Pseudo buffers

Strongly acidic or basic solutions show little change in pH when acid or alkali is added. This is a consequence of the nature of the pH scale and of the ionization of water as a weak base (pA a 0) and as a weak acid (pATj 1 ). Such solutions, in which the buffer action is due to the solvent rather than to any added solute, are not ordinarily described as buffers but it is convenient to speak of them as pseudo buffers . 

The buffer capacity of water containing completely dissociated acids or alkahs is 

Because of the high concentrations of solvent molecules, this buffer capacity may be much larger than for conventional buffers. 

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Solutions in which the buffering action is due to the solvent rather than any added solute Strongly acidic or basic aqueous solutions will show httle change in pH when additional increments of acid or base are added (recall that the pK value for H3O+ is -1.74, and that for H2O is 15.74) . Because the solvent is in such high concentration, the buffering capacity for pseudo buffers is larger than for conventional buffers. See Buffer Capacity... 

The concentration of phenylacetate can be determined from the kinetics of its pseudo-first-order hydrolysis reaction in an ethylamine buffer. When a standard solution of 0.55 mM phenylacetate is analyzed, the concentration of phenylacetate after 60 s is found to be 0.17 mM. When an unknown is analyzed, the concentration of phenylacetate remaining after 60 s is found to be 0.23 mM. What is the initial concentration of phenylacetate in the unknown ... 

An effective experimental design is to measure the pseudo-first-order rate constant k at constant pH and ionic strength as a function of total buffer concentration 6,. Very often the buffer substance is the catalyst. Let B represent the conjugate base form of the buffer. Because pH is constant, the ratio (B]/[BH ] is constant, and the concentrations of both species increase directly with 6 where B, = [B] -t-[BH"]. 

Throughout this section the hydronium ion and hydroxide ion concentrations appear in rate equations. For convenience these are written [H ] and [OH ]. Usually, of course, these quantities have been estimated from a measured pH, so they are conventional activities rather than concentrations. However, our present concern is with the formal analysis of rate equations, and we can conveniently assume that activity coefficients are unity or are at least constant. The basic experimental information is k, the pseudo-first-order rate constant, as a function of pH. Within a senes of such measurements the ionic strength should be held constant. If the pH is maintained constant with a buffer, k should be measured at more than one buffer concentration (but at constant pH) to see if the buffer affects the rate. If such a dependence is observed, the rate constant should be measured at several buffer concentrations and extrapolated to zero buffer to give the correct k for that pH. 

Danckwerts et al. (D6, R4, R5) recently used the absorption of COz in carbonate-bicarbonate buffer solutions containing arsenate as a catalyst in the study of absorption in packed column. The C02 undergoes a pseudo first-order reaction and the reaction rate constant is well defined. Consequently this reaction could prove to be a useful method for determining mass-transfer rates and evaluating the reliability of analytical approaches proposed for the prediction of mass transfer with simultaneous chemical reaction in gas-liquid dispersions. 

Pectins were incubated in buffered medium in mild alkaline conditions (pH 8.5 to 11.2) at room temperature, leading to both demethylation and P-elimination. At higher pHs p-elimination had increased initial speed but soon plateaued. Demethylation was slower but proceeded until completion. It followed a (pseudo)-first order kinetics with respect to concentration of methylesterified carboxyl groups. A rate constant of 27.2 9.0 moT 1 min was calculated after correction for the pH variation during the course of the reaction. 

In mild alkaline conditions, highly methylated pectin was demethylated following a (pseudo)-first order kinetics with respect to the concentration of methoxylated galacturonate moieties. Investigation in this pH range, where the initial concentration of methylesters was higher than the initial concentration of OH ions, was complicated by the necessity to use a buffer. This led to deviations from the theoretical behavior as the concentration of OH ions still varied in proportions which could not be neglected in the equations of the kinetics. However these deviations could be accounted for be the pH variation, and the pH variation itself predicted from the amount of liberated methanol. The constant we found was similar to previously reported data (Scamparini Bobbio, 1982). 

MEKC is a CE mode based on the partitioning of compounds between an aqueous and a micellar phase. This analytical technique combines CE as well as LC features and enables the separation of neutral compounds. The buffer solution consists of an aqueous solution containing micelles as a pseudo-stationary phase. The composition and nature of the pseudo-stationary phase can be adjusted but sodium dodecyl sulfate (SDS) remains the most widely used surfactant. 

