Ampholytes and zwitterions

Tab. 2.6. Measured versus literature values for pKas of ampholytic and zwitterionic compounds.

Ampholytes (amphoteric electrolytes) can function as either weak acids or weak bases in aqueous solution and have plC values corresponding to the ionisation of each group. They may be conveniently divided into two categories - ordinary ampholytes and zwitterionic ampholytes - depending on the relative acidity of the two ionisable groups. 

With respect to the properties of polar groups, surfactants can be subdivided into ionic (cation- and anion-active, ampholytic, and zwitterionic) and nonionic surfactants. If the effect produced by the polar group of the surfactant molecule is more significant than that of the lipophilic group, this substance is soluble in water. It is less surface active as compared to any substance characterized by an optimum balance between the activities of hydrophilic and lipophilic groups. Similar conclusions can be drawn also with respect to the solubility in oil here, the role of the lipophilic group is determining. Clearly, the efficiency of a surfactant is not determined solely by the amphiphilicity, but depends on the hydrophilic/lipophilic balance (HLB) characteristic for this compound. Therefore, this balance is an important characteristic of both the surfactant and the interface. 

The following sections have been rewritten activity corrections, temperature corrections, overlapping constants, polyelectrolytes, ampholytes and zwitterions, buffers and acidity functions. The tables of about 400 typical ionization constants, in C hapter 8, have been re-compiled so as to present only the most reliable of the available values for each substance listed. 

Ampholyte or zwitterions. An ampholyte is a molecule that contains both acidic and basic groups. At a particular pH, known as the isoelectric point, the charge on the groups is balanced and the molecule is neutral. If an ampholyte is placed in a pH gradient and electrophoresed it will migrate to the point at which it is uncharged and then stops moving. 

Zwitterionic surfactants have positive and negative charges on the head group. Amphoteric surfactants have a head group with a pH-dependent charge. The amine oxide shown in Fig. 3 is zwitterionic at high pH, but becomes cationic as protonation occurs at low pH. Because amphoteric surfactants are generally zwitterionic at some pH. and zwitterionic surfactants are often amphoteric, in practice, the terms zwitterionic and amphoteric are used as synonyms, and the term ampholytic is used to describe both surfactant types. 

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. 

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. 

Figure 6. Histogram comparing the isoelectric points of both ordinary and zwitterionic ampholytes. In this case the frequencies of the distributions were shown to reflect the differing number of ordinary ampholytes (44 compounds) and zwitterionic ampholytes (21 compounds). Compounds were binned into 1 log unit ranges as per Figure 3.

Niflumic acid, which has two pKa values, was studied both pH-metrically and spectroscopically using the shake-flask method [224]. The monoprotonated species can exist in two forms (1) zwitterion, XH 1 and (2) ordinary (uncharged) ampholyte, XH°. The ratio between the two forms (tautomeric ratio) was measured spectroscopically to be 17.4. On assuming that a negligible amount of zwitterion XH partitions into octanol, the calculated micro-log/1 for XH° was 5.1, quite a bit higher than the macro-log/1 3.9 determined pH-metrically in 0.15 M NaCl. It is noteworthy that the distribution coefficient D is the same regardless of whether the species are described with microconstants or macroconstants [275]. 

P. I. Nagy and K. Takacs-Novak, Theoretical and experimental studies of the zwitterions neutral form equilibrium of ampholytes in pure solvents and mixtures, J. Am. Chem. 

Figure 4. Scheme of lipophilicity profile of zwitterionic compounds. The line drawn represents the case where the neutral tautomer predominates or the zwitterion is rather hydrophobic, resulting in a bell-shaped profile. The dashed line represents the case where the zwitterion predominates and intramolecular interactions are not possible, resulting in a U-shaped profile. Adapted with permission from [133] Pagliara, A. et al. (1997). Lipophilicity profiles of ampholytes , Chem. Rev., 97, 3385-3400 copyright (1997) American Chemical Society... 

Particularly interesting examples are also the lipophilicity profiles of ampholytes. Depending on the ratio between the neutral tautomer and the zwitterionic tautomer, the log Dow versus pH profile may be bell-shaped or U-shaped [133] (Figure 4). For zwitterions, the shape of the lipophilicity profile depends upon the structure and conformation of the molecule. If the charged groups are situated in proximity and can interact with each other, the zwitterion might be more hydrophobic than the anionic and the cationic species, resulting in a bell-shaped lipophilicity profile. If, however, intramolecular interactions are not possible for steric reasons, the lipophilicity profile is U-shaped [133],... 

The pH gradient in cIEF is produced by the use of reagents, known as carrier ampholytes, that are zwitterionic and are chosen so that the... 

A chemical structure having both anionic and cationic charges on the same molecule is called a zwitterion. At a specific pH, the degree of the ionization of the zwitterion to an anionic electrolyte or to a cationic electrolyte is the same. The pH is called the isoelectric point (IEP). At the IEP, the same amount of anionic and cationic electrolytes exist. The zwitterion has the lowest solubility, denoted as SG. One can write the ionization of an ampholyte simply as ... 

Ampholyte — A substance that can react both as an acid and as abase is called an ampholyte, or amphoteric compound. Usually this property refers to the - Bronsted acid-base theory. An example is HCOj which can act as a proton acceptor and as a proton donator. An ampholyte can be a zwitterion, as in case of amino acids in the range between pH = pJCai and pH = pfCa2, they exist as [+(H3N)HRC-COO-]. 

The ideal sample run on the Rotofor cell would contain only the protein mixture, water, and ampholytes or buffers. However, pi precipitation may require that 3 M urea be included for solubility. When higher urea concentrations are needed, the Rotofor cell is run at 12°C. Detergents (1-2% w/v) may also be added to samples. Zwitterionic detergents, such as CHAPS, CHAPSO, and nonionic octyl-glucoside are satisfactory. 

Depending on their chemical structure, surfactants capable of forming micelles are usually classified into cationic e.g. ammonium salts), anionic e.g. sulfates, carboxylates), ampholytic e.g. zwitterionic salts), and non-ionic surfactants (usually containing polyoxyethene chains) cf. Table 2-10 in Section 2.5. 

The zwitterionic or ampholytic surfactants shown in Box 6.1 possess both positively and negatively charged groups and can exist as either an anionic or a cationic surfactant depending on the pH of the solution. A typical example is N-dodecyl-N,N-dimethylbetaine (Ci2H25N"(CH3)2CH2COO). 

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