2.3. The Evaluation of Calcium and Magnesium Ion Content in Experimental Buffers

The assay of Ca²⁺ and Mg²⁺ in every buffer solution was evaluated via complexometric titration with standardized Na₂-EDTA solution in a pH-controlled environment [31,32,33]. Here we determined the buffer solutions used in the electroporation experiments, i.e.,: Milli-Q class deionized water (control); 10 mM HEPES, 10 mM HEPES with sucrose and 1 mM Mg²⁺; and 10 mM HEPES with sucrose, 1 mM Mg²⁺, and 2 mM CaCl₂. At first, the sum of dissociated Ca²⁺ and Mg²⁺ ions was measured at pH 10 against eriochrome black T (solochrome black) as an indicator. A total color change from wine red to blue or greenish-blue (since it was the final color being inflected by the buffer composition) was considered as a titration end point. Secondly, the assay of Ca²⁺ alone was evaluated from a separate aliquot, at pH 12, against murexide as an indicator. A total color change from pink to violet was considered as a titration end point. The assay of Mg²⁺ alone was calculated as a difference between the result of the first titration (Ca²⁺ + Mg²⁺) and second titration (Ca²⁺ only). Every titration was performed in triplicate using an over-titrated sample as an end point color reference. The “blank” titration was also performed in each of the above methods and the final results were corrected upon it. Every solution or dilution was performed using Milli-Q class deionized water which was free from measurable traces of Ca²⁺ or Mg²⁺. The pH was controlled by addition of concentrated ammonium chloride buffer (final pH 10) or 1 mol/L NaOH solution (final pH 12–13). The measurements are presented in Table 1.

The results of the titration for calcium and magnesium ions in experimental buffers.