Orange I

Orange I (α-Naphthol Orange) is an anionic azo dye that can form a complex with Methylene blue (HY-14536) and cause fluorescence quenching. The equilibrium constant of the complex of Orange I and Methylene blue is 79900 mol^-1·dm³. Orange I can change the absorption spectrum of itself and Methylene blue^[1][2]. In Vitro:Guideline (The following are our recommended procedures. This guideline is for informational purposes only and should be modified according to your specific needs.)
1. Solution Preparation
Stock Solution: Accurately weigh an appropriate amount of Orange I powder and dissolve it in deionized water or a suitable buffer (such as acetate-sodium acetate buffer at pH 4.9) to prepare a 1-10 mM stock solution. Use sonication to assist dissolution and ensure complete dissolution.
Working Solution: Dilute the stock solution with the same buffer or solvent according to the final concentration required for the experiment. Common working concentrations range from 1-100 μM, depending on the properties of the stained material and the experimental purpose.
2. Staining/Treatment Procedure (Taking the study of its interaction with cationic substances, such as methylene blue (MB) (HY-14536), as an example)
Sample Preparation: Prepare the sample to be treated. 3. Staining/Reaction: Solution Complexation Experiment: Add the prepared Orange I working solution dropwise or in a gradient to a cuvette containing MB solution, and mix well.
Material Adsorption Experiment: Immerse a certain amount of material in a known volume and concentration of Orange I working solution, and shake or stir at a certain temperature.
4. Incubation: Incubate the reaction system at a constant temperature (e.g., 25°C) for a sufficient time (usually 15-60 minutes) to ensure that the reaction or adsorption equilibrium is reached.
5. Detection/Analysis: Spectroscopy: Use a UV-Vis spectrophotometer to scan the absorption spectrum in the range of 300-700 nm. The characteristic absorption peak of Orange I is usually around ~480-490 nm (the specific position is affected by pH and environment). After complexation with MB, the absorption peak of MB red-shifts and changes in intensity. When reacting with surfactants, the intensity and position of its absorption peak change regularly with concentration.
Visual/Colorimetric Method: Observe the color change of the solution. Free Orange I is orange-yellow; the color may deepen or change after binding with cationic substances.
Adsorption Capacity Determination: For material adsorption experiments, after incubation, separate the material by centrifugation or filtration, measure the absorbance of the supernatant, and calculate the concentration of the remaining Orange I in the solution using a standard curve, thereby calculating the adsorption capacity.