Charles F. Kettering Research Laboratory, Yellow Springs, Ohio 45387.
Appl Environ Microbiol. 1984 May;47(5):1027-30. doi: 10.1128/aem.47.5.1027-1030.1984.
The analytical potential of the reaction of ammonia with o-phthalaldehyde mercaptoethanol reagent at pH 7 (an atypical fluorescence) has already been demonstrated. This, coupled with additional findings reported here, has led to an ammonia determination well suited to nitrogenase studies. As a result, large numbers of samples can be rapidly analyzed by high-pressure liquid chromatrography methods under mild conditions and without prior microdiffusion. Neither sodium dithionite (or other components of the usual nitrogenase assay), nor alternative substrates (cyanide, azide, methyl isonitrile), nor their products (methylamine, dimethylamine, hydrazine) interfere. High-pressure liquid chromatography showed that the fluorescent "product" of the o-phthalaldehyde mercaptoethanol reagent-ammonia reaction was, in fact, more than just a single compound. Despite this, once the proper solvent composition was found, high-pressure liquid chromatography with a small inexpensive C(18) "guard" column proved quite fast and reproducible for this measurement. Fluorescence response to ammonia was linear to at least 40 nmol/ml. A previous problem, long-term stability of the fluorescence, was solved by running the reactions in the dark. Background ammonia in the buffer could be substantially reduced by an analogous o-phthalaldehyde mercaptoethanol reagent reaction, using t-butyl mercaptan, and solvent extraction.
氨与邻苯二醛巯基乙醇试剂在 pH 7 下的反应（非典型荧光）的分析潜力已经得到了证明。再加上这里报告的其他发现，这种方法非常适合氮酶研究中的氨测定。因此，大量样品可以在温和的条件下通过高压液相色谱法快速分析，而无需预先进行微量扩散。连二亚硫酸钠（或通常氮酶测定中的其他成分）、替代底物（氰化物、叠氮化物、甲基异腈）及其产物（甲胺、二甲胺、联氨）都不会干扰。高压液相色谱表明，邻苯二醛巯基乙醇试剂-氨反应的荧光“产物”实际上不只是一种单一的化合物。尽管如此，一旦找到了合适的溶剂组成，带有小型廉价 C(18)“保护”柱的高压液相色谱对于这种测量就非常快速且重现。对氨的荧光响应至少在 40 nmol/ml 范围内是线性的。以前的一个问题，即荧光的长期稳定性，通过在黑暗中进行反应得到了解决。通过使用叔丁基硫醇和溶剂萃取，缓冲液中的背景氨可以通过类似的邻苯二醛巯基乙醇试剂反应大大降低。
