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Water was used to prepare stock solution of PG and TXA because of the free solubility of the drugs and working range solutions were prepared in acetonitrile. It has been known that methanol interferes in charge transfer complex formation so acetonitrile was used in working range [http://www.selleckchem.com/products/r428.html this website] solutions in subsequent analysis [25]. Dichloromethane was found to be suitable for preparation of DNP and TNP solutions. The solutions were scanned in visible range (800 to 350?nm) by spectrophotometer using the corresponding blank. It was found that, for TNP complex, ��max was 425?nm, while for DNP it was 418?nm which justifies intensity in color of complex derived from TNP as compared to that of DNP as in Figure 3. In view of these results, all working solutions to reaction were prepared in acetonitrile in sequence of drug-reagent solvent, [https://en.wikipedia.org/wiki/E-64 E-64] maintained at 25 �� 2��C. Absorbance is measured at 418 and 425 nm for DNP and TNP complexes, respectively, after 10 minutes of mixing. Figure 3 Spectrum of complexes and blanks. 3.2. Stoichiometry Job's continuous variations method suggested a 1?:?1 molar ratio for both reagents with both drugs. However, experimental design considered the drug molecules to be limiting reactants, so the chromogenic reagent was taken in slight excess in subsequent analysis in order to make the reaction drug concentration dependent and to counter any possible interference. 3.3. Thermodynamic Study of the Complex Complexes were studied in detail by applying Bensi-hilbrand theory. Association constant (Kc) was in the range of 1.51�C1.62 �� 103 for DNP, while it was in the range of 2.74�C2.86 �� 103 for TNP and standard free energy changes were ?2.336 to ?2.358 for complex derived from DNP and ?2.498 to ?2.508 for TNP-based complex. Results are portrayed in Table 1. Standard free energy changes value indicates spontaneity of the reaction. High wavelength, high association [http://www.selleckchem.com/products/jq1.html JQ1 concentration] constant, and low standard free energy changes with complex involving TNP as compared to DNP justify the resonance phenomenon in the phenolate ion derived from TNP compared to DNP [26]. Table 1 Thermodynamic study. 3.4. Validation Studies After development method validation studies were conducted in line with the above-mentioned protocol. Using linear regression equation, methods showed linear response in concentration range from 0.02 to 200??��g?mL?1 with correlation coefficient of more than 0.9990 in both cases. In all the cases studied, Beer's law was obeyed with small intercept values (?0.0028 to 0.0021). Slopes were found to be in the range of 0.01018 to 0.0146. Small intercept and insignificant variation of slope are attributes of the excellent response of the methods. Low LOQ values of (0.095�C0.109)?��g?mL?1 explained sensitivity of the method as in Table 2.