Calcein is used for determination of Ca in biological systems. Indicator for the fluorimetric determination of calcium in presence of Mg by complexometric titration.
Purification Methods
Dissolve it in distilled H2O and acidify with dilute HCl to pH 3.5. Filter off the solid acid and wash it well with H2O. Redissolve ca 10g in 300mL H2O containing 12g of NaOAc. Precipitate it again by adding HCl, filtering and washing with H2O. Add the solid to 200mL of EtOH stir for 1hour and filter. Repeat the EtOH wash and dry the bright yellow solid in a vacuum. This acid decomposes on heating at ca 180o. See below for the preparation of the Na salt. [Diehl & Ellingboe Anal Chem 28 882 1956]. Altenatively dissolve it in H2O and acidify with 3N HCl to pH 3.5. Collect the solid and wash it with H2O. The air-dried precipitate is extracted with 70% aqueous EtOH, filtered hot and cooled slowly. Fine yellow needles of the acid crystallise out; they are filtered off and dissolved in the minimum quantity of 0.01N NaOH and re-precipitated by adding N HCl to pH 3.5. It is then recrystallised from 70% aqueous EtOH (3x). The final product (acid) is dried at 80o in a vacuum for 24hours, m >300o(dec). It contains one molecule of water per molecule of acid (C30H36N4O13.H2O). The product is pure as revealed by electrophoresis at pH 5.6 and 8.6, and by TLC in i-BuOH/i-PrOH/AcOH/H2O (60:60:5:5 by volume) or i-PrOH or pH 8.0 borate buffer. [Wallach et al. Anal Chem 31 456 1959.] The Na salt is prepared by dissolving the pure acid in H2O containing 2 mols of NaOH per mol of acid reagent and lyophilising. It complexes with Ca and Mg ions. [Beilstein 19 III/IV 4338.]
