Acid and base ampholyte

Isoelectric focusing is a procedure used to determine the isoelectric point (pi) of a protein (Fig. 3-21). A pH gradient is established by allowing a mixture of low molecular weight organic acids and bases (ampholytes p. 81) to distribute themselves in an electric field generated across the gel. When a protein mix-... 

Equations have been published [16] which relate pKa and p0Ka values to partition coefficient (P) values for monoprotic acids and bases, and diprotic acids, bases and ampholytes. For example, P1 for a monoprotic acid is calculated from... 

Amphoteric substances, or ampholytes, are able to engage in neutralization reactions both with acids and bases (more precisely, both with hydrogen and hydroxyl ions). Aluminium hydroxide, for example, reacts with strong acids, when it dissolves and aluminium ions are formed ... 

Some substance (like HS04, H2P04 , HP04-, NH3, H20 etc.) can function both as acids and bases, depending on the circumstances. These substances are called amphoteric electrolytes or ampholytes. 

IEF was first successfully applied to proteins in 1938, when it was used to separate the protein hormones vasopressin and oxytocin from tissue extracts. Twenty years later, ampholytes were first focused in a continuous pH gradient, stabilized by a dense sucrose medium, as an alternative to the multicompartment method. The continuous pH gradient in the sucrose medium was established by allowing acid and base to diffuse into opposite ends of the sucrose medium, held in a U-cell, from their respective electrode chambers. The stabilization of this continuous pH gradient with carrier ampholyte species led to modern IEF methods. 

Values of p/f =-log at 25°C for some important dissolved acids and bases in natural waters are given in Table 5.1. In the table Fe " and AP" have been written as aquocomplexes, to show the role of water in their acid-base behavior. The convention is generally not to write the waters of hydration. Notice that the species HSO4, H2PO4, Fe0H(H20), HCO3, HPO, and H SiOi can act either as acids or as bases. Thus they are called ampholytes or amphiprotic. 

Solutions of any desired pH may be obtained simply by mixing a weak acid or base with one of its salts in various proportions. It is evident from equation (48) that even small amounts of strong acids and bases have only a slight effect on the pH of such mixtures. Certainly the small quantities of alkali from glass and carbon dioxide from the atmosphere can exert no perceptible influence. Such mixtures which are resistant to a change in reaction were called Buffer Mixtures by S. P. L. SOkensen. L. Michaelis coined the term Regulators. They may also be referred to as Ampholytes because of the amphoteric character of such mixtures. AU mixtures of weak adds and their salts,... 

An equa tion corresponding to (14) likewise may be derived for the case in which a base is added to a solution of an ampholyte. The complicated expression (14) need be used only in the neighborhood of the isoelectric point. The simple equations for monovalent acids and bases (cf. Chapter One) may be employed at other hydrogen ion concentrations. 

Calculated pKa values are used to determine at which pH the compound is neutral. LogP (neutral) is then used to calculate back the membrane log P partition coefficient. This way one calibration curve covers neutral, acids, and bases (Figure 15.8). One difference with the RP-HPLC based method is that the approach described here measures logP, while the former measures logZ) values. Potentially the method can be relatively easily extended to other water/solvent systems. The limitation of the approach is with some ampholytes and with zwitterions, which are not >95% neutral at any pH value througout the 2 to 11 range. 

A solution containing Q.5-2.0% carrier ampholytes and 0.1-0.4% methylcellulose (1500 cP for a 2% solution) is filled into the capillary. A coated capillary is used to suppress the electro-osmotic flow (EOF) in conjunction with the methylcellulose solution. The sample (protein) concentration in the ampholyte blend is usually between 50 and 200 p.g/ml. The inlet reservoir (anolyte) is filled with 10 mM phosphoric acid in methylcellulose solution. The outlet reservoir (catholyte) contains 20 mM sodium hydroxide. Higher concentrations of acid and base may be used to further stabilize the pH gradient. 

