Dye Extraction Method: Stability of Methylene Blue against Nitric Acid

Objective: The dye extraction experiment (storage of test specimens in methylene blue, dissolving of roots in nitric-acid, spectrophotometric determination of extracted dye) is a common method applied to study sealing behavior of endodontic filling materials. Stability of methylene blue against nitric-acid is a prerequisite for achieving informative results. Purpose of this study was to evaluate the stability of methylene blue (MB) and alizarin red s (AR) against nitric- and hydrochloric-acid. Methods: 80mg/l of AR and 2mg/l of MB were dissolved in water (control groups), hydrochloric- (29%) and nitric-acid (52%), respectively. For pH-indicator AR, one droplet of concentrated phosphoric-acid was added to control group. For the next days potential differences in color between solutions were followed. Additionally, for AR (80mg/l in hydrochloric-acid) and MB (2mg/l in nitric-acid) stability was observed via UV/VIS-spectroscopy for seven days. No statistical analysis was performed because of using a validated absolute method. Results: Only after two days, it was already clearly visible that both nitric-acid solutions paled compared to control groups. For hydrochloric-acid solutions, AR showed no detectable difference to aqueous solution, whereas MB solution was nearly colorless. Results of UV/VIS-measurements (wavelength of maximum λ, absorption A) of AR in hydrochloric- and MB in nitric-acid are summarized in the table below. In contrast to AR, MB showed both a significant difference in absorption and a shift in maximum after at least four days (significance based on device accuracy).

*Stated errors based on device accuracy.

  Conclusions: It is highly questionable that the combination methylene blue/nitric-acid is suitable for dye extraction measurements. A promising alternative is alizarin red s/hydrochloric-acid which should be chosen instead.