What Is Happening When the Blue Bottle Bleaches: An Investigation of

Article pubs.acs.org/jchemeduc

What Is Happening When the Blue Bottle Bleaches: An Investigation of the Methylene Blue-Catalyzed Air Oxidation of Glucose Laurens Anderson,*,† Stacy M. Wittkopp, Christopher J. Painter,‡ Jessica J. Liegel,§ Rodney Schreiner, Jerry A. Bell, and Bassam Z. Shakhashiri Wisconsin Initiative for Science Literacy, Department of Chemistry, University of WisconsinMadison, Madison, Wisconsin 53706-1322, United States S Supporting Information *

ABSTRACT: An investigation of the Blue Bottle Experiment, a well-known lecture demonstration reaction involving the dye-catalyzed air oxidation of a reducing sugar in alkaline solution, has delineated the sequence of reactions leading to the bleaching of the dye, the regeneration of color, and so forth. Enolization of the sugar is proposed as a key step in the sequence. The first-order rate constant for this step was found to be ∼2.3 × 10−3 min−1 with respect to total sugar concentration under the conditions used (0.184 M glucose, pH 13.3, 25 °C). Measurements with an oxygen-sensing electrode on Blue-Bottle mixtures containing varying quantities of methylene blue indicated that in a usual demonstration the rate of O2 consumption is ∼60% of the enolization rate. Small samples of the organic oxidation product were isolated and found by chromatographic and NMRspectroscopic examination to consist primarily of the 5-carbon sugar acid arabinonic acid. KEYWORDS: Graduate Education/Research, Upper-Division Undergraduate, Analytical Chemistry, Problem Solving/Decision Making, Carbohydrates, Catalysis, Mechanisms of Reactions, Oxidation/Reduction gluconic acid, glucuronic acid, and δ-gluconolactone. We undertook the studies described in the present paper with the aim of more fully elucidating the details of the reaction and clarifying the relation of its kinetics to these details. With this information, in addition to the usual kinetic investigations1−3 associated with the macroscopic observations on the system, instructors who wish to do so can introduce their students to interesting sugar chemistry that is almost never included in standard chemistry courses. The sugar acid−base chemistry alone is likely to be eye opening for students who probably never think of sugar as acidic. A few publications4 from the Japanese literature in the 1950s suggested that the oxidation of reducing sugars by methylene blue in alkaline solutions yields mixtures of acids, some with shortened carbon chains. However, most widely available descriptions of the Blue Bottle Experiment describe it as a simple oxidation of glucose at its reactive center (C-1) to give the corresponding six-carbon acid, gluconic acid. In the reoxidation of the reduced dye, oxygen is said to be converted into water. As to kinetics, it is generally held that the reaction (debluing) shows first-order dependence on the concentrations of glucose and methylene blue.3,5 Authors differ on the relationship of the rate to [OH−]. However, it is agreed that the rate of regeneration of the blue color is zero order in dissolved oxygen.5 There is no mention in the Blue Bottle literature of the primary process believed by carbohydrate chemists to be

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he popular chemistry lecture demonstration known as the Blue Bottle Experiment1 is performed with an aqueous mixture of glucose or a similar reducing sugar, alkali, and methylene blue or other redox dye. Shortly after preparation, the solution bleaches, but color can be restored by shaking the mixture vigorously to dissolve oxygen from the air, and the cycle of bleaching and color restoration can be repeated a number of times. After a half hour or so, the solution begins to yellow, and after several hours, it becomes deep reddish brown. The essential reaction responsible for the coloring and bleaching cyclereduction of the dye by glucose under basic conditionswas discovered soon after Caro’s synthesis of methylene blue in 1876. Early on, several chemists advocated the use of the reaction for the detection, or even the quantitative assay, of invert sugar in commercial table sugar and of glucose in urine. Better procedures were developed for such analyses, but in the mid-20th century the phenomenon attracted attention for use as a lecture demonstration. Campbell, in a paper published in 1963 in this Journal,2 reported that he found directions for the Blue Bottle Experiment in the demonstrations catalog at the Chemistry Department of the University of WisconsinMadison, and he promoted the experiment as a vehicle for teaching kinetics. The considerable literature that has appeared since Campbell’s original article has emphasized this feature, with rather less attention to the actual chemistry that is taking place. In our description of this demonstration,1 we indicated that dextrose (D-glucose) is oxidized to a variety of products, including © XXXX American Chemical Society and Division of Chemical Education, Inc.

