In the Laboratory
Glowmatography Thomas S. Kuntzleman,* Anna E. Comfort, and Bruce W. Baldwin Department of Chemistry, Spring Arbor University, Spring Arbor, MI 49283; *
[email protected] Chemical reactions involving chemiluminescence are some of the most exocharmic reactions known (1, 2). In addition, experiments that incorporate the chromatographic separation of the colorful components of a mixture are of great interest to scientists of all ages (3–10). A simple exercise is described featuring the separation of the contents of an activated lightstick using a chromatography column prepared as described previously (11). Because the chemiluminescent reactions are clearly visible during a large portion of the chromatographic separation, this exercise can be used as a demonstration to a classroom audience of approximately 50 spectators. In addition, this exercise can also be used in a laboratory setting. This process has been presented to observers varying in chemical sophistication from elementary school students to upper-level undergraduate chemistry majors, but it is most often used when discussing intermolecular forces in general chemistry class. As a general chemistry demonstration or laboratory exercise, this procedure reinforces the role played by intermolecular forces in the separation of components from a mixture. In the upper-level undergraduate laboratory curriculum, this process may be used to introduce the principles of chromatography in analytical chemistry and in physical chemistry to introduce electronic absorption and emission spectroscopy. Two separations are described: one involving a lightstick that yields emission of light of a single color (procedure A) and a second involving a color-changing lightstick that appears to emit different colors as time progresses (procedure B).
Background The polar and nonpolar components of commercial lightsticks are chromatographically separated in this experiment. The chemical constituents of a lightstick may be classified into broad groups: hydrophobic (nonpolar organic) and hydrophilic (polar or ionic) species. Common nonpolar components of lightsticks include dibutyl phthalate (the solvent) and a phenyl oxalate, usually bis(2,4,5-trichloro-6-carbopentoxyphenyl) oxalate (12–16). The hydrophilic species include H2O2 and sodium salicylate, the latter serving as a catalyst (12–16). A wide range of fluorescent dyes are used to produce a variety of light emission colors from lightsticks (Table 1); the dyes vary in polarity. Usually, the fluorescent dyes and nonpolar components are contained in a glass ampoule in the center of a lightstick. This ampoule is encased within a plastic, outer tube. The plastic tube houses this ampoule and additionally contains a mixture of H2O2 and sodium salicylate in dibutyl phthalate. To initiate the glowing reaction, the inner glass ampoule is broken and the two classes of compounds are mixed (Scheme I). Once mixed, the H2O2 oxidizes the phenyl oxalate to form phenol and a high energy CO2 dimer, also known as 1,2-dioxetanedione (2, 12, 26, 27). The CO2 dimer transfers energy to the fluorescent dye(s), and is concomitantly cleaved into 2 molecules of CO2. The energy gained by the dye promotes electrons within the dye molecule to an excited state, which relaxes back to the ground
Table 1. Fluorescent Dyes Contained in Commercial Lightsticks Color(s)
Possible Dye(s) Responsible for Colors
References
Red
5,12-Bis(phenylethynyl)naphthacene* or 5,16,11,12-tetraphenylnaphthacene* or perylene dicarboximide derivatives
13–19, 22–25
Orange
Rhodamine derivatives, perylene dicarboximide derivatives, or mixtures of red, yellow, and green fluorescers
18, 22–25
Yellow
Perylene dicarboximide derivatives or 1,5-dichloro-9,10-bis(phenylethynyl)anthracene or 1,8-dichloro-9,10-bis(phenylethynyl)anthracene
18, 19, 22–25
Green
9,10-Bis(phenylethnyl)anthracene or 2-chloro-9,10-bis(phenylethnyl)anthracene or 2-methyl-9,10-bis(phenylethnyl)anthracenes
1, 16, 18, 22–25
Blue
9,10-Bisphenylanthracene or 9,10-bis(4-methoxyphenyl)-2-chloroanthracene or perylene
16, 18–20, 22–25
Purple
Mixture of 9,10-bis(4-methoxyphenyl)-2-chloro-anthracene and a perylene dicarboximide derivative
18, 22–25
White
Mixture of blue fluorescer and a perylene dicarboximide derivative
20, 21
Aqua
Mixture of blue and green fluorescers
16
Hot pink
Mixture of red and blue fluorescers
16
Pink-to-Blue
Mixture of 9,10-bis(4-methoxyphenyl)-2-chloro-anthracene and a red, peroxide unstable fluorescer
18, 20
Orange-to-Green
Mixture of 2-methyl-9,10-bis(phenylethnyl)anthracene and 5,6,11,12tetraphenylnaphthacene*
18, 20
*These compounds are unstable in the presence of peroxide.
