Isobestic point

Prepare solution B by diluting a mixture of lO.OmL of the standard solution of the indicator and 25.0 mL of 0.04M sodium acetate to 100 mL. The pH of this solution is about 8, so that the indicator MR is present entirely as MR-. Measure the absorbance of solution B over the range 350-600nm as detailed for solution A with a manual spectrophotometer use 25 nm steps except for 400 450 nm, where 10 nm steps are recommended. Determine the wavelength 2B of maximum absorbance as above this is about 430 nm. The type of plots obtained for solutions A and B is shown in Fig. 17.21. The absorption peaks are not completely separated, but cross at a wavelength of about 460 nm. This point is known as the isobestic point . If the absorbance of a solution containing both HMR and MR - is measured at this wavelength, the observed absorbance is independent of the relative amounts of HMR and MR - present and depends only on the total amount of the indicator MR in the solution. 

Tautomerism of the Dye. The complete visible absorption spectra were secured for dyed parathion at several pH values in both 20 and 60% ethyl alcohol. These curves are plotted in Figures 9 and 10. The appearance of an isobestic point in both instances indicates that tautomeric forms of the dye are involved (8). 

The basicity of OLOA 1200 has been evidenced by its interaction with the oil-soluble acidic indicator dye, Brom Phenol Magenta E (EK 6810) which is normally yellow but turns blue and then magenta with increasing bacicity. The acidic form has an adsorption peak at 390 nm, the basic at 610 nm, and the isobestic point is at 460 nm. These spectra have be used to determine the concentration of OLOA 1200 in solution for adsorption isotherms. 

When p-nitrophenolate is incrementally added to a-cyclodextrin in water, the ultraviolet spectrum of this anion changes such that successive spectra give rise to two isobestic points (Figure 5.2). Such behaviour is in accord with the formation of a 1 1 species. The spectral changes may be used for the direct calculation of K, which in this case was found to be approximately 104 dm3 mol-1 (Cramer, Saenger Spatz, 1967). 

It is frequently necessary to subtract from the total absorption a part owing to the un-ionized molecule in measuring the amount of ion present. If a given solute exists in two forms, such as a molecule and an ion, it is quite likely that the absorption curves of the two forms will intersect at some point. The intersection is known as an isobestic point, and at the wave length of the isobestic point the extinction depends only on the total amount of solute and not on the proportion of the two forms.166 One investigator who had the bad luck to pick the isobestic wave length for all his experiments came to the mistaken conclusion that it was not possible to determine the position of his particular equilibrium by optical means. A medium effect will cause a family of absorption curves to intersect in a small region rather than in a point, but failure to show an isobestic point even approximately means that the solute exists in more than two forms. 

Titrations of Ag+ with porphyrazines 81c and 81h resulted in a decrease and broadening of the Q band up to 2 equiv of metal coupled with the appearance of at least five isobestic points. Further addition of Ag+ resulted in a visible change from purple to blue and a decrease of the 675-nm band and appearance of a sharp peak at 650 nm, which reached a maximum at 6 equiv of Ag+. The n-n transition disappeared between 0 and 6 equiv. The Hg+ ion gave similar results. Compound pz 81f displayed more complex interactions in titrations with AgC104. 

Oxy-Hb Crosses the y axis near the deoxy-Hb line but falls steeply around 600 nm to a trough around 660 nm. It then rises as a smooth curve through the isobestic point where it flattens out. This curve must be oxy-Hb as the absorbance of red light is so low that most of it is able to pass through to the viewer, which is why oxygenated blood appears red. 

Deoxy-Hb Starts near the oxy-Hb line and falls as a relatively smooth curve passing through the isobestic point only. Compared with oxy-Hb, it absorbs a vast amount of red light and so appears blue to the observer. 

The naphthopyran ring-opening reactions have not been as well studied as they have for the spiropyrans and spiro-oxazines. Aubard et al. [75] recently reported that in acetonitrile and hexane, irradiation of CHRl resulted in a broad transient spectrum after 0.8 psec, having three maxima at 360-370 nm, 500 nm, and 650 nm. At 1.8 psec, a well-defined band forms at 425 nm. From 10 to 100 psec, there is little further evolution except for a continued growth in the peak at 425 nm of about 15%. There is also a decrease in the overall bandwidth. The mechanism in Scheme 9 has been proposed, where B2 and B, are isomers of the mero-form. Three isobestic points were identified in the transient spectra at different times, suggesting four transient states. Forming between 0.8 and 1.6 psec, the Bi state was assigned as the cis isomer. This had a spectrum similar to that obtained for Tamai s X transient of the spiro-oxazine NOSH, which was obtained at subpicosecond time scales [26]. 

Isobestic points. Consider compound A, which can be transformed by a first order reaction into compound B. Assume that the absorption spectra obtained under the same conditions of concentration cross over at a point I when they are superimposed (Fig. 11.21). That is, the absorbances of the two solutions are the same for the wavelength at point I. Consequently, the coefficients cA and eB are identical. In this type of experiment, A is initially pure and at the end of the experiment B is pure. For all the intermediate solutions, mixtures of A and B can be prepared but the global concentration does not change (CA + CB = constant). This leads to the following relationship ... 

All spectra of the mixtures A + B will pass, over the course of time, through point I, called the isobestic point, where the absorbance of A will be constant. 

Figure 11.21—Isobestic point. Alkaline hydrolysis of methyl salicylate at 25 C. Superposition of the successive spectra recorded between 280 and 350 nm at 10 min intervals.

After each addition, plot the absorption spectrum of the solution from 300 to 500 nm. You will have 10 spectra characterizing absorption of methylnitrophenol at the different pHs (2-11). What do you notice about the absorption peak position Is there any correlation between the absorption peak position and the color of the methylnitrophenol solution Do you observe an isobestic point If yes, explain its meaning. 

Upon binding of ethidium bromide to DNA, the maximum OD from its absorption spectrum decreases, together with a shift to higher wavelengths (Figure 12.3). An isobestic point is observed at 510-511 nm, indicating that the fluorophore molecules... 

Figure 12.3 Absorption spectra of 50 /

Figure 13. Infrared absorption spectra of dilute solutions of HOD in H20 at four temperatures (a) 302 K, (b) 320 K, (c) 340 K, and, (d) 360 K, with isobestic point at ca. 2575 cm-1 (Senior and Verrall, 1969).

Quantitation was carried out by the simultaneous equation (Method 1) and the absorbance ratio (Method 2). The wavelengths selected for Method 1 were 257.10 and 288.66 nm of both drugs, respectively. In Method 2, two wavelengths 257.10 nm of paracetamol and 284.36 mn, the isobestic point, were selected. Both the methods were validated for linearity, accuracy, and precision. 

It is concluded that below the transition temperature the monomer intensity increases with increasing temperature due to excimer dissociation to excited-state monomer. Above the transition temperature the excited-state equilibrium is apparently broken because the thermal energy is sufficient to activate excimer non-radiative decay by dissociation to ground-state monomer. Consequently, the monomer emission no longer increases with increasing temperature and the isobestic point disappears. Pyrene has been used as a fluorescent probe to monitor the conformational state of maleic acid with... 

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