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dx.doi.org/10.1021/ed200511d | J. Chem. Educ. XXXX, XXX, XXX−XXX

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peroxide is produced. The system is now set up for the cleavage of the glycosulose, a vicinal dicarbonyl compound, by the H2O2, a reaction well-known at the time of the AQ2S work.11 Thus, the final products are water, formate, and the ion of a carboxylic acid with its substitution pattern corresponding to that of the original sugar, but having a carbon chain one atom shorter10 (arabinonic acid when the sugar is glucose). The Blue Bottle reaction mixture is completely analogous to the one just described, with methylene blue (MB+) instead of anthraquinone-2-sulfonate as the redox catalyst, eq 3. Thus, the

involved in most of the reactions of reducing sugars in alkaline solutions, namely enolization.6

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THE ROLE OF ENOLIZATION The enolization of a carbonyl compound is accomplished by removal of a carbon-bound proton from a position adjacent to the CO group, as represented by eq 1. When this position

carries an OH group, which is characteristic of sugars, the product is an enediol or enediolate ion, as illustrated by 4 in Scheme 1. The possible role of enolization in Blue Bottle chemistry was adumbrated by the 1983 work of Vuorinen7 on the oxidation of glucose by anthraquinone-2-sulfonate (AQ2S) in alkaline solution. The reduction of AQ2S to an anthracenediol derivative is shown in eq 2:

Blue Bottle reaction could comprise two stages, closely paralleling those of an aerobic AQ2S oxidation. In accordance with the foregoing discussion the reactions are formulated as shown in Scheme 1. The first stage involves the initial ionization and enolization of the glucose,12 followed by the redox reaction of the enediolate with methylene blue to yield the glycosulose D-arabino-hexos-2-ulose (5) (traditional name: glucosone) and methylene white (MBH). In the second stage, beginning immediately, dissolved oxygen reoxidizes the methylene white as fast as it is formed, thus maintaining the dye largely in the blue MB+ form. Similar to the reoxidation of reduced AQ2S, this reaction yields hydrogen peroxide. The final step is again the cleavage of the glycosulose. When the oxygen is exhausted, the methylene white persists (the bleached solution), and further oxidation of the sugar awaits the reintroduction of oxygen by shaking the mixture.

Vuorinen studied the glucose oxidation in water−ethanol mixtures and noted that its energy of activation is almost the same as that of the base-catalyzed isomerization of glucose to fructose. Moreover, he found that the effects of hydroxide ion concentration, ethanol concentration, and temperature are closely similar for the two processes. Because extensive studies by previous investigators6,8 had led to the conclusion that enolization of the sugar is the key step in the isomerization, Vuorinen suggested that the AQ2S oxidation proceeds via ratedetermining enolization. He found that the reaction products are a dicarbonyl sugar (generic term: glycosulose, structure 5 in Scheme 1) and reduced anthraquinonesulfonate, (AQ2S2−). Further studies of the alkaline sugar−AQ2S system by Vuorinen,9 and by Hendriks et al.,10 showed that when oxygen is added to reoxidize the AQ2S2− to the quinone form, hydrogen

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THE RATE OF ENOLIZATION OF GLUCOSE If the mechanism of Scheme 1 is accepted, an obvious candidate for the rate-controlling step in the sequence is the enolization of the sugar. A major limitation on the rate of this process is the extremely low maximal concentration achieved by its presumptive open-chain precursor (3). The open-chain forms of the sugar are regarded as being in a facile equilibrium with the mixture of the cyclic forms (2a and 2b at the pH of the Blue Bottle Experiment), which heavily predominate. The likely immediate product of ring-opening, ionic 3 (O− on C-5)