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Journal of Chemical Education • Vol. 86 No. 1 January 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
state by the emission of light. The excitation energy is supplied by a [2+2] cycloreversion reaction of the CO2 dimer. As stated previously, different dyes or mixtures of dyes are used to produce a variety of colors in commercial lightsticks (Table 1). It should be noted that the colors of light emitted by these dyes can vary somewhat depending upon the type of solvent or dye concentration (18, 28). In addition, the perceived color of light emitted by a mixture of dyes varies with their ratio of concentrations (18). The sodium salicylate functions to catalyze the lightstick reaction, ensuring that the steps of the chemiluminescent reaction sequence go fast enough so that light emission is easily observed, but slow enough that the light is emitted for several hours (12–16, 26, 29).
O O
C O
O
O O
C 7 H 5 O3
H 2 O2
2
C C O O
OH
O O
C C
2 CO 2
dye
dye*
O O
Exercise Description and Discussion
dye*
dye
hO
Emission
Scheme I. Proposed reaction sequence for the reaction between bis(phenyl) oxalate and H2O2 in the presence of a fluorescent dye to generate visible light (2, 12, 26, 27). The first step is base-catalyzed (26, 27). Note that in a commercial lightstick a substituted bis(phenyl) oxalate is used.
450
500
550
600
650
Wavelength / nm
Figure 1. Emission intensities recorded for dye eluted from column (bottom trace); dye eluate to which H2O2 has been added (small peak); dye eluate to which both H2O2 and sodium salicylate have been added (medium peak); and an activated lightstick (large, slightly red-shifted peak). Dye eluate intensities recorded with a 500 ms integration time; lightstick intensity recorded with a 100 ms integration time (actual student data).
Emission
Absorbance
Based on Scheme I, a lightstick will only produce light if phenyl oxalate and hydrogen peroxide are physically capable of contacting one another in the presence of fluorescent dye(s). In addition, light output is significantly enhanced in the presence of base. In procedure A, the glowing lightstick1 mixture that is added to the chromatography column initially contains all four components necessary for facile observation of light emission. The stationary phase of the chromatography column is packed with silica gel containing a number of polar surface Si–OH groups. The mobile phase is comprised of nonpolar hexanes. As the glowing lightstick mixture passes through the column, the hydrogen peroxide strongly interacts with the Si–OH groups of the silica gel through hydrogen bonding interactions. Similarly, the salicylate ion is strongly attracted to the stationary phase through ion– dipole and hydrogen bonding interactions. Because both salicylate ion and hydrogen peroxide are strongly attracted to the stationary phase, these components tend to move very little through the column. In contrast, the hydrophobic phenyl oxalate and fluorescent dye do not interact as strongly with the polar Si–OH groups on the column and as a result spend most of the time traveling with the hexanes down the column. Therefore, as more hexanes are added to the column, the hydrophobic components are separated from the hydrophilic components, the former being eluted from the column and the latter being “stuck” on the column. Both the hydrophobic and hydrophilic components are necessary for light emission, and the glowing mixture is observed to progressively lose its intensity of light emission as increasing quantities of hexanes are passed through the column. The fluorescent dye that is no longer emitting is collected in the eluate. To further demonstrate that the hydrophobic and hydrophilic components have been separated in the chromatographic process, the hydrophilic H2O2 and salicylate ion are added, in turn, to the eluate (which contains the hydrophobic species), restoring the chemiluminescent reaction (Figure 1). While the addition of H2O2 alone restores the lightstick reaction, the addition of the salicylate catalyst in addition to H2O2 re-establishes the emission intensity close to that of the original lightstick. Of note, if sodium salicylate alone is added to the dye eluate, the luminescent reaction is barely perceptible in a completely darkened room; no emission above baseline is recorded (data not shown). Once a relatively pure sample of dye is collected from the column, a simple extension to the chromatographic procedure is to obtain fluorescence and absorption spectra of the dye, demonstrating their approximate “mirror image” relationship (Figure 2). From these spectra, the relationship between the
C
400
450
500
550
Wavelength / nm Figure 2. Comparison of emission from dye eluate treated with H2O2 and sodium salicylate with the absorbance of dye eluate with no H2O2 or sodium salicylate added. Spectra have been manipulated so that their maxima coincide at about the same intensity (actual student data).
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 1 January 2009 • Journal of Chemical Education
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In the Laboratory
O C
O
C O
O
H 2 O2
k2
C C O O
1 min 2 min 3 min 4 min 12 min 26 min 42 min
400
450
500
550
600
650
700
750
Wavelength / nm Figure 3. Time-dependent emission from a pink-to-blue color-changing lightstick (actual student data).
2 CO 2 dye1*
k3 dye2
k4 k5 k6
2 CO 2 dye2*
dye1 h O (blue) dye2 h O (pink–red) non-fluorescing products
Scheme II. Proposed reaction sequence occurring within a pink-toblue color-changing lightstick (implied from ref 18). Note that in a commercial lightstick a substituted bis(phenyl) oxalate is used.
