Buffer Components, Effect

Properties of the solvent. Reaction rates will differ with the solvent s polarity, viscosity, donor number etc. Added electrolytes may lower or raise the rates ( salt effects ), and buffer components may do so as well. 

Probably the most effective use of XRF and TXRF continues to be in the analysis of samples of biological origin. For instance, TXRF has been used without a significant amount of sample preparation to determine the metal cofactors in enzyme complexes [86]. The protein content in a number of enzymes has been deduced through a TXRF of the sulfur content of the component methionine and cysteine [87]. It was found that for enzymes with low molecular weights and minor amounts of buffer components that a reliable determination of sulfur was possible. In other works, TXRF was used to determine trace elements in serum and homogenized brain samples [88], selenium and other trace elements in serum and urine [89], lead in whole human blood [90], and the Zn/Cu ratio in serum as a means to aid cancer diagnosis [91]. 

For sufficient retention of these very polar sulfonated carboxylates on RP columns, the addition of an ion-pairing (IP) agent such as tetraethylammonium acetate (TEAA) to the LC buffer was compulsory [13]. To maintain the compatibility of the eluent with the MS interface, the use of such an involatile cationic additive entailed a cation exchanger to be placed between the column and the interface [13]. Alternatively, equimolar amounts (5 mM) of acetic acid and triethyl-amine, which form the volatile IP agent triethylammonium, were added to the mobile phase in order to improve the retardation of very polar SPC [14]. While the first approach with TEAA was effective in retaining even the very short-chain C3- and C4-SPC (Fig. 2.10.4), the weaker IP agent triethylammonium notably increased the retention of C5-SPC and higher, whereas C4-SPC elutes almost with the dead volume of the LC (Fig. 2.10.5). Addition of commonly used ammonium acetate as buffer component led to the co-elution of the short-chain SPC ([Pg.322]

In studies on solvent effects involving variation in the composition of two component mixtures, similar types of outer-sphere interactions yield preferential solvation wherein the solvent composition of the outer-sphere may differ markedly from the bulk solvent composition. Supporting electrolyte species and buffer components may also participate in outer-sphere interactions thereby changing the apparent nature (charge, bulk, lability) of the reacting solvated metal ion or metal complex as perceived by a reacting ligand in the bulk solvent. 

Figure 7.6 Effect of pH on the stability of AIC after incubation at 30°C for 12 h in citrate buffer with the pH adjusted to 5 (panel A), 6 (panel B), and 7.4 (panel C). The peak with a retention time >10 min is due to a buffer component. Panel C consist mainly of monomer, and the front peak observed in panels A and B is due to aggregation/unfolding of the conjugate. Analysis conditions as described in Figure 7.5.

NMR is a remarkably flexible technique that can be effectively used to address many analytical issues in the development of biopharmaceutical products. Although it is already more than 50 years old, NMR is still underutilized in the biopharmaceutical industry for solving process-related analytical problems. In this chapter, we have described many simple and useful NMR applications for biopharmaceutical process development and validation. In particular, quantitative NMR analysis is perhaps the most important application. It is suitable for quantitating small organic molecules with a detection limit of 1 to 10 p.g/ml. In general, only simple one-dimensional NMR experiments are required for quantitative analysis. The other important application of NMR in biopharmaceutical development is the structural characterization of molecules that are product related (e.g., carbohydrates and peptide fragments) or process related (e.g., impurities and buffer components). However, structural studies typically require sophisticated multidimensional NMR experiments. 

Ad values) (4) effect on the pH function of any or all components of the reactions (including the buffer) (5) effect on the affinity(ies) of enzyme effector(s) (6) an alteration in the rate determining (or rate contributing) step(s) (7) effect on the coupling enzymes of the assay and (8) effect on physical properties e.g., solubility of substrates, particularly gas substrates such as O2 and N2, dielectric constant of the solvent, etc.). 

A buffered solution may contain a weak acid and its salt, e.g. acetic acid and acetate ion, or a weak base and its salt, e.g. NH3 and NH4CI. By choosing the appropriate components, a solution can be buffered at virtually any pH. The pH of a buffered solution depends on the ratio of the concentrations of buffering components. When the ratio is least affected by adding acids or bases, the solution is most resistant to a change in pH. It is more effective when the acid-base ratio is equal to unity. The pK of the weak acid selected for the buffer should be as close as possible to the desired pH, because it follows the following equation ... 

Fluorescent compounds are sensitive to changes in their chemical environment. Alterations in media pH, buffer components, solvent polarity, or dissolved oxygen can affect and quench the quantum yield of a fluorescent probe (Bright, 1988). The presence of absorbing components in solution that absorb light at or near the excitation wavelength of the fluorophore will have the effect of decreasing luminescence. In addition, noncovalent interactions of the probe with other components in solution can inhibit rotational freedom and quench fluorescence. 

Although buffers were used by Dixon and Greenhill to control the pH of their reaction solutions, the concentration of buffer components was very low (total [buffer] 10"2 M) and, perhaps for this reason, catalysis by buffers was not detected. Nor were experiments carried out in D20. Therefore two of the major criteria for rate-controlling proton transfer, general-acid catalysis and a primary kinetic solvent isotope effect were not demonstrated. Nevertheless the pH-rate profiles are indicative, for the most part, of a mechanism much like Scheme 1. 

Figure 9.28. The Effect of Buffer on Deprotonation. The deprotonation of the zinc-bound water molecule in carbonic anhydrase is aided by buffer component B.

Eq. (18) allows one to calculate the protein solubility in a wide range of cosolvent concentrations if information regarding (i) the composition dependence of the preferential binding parameter and (ii) the properties of the protein-free mixed solvent such as the molar volume and the activity coefficients of the components are available. In addition, one should mention that Eq. (18) was obtained for ternary mixtures (water (l)-protein (2)-cosolvent (3)). However, those mixtures contain also a buffer, the effect of which is taken into account only indirectly through the preferential binding parameter. Another limitation of Eq. (18) is the infinite dilution approximation, which means that the protein solubility is supposed to be small enough to satisfy the infinite dilution approximation (y2 = where is the activity coefficient of a protein at infinite dilution). 

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