A formazan belongs to the family defined by the parent connectivity and derivatives formed by substitution at carbon or nitrogen. The chain contains four nitrogen atoms and one central carbon atom. A particular substituted formazan is a chemical compound; the family name does not identify one universal dye.[ref]
The linked nitrogen–carbon framework provides a conjugated system. Substitution and chemical conditions affect its optical behavior, so a measured formazan optical response should identify the derivative and its state. The strongly colored product obtained by reducing MTT is a specific example, not the definition of every member of the class.
In the manufacturer's MTT test principle, yellow tetrazolium salt is reduced to purple formazan crystals, then the product is dissolved for optical measurement.[ref] Product formation, crystal recovery and final dissolved-product absorbance are different stages; incomplete dissolution or optical scattering can change their relationship.
For an original dissolved-product model, the Beer-Lambert law giveswhere is product amount, solution volume, optical path length and the appropriate molar absorption coefficient. At fixed , doubling while keeping unchanged halves the ideal net absorbance. Doubling the amount and the volume together preserves concentration and therefore preserves that ideal signal.
The equation requires a suitable solution measurement regime and a specified product. A tetrazolium-derived color can arise from cellular or noncellular reduction; identifying a formazan product does not by itself identify its electron source. This is why tetrazolium-reduction interference matters when formazan is used as a readout of cell viability.
New to topics? Read the docs here!