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Sodium salicylate may cause eye, skin, and respiratory tract irritation. The hydrogen peroxide in fresh lightsticks is a strong oxidizing agent, and it may cause eye, skin, and respiratory tract inflammation. Hexanes are flammable. Silica gel causes respiratory tract irritation and the chromatography column should be packed in a hood. Exposure to or ingestion of the contents of lightsticks is unlikely to cause severe harmful effects; in rare cases irritation of the skin or eyes at the site of exposure may be experienced (30). The contents of lightsticks are claimed to be nontoxic by the manufacturer.
Emission
C C
dye2*
2
Hazards
dye1
O O
dye2*
C 7 H 5 O3
OH
O O
dye1*
O O
k1
emission is less polar than the dye responsible for red emission. In addition, it can be noted that the blue-emitting dye ceases to emit light, and thus appears yellow, as this nonpolar dye is separated from the polar H2O2 and salicylate ion. Because only the nonpolar, fluorescent yellow dye that emits blue light is eluted from the column, only blue chemiluminescence is observed after the addition of salicylate ion and hydrogen peroxide, further demonstrating that the polar and nonpolar components of the lightstick are separated (Figure 4).
Emission
perceived color of the dye when it is, and is not, emitting light may be discussed as described previously (2). The color-changing lightstick2 used in procedure B contains the same chemical components as those described for the lightsticks used in procedure A with the exception that two different fluorescent dyes, which happen to have substantially different polarity, are used to bring about the color-changing effect. A probable reaction sequence for the reactions occurring in the color-changing lightstick is shown in Scheme II. A stable dye that emits blue light (dye1) and a “peroxide unstable” dye that emits pink–red light (dye2) are included in the color-changing lightstick (18). At first, the lightstick is observed to emit a pink– red color, simply because much more pink–red light is emitted than blue (Figure 3). The greater output of red versus blue light may be due to a greater quantum fluorescence yield of dye2 compared to dye1, a faster reaction of dye2 than dye1 with the CO2 dimer (k3 >> k2), or a combination of these two effects. However, dye2 is unstable in the reaction mixture and decomposes over time, leading to a faster decrease in fluorescence from this dye as compared to dye1. As a result, the perceived color of the lightstick changes to a purple color at intermediate timescales, and finally to blue at long time scales when dye2 has been consumed. Nevertheless, the pink fluorescence is observed to persist on the column throughout the entire chromatography procedure described herein. In the initial phase of this demonstration, two glowing bands of light are simultaneously observed on the chromatography column. Considering that the blue-emitting band is observed to travel further down the column than the redemitting band, it can be noted that the dye responsible for blue
400
450
500
550
600
650
700
Wavelength / nm Figure 4. Comparison of light emission from blue-emitting fluorescent dye on the column as described in procedure B (trace with two peaks) with that of the contents removed from an activated pink-to-blue color-changing lightstick (trace with only one peak). Spectra have been manipulated so that maxima of the peaks at ~450 nm coincide (actual student data).
Journal of Chemical Education • Vol. 86 No. 1 January 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
Summary A simple exercise that interests and motivates observers from all levels of chemical sophistication is described. As such, this exercise can be used to teach a variety of topics in a variety of settings. Whether used in summer camp or in the physical chemistry laboratory, this exercise will appeal to chemists of various chemical skill levels. Whether illustrating the process of chromatographic separations, presenting lecture demonstrations, or conducting laboratory experiments, this exercise will illuminate important concepts and techniques in chemistry. Acknowledgments We wish to thank Ocean Optics, Inc. for an educational grant to support this work; Thomas McEldowney, Jacob Atem, Emily Wittenberg, Toyin Rotibi, Seth King, and Christine Becker for technical assistance; Stephany Briceno and Stephanie Ling for photography; and Deborah K. Sayers for helpful discussion. Notes 1. Lightsticks (Crazy Glow brand) were purchased from Windy City Novelties. http://www.windycitynovelties.com (accessed Jun 2008). 2. Similar separation of glowing bands on the column are obtained for procedure B with orange-to-green, white, purple, orange, hot pink, and aqua lightsticks. We report here the results of the pink-toblue lightsticks because this type displays the best separation of bands and contrast of colors. The color-changing lightsticks were bought in bulk (not individually wrapped), therefore the specific brand of these lightsticks is not known.
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Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2009/Jan/abs64.html Abstract and keywords Full text (PDF) Links to cited URLs and JCE articles Supplement Instructions for the preparation and implementation of this procedure JCE Featured Molecules for January 2009 (see p 128 for details) Structures of some of the molecules discussed in this article are available in fully manipulable Jmol format in the JCE Digital Library at http://www.JCE.DivCHED.org/JCEWWW/Features/ MonthlyMolecules/2009/Jan/. JCE Cover for Jaunary 2009 This article is featured on the cover of this issue. See p 3 of the table of contents for a description of the cover.
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