In order to analyze the distribution of simple ampholytes (i.e. single acid and base) they were first classified as either ordinary or zwitterionic ampholytes and the isoelectric points were calculated. Figure 6 illustrates the range of isoelectric points for both the ordinary and zwitterionic ampholytes. While no clear pattern emerges this may be a reflection of the limited number of compounds (65) available for this analysis. The larger number of ordinary ampholytes at the high end of the scale represent simple phenols with alkylamine side chains (e.g. phenylephrine). If these compounds are left aside, those that remain tend to have isoelectric points between 3.5 and 7-5. 

In a similar way one may define conjugate acids of appropriate bases. It is apparent that some species, known as ampholytes, are capable of behaving as both acids and bases. In this respect the nature of water when acting as a solvent is of particular significance, as it means that all acidic and basic properties in aqueous solution will set up equilibria involving the solvent. 

The thermodynamic ionization constants for ampholytes and diacidic bases can be calculated by a similar method. For these determinations, ampholytes are dissolved in one equivalent of hydrochloric acid and bases in two equivalents. 

In the case of our initial and unsuccessful TLC attempts to enantioseparate the 5,/ -( )-ibuprofen and the 5,/ -( )-2-phenylpropionic acid antipodes [1-3], we kept our test samples for a longer period of time dissolved in 70% ethanol (and also in dichloromethane and physiological salt). Evidently, none of these solvents can be considered as a base or an acid, at least not in the spirit of the acid and base definitions introduced by Arrhenius. In other words, none of these solvents can catalyze or hamper transenantiomerization of the chiral APAs. However, the 70% ethanol solvent can easily be viewed as a weak ampholyte, able to simultaneously exert the catalytic and inhibiting effect on transenantiomerization of the chiral analytes considered. Perhaps, this perceptible ampholytic nature of 70% ethanol (combined with a change in viscosity of the APA solutions, as related to that of... 

Figure 3.2(c) shows an example of an ampholyte, labetolol. The log S versus pH shake-flask data were taken from the Hterature [49]. Ampholyte paraboHc-shaped curves show features of both an acid and a base profile. 

Figure 5.7 shows lipophilicity profiles (log D vs. pH) for an acid (warfarin), a base (tetracaine), and an ampholyte (morphine). The dashed curves correspond to the values determined in octanol-water and the solid curves, to values in liposome-water. As is readily apparent, the major differences between octanol and liposomes... 

Figure 5.7 Comparison of liposome-water (solid lines) to octanol-water (dashed lines) lipophilicity profiles for a weak acid, a weak base, and an ampholyte. [Avdeef, A., Curr. Topics Med. Chem., 1, 277-351 (2001). Reproduced with permission from Bentham Science Publishers, Ltd.]...

Figure 6.8 shows the Bjerrum plots for an weak acid (benzoic acid, pKa 3.98, log So — 1.55, log mol/L [474]), a weak base (benzydamine, pKa 9.26, log So —3.83, log mol/L [472]), and an ampholyte (acyclovir, pKa 2.34 and 9.23, log So — 2.16, log mol/L I/40N ). These plots reveal the pKa and pA pp values as the pcH values at half-integral % positions. By simple inspection of the dashed curves in Fig. 6.8, the pKa values of the benzoic acid, benzydamine, and acyclovir are 4.0, 9.3, and (2.3, 9.2), respectively. The pA pp values depend on the concentrations used, as is evident in Fig. 6.8. It would not have been possible to deduce the constants by simple inspection of the titration curves (pH vs. volume of titrant, as in Fig. 6.7). The difference between pKa and pA pp can be used to determine log So, the intrinsic solubility, or log Ksp, the solubility product of the salt, as will be shown below.

As can be seen in Fig. 6.8, the presence of precipitate causes the apparent pKa, pA jjPP, to shift to higher values for acids and to lower values for bases, and in opposite but equal directions for ampholytes, just as with octanol (Chapter 4) and liposomes (Chapter 5). The intrinsic solubility can be deduced by inspection of the curves and applying the relationship [472]... 

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