Aqueous Buffer Systems

The choice of aqueous buffer systems depends on different factors protein stability, resin performance, waste water treatment, and standard operation conditions, for example, standardization of solvents in a production facility. 

The most common buffer systems used in biochromatography are listed in Table 3.23. 

Positively charged buffering ions should be used on anion exchangers to avoid an interaction or binding to the functional group. Therefore Tris (pKa 8.2) is preferred 

Common name pffa at 25 °C Buffer range Molecular weight Full compound name 

TAPS 8.43 7.7-9.1 234.3 3- [(tris(hydroxymethyl)methyl]amino propanesulfonic acid 

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The role that acid and base catalysts play can be quantitatively studied by kinetic techniques. It is possible to recognize several distinct types of catalysis by acids and bases. The term specie acid catalysis is used when the reaction rate is dependent on the equilibrium for protonation of the reactant. This type of catalysis is independent of the concentration and specific structure of the various proton donors present in solution. Specific acid catalysis is governed by the hydrogen-ion concentration (pH) of the solution. For example, for a series of reactions in an aqueous buffer system, flie rate of flie reaction would be a fimetion of the pH, but not of the concentration or identity of the acidic and basic components of the buffer. The kinetic expression for any such reaction will include a term for hydrogen-ion concentration, [H+]. The term general acid catalysis is used when the nature and concentration of proton donors present in solution affect the reaction rate. The kinetic expression for such a reaction will include a term for each of the potential proton donors that acts as a catalyst. The terms specific base catalysis and general base catalysis apply in the same way to base-catalyzed reactions. 

Organic modifiers have been frequently employed in CE to increase the solubility of hydrophobic solutes in the aqueous buffer system. Unfortunately, many organic modifiers are UV absorbent and cannot be used without considerable loss of sensitivity of detection. A contactless conductivity detection system has been developed which extends the application range of UV-absorbing solvents [ 119]. As both natural pigments and synthetic dyes absorb in the visible part of the spectra, the application of UV-absorbing organic modifiers in their CE analysis does not cause detection problems. 

Unlike capillary electrophoresis, wherein absorbance detection is probably the most commonly utilized technique, absorbance detection on lab-on-a-chip devices has seen only a handful of applications. This can be attributed to the extremely small microchannel depths evident on microchip devices, which are typically on the order of 10 pm. These extremely small channel depths result in absorbance pathlengths that seriously limit the sensitivity of absorbance-based techniques. The Collins group has shown, however, that by capitalizing on low conductivity non-aqueous buffer systems, microchannel depths can be increased to as much as 100 pm without seeing detrimental Joule heating effects that would otherwise compromise separation efficiencies in such a large cross-sectional microchannel [38],... 

Partition coefficients for indinavir sulfate, as determined using buffered solutions of indinavir free base monohydrate, have been obtained in / -octanol / aqueous buffer systems at various pH values. The results, expressed as log of the ratio of the concentrations of the compound in the organic phase versus the concentrations in the aqueous phase (log P), are shown in Table 5 [7]. 

A number of soluble oxymetal salts were screened, particularly those of Mo, W, V, Ti and Zr, which are known to act via peroxometal and/or oxometal mechanisms (8). Unfortunately, no alternative to the existing KBr based protocol was found, so it was decided to, at least, improve the aqueous buffer system to significantly reduce the volume of the aqueous phase. 

Emulsification properties in model food systems. Pearson et al. (25) investigated the emulsification properties of caseinate and NFDM in model emulsion systems produced by blending soybean oil into an aqueous buffer system as a function of pH and ionic strength (Figures 7 and 8). They found that caseinate exhibited good emulsification properties under all pH and ionic strength conditions studied, but was particularly effective at pH 10.4. 

Release the protein from the source and solubilize it in an aqueous buffer system. 

Form I is the more stable form above the transition point up to its melting point of 191°C which is about five degrees above the metastable melting point of Form II. Although Form I is metastable with respect to Form II at room temperature, the rate of conversion of Form I to Form II is dependent upon the conditions. With solid Form I alone, the rate is so slow that it cannot be conveniently measured. In contact with water or aqueous buffered systems, the conversion requires weeks or months. In contact with ethanol Form I converts to Form II within 16 hours or less. 

The effect of surface polarity is even more important in separations where two or more simultaneous interactions must occur in order to achieve the desired selectivity. This is particularly true in chiral separations. Since aqueous buffer systems are almost universally used as CEC mobile phases, enantioseparations are often run under re-versed-phase conditions as opposed to the normal-phase mode typically used in chiral HPLC. Therefore, non-specific hydrophobic interactions would be highly detrimental to the discrimination process that involves subtle differences between the enantiomers. 

Lipophilic materials (Sephadex LH-20 and LE-60) are used when organic solvents are required. They are prepared from Sephadex G-25 and G-50 by hydroxypropylation. They are designed for use in aqueous buffer systems, polar organic solvents, and aqueous solvent mixtures. In mixed solvents, the gel preferentially takes up the more polar component. They have wide applications in the fractionation of lipids, steroids, fatty acids, hormones, vitamins, and other small molecules. 