Scheme 1. Reactions Occurring in the Blue Bottle Experimenta

1a, α-D-glucopyranose; 1b, β-D-glucopyranose; 2a, α-D-glucopyranosyloxy anion; 2b, β-D-glucopyranosyloxy anion; 3, open chain form of glucose; 4, enediolate anion; 5, D-arabino-hexos-2-ulose (glycosulose, open chain form); 6, D-arabinonate anion; MB+, methylene blue; MBH, methylene white; HO2−, hydroperoxide anion; HCO2−, formate anion. a

B



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instantly protonated to neutral 3 (OH on C-5), has the free aldehyde group required to activate the sugar H-2 for removal by base to form the enediolate ion 4. However, the aldehyde group has a strong tendency to add water, diminishing the concentration of the active enediolate precursor 3. Once the enediolate is formed, several pathways are open to it. Characteristically, it undergoes rapid reprotonation, which can happen in three different ways: (1) to regenerate the original glucose; (2) to form the epimeric aldose13 (mannose in this case); and (3) to form the ketose13 related to the two aldoses (fructose in this case).6 The short-term result is a quasisteady state involving the formation of the enediolate and its reactions of reversion to the three related sugars. Prolonged exposure to alkali of the concentrations used here promotes the degradation of the sugars by pathways involving eliminations, reverse aldol condensations, and so forth. These degradation processes can be ignored only if manipulations such as the Blue Bottle Experiment are accomplished immediately after the reaction mixture is prepared. To quantitatively assess the role of the enediolate in the oxidative pathway, one needs data on the rate of enolization of the sugar under various conditions, as exemplified by the results reported by Vuorinen7 from his work with AQ2S. Vuorinen used low concentrations of glucose and excess AQ2S to maximize the fraction of enediol oxidized by AQ2S as it formed and made rate estimates by following the consumption of glucose. However, the conditions of Vuorinen’s measurements do not fully parallel those characteristic of the Blue Bottle Experiment. Thus, it was desirable to do rate studies at higher concentrations of glucose and OH−. In the experiments, spectrophotometry was used to follow the increase in reduced AQ2S (AQ2S2−) as it was formed in the anaerobic reaction of the anthraquinone with the sugar. By blending oxygen-free component solutions in situ, in cuvettes that were thermostatted at 25 °C and continuously flooded with nitrogen, mixtures were generated conforming to Shakhashiri’s directions1 for the Blue Bottle Experiment (see the Supporting Information). As constituted, these mixtures contained glucose at 0.184 M and, to serve as oxidant, various levels of AQ2S in the fractional millimolar range. Potassium hydroxide was included at 0.407 mmol mL−1, a quantity sufficient to convert over 90% of the sugar into its ionic form and establish a pH above 13.3. Monitoring absorbance at a fixed wavelength,14 beginning with the moment of mixing, gave plots that leveled off when the AQ2S was exhausted, as illustrated in Figure 1. For the approximately straight-line middle segment of each curve, the slope, which is proportional to the maximal reaction rate achieved in the mixture being examined, was recorded. Straightforward evidence for the role of enolization in the reduction of AQ2S by alkaline sugar was obtained by comparing the rate of reduction shown by ordinary glucose with that shown by (2-2H)glucose.15 The rate was markedly depressed by the substitution of deuterium for hydrogen on C2 of the labeled sample. Under the conditions used, the rate of AQ2S reduction by unlabeled glucose was 7.3 × 10−5 mol L−1 min−1 and by (2-2H)glucose was 2.1 × 10−5 mol L−1 min−1. Because only one measurement was made on each glucose sample, the ratio of the two rates does not provide a quantitative value for the isotope effect of the deuterium substitution, as may be understood from the following. Determination of the rate of reduction of AQ2S at a single concentration provides no information about the fraction of

Figure 1. The rate of reduction of AQ2S by glucose. Spectrophotometer trace at 575 nm with [AQ2S]0 = 2.5 × 10−4 M.