In PAMPA measurements each well is usually a one-point-in-time (single-timepoint) sample. By contrast, in the conventional multitimepoint Caco-2 assay, the acceptor solution is frequently replaced with fresh buffer solution so that the solution in contact with the membrane contains no more than a few percent of the total sample concentration at any time. This condition can be called a physically maintained sink. Under pseudo-steady state (when a practically linear solute concentration gradient is established in the membrane phase see Chapter 2), lipophilic molecules will distribute into the cell monolayer in accordance with the effective membrane-buffer partition coefficient, even when the acceptor solution contains nearly zero sample concentration (due to the physical sink). If the physical sink is maintained indefinitely, then eventually, all of the sample will be depleted from both the donor and membrane compartments, as the flux approaches zero (Chapter 2). In conventional Caco-2 data analysis, a very simple equation [Eq. (7.10) or (7.11)] is used to calculate the permeability coefficient. But when combinatorial (i.e., lipophilic) compounds are screened, this equation is often invalid, since a considerable portion of the molecules partitions into the membrane phase during the multitimepoint measurements. 

Fig. 29. Equilbrium unfolding of C40A/C82A/P27A (pseudo-wild-type) barstar monitored by A R, mean residue circular dichroism. Conditions for near-UV CD were 50 /xM protein in 50 mM Tris-HCl buffer, pH 8, 0.1 M KC1, path length 1 cm. (A) Urea-induced unfolding at 25°C at urea concentrations as indicated. (B) Cold-induced unfolding in...

The experimentally observed pseudo-first order rate constant k is increased in the presence of DNA (18,19). This enhanced reactivity is a result of the formation of physical BaPDE-DNA complexes the dependence of k on DNA concentration coincides with the binding isotherm for the formation of site I physical intercalative complexes (20). Typically, over 90% of the BaPDE molecules are converted to tetraols, while only a minor fraction bind covalently to the DNA bases (18,21-23). The dependence of k on temperature (21,24), pH (21,23-25), salt concentration (16,20,21,25), and concentration of different buffers (23) has been investigated. In 5 mM sodium cacodylate buffer solutions the formation of tetraols and covalent adducts appear to be parallel pseudo-first order reactions characterized by the same rate constant k, but different ratios of products (21,24). Similar results are obtained with other buffers (23). The formation of carbonium ions by specific and general acid catalysis has been assumed to be the rate-determining step for both tetraol and covalent adduct formation (21,24). 

Fig. 14 Plots of observed pseudo-first-order rate constants for the methanolysis of increasing and equimolar [La3 + ] = [32, HPNPP] at 25 °C and pH 5.0 (iV,jV-dimethylaniline buffer, , right axis) or pH 6.7 (2,6-lutidine buffer, , left axis). Lines through the data computed from fits to a standard one-site binding model. Reproduced from ref. 81 with permission.

While the acceleration afforded to the cyclization of 32 by La3 + in methanol is certainly spectacular, this is not a biologically relevant metal ion and its charge exceeds that of the natural metal ion Zn2+. Very recent investigations of Zn2+-catalysis of the methanolysis and ethanolysis of 32 indicated that there were indeed interesting catalytic effects, and that the situation in pure ethanol is quite different.85 Shown in Figs 15 and 16 are plots of the pseudo-first-order rate constant (kobs) for ethanolysis and methanolysis of HPNPP (32) as a function of [Zn2+]total when the [ OR]/[Zn2+] ratio is 0.5. This ratio was chosen to buffer the system at the half neutralization jjpH of 7 in ethanol8 and 9.5 in methanol at [Zn2+]to)ai = l-2 mM7... 

In previous chapters, we discussed the hydrolysis of a number of esters of A-(hydroxymethyl)phcnytoin, namely esters of organic acids (7V-acyloxy-methyl derivatives, Sect. 8.7.3) or inorganic acids (Sect. 9.3.2). Hydrolysis of these potential prodrugs released 3-(hydroxymethyl)phenytoin (11.45), whose breakdown to phenytoin and formaldehyde was also investigated per se [79], The latter reaction followed pseudo-first-order kinetics. At pH 7.4, the f1/2 values were 4.7 and 1.6 s at 25° and 37°, respectively. The tm values decreased tenfold for each increase of pH by one unit, which, together with the absence of any buffer catalysis, indicates catalysis by the HO- anion. 

Chemical. Although no products were identified, p-chloronitrobenzene (1.5 x 10 M) was reduced by iron metal (33.3 g/L acid washed 18-20 mesh) in a carbonate buffer (1.5 x 10 M) at pH 5.9 and 15 °C. Based on the pseudo-first-order disappearance rate of 0.0336/min, the half-life was 20.6 min (Agrawal and Tratnyek, 1996). 

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