Extended pH Gradients

For wider pH intervals, several buffering species have to be mixed and the situation becomes considerably more complex. This has been solved with the aid of computer programs designed specifically for this purpose. The basic findings are: first for generating a linear pH gradient the buffering power has to be

Figure 5 Effect of changes in the number of (evenly spaced) buffering components. The optimal concentrations of fictitious buffers (bases) with pKs differing by 1, 1.25, 1.66 and 2.5 pH units, were calculated so as to cover the pH 4.5-8.5 range. The resulting courses of ß powerare shown as a function of ApK The insert is a plot of percentage variation, in comparison with the case ApK= 1, of the ranges of deviation of pH (left scale) and of ß (right scale). Note that the smoothest ß power is obtained with ApK= 1 (from Gianazza etal., 1983, with permission of Elsevier Science Publishers).

Figure 6 Effect of changes in the pKof the acidic titrant. A reference Immobiline mixture was titrated to the same pH value with fictitious acids whose pKwas 0.5, 1.0, 1.5, 2.0 and 2.5 pH unit lower than the gradient's limit (in this case, pHmin = 3.5) and the pH course was calculated for the five cases. The insert is a plot of the percentage variation of deviation from linearity as the titrant's pK increases (from Gianazza etal., 1983, with permission of Elsevier Science Publishers).

Figure 5 Effect of changes in the number of (evenly spaced) buffering components. The optimal concentrations of fictitious buffers (bases) with pKs differing by 1, 1.25, 1.66 and 2.5 pH units, were calculated so as to cover the pH 4.5-8.5 range. The resulting courses of ß powerare shown as a function of ApK The insert is a plot of percentage variation, in comparison with the case ApK= 1, of the ranges of deviation of pH (left scale) and of ß (right scale). Note that the smoothest ß power is obtained with ApK= 1 (from Gianazza etal., 1983, with permission of Elsevier Science Publishers).

Figure 6 Effect of changes in the pKof the acidic titrant. A reference Immobiline mixture was titrated to the same pH value with fictitious acids whose pKwas 0.5, 1.0, 1.5, 2.0 and 2.5 pH unit lower than the gradient's limit (in this case, pHmin = 3.5) and the pH course was calculated for the five cases. The insert is a plot of the percentage variation of deviation from linearity as the titrant's pK increases (from Gianazza etal., 1983, with permission of Elsevier Science Publishers).

constant throughout the desired pH interval (this is best achieved when the pK values are spaced at 1 pH unit intervals, see Figure 5). Secondly, to avoid deviations from linearity, the titrants should have pKs well outside pHmin and pHmax of the wanted pH range (in general, at least 2 pH units removed from the limits of the pH interval) (see Figure 6). As a consequence of this, for pH ranges wider that 3 pH units, two additional Immobilines are needed as titrants: one strongly acidic (pK < 1) and one strongly basic (pK > 12). There are two ways of generating extended pH intervals. In one approach the concentration of each buffer is kept constant throughout the span of the pH gradient and 'holes' of buffering power are filled by increasing the amounts of the buffering species bordering the largest ApKs; in the

Gel length

Figure 7 Non-linear pH 4-10 gradient. Ideal (dotted line) and actual (solid line) formulation courses. The shape for the ideal profile was computed from data on the statistical distribution of proteins p/s. The relevant histogram is redrawn in the figure inset (from Gianazza et al., 1985; by permission of VCH).

Gel length

Figure 7 Non-linear pH 4-10 gradient. Ideal (dotted line) and actual (solid line) formulation courses. The shape for the ideal profile was computed from data on the statistical distribution of proteins p/s. The relevant histogram is redrawn in the figure inset (from Gianazza et al., 1985; by permission of VCH).

other approach (varying buffer concentration) the variation in concentration of the various buffers along the width of the desired pH gradient results in a shift of their apparent pKs with a concomitant evening-out of the ApK values. With the available recipes, preparation of any Immobiline gel is now a trouble-free operation, as all the complex computing routines have already been performed and no further calculations of any type are required.

Solar Panel Basics

Solar Panel Basics

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