Glucose Oxidation: The Blue Bottle Experiment

This fun exercise elegantly demonstrates the role of oxidizing coenzymes such as NAD+ and FAD in metabolism. The blue bottle experiment is, I gather, an old favorite "chemical magic" demonstration. Under alkaline conditions, glucose slowly reduces methylene blue to a colorless form. The blue form is rapidly regenerated by introduction of air into the solution (shaking). Thus, methylene blue is alternately oxidizing glucose and reducing oxygen.

The introduction to the exercise shows students the three relevant reactions, oxidation of glucose to gluconic acid, reduction of oxygen to water, and reversible oxidation/reduction of methylene blue. Glucose oxidation in alkaline media undoubtedly produces other products and reduction of oxygen may in fact form hydrogen peroxide (?) but I want to keep complexity minimal. I'm guessing from the structure of methylene blue that, like NAD+, it is reduced by accepting a hydride ion.

Students do the reaction in 125-mL flasks. My original recipe calls for mixing 20 mL of 5% KOH with 20 mL of 7% glucose and adding one drop of 0.2% methylene blue. Glucose isn't cheap, and one year I figured out a different recipe using corn syrup. Just before lab, I mixed up a solution of 10% (w/v) corn syrup and 1% NaOH. Students measured out 40 mL of this and added 4 drops of methylene blue. The alkaline corn syrup turned yellow pretty quickly, but that didn't interfere at all with the exercise: the yellow was hardly noticeable against the blue solution.

I pose a question to the students: is glucose also oxidized in the absence of the dye? Since there's no signaling color change and the solution is too alkaline to measure acid production, the question can only be answered by measuring oxygen uptake. Now, I'm not seventy years old, but I can still rig up a serviceable manometer. The tips of two graduated pipets (5- and 10-mL) are connected by an 8-inch length of rubber tubing, and then filled about halfway with water. I've made a simple support with clothespins that holds the two pipets close to each other but allows easy adjustment to level the water in the two pipets.

Students have their solutions in 125-mL flasks stoppered and connected to the top of the 5-mL pipet by a rubber hose. After adjusting the pipets so the water levels are the same, pairs of students, one with and one without dye, shake each flask 20 times, waiting each time until the blue color goes away. Finally, they level the water in the pipets and determine the change in volume. It is important that the flasks not be warmed by their hands, so I ask them to bring gloves or mittens to lab and hold the flask by the top when they shake it. If done carefully, the flask with dye consumes 2-3 mL of gas, whereas the flask lacking dye consumes less than 0.5 mL.

For both color changes (bleaching and reoxidation of the dye), students are asked what is happening to the dye and what is the electron donor or acceptor. At the end, they are asked to describe the role of the dye and to depict the electron transfer reactions in pathway form.

Return to CHM 201

Return to Barry Ganong's Homepage