nascent enediolate being captured by oxidation instead of being reprotonated as described above. A simplified reaction scheme for the glucose−AQ2S system involves glucose, G, the enediolate, E, the reversion of the enediolate to give product(s), R, and the oxidation of the enediolate to the glycosulose, U, by the oxidant, A. The three relevant reactions are ke

kr

G→E→R ko

E + A → U + A2 −

The rate of enolization is ke[G], the rate of reversion is kr[E], and the rate of oxidation is ko[E][A]. In the experiments reported here, the concentration of enediolate reaches a steady state, where its rate of formation equals its rate of disappearance, eq 4: ke[G] = k r[E] + ko[E][A]

(4)

For the graphical analysis of the effect of varying [A], one needs a value for the concentration of E, which is not experimentally accessible. However, an expression for it may be written by rearranging eq 4: [E] =

ke[G] k r + ko[A]

(5)

Substituting the right side of eq 5 into the expression ko[E][A] leads to eq 6 for the rate of oxidation as a function of [A], the concentration of oxidant. ⎛ ko[A] ⎞ rate of oxidation = ke[G]⎜ ⎟ ⎝ k r + ko[A] ⎠

(6)

For an estimate of the rate of enolization, the runs were made under Blue Bottle conditions, as described above, with four different initial concentrations of AQ2S. To fit the resultant data to eq 6 by nonlinear regression, the equation is first simplified. ⎛ k [G]ko[A] ⎞⎛ 1/ko ⎞ rate of oxidation = ⎜ e ⎟⎜ ⎟ ⎝ k r + ko[A] ⎠⎝ 1/ko ⎠ ⎛ k [G][A] ⎞ rate of oxidation = ⎜ e ⎟ ⎝ k r /ko + [A] ⎠ C


Journal of Chemical Education ⎛ p[A] ⎞ rate of oxidation = ⎜ ⎟ ⎝ q + [A] ⎠

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consumption with an oxygen-sensing electrode, using the stationary reactor shown in Figure 3. To avoid any possible

(7)

where p = ke[G] and q = kr/ko. Equation 7 is the equation for a hyperbola. Nonlinear regression16 on our data using this equation yielded p = 1.05 ± 0.03 AU min−1, and q = 2.2 ± 0.1 × 10−4, with an R2 value of 0.993. A plot of the data and fitted curve are shown in Figure 2.

Figure 3. Reactor for determination of the rate of oxygen consumption, with oxygen electrode on the left. Figure 2. The rate of reduction of AQ2S by glucose as a function of [AQ2S]0. Each point is the average of 2 or 3 determinations, with range of values indicated by error bars. For each determination, the measured slope of the spectrometer trace is the value of interest, as it shows the maximal rate achieved. However, it was convenient to record the AQ2S levels as initial concentrations, which govern the eventual maximal rates even though the actual concentrations of unreduced oxidant are only about half of the original by the time the rate maxima are reached. The solid curve is the nonlinear regression fit to eq 7.

complication that might result from using a “non-standard” scale of operation, the reactor was sized to contain a little over 300 mL, which is the full volume of mixture used in a normal Blue Bottle Experiment.1 Solutions of glucose and KOH were made up in house deionized water, which routinely had an oxygen content (∼8 mg L−1) in approximate equilibrium with the air. After the reactor had been charged with the glucose solution, the desired quantity of methylene blue (MB+) was added, the temperature was adjusted, and the dense KOH solution, which had a relatively small volume, was run in as a layer below the glucose solution. Then, stirring was initiated to mix the layers, and oxygen disappearance was recorded. A typical plot is shown in Figure 4. The slope of the straight-line portion of the curve was recorded as the rate of oxygen consumption. Replicate runs were done at 10 concentrations of methylene blue in the range 0.1−5.0 mg per reactor volume, plus one run without dye. The run without dye gave a rate for the noncatalyzed oxidation, namely, 0.043 mg L−1 s−1. This rate was subtracted from the rates determined in the presence of methylene blue to

