The substituents on the diazirine affect which carbene species is generated upon irradiation and subsequent photolytic cleavage. Diazirine substituents that are electron donating in nature can donate electron density to the empty p-orbital of the carbene that will be formed, and hence can stabilize the singlet state. For example, phenyldiazirine produces phenylcarbene in the singlet carbene state whereas p-nitrophenylchlorodiazirine or trifluorophenyldiazirine produce the respective triplet carbene products.
Electron donating substituents can also encourage photoisomerization to the linear diazo compound , rather than the singlet carbene, and hence these compounds are unfavorable for use in biological assays. On the other hand, trifluoroaryldiazirines in particular show favorable stability and photolytic qualities and are most commonly used in biological applications. Carbenes produced from diazirines are quickly quenched by reaction with water molecules, and hence yields for photoreactive crosslinking assays are often low. Yet, as this feature minimizes unspecific labeling, it is actually an advantage of using diazirines.