Ampholytes conductance

An example of isoelectric focusing is shown at the left below. A mixture of seven proteins (and some impurities) was applied to a polyacrylamide gel containing a mixture of polyprotic compounds called ampholytes. Each end of the gel was in contact with a conducting solution and several hundred volts were applied across the length of the gel. The ampholytes migrated until they... 

IEF buffer pairs are intended for use as a final purification step when it is desirable to obtain protein preparations that are demonstrably free of carrier ampholytes, such as for use as pharmaceuticals or in sensitive bioassays. With the buffer pairs, it is not necessary to carry out lengthy dialysis or other procedures for ampholyte removal. The correct choice of buffer pair is that combination for which the pi of the protein of interest falls in the middle of the pH gradient (which, of course, means that the pi must be determined beforehand). The desired proportion of the buffers (Table 2), at 100 mM final total concentration, is mixed with the sample solution prior to being placed the separation chamber and the IEF run is conducted according to the instructions of the manufacturer of the chamber. 

ZIL monomers, having both cation and anion in the same monomer unit, were prepared in order to give different types of ampholyte polymers. Figure 30.4 shows the structure of the monomers used here [6]. Four different monomers were synthesized to study the relation between structure and such properties as ionic conductivity and Tg. 

Ampholyte-type polymers should become attractive materials once successive and effective ion conductive paths are successfiiUy designed in the matrix. 

Isoelectric Points.—An ampholyte is at its isoelectric point when the concentration of positive ions is equal to that of the negative ions, i.e., when CrhJ is equal to crh . Since these ions have almost the same equivalent conductances, because of their size, equal amounts of positive and negative ions of the ampholyte will migrate in opposite directions. At the isoelectric point, therefore, an amino-acid, or more complex ampholyte, will appear to remain stationary in an electrical field, although the solution may have an appreciable conductance. According to equation (1),... 

The run was made at constant potential, and the current intensity was recorded. After passing through a maximum after 30 minutes of focusing, it decreased to a stable value of 0.6 mA by the end of the experiments. In usual ampholytes, this variation of conductance is an... 

The first studies of carrier ampholytes were conducted using amino acids and dipeptides, but these species did not work well because their pK values for the amino and carboxylate groups are too far removed from their pi values. After they were prefocused, these species had very low buffering capacity. Good... 

Carrier ampholyte-based IEF methods are commonly used in situations where very high resolution of proteins according to their pi values is not required. Several problems exist with the use of carrier ampholytes that limit their resolving power. These include the low and uneven ionic strength that results in smearing of the most abundant proteins in the sample, the uneven buffering capacity and conductivity, the unknown chemical environment, a low sample loading capacity, and a... 

For isoelectric focusing (IFF) (see later section), a power supply that provides constant power is advisable. During electrophoresis, current drops significantly because of lower conductivity as carrier ampholytes focus at their isoelectric points and because of creation of zones of pure water. If a constant-voltage supply is used, frequent voltage adjustments may be necessary. Constant-current power supplies are not customarily used in lEF. Pulsed-power or pulsed-field techniques (see later section) require a power supply that can periodically change the orientation of the applied field relative to the direction of migration. 

As the ampholytes and the sample molecules become neutral, they do not conduct current. 

A problem that manifests itself with the use of certain ampholyte ranges and high field strengths is the appearance of "hot spots" (42). These "hot spots" are conductivity gaps in the gradient and serve as a limiting factor for the field strength that may be applied to the gel. 

For good separation results, a stable pH gradient with constant conductivity is required. This can be achieved by carrier ampholytes or immobilised pH gradients. 

Fig. 3.10. A pH gradient with increasing pH over the gel length formed by a mixture of hundreds of ampholytes each with a different pi. The concentration of each ampholyte is the same to ensure a homogeneous conductivity.

All the isoelectric fractionations previously described took place in water. Thus they all contain the ampholyte water, which is isoelectric at pH 7. An ampholyte with pi less than 7 can thus be completely separated from another with pi larger than 7. A zone of pure water will be created between them. But if a pair of ampholytes both have a pi lying on the same side of pH 7, that is, both are less than 7, or both exceed 7, they can never be separated unless other ampholytes are present. Water as an ampholyte furthermore has the drawback of having an extremely low conductivity at its isoelectric point pH 7. 

Since pi is always larger than pK+, the second term in the denominator will always be larger than unity. This means that the upper limit for a is equal to Obviously a becomes larger as the difference between pK+ and the isoionic point diminishes. The conductivity of an ampholyte is directly dependent of the extent of its ionization. This means that the condition for high conductivity at the isoionic and isoelectric point is that the pair of pK values lying on either side of the pi and nearest to it are close together. 

Svensson (2) has calculated the conductivities of several ampholytes of low molecular weight. We refer the reader to his works. For example, histidine has pi = 7.81, pi — pK+ = 1.50, and conductivity 3C = 158 mhos cm 10 . At this pH, the contribution from the conductivity of the water is zero. Another example is lysine, with pi = 9.96, pi — pK+ = 0.79 and 3C(corr) = 2.162 mhos cm 10 . Thus lysine has a considerably larger conductivity at its isoelectric point than histidine. This results... 

Figure 4. Titration curves of glutamic acid, glycine, histidine, and lysine. The three amino acids whose titration curves sharply cut the zero-charge-horizontal-line also have a net charge apart from zero in the vicinity of the pH where they cut the zero line, i.e., the pi. That means that they have buffering capacity and conductance in the neighborhood of the isoelectric point and therefore are useful as carrier ampholytes. Glycine, on the other hand, with its extended horizontal part of the curve is not suitable. (Svensson, 2.)...

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