Binding constants, determination

Both of the above methods require that the stoichiometry is known prior to manipulation of the data. Most experimentalists assume 1 1 stoichiometry, though this is not always the case, and that self-association of the host does not occur. These two possibilities may be checked, in the first case, by constructing a Job plot and, in the second, by undertaking dilution experiments of the host alone. It may also be pertinent to attempt spectroscopic studies in a solvent where selfassociation may be enhanced and another that would inhibit the effect. For 

For slow exchange systems this allows the original concentrations to be used to calibrate the system. Combining the integrals of sets of peaks generated by specific 

For fast exchange the calculation is more complicated and requires that averaged signals for protons that are affected by complexation are distinct from the signals those same protons generate in the unbound state. It is also desirable to reduce the number of variables in the equation. This can be achieved by using dilute solutions of either host or guest. In dilute solutions where [G]o 2 [H]o then  

These relationships allow the determination of the binding constant by monitoring the chemical shift of protons on either the host or guest. Ideally high field instruments (500 or 600 MHz) should be used for their high resolution although good quality data can still be obtained on lower field instruments. Proton NMR is far more informative for this purpose than carbon-13 as the nuclei of the latter rarely shift far upon complexation. Proton shifts, on the other hand, may be of the order of 1 to 2 ppm and easy to detect even on low field instruments. 

Substituting for [G]0 — [H G] in equation (6) above, in which a large excess of guest is present, the following is obtained  

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K. Saha, F. Bender, and E. Gizeli, Comparative study of IgG binding to proteins G and A nonequilib-rium kinetic and binding constant determination with the acoustic waveguide device. Anal. Chem. 75, 835-842 (2003). 

The first, 7(0, can be determined from the experimental data for any finite value of C. The second, 7,<0, may be interpreted as the same integral 7(0, but computed for a hypothetical system of independent sites. As we shall see below, this interpretation is somewhat risky and should be avoided. The function 7,.(Q is better viewed as defined in Eq. (5.8.10), with the binding constants determined in Eq. (5.8.8). 

The values of the binding constants determined with different salt concentrations by equilibrium dialyses [43, 48], luminescence titrations and electrochemiluminescence [82], are all 2 or 3 orders of magnitude lower than for ethidium bromide. Therefore, a priori, they do not indicate contribution of classical intercalation into DNA as described for organic molecules and for the DPPZ, HAT and PPZ complexes. 

J Sowell, JC Mason, L Strekowski, G Patonay. Binding constant determination of drugs toward subdomain IIIA of human serum albumin by near-infrared dye-displacement capillary electrophoresis. Electrophoresis 22 2512-2517, 2001. 

Binding constants determined for ternary complex formation between capped and uncapped zinc porphyrins and small molecules such as imidazole and (substituted) pyridine contribute to the understanding of capped zinc porphyrins in molecular recognition, to which topic ternary zinc-cyclen complexes are also relevant. ... 

A Practical Course of Binding Constant Determination by UV/vis Spectroscopy... 

A Practical Course of Binding Constant Determination by UV/vis Spectroscopy 31 From Eq. (2.29) it is possible to express the -coordinate using a and f as follows,... 

Practical Course of Action for NMR Spectroscopic Binding Constant Determination... 

This chapter describes in detail the theoretical principles underlying binding constant determinations using only a basic level of mathematics, statistics, and programs of spreadsheet software. As a concrete example, a practical measurement and a practical data treatment of an UV/vis and NMR titration experiment are discussed. The programs attached as Appendices would function with commonly available spreadsheet software on personal computers and provide another way to understand the contents described in this chapter. The appendices are also useful for the reader when running an actual experiment. 

The apparent binding constants determined for the mass-law adsorption equations are empirical parameters related to thermodynamic constants (so-called intrinsic constants) via activity coefficients of the surface species. 

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