Sephacryl can be used in aqueous buffer systems, pH 2-11, in concentrated urea or guanidine HCl, and in a number of organic solvents. The fractionation ranges of the five types of Sephaciyl (Table 17-1, p. 174) cover molecules from small polypeptides to particles of diameter 300-400 nm. Sephacryl S-200 and S-300 are useful for most proteins. 

The study of membrane and nucleic acid systems presents greater complexities. The study of lipids and insoluble membrane proteins in association with them can be achieved in solution scattering [75,76] by the use of lipid vesicles to solubilize the system of interest. Membrane proteins can alternatively be studied by solubilization using detergent micelles. The exception to this generalization is the group of plasma hpoproteins which are readily soluble in aqueous buffers. Systems with nucleic acids (i.e. protein-nucleic acid complexes, viruses, ribosomes and chromatin) [24-27,77,78] are not affected by these solubihty problems. However, lipid and nucleic acid systems are both further complicated in their analyses by the polyionic character of these macromolecules. Particular care is required concerning the partial specific volumes of the individual components to be used within the system of interest. 

Literature proposed CZE methods for phenols and derivatives using test mixtures based on aqueous buffered systems (phosphate-borate and borate " ), volatile electrolytes (ammonium hydrogencarbonate, diethylmalonic acid/dimethylamine in isopropanol and L-cysteic acid, 3-amino-1-propanesulfonic acid, aminomethanesulfonic acid, and diethylmalonic acid ), andnon-aqueous media (ammonium acetate in ACN/acetic acid in MeOH acetate, bromide, chloride, and malonate in ACN and diprotic acids/tetrabutylammonium hydroxide and maleate in MeOH ). 

The bioeatalytie resolution of epoxides has mainly been performed in single aqueous buffer systems. In our laboratory, B/nEH has been proved to be very useful for ehiral synthesis of (i )-GPE on a preparative scale. More importantly, this enzyme exhibited a eomplementary enantiospecificity as compared with those deseribed so far, affording the unreacted epoxide in (S)-configuration which is the solely useful enantiomer for synthesis of bioactive p-blockers. In a single aqueous phase, however, most epoxides including GPE may be spontaneously hydrolyzed into vicinal diols without any enantiospecificity. In addition, only at a very low concentration can epoxides be dissolved in aqueous media. The instability and low solubility of epoxides in the aqueous phase may result in a remarkable decrease in the yield of kinetic resolutions, thus limiting the application of these resolution processes on a practical scale. 

An incompatability that does need to be considered in CE/MS method development is the use of certain CE buffer systems and additives which are detrimental to the ESI process. For example, although sample concentration can be increased by the use of more conductive buffers, this approach is not advantageous for ESI/MS detection. These characteristics result in a significant demand upon ESI interface efficiency. Ideally, the chosen CE buffer should be volatile, such as ammonium acetate or formate. The use of pure acids or bases rather than a true buffer has also been shown to be advantageous for certain molecules. Non-aqueous buffer systems are also being employed more widely. 

The selectivity enhancement of PLE-mediated hydrolyses upon the addition of methanol, tcrt-butanol, and dimethyl sulfoxide to the reaction medium is exemplified in Scheme 2.46. The optical purities of products were in a range of 20-50% when a pure aqueous buffer system was used, but the addition of methanol and/or DMSO led to a significant improvement [287]. 

Variation of pH. Reactions catalyzed by hydrolases are usually performed in aqueous buffer systems with a pH closer to that of the pH optimum of the enzyme. Because the conformation of an enzyme depends on its ionization state (among others), it is reasonable to assume that a variation of the pH and the type of buffer may influence the selectivity of a given reaction. Such variations are facilitated by the fact that the pH activity profile of the more commonly used hydrolytic enzymes is rather broad and thus allows pH variations while maintaining an adequately high... 

This book, the primary topic of which Is the establishment of an operational pH scale, contains several tables of Interest. Tabulated are the Ion product of water from 0 to 60 C, the vapor pressure, density, and dielectric constant of water from 0 to 100 C, dielectric constants of pure liquids, and pH Values of several aqueous buffer systems. 

The third point, and related to the second point, is when samples are prepared in solvents weaker than the mobile phase. A classic example involves proteins. Here, the protein is often dissolved in an aqueous buffer system and subsequently injected into a mobile phase containing a significant level of organic modifier. When the level of organic is too high (e.g., acetonitrile >20% v/v), the protein may denature and precipitate out of the mobile phase onto the column support material [36]. 

MAA-EGDMA copolymers were imprinted using a chiral oxazine-based TSA, and selectivity for the TSA over both the substrate and the product was observed in both aqueous and nonpolar media. A 15-fold rate enhancement (fecat 0.1 s ) was obtained using the L-form of the TSA, and enantioselective preparation of phenylalanine was observed (32% 4% ee) for reactions carried out in an aqueous buffer system. This study was the first example of a sigmatropic shift in aqueous media catalyzed by MIPs. It was also one of a number of early examples that displayed only modest rate enhancements when compared to enzymes. 

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