The value of the parameter p in AU min−1 is converted into molar terms by dividing by 2500 (ε575 for AQ2S2−), giving ke[G] = 4.2 ± 0.1 × 10−4 mol L−1 min−1 for the reduction at infinite [AQ2S]. This corresponds to a first-order rate constant, ke = 2.3 × 10−3 min−1 based on total sugar, 0.184 M for glucose at 25 °C in the presence of 0.24 M OH−. In comparison, a plot in Vuorinen’s 1983 paper (Figure 2 in ref 7) shows k = ∼1 × 10−3 min−1 in 0.1 M NaOH at 25 °C, which, allowing for his lower OH− concentration, compares well with our result. Our value is subject to some uncertainty because the reduction of AQ2S by the sugar enediolate gives in part a free radical anionic form of the anthraquinone [AQ2S•−, eq 2], which has a very low optical absorbance at 575 nm.14 The rapid dismutation of this species under our conditions allows all of the anthraquinone to be fully reduced at the termination of the reaction.

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ENOLIZATION RATE AND BLUE BOTTLE KINETICS When presenters or students make kinetic observations on the Blue Bottle reaction, they usually record the debluing time, the time taken by the solution to bleach after having been oxygenated by shaking. For a given mixture, this time is subject to variation because of variation in the degree of oxygenation achieved by the shaking operation. Oxidation of the sugar by the dye in the blue solution continues to generate methylene white, but this is immediately reoxidized to the blue form until the supply of oxygen is exhausted. The solution then bleaches. A more accurate estimate of the rate of the overall reaction could be obtained by measuring the rate of oxygen

Figure 4. Oxygen consumption under Blue-Bottle conditions with 0.75 mg methylene blue in the full-scale reaction mixture. D



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To test the supposition that arabinonic acid is the product of the Blue Bottle oxidation when glucose is the sugar, two different reaction mixtures were examined. The first was from a small-scale run using arbitrarily chosen proportions of reactants (see the Supporting Information) in which the oxidation of the sugar was allowed to go to completion. Analysis of a sample of the final mixture by high-performance liquid chromatography revealed one major component having the mobility of arabinonic acid; there were only traces of minor components. In the usual Blue Bottle Experiment, the oxidation of glucose proceeds intermittently, during the period in each bluing− debluing cycle when oxygen is present to maintain the dye in its blue state. For an accurate account of the chemistry of the demonstration, it seemed important to have a sample of the intermittently formed product, of which a small quantity (possibly 10−12 mg) is generated in each cycle. Accordingly, a standard Blue Bottle Experiment was run, putting the solution through several cycles by manual shaking. To isolate the oxidation product(s),7 the solution was treated with sufficient Dowex 50-H+ to neutralize the alkali and the resin was filtered off. On putting the filtrate through a small column of Dowex 1 anion exchange resin in its acetate form, the acidic components were retained, and the sugar was washed through to waste. The Dowex 1 column was eluted with strong acetic acid, the residue from the evaporation of the eluate was neutralized with KOH, and the resulting potassium salt was taken up in D2O. This isolate was found by NMR examination to contain residual glucose, so it was subjected to a second round of adsorption on Dowex 1 and acetic acid elution. A periodate assay of a sample of the final D2O solution (see the Supporting Information) was consistent with the assumption that ∼40 mg of pentonic acid salts was isolated. A 1H−13C one-bond correlation NMR spectrum of the product showed a five-carbon acid. For rigorous identification by NMR analysis, a prepared or stock sample of the potassium salt of each of the four diastereomeric pentonic acids, arabinonic, lyxonic, ribonic, and xylonic, was examined. The proton spectra of the four authentic samples are easily distinguishable from each other, and on comparison, Figure 6, the spectrum of the Blue Bottle product matched that of potassium arabinonate; the two spectra are superimposable.

determine rates for the catalyzed reactions. As with the determinations made with AQ2S, the data gave a hyperbolic plot, Figure 5. A nonlinear regression on the data plotted in

Figure 5. Catalyzed rate of oxygen consumption as a function of amount of MB+ under Blue-Bottle conditions. Each point is the average of 2 or 3 determinations. The solid line is the nonlinear regression fit to a hyperbola, giving a rate at infinite MB + concentration of 0.0996 ± 0.002 mg L−1 s−1, a ratio kr/ko of 0.205 ± 0.016, and an R2 value of 0.991.

