The mechanism of the blue bottle experiment requires an understanding of rates and mechanisms of complex interacting chemical reactions. In complex chemical reactions, individual sub-reactions can occur simultaneously but at significantly different rates. These, in turn, can be affected by reagent concentration and temperature. In most cases, the overall reaction rate is determined by the fastest single component reaction. However, when some processes form intermediate molecules which then react in other processes to form the end product, the rate of the overall reaction is determined by the rate of the slowest reaction. In such circumstances the intermediate products are usually in a steady state at low concentrations because they are highly reactive. Equilibrium state requires that all reaction forward and backward mechanism happens at the same rate. Thus, the overall net reaction is determined by the sum of all the mechanism steps where the rate depends on the concentration and temperature. The blue bottle experiment illustrates this principle of interacting reactions with different rates.
The blue bottle experiment requires only three reagents: potassium hydroxide solution, dextrose solution, and dilute methylene blue solution. These reagents are added to a flask, mixed, and the flask is stoppered. The initial color of the solution is blue, but upon standing for a short interval it spontaneously fades to colorless, as the alkaline dextrose solution reduces the methylene blue to colorless leuco-methylene blue. Shaking the flask causes oxygen present in the head space air to dissolve in the solution and oxidize the leuco-methylene blue back to its colored form again. Another variation uses methylene blue in water, glucose, and caustic soda (NaOH). There are many versions of the experiment, however, unlike the classical version where dye is necessary to use as a catalyst for the reaction, the green and rapid versions undergo autoxidation even in the absence of the dye.
In the past, it was thought that the reaction occurred by the oxidation of an aldehyde group to a carboxylic acid under alkaline conditions. For instance, glucose would be oxidized to gluconate by oxygen. However, the experiment also works with compounds such as vitamin C and benzoin, which do not contain an aldehyde group. Thus, the reaction is actually the oxidation of an acyloin or related α-hydroxycarbonyl group, which is a structural feature of glucose, to a 1,2-diketone. The reduced redox dye (colorless state) is formed from oxidized redox dye (blue). The color-change that occurs in the blue bottle experiment has features of a clock reaction, in which a visible change in the concentration of one or more reagents suddenly occurs upon the exhaustion of a limiting reagent. For example, the limiting reactant, oxygen, is consumed by another reactant, benzoin, with the help of safranin as a catalyst. Once the limited amount of oxygen has been used up, the catalyst is unable to change forms, and as a result, the solution changes color.