The kinetics of the transfer of the chelate, ethylenediamine tetraacetate (EDTA), from Calcium(II) to Copper(II) in imidazole (Im) buffers near neutral pH, corresponding to the conversion, [Cu(II)Im₄]²⁺→ [Cu(II)EDTA]²⁻, are characterized with stopped-flow absorption spectroscopy and implemented as a tool for calibrating the interval between mixing and freezing, the freeze-quench time (t_Q), of a rapid freeze-quench (RFQ) apparatus. The kinetics of this reaction are characterized by monitoring changes in UV–visible spectra (300 nm) due to changes in the charge-transfer band associated with the Cu²⁺ ions upon EDTA binding. Stopped-flow measurements show that the rates of conversion of the Cu²⁺ ions exhibit exponential kinetics on millisecond time scales at pH values less than 6.8. In parallel, we have developed a simple but precise method to quantitate the speciation of frozen solution mixtures of [Cu(II)(EDTA)]²⁻ and tetraimidazole Cu(II) ([Cu(Im)₄]²⁺) in X-band EPR spectra. The results are implemented in a simple high-precision ‘recipe’ for determining t_Q. These procedures are more accurate and precise than the venerable reaction of aquometmyoglobin with azide for calibrating RFQ apparatus, with the benefit of avoiding high-concentrations of toxic azide solutions.

螯合物乙二胺四乙酸 (EDTA) 在接近中性 pH 的咪唑 (Im) 缓冲液中从钙 (II )转移到铜 (II) 的动力学，对应于转化 [Cu(II)Im ₄ ] ²⁺ → [Cu(II)EDTA] ²⁻，采用停流吸收光谱进行表征，并作为校准混合和冷冻之间的间隔、快速冷冻骤冷 ( t _Q ) 之间的时间间隔的工具 ( RFQ）装置。该反应的动力学特征是通过监测由于EDTA 结合时与 Cu ²⁺离子相关的电荷转移带的变化而引起的紫外可见光谱 (300 nm) 的变化。停流测量表明，当 pH 值小于 6.8 时， Cu ²⁺离子的转化率在毫秒时间尺度上表现出指数动力学。与此同时，我们开发了一种简单但精确的方法来定量X 中[Cu(II)(EDTA)] ²⁻和四咪唑 Cu(II) ([Cu(Im) ₄ ] ²⁺ )冷冻溶液混合物的形态形成带 EPR 谱。结果以简单的高精度“配方”实现，用于确定t _Q。这些程序比用于校准 RFQ 装置的水合肌红蛋白与叠氮化物的古老反应更加准确和精确，并具有避免高浓度有毒叠氮化物溶液的优点。