by David Mazawa
Ninhydrin reacts with primary amines and hydrindantin to form Ruhemann’s Purple which is detectable at 570nm. Ninhydrin reacts with secondary amines to form a yellow complex detectable at 440nm. The ninhydrin reaction is carried out at 130°C with a reactor volume of 500uL. The elevated temperature is required because at room temperature, the ninhydrin reaction is very slow and takes hours to go to completion.
Trione is a proprietary formulation containing ninhydrin, hydrindantin (reduced ninhydrin), a lithium acetate buffer, and sulfolane, a water-miscible organic solvent. The solvent is necessary to maintain the solubility of both the hydrindantin and the primary amine product, Ruhemann’s Purple. The buffer is required because the reaction is pH dependent. T100 and T100C (Case of 4 bottles of T100) are premixed and have a shelf life of 4 months at room temperature. T200 comes in 2 bottles and must be mixed together before use.
Trione will oxidize when exposed to air. Depending on the exposure, part or all of the hydrindantin will be oxidized to ninhydrin. Only primary amine development requires hydrindantin. Consequently, as the hydrindantin disappears, so will the primary amine signal. Secondary amines, such as Proline, will appear to be larger than normal because the signal for primary amines will so low.
The Trione aging process is different from Trione oxidation. The concentration of hydrindantin will increase as Trione ages. Consequently, the signal for primary amines will increase. The increase in hydrindantin can cause changes in reagent color and increase the risk of precipitation in the heated reactor. For this reason we have set the shelf life of T100 and T100C to 4 months at room temperature. The aging process begins as soon as T100 is bottled. The aging process for T200 is slower; however, once mixed together it will age at the same rate as T100. Unmixed T200 has a shelf life of 12 months at room temperature. Never top-off your old Trione with new Trione, as the old Trione acts as a catalyst and will make the new Trione age very fast. Always use a clean reagent bottle when using a new bottle of Trione.
Hydrindantin decreases. Low primary amine signal.
Hydrindantin increases. High primary amine signal. Increased risk for precipitation in the heated reactor.