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Apparent stability constants and purity of Ca-chelating agents evaluated using Ca-selective electrodes by the
1Department of Cellular and Molecular Physiology, National Institute for Physiological Sciences, Okazaki, Japan.
Cell Calcium
|March 1, 1994
Summary
This study assessed calcium-chelating agent purity and stability using Ca(2+)-selective electrodes. EGTA demonstrated high purity and stability, while BAPTA showed decreased purity upon storage, both improved by drying.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Electrochemistry
Background:
- Accurate measurement of free calcium ion (Ca2+) concentrations is crucial in biological and chemical research.
- Calcium-chelating agents like EGTA and BAPTA are widely used but their purity and stability can affect experimental results.
- Existing methods for evaluating chelator purity and calcium contamination may require refinement.
Purpose of the Study:
- To evaluate the apparent stability constants and purity of calcium-chelating agents, specifically EGTA and BAPTA.
- To adapt the double-log optimization method for assessing free Ca2+ contamination in electrolyte solutions.
- To determine the impact of storage and drying procedures on the purity of EGTA and BAPTA.
Main Methods:
- Utilized Ca(2+)-selective electrodes and the double-log optimization method for determining apparent calcium stability constants.
- Amended the double-log method to quantify free Ca2+ contamination in electrolyte solutions.
- Employed atomic absorption spectroscopy to measure total contaminating Ca content.
- Assessed the purity of EGTA and BAPTA before and after storage and drying (baking at 150°C for 3 h).
Main Results:
- The amended double-log method accurately estimated free Ca2+ contamination (3.7 µM), consistent with atomic absorption spectroscopy.
- Commercial EGTA purity ranged from 95.5-98.0% and remained stable during storage; impurities were eliminated by drying.
- BAPTA purity decreased from 85.8% to 77.2% after 3 months of storage at -20°C, but drying also removed its impurities.
- Apparent stability constants (K's) for EGTA at pH 7.30 were 7.13 (0.10 M ionic strength) and 6.97 (0.16 M ionic strength).
- Apparent stability constants (K's) for BAPTA at pH 7.30 were 6.50 (22°C, 0.20 M ionic strength) and 6.69 (37°C, 0.20 M ionic strength), increasing with decreasing ionic strength.
Conclusions:
- The double-log optimization method, when amended, provides a reliable assessment of free Ca2+ contamination in solutions.
- EGTA exhibits high purity and stability, making it a robust choice for calcium chelation applications.
- BAPTA's purity degrades over time, necessitating careful storage and potential purification before use.
- Drying is an effective method for removing impurities from both EGTA and BAPTA, restoring their purity.