Figure 5 gives a catalyzed O2-consumption rate at infinite MB+ concentration of 0.0996 ± 0.002 mg L−1 s −1, which corresponds to a total rate of 0.143 mg L−1 s−1 or, in molar terms, 2.68 × 10−4 mol L−1 min−1. Thus, the maximal expected rate of oxygen usage at 25 °C in a Blue Bottle reaction mixture having methylene blue as the redox reagent is only two-thirds of the apparent rate of enolization of the sugar. If one assumes 0.7 mg of MB+ per reactor volume, a plausible concentration for an actual Blue Bottle Experiment, the calculated rate of O2 consumption would be 2.5 × 10−4 mol L−1 min−1, which is 93% of the maximum. This would account for the capture of about 60% of the enediol being produced. Given the O 2 concentration of a newly mixed solution, one would predict a debluing (bleaching) time of a little over a minute, somewhat less for a solution reblued by quick shaking, in accord with actual observations.

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CONCLUSIONS The results presented here solidly establish the phenomena observed during a Blue Bottle Experiment as resulting from a two-stage process. The key step in the process is the enolization of the sugar that is the active component of the solution being manipulated. Some details remain unclarified, but for the present, the reaction sequence can be formulated as in Scheme 1. As depicted in the scheme, the principal end product of the Blue Bottle reaction sequence is arabinonic acid, formed by loss of one carbon atom from each molecule of the C6 sugar glucose. This acid was shown by Ericsson et al.17 to be the major result of the action of hydrogen peroxide on the dicarbonyl sugar arabino-hexos-2-ulose (5), and its preponderance in the final product is evidence for the role of this osulose in the Blue Bottle Experiment. Although the glycosulose molecule is presumably born with the linear structure shown, it is known to be readily susceptible to cyclization in several ways. One might expect these rearrangements to interfere with its cleavage by hydrogen peroxide, but a synthetic sample of the compound, presumably a mixture of cyclized forms, was shown

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THE FINAL STAGE OF THE REACTION As indicated above, we supposed that the rebluing that occurs during parts of the Blue Bottle cycle produces hydrogen peroxide, which does not accumulate but instead cleaves the glycosulose derived from the original sugar, yielding arabinonic acid (6) when the sugar is glucose. To confirm the involvement of hydrogen peroxide, methylene white was prepared on a small scale by the reduction of 0.33 mmol of methylene blue with excess sodium dithionite. The reaction was done in a large test tube continuously flushed with nitrogen, the precipitated methylene white was washed in situ to remove excess dithionite, and then oxygen was bubbled into the suspension, which regenerated the blue dye. After removal of the dye and alkali from the solution with Dowex 50 ion-exchange resin and filtration, an assay of the filtrate by a standard iodine titration revealed 0.16 mmol of peroxide, ∼50% of the theoretical based on the starting amount of methylene blue. The low yield was expected in view of obvious losses of material during the washing of the methylene white. E



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The weighted average19 of these values is ∼12.3, in good agreement with numbers reported in the literature. Given this pKa, much of the alkali used in preparing the mixture is consumed in creating the sugar anions. To relate reaction rates to hydroxide concentration requires knowledge of the actual value of [OH−], which cannot be obtained by simply considering the quantity of hydroxide used to prepare the solution. It is necessary to measure the pH of the mixture and from this calculate [OH−]. Although uncertainties remain about some details of the series of reactions that are responsible for the phenomena exhibited in the Blue Bottle Experiment, the present research clarifies the overall chemistry. Identifying the source of the yellow and brown colors is still under investigation.

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ASSOCIATED CONTENT

S Supporting Information *

Figure 6. H NMR spectra at 300 MHz in D2O: (A) potassium salt of isolated Blue-Bottle product and (B) authentic potassium arabinonate. The singlet at δ 3.93 and the multiplets at δ 4.07 and 4.16 in the product spectrum are due to a minor component. 1

Enolization rate by spectrophotometry; rate of oxygen consumption; isolation of the product; authentic aldonic acid potassium salts; the pKa’s of the anomers of glucose. This material is available via the Internet at http://pubs.acs.org.

