Ninhydrin solutions, which are used in automatic amino acid analysis (I), are light sensitive and develop a brownish tinge when exposed to air. It is, therefore, necessary to add antioxidants and store the ninhydrin reservoir in a dark container under nitrogen at low temperatures in order to preserve this reagent. Stannous chloride has been routinely used as a reducing agent. However, as a consequence of the addition of stannous chloride to ninhydrin reagent in the reservoir of a Beckman 121 automatic amino acid analyzer, a fine white precipitate progressively accumulates in the analyzer fluid lines and cuvettes and seriously interferes with quantification of amino acids. The precipitate is sparingly soluble or almost insoluble in water, which is generally used to flush the coil in most amino acid analyzers. Flushing the coil with 0.2 to 1 N NaOH does not reduce or eliminate the precipitate formed in the lines. Washing the fluid line system with methyl Cello-solve or an aqueous solution of Methyl Cellosolve (IO-50%) or ethyl alcohol (70-95%) partially frees the lines of the precipitate.
However, washing with aqueous ethanol creates tiny air bubbles inside the lines, which eventually interfere with quantitative measurements.
It was reported that titanous chloride (2,3), when used in ninhydrin solution, was beneficial in reducing the interfering precipitate in fluid lines and in stabilizing the baseline of the recorder. We have tried to replace stannous chloride with titanous chloride (Pierce Chemical Co.) as a reducing agent in ninhydrin solutions with little success. During the first 4 to 6 days, the color yield of ninhydrin solution is almost the same as that of stannous chloride, and there is only a minimal amount of color quenching. However, after 5 to 6 additional days, the reagent turns to dark brown, which seriously quenches the color development in the reaction coil. Sometimes the color yield drops as low as 50% that of the fresh ninhydrin solution.
Occasional precipitation and subsequent blocking of fluid lines have also been observed, even when hydrindantin and methyl Cellosolve are used in the ninhydrin reagent (4,5). This problem can be reduced if dimethyl sulfoxide is used instead of methyl Cellosolve, but some equipment modifications are required to accommodate this solvent system (6).
We have found that flushing the coil with 0.1 to 2 N HCl for 1 to 2 hr after the end of the run and/or before shutting down the instrument helps to ' Supported in part by NIH Grants 5S07RR07097-13 and GM-19,972.