**4. The general methodology for the development of spectrophotometric methods**

To develop a quantitative method for an unknown concentration substance using spectroscopic methods, the first step is by chooses the appropriate wavelength that corresponds to the highest absorbance. We can also choose the appropriate wavelength from the literature.

#### **4.1 Step 1: method development**

#### *4.1.1 Optimization conditions*

Several experimental variables such as the pH of the buffer system, choice of organic solvent, the volume of dye, and shaking time for the extraction of the ion-pair complex were tested with respect to their effect on complex formation.

For the development of spectroscopic methods, oxidation and reduction reactions, and complex formation are considered the backbone of these methods. These types of reactions produce colored products whose absorbance is measured. The sensitivity of the method and the degree of color stability are studied through several variables, the most important of which is the acid concentration/pH reagent concentration, nature of solvent, temperature, etc. until we reach the optimum conditions for the method. Before all that, the wavelengths of the colored products are scanned until the maximum wavelength (λmax) is obtained. The range at which the attainment of maximum color and stability occurs in the color species formation is termed as an optimum condition of each parameter.

#### **4.2 Study of the composition of the complex (stoichiometry)**

Job's method of continuous variation [61] was followed for finding out the composition of the ion-association complex formed between the studied drugs and selected dyes.

#### **Figure 4.**

*Reaction pathway for the formation of electron donor-acceptor complex due to Albendazole and picric acid interaction [58].*

#### **4.3 Chemistry of the colored species formed**

The chemistry of the colored species formed in each method is ascertained either through probability with the existing experimental evidence or through analogy with the literature methods.
