When 3-nitrophthalic acid, a dicarboxylic acid, is combined with hydrazine and heated, 3-nitrophthalhydrazine, a cyclic diamide is produced. The nitro group is then reduced with sodium dithionite, producing luminol.
The nucleophile in this reaction is the nitrogen of the amine and the electrophile is the carbonyl carbon of 3-nitrophthalic acid. Since the carbonyl carbon is not a good electrophile, under normal conditions, the two reagents will not react. In this case, Fischer’s approach of running the reaction under acidic conditions would not work due to the basic nature of the amine. This would cause the amine to be protonated, making the ammonium ion, which is no longer a nucleophile.
The problem can be solved by simply adding energy (heat) to the reaction.
Because the high temperature used (heating to 230ºC), we need take to make sure everything, including the solvent, stays in solution. This can be done by running the reaction under pressure, but this poses a potential safety hazard. Instead, we can use a high boiling point solvent, such as triethylene glycol. Triethylene glycol has a boiling point of 285ºC and has similar solubility properties as water. At low temperatures, it is quite viscous, and doesn’t act as a good solvent. However, at high temperatures, it is ideal.
A second problem involves obtaining such a high temperature. We use a hot plate, along with an aluminum heating block. Aluminum foil is placed around the heat block and conical vial to direct more heat to the conical vial.
Once we make the cyclic diamine, we reduce this product with sodium dithionite to produce 3-aminophthalhydrazide (luminol). This two-step process is necessary, instead of directly starting with 3-aminophthalic acid because of reaction between the carboxylic acid and amine. If we were to start with 3-aminophthalic acid, there would be a competing amine present in the synthesis. This would produce a lower product yield and an additional purification step would be necessary.
When luminol is in the presence of a strong base and an oxidizing agent, a dicarboxylic acid is formed, along with nitrogen release, and the formation of a photon product. In other words, light is produced, a chemiluminescent reaction.
Luminol can even be used to detect blood. Instead of using an oxidizing agent such as hydrogen peroxide, we can use potassium ferricyanide, a blood mimic (due to the presence of the same iron present in blood). The use of this oxidizing agent allows the chemiluminescent reaction to work successfully.
How the energy is being absorbed and released in this reaction is what causes the light to be generated. Electrons typically exist in the ground singlet state, S0. During the reaction, the electron will absorb energy, and as a result, goes to an exited singlet state, S1 or S2. The electrons in this excited state want to release the extra energy and return to the ground state. Usually, the energy is released through slow vibrations (in the form of heat). However, it can also go directly to the ground state. All of the energy is released at once in the form of a photon, a process called fluorescence.
The electrons can also do an intersystem crossing, going from the excited singlet state to an excited triplet state. In S0 and S1, electrons are normally spin paired, but in the triplet state, the electron spins are flipped. However, there is still extra energy that needs to be released. When the triplet state is directly dropped to the ground singlet state, with the release of a photon, the process is termed phosphorescence.
In order for an electron to flip its spin, it must absorb energy. Because of this, the average triplet state is lower in energy than the average singlet state, and thus the energy of the photon released is lower. Phosphorescent reactions should then be redder in color because a smaller amount of energy is involved, therefore a smaller frequency, and a longer wavelength in the EM visible light spectrum. And in correlation, ground singlet states are higher in energy, so the fluorescent molecule is bluer in color.
Technically, the reaction with luminol is a fluorescent one. When its electrons get excited, they go to an excited triplet state, an intersystem crossing is observed where they go to an excited singlet state, and drop back down to the ground singlet state. This produces a bluish green molecule.
During the course of the reaction, when hydrazine and 3-nitrophthalic acid were mixed and heated, a dark red color was produced. The precipitate that resulted was a dark reddish brown color. After reduction to luminol, the precipitate was a dark orange color. When the chemiluminescent reaction was carried out, a bright blue/cyan color was produced for about 20 seconds. This indicates that the luminol could also be used to detect blood.