narra solution fluorescence excitation spectrum, solid line (the fluorescence emission as it depends on excitation wavelength); (C) the narra solution fluorescence emission spectrum, dot–dash line; and (D) the percent transmission spectrum of the yellow filter (Roscolene Medium Lemon #806), dashed line. The excitation spectrum and both emission spectra are normalized and refer to the left axis; the filter spectrum refers to the right axis. The yellow filter absorbs the black light emission, but passes the narra solution emission.
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Figure 3. Structure of 7-hydroxy-4’-methoxyisoflavone or formononetin.
the larger narra logs, which were imported to Spain from the Phillipines via Mexico, no doubt contributing to the confusion of origins. The two species are in the same botanical family, Fabaceae (formerly known as Leguminosae). Published studies of the constituents of the woods, narra and Mexican kidneywood, have suggested the principal fluorescent component is an isoflavone. Cooke and Rae examined the heartwood of narra (8) and, while their study made no mention of the fluorescence characteristics, they did identify 7-hydroxy-4´-methoxyisoflavone or formononetin (Figure 3) as one of several principal components in successive petroleum and acetone extractions. Burns et al. sought Robert Boyle’s fluorescent indicator in their study of Mexican kidneywood (9). They assigned this role to 7-hydroxy2´,4´,5´-trimethoxyisoflavone, which they identified in a methanol extract. In the Mexican kidneywood study, they specifically examined the constituents for their fluorescent characteristics, including pH dependence. Because the two isoflavones are so closely related, Burns et al. assume that formononetin is the fluorescent constituent in narra and that the similarity explains the like accounts of blue fluorescence from the two woods. Preliminary studies of narra and formononetin fluorescence done in our laboratories, however, suggest that the identity of the fluorescent component is not completely known. We have confirmed that formononetin, a substance that is only partially soluble in room-temperature water, does give a blue fluorescent aqueous solution with a similar pH-dependence to narra. Our results are in agreement with published studies on formonentin fluorescence (10, 11). On the other hand, an aqueous formonentin solution absorbs light only in the UV region and hence is colorless. In our study, the fluorescence excitation spectrum of narra extract, shown in Figure 2, peak B, clearly indicates that the fluorescent component absorbs in the region 370 to 450 nm with peak absorbance at 430 nm accounting for some if not all of the extract’s yellow appearance. The UV–vis absorption spectrum of narra extract also shows the absorption band at 430 nm along with another band in the ultraviolet that does not lead to fluorescence; both absorption bands are pH dependent. While formononetin may be related to the fluorescent substance in narra wood, formononetin cannot by itself account for the fluorescence in narra solutions. Fluorescence within the family Fabaceae is not limited to the three species of wood already mentioned. In his book on identifying woods, Hoadley (12) lists nine North American species in the family Fabaceae in which the wood is known to be fluorescent. Other members of the Fabaceae family are known for containing isoflavones, notably red clover and soybeans. We have confirmed in our laboratories that red clover extract, which contains four isoflavones including
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formononetin, also exhibits a pH-dependent fluorescence similar to the narra extract. The focus of this article however is on the aqueous extract of narra wood since that was the historical observation. Summary This article describes an easy demonstration of the first recorded observation of fluorescence, noted over 400 years ago. The narra wood is inexpensive and easily obtained. Its aqueous extract gives a beautiful, strong, blue fluorescence in sunlight and under ultraviolet light. Narra extract is one of the more easily demonstrated examples of fluorescence in natural substances since the extract is simple to prepare. The fact that the fluorescence is strongly pH dependent provides an additional dimension of interest to the demonstration. Highlighting the historical significance of Lignum nephriticum and discussing some of the relevant studies suggesting the chemical origin of the fluorescence provides a context for this demonstration. Acknowledgments MM thanks Holly Hoffman, Ashlee Hardy, Sarah Jelsema, and Rachael Glassford for their work on this project, and the students of two particular classes: the Calvin College Interim 2004 course on fluorescence, and the fall 2005 Honors General Chemistry Laboratory course. W
Supplemental Material
A photo of narra wood and information on wood sources, ultraviolet LEDs, and additional spectral data on narra solutions are available in this issue of JCE Online. Notes 1. One source is Curious Woods division of Curtis Lumber Company, Ballston Spa, NY. 800/724-WOOD, http:// www.curiouswoods.com/ (accessed Feb 2006). Information on additional vendors of narra used by the author is available in the Supplemental Material.W 2. It is important to keep in mind that the fluorescence is pH dependent. While using tap water of sufficient hardness will normally produce fluorescence without pH adjustment, it is possible that raising the pH will be necessary to see the full effect.
3. The author has used UV light-emitting diodes with maximum emission at 395, 400, and 405 nm; all work well. See the Supplemental MaterialW for more information. As with any light source containing UV wavelengths, appropriate caution should be used to minimize direct exposure. Never look directly at a bright light source. 4. See filter manufacturer for a directory of dealers, Rosco USA, 800/767-2669, http://www.rosco.com/us/ (accessed Feb 2006). You can also search on the Internet using “roscolene filter sheets” to find vendors. See the Supplemental MaterialW for additional information. Note that other yellow filters are effective, particularly with the UV light-emitting diodes, but the Roscolene #806 filter is clearly the best at completely blocking fluorescence from a black light. 5. The online supplement has more information about the excitation spectrum of narra extract. 6. Authors give slightly different dates for Monardes’ writings: 1565 according to Partington (4), Valuer (6) and Safford (7); 1574 according to Harvey (5), which according to Partington corresponds to the first appearance of the Latin translation from the earlier Spanish version.
Literature Cited 1. Muyskens, M. A. J. Chem. Educ. 2006, 83, 768A–768B. 2. Hicks, B. W. J. Chem. Educ. 1999, 76, 409–415. 3. O’Hara, P. B.; Engelson, C.; St. Peter, W. J. Chem. Educ. 2005, 82, 49–52. (b) 2005, 82, 48A–48B. 4. Partington, J. R. Ann. of Sci. 1955, 11, 1–26. 5. Harvey, E. N. A History of Luminescence from the Earliest Times Until 1900; The American Philosophical Society: Philadelphia, 1957; pp 391–392. 6. Valeur, B. Molecular Fluorescence: Principles and Applications; Wiley–VCH: Weinheim, Germany, 2002; p 6. 7. Safford, W. E. Annual Report of the Board of Regents of the Smithsonian Institution 1915, 271–298. 8. Cooke, R. G.; Rae, I. D. Aust. J. Chem. 1964, 17, 379–384. 9. Burns, D. T.; Dalgarno, B. G.; Gargan, P. E.; Grimshaw, J. Phytochemistry 1984, 23, 167–169. 10. Dunford, C. L; Smith, G. J.; Swinny, E. E.; Markham, K. R. Photochem. Photobiol. Sci. 2003, 2, 611–615. 11. de Rijke, E.; Joshi, H. C.; Sanderse, H. R.; Ariese, F.; Brinkman, U. A. T.; Gooijer, C. Anal. Chem. Acta 2002, 468, 3–11. 12. Hoadley, B. R. Identifying Wood: Accurate Results with Simple Tools; Taunton Press: Newtown, CT, 1990; p 53.
This article has been developed into a JCE Classroom Activity: “pHantastic Fluorescence” by Mark Muyskens. See pages 768A–768B in this issue.
JCE Classroom Activities
Find all 81 Activities at JCE Online
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