Ampholyte and

Isoelectric focusing takes along (from ca 3 to 30 h) time to complete because sample compounds move more and more slowly as they approach the pH in the gel that corresponds to their isoelectric points. Because the gradient ampholytes and the samples stop where they have no mobiHty, the resistivity of the system increases dramatically toward the end of the experiment, and the current decreases dramatically. For this reason, isoelectric focusing is usually mn with constant voltage. Constant current appHcation can lead to overheating of the system. 

Scherrer, R. A. Biolipid pK values and the lipophilidty of ampholytes and ion pairs. In Pharmacokinetic Optimization in Drug Research Biological, Physicochemical, and Computational Strategies, Testa, B., Van de Waterbeemd, H., Folkers, G., Guy, R. (eds.), Wiley-VCH, Weinheim, 2001, pp. 351-381. 

Figure 4.8 Octanol-water Bjerrum plots for a diprotic (a) acid, (b) ampholyte, and (c) base. The volumes of octanol and water are equal, so that the difference between the apparent pKa and the true )Ka is about equal to the partition coefficient. [Avdeef, A., Curr. Topics Med. Chem., 1, 277-351 (2001). Reproduced with permission from Bentham Science Publishers, Ltd.]...

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

In 1952 Carsten (Cl) developed a method, which allowed him to isolate and characterize several lower peptides contained in normal and pathological urine. According to this procedure, urine was desalted on the Amberlite IR-100 column and the adsorbed substances washed out with 2 M ammonia solution. The eluate was then passed through the column of Amberlite IRA-400. This column retained the ampholytes and rejected the weak bases. The former were recovered by elution with 1 M hydrochloric acid and the eluate was subsequently fractionated on Dowex 50 resin with 2M and later 4M hydrochloric acid as the eluents. By applying two-dimensional paper chromatography to further analysis of... 

IPGs into mirrors. An easy way to stain IPGs is to immerse them for 1 h in colloidal CBB G-250 followed by two 10-min water washes. There is also a version of SYPRO Ruby stain specifically formulated for use with both carrier ampholyte and IPG-IEF gels. 

IEF is similar in concept to conventional gel IEF a stable pH gradient is formed in the capillary using carrier ampholytes, and proteins are focused in the gradient at their pis. The major difference in performing IEF in the capillary format rather than slab gel is the requirement for mobilizing focused protein zones past the detection point. IEF is described in Capillary Isoelectronic Focusing. ... 

The use of urea must be approached with caution, because urea solutions often contain ammonium cyanate, the concentration of which increases with temperature and pH. This contaminant can react with the amino group of lysines and the amino terminus of the polypeptide chain, thus leading to artifact peaks. This effect is minimized by the presence of ampholytes, whose primary amines are cyanate scavengers, and by deionizing the urea solution with a mixed-bed resin prior to adding the ampholytes and detergent. 

When particles or large molecules make contact with water or an aqueous solution, the polarity of the solvent promotes the formation of an electrically charged interface. The accumulation of charge can result from at least three mechanisms (a) ionization of acid and/or base groups on the particle s surface (b) the adsorption of anions, cations, ampholytes, and/or protons and (c) dissolution of ion-pairs that are discrete subunits of the crystalline particle, such as calcium-oxalate and calcium-phosphate complexes that are building blocks of kidney stone and bone crystal, respectively. The electric charging of the surface also influences how other solutes, ions, and water molecules are attracted to that surface. These interactions and the random thermal motion of ionic and polar solvent molecules establishes a diffuse part of what is termed the electric double layer, with the surface being the other part of this double layer. 

The status of the isoelectric focusing process can be followed by the current reading. When steady state is reached where no sample migration occurs anymore, the current drops to zero. After focusing, the ampholytes and solutes are mobilized again in order to pass the detector. Mobilization can be accomplished by replacing one of the solutions in the reservoirs at the capillary end with a salt (e.g., sodium chloride), or the volume in the capillary is pushed out by applying pressure. 

Surfactants in broad use may be classified into three general types (I) anionics, in which the hydrophilic portion of the molecule carries a negative charge (2) cationics. in which the charge of this portion is positive and (3) nonionics, which do not dissociate but commonly derive their hydrophilic portion from polyhydroxy or polyelhoxy structures. Ampholytic and it itlerionic surfactants are also known and arc starting to be of commercial importance. 

In the most typical format, cIEF separations are performed in the absence of EOF in three distinct steps the sample is introduced into the capillary, the analytes are focused or separated into distinct zones, and, finally, the zones are mobilized so that they pass by the detector. If the EOF is carefully controlled, it is also possible to perform cIEF separations in the presence of low EOF, in which case the mobilization step becomes unnecessary. To load the analytes, the desalted sample is usually mixed with a 1-2% (w/v) solution of the carrier ampholytes and the entire capillary is filled with the mixture. The procedure, however, requires the use of a relatively large amount of sample. By pretreating the capillary to eliminate the EOF (Section 4.3.3), it is possible to introduce smaller volumes of sample into the capillary.4344... 

The same approach can be employed in the solution of ampholytes, and one can derive a similar solution to Equation (2.112). In this case, the ampholyte is represented by NHjRCOCT (HY=). The dissociation occurs as follows ... 

All three methods for generating pH gradients depend on electrolytic, or protolytic, mechanisms to form pH gradients. The pH gradients obtained with carrier ampholytes and the buffer pairs are linear, but IPGs can be constructed that are nonlinear.2... 

The first practical IEF experiments were carried out with the use of synthetic molecules, called carrier ampholytes, to generate the pH gradients.1,26 Carrier ampholytes are amphoteric electrolytes that carry both current and buffering capacity. Much of the early theoretical activity in electrofocusing dealt with the properties required of carrier ampholytes and is more or less irrelevant to a current discussion.1,3,9 Different varieties of... 

Electroosmotic flow can be reduced and even eliminated through coating of the internal surface of the capillaries. Capillaries coated with, e.g., methylcellulose or non-cross-linked acrylamide have negligible EOF [18]. As a result, the focusing of substances will depend principally on the quality of the ampholytes and... 

The metal complexation of ampholytes may cause unwanted results when iron-containing tools are used for handling the sample (e.g., syringes with metal needles, metal tubing, etc.). This was demonstrated with transferrin, a metalbinding protein, when iron-complexed forms of transferrin appeared in the mobilization pattern of an iron-free sample upon mixing the ampholytes and the protein solution with a Hamilton syringe equipped with metal needle [36]. 

Hydrated oxides with clearly exhibited basic and acidic properties are well soluble in water. They are prepared by the interaction of the corresponding oxides or metals with water. Weak bases, ampholytes and weak acids are practically insoluble or poorly soluble. Exchange reactions between the solutions of the corresponding salts and strong bases are used to synthesize them. 

Since temperature has a marked effect on the pK of the carrier ampholytes and immobilines, and on the pi values of proteins, IEF gel runs should be thermostatted to achieve reproducible results. Also, for rapid IEF separations, it is necessary to use higher voltages. This produces a lot of heat which is best removed by running the gel on a cooled plate (10 °C) it is also desirable to even out thermal fluctuations by using stabilised power packs such as those used for DNA sequencing. 

In the case of non-electrolytes, such as urea, mannitol, sucrose, glycine and other amino-acids (ampholytes), and morpholine, the apparent molal heat content is a linear function of the molality m and not of or is a more complicated function, quadratic or cubic, of m. 

In our experiments the commercial type of ion exchangers CB-4 (carboxylic), VPC (vinylpyridine carboxylic), ANKB (ampholyte), and AN-13 (ampholyte) were used. 

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