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by Vuorinen9 to undergo rapid scission. Still, it is remarkable that the reaction is efficient under Blue Bottle conditions, where the achievable concentrations of the reactants are very low. As mentioned in the introduction, it has frequently been stated that the rate of the Blue Bottle reaction is directly proportional to the concentrations of sugar and methylene blue, but nonlinearly related to the hydroxide ion concentration. Suggestions of more complex rate-concentration relationships came from a technically excellent study of Blue ́ Bottle kinetics carried out by Adamčiková et al.,5 but these authors’ interpretation of their results is confused by their incorrect formulation of the component reactions. In the present work, it was found that increasing the concentration of oxidant (redox reagent, AQ2S or MB+) in Blue Bottle reaction mixtures does cause increases in the rate of oxidation of the sugar. However, the magnitude of the rate increase per unit of added oxidant diminishes with each addition. This behavior, expressed in hyperbolic plots of rate versus oxidant concentration (Figures 2 and 5), is understandable as a result of the fact that the rate of production in the reaction mixture of the sugar enediolate, which in this scheme is the co-reactant in the oxidation step, does not change with increases in the quantity of oxidant. When methylene blue is the oxidant a further weakening of the oxidation rate response may stem from the tendency of this dye to form molecular aggregates (dimers, trimers) at elevated concentrations.18 However, our finding that the maximal oxidation rate achievable with methylene blue is considerably short of the measured enolization rate remains surprising. The elucidation of the rate-concentration relationships of the sugar and hydroxide components of the Blue Bottle reaction mixture will require further research. It may not be possible to ascertain the concentration of the true aldehydo form of the sugar, which undergoes deprotonation at C-2 to give the enediolate ion. However, it is useful to know the proportions of the major forms of the sugarthe ring formsthat are ionized at O-1, because their pKa’s are in the pH range of the mixtures normally used in Blue Bottle Experiment. The pKa of glucose was measured (see the Supporting Information), arriving at the values of 12.5 for the α- and 12.2 for the β-pyranose anomer.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Addresses ‡

Emergency Medicine, University of Wisconsin Hospital and Clinics, Madison, WI 53792 § Medicine Hematology Office, MMC 480 Mayo 8480A, 420 Delaware St SE, Minneapolis, MN 55455 Notes

The authors declare no competing financial interest. † Laurens Anderson is an emeritus professor of Biochemistry, University of Wisconsin-Madison

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ACKNOWLEDGMENTS We acknowledge the assistance, via undergraduate research projects, of Chris Clark, Brian Butzen, Lynn Ghermann, and Richard Horak. Our thanks go to Anthony Serianni of Omicron Biochemicals, Inc. for gifts of isotopically substituted glucose, to colleagues at the USDA-ARS Dairy Forage Research Center in Madison, who arranged for chromatographic analyses (Ron Hatfield) and NMR measurements of pH samples (John Ralph and Paul F. Schatz), and to Monika Ivancic of the Magnetic Resonance Facility in the University of WisconsinMadison Chemistry Department for the initial NMR examination of the reaction product. We also thank W. W. Cleland, University of WisconsinMadison Biochemistry Department, for discussions of kinetic theory, and M. Francisca Jofre of the National Magnetic Resonance Facility (Biochemistry Department, University of WisconsinMadison) for collecting full NMR data on the product and related standards with support from NIH grant RR02301 (John Markley, PI). With deep appreciation, we acknowledge the generous support of the Evjue Foundation, and at the University of Wisconsin Madison, from the Department of Chemistry, the College of Letters and Science, the Graduate School, and the Office of the Chancellor. F



