Isosbestic Points Often one absorbing species, X, is converted to another absorbing species, Y, during the course of a chemical reaction. This transformation leads to a very obvious and characteristic behavior, shown in Figure 19-6. If the spectra of pure X and pure Y cross each other at any wavelength, then every spectrum recorded during this chemical reaction will cross at the same point, called an isosbestic point. The observation of an isosbestic point during a chemical reaction is good evidence that only two principal species are present. Consider methyl red, which is an indicator that changes between red (HIn) and yellow (In-) near pH 5.1: - O2C N(H3C)2 N N H pK2=5.1 - O2C N(H3C)2 N N HIn (red) In(yellow) Because the spectra of HIn and In- (at the same concentration) happen to cross at 465 nm, all spectra in Figure 19-6 cross at this point. (If the spectra of HIn and In- crossed at several points, there would be several points.) To see why there is an isosbestic point, we write an equation for the absorbance of the solution at 465 nm: 465 465 A465 = ε HIn b[ HIn] + ε In b[ In − ] − (19-8) But because the spectra of pure HIn and pure In- (at the same concentration) cross at 465 nm, 465 ε HIn must be equal to 465 465 465 ε In . Setting ε HIn = ε In = ε 465 , we can factor − − Equation 19-8: A465 = ε 465b([ HIn] + [ In − ]) (19-9) In figure 19-6, all solution contain the same total concentration of methyl red ( = [ HIn] + [ In − ] ). Only the pH varies. Therefore the sum of concentrations in Equation 19-9 is constant, and A465 is constant.