The Purification of Methylene Blue and Azure B by Solvent Extraction and Crystallization

Abstract

Detailed schemes are described for the preparation of purified methylene blue and azure B from commercial samples of methylene blue. Purified methylene blue is obtained by extracting a solution of the commercial product in an aqueous buffer (pH 9.5) with carbon tetrachloride. Methylene blue remains in the aqueous layer but contaminating dyes pass into the carbon tetrachloride. Metal salt contaminants are removed when the dye is crystallized by the addition of hydrochloric acid at a final concentration of 0.25 N. Purified azure B is obtained by extracting a solution of commercial methylene blue in dilute aqueous sodium hydroxide (pH 11-11.5) with carbon tetrachloride. In this pH range, methylene blue is unstable and yields azure B. The latter passes into the carbon tetrachloride layer as it is formed. Metal salt contaminants remain in the aqueous layer. A concentrated solution oa azure B is obtained by extracting the carbon tetrachloride layer with 4.5 X 10(-4)N hydrobromic acid. The dye is then crystallized by increasing the hydrobromic acid concentration to 0.23 N. Thin-layer chromatography of the purified dyes shows that contamination with related thiazine dyes is absent or negligible. Ash analyses reveal that metal salt contamination is also negligible (sulphated ash less than 0.2%).

Effect of alkali on methylene blue (C.I. Basic Blue 9) and other thiazine dyes

“…This was not surprising, given others have noted MB is 'indefinitely stable' in aqueous solution at pH 9.5 [29,30]. Indeed, mixing MB with 0.1 mM alkali and shaking with a water-immiscible solvent (usually dichloromethane) is a published method for purifying MB of the less methylated thiazines, such as azure B (5;usually the most prevalent species), azure A (6) and azure C (7), which are common impurities in most past commercial samples of MB [13,31]. It is generally accepted that this purification procedure is effective because the latter thiazines are readily deprotonated by the alkali to their neutral, lipophilic orange or red-coloured forms [31] and MB itself is stable in 0.1 mM alkali.…”

“…Structures are given in Table 1. MB was further purified by continual extraction of red impurities from its buffered aqueous solution by CCl 4 until the latter washings were colourless, followed by crystallization according to a published method [13]. All other chemicals were used without further purification.…”

Purified azure B as a reticulocyte stain.

“…Purified azure B was prepared by the method of Marshall and Lewis (1975 solution (1 volume of aqueous sodium citrate (30 g/l) and 4 volumes of aqueous sodium chloride (9 g/l)). This solution is used as in the standard new methylene blue technique described by Dacie and Lewis (1968 Dacie and Lewis (1968).…”

“…In order to compare the reticulocyte counts obtained by the two methods, 12 specimens of blood were Marshall and Lewis (1975) selected so as to provide a range of reticulocyte counts. Duplicate preparations were stained with purified azure B and new methylene blue (R. A.…”

Cien años del colorante de Giemsa.

“…Esta síntesis tiene una fase final de oxidación con bicromato de potasio, por lo cual se produce algo de azur B (8 muestras tuvieron un promedio de 4,1% con mínimo de 3,0% y máximo de 5,1%) (20), que acompaña al azul de metileno del comercio, así se trate del más 'puro' y los intentos para purificarlo determinan casi siempre un incremento en su contenido de azur (21). Su purificación (22) por extracción es laboriosa y no se justifica salvo para estudios o usos que ameriten su homogeneidad, en particular los que se hacen con seres humanos vivos. Ehrlich, citado en primer lugar por Woronzoff-Dashkoff (2), realizó los primeros trabajos biológicos con azul de metileno (23), como lo reconoce elegantemente Bernthsen. justo homenaje, los nombres del primero y el de Klebs-Loeffler, con el azul policromo que aún se conoce como azul de metileno alcalino de Loeffler (27).…”