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REFERENCES

(1) Shakhashiri, B. Z. Chemical Demonstrations; University of Wisconsin Press: Madison, WI, 1985; Vol. 2, pp 142−146. Cook, A. G.; Tolliver, R. M.; Williams, J. E. J. Chem. Educ. 1994, 71, 160−161. Engerer, S. C.; Cook, A. G. J. Chem. Educ. 1999, 76, 1519−1520. (2) Campbell, J. A. J. Chem. Educ. 1963, 40, 578−583. (3) Campbell, J. A. Why Do Chemical Reactions Occur?: Prentice-Hall: Englewood Cliffs, NJ, 1965; Chap. 2, pp 8−23. (4) Ashida, K. Mem. Inst. Sci. Ind. Res., Osaka Univ. 1953, 10, 205− 207 CAPLUS AN 1953:65556. Kurasawa, H.; Igaue, I. Nippon Nogei Kagaku Kaishi 1955, 29, 168−174 CAPLUS AN 1957:99919. Kurasawa, H.; Igaue, I. ibid. 1955, 29, 205−210 CAPLUS AN 1957:99920. (5) Adamčíková, L.; Pavlíková, K.; Ševčík, P. Int. J. Chem. Kinet. 1999, 31, 463−468. (6) Speck, J. C., Jr. Adv. Carbohydr. Chem. 1958, 13, 63−103. (7) Vuorinen, T. Carbohydr. Res. 1983, 116, 61−69. (8) Isbell, H. S.; Linek, K.; Hepner, K. E., Jr. Carbohydr. Res. 1971, 19, 574−582. (9) Vuorinen, T. Carbohydr. Res. 1984, 127, 319−325. (10) Hendriks, H. E. J.; Kuster, B. F. M.; Marin, G. B. Carbohydr. Res. 1991, 214, 71−85. (11) Bunton, C. A. Nature 1949, 163, 444. (12) A referee suggested the possibility of an alternative mechanism for the oxidation step, involving the loss of O-2 bound H of the sugar as a proton (H+) and removal by the oxidizing agent of carbon-bound H-2 as a hydride ion. The data collected in the present study do not permit a distinction between an enolization based mechanism and the alternative, but the route via enolization seems more plausible in view of the fact that, regardless of the presence of a redox catalyst, sugar enediolate will be generated steadily. The formation of enediolate requires only the alkaline medium and the presence of the sugar. As shown by a study of a simple sugar analogue (hydroxyacetaldehyde, Fedoroňko, M.; Temkovic, P.; Konigstein, J.; Kovácǐ k, V.; Tvaroška, I. Carbohydr. Res. 1980, 87, 35−50), enediolate is rapidly oxidized by methylene blue. Thus, the mechanism shown in Scheme 1 must be a major feature of the Blue Bottle reaction. (13) Glucose and mannose are polyhydroxy aldehydes in their open chain forms, hence the designation aldose. Similarly, open chain fructose is a ketone, and so it is classified as a ketose. (14) Reduced anthraquinone-2-sulfonate dianion (AQ2S2−), the predominant AQ2S species at pH 13, is actually triply charged, eq 2, but the charge on the sulfonate group is irrelevant for the redox properties of the compound. The dianion has its principal absorption maximum at 432 nm, ε 14000 [our measurement; literature values are 435 nm, ε 17000] ( Gamage, R. S. K. A.; McQuillan, A. J.; Peake, B. M. J. Chem. Soc. Faraday Trans. 1991, 87, 3653−3660. For monitoring, we used the off-maximum wavelength 575 nm so that the absorbance of the most concentrated samples would be in the reading range of the spectrophotometer. From measurements of the absorptivity of AQ2S2−, we could assign the value 2500 to ε575 for this species. (15) Painter, C. J. Enolization of Glucose in Strongly Alkaline Conditions; M.S. Thesis, Department of Chemistry, University of WisconsinMadison, 2005. (16) Bethea, R. M.; Duran, B. S.; Boullion, T. L. Statistical Methods for Engineers and Scientists, 3rd ed.; Marcel Dekker: New York, 1995; pp 352−362. (17) Ericsson, B.; Lindgren, B. O.; Theander, O. Cell. Chem. Technol. 1973, 7, 581−591. (18) Rabinowitch, E.; Epstein, L. F. J. Am. Chem. Soc. 1941, 63, 69− 78. (19) Assuming a β/α ratio of ∼2/1, i.e., not far from the known equilibrium ratio of the neutral species in water at 25 °C.

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What Is Happening When the Blue Bottle Bleaches: An Investigation of

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