Conductivity, buffering capacity, concentration and pH profiles of carrier ampholytes focused in narrow-range immobilized pH gradients

It is possible to measure pH values in ultra-narrow immobilized pH gradients (IPGs) when the polyacrylamide matrix contains a secondary, carrier ampholyte generated pH gradient. After an IPG run, the buffering ampholytes, contained in 5 mm gel segments, along the focusing axis, are eluted and the following measurements are made: pH, conductivity, buffering capacity and concentration profiles of carrier ampholytes. Local concentration maxima of buffering ampholytes correspond to maximum conductivity and buffering capacity. The same relationship applies to local minima of the same parameters. No single commercial carrier ampholyte mixture ensures smooth conductivity and buffering capacity profiles and even pH gradients over very narrow pH intervals (0.3 pH unit spans). The best profiles are obtained only with mixtures of different commercial carrier ampholytes. Across neutrality, ca. 10% total buffering ampholytes should be added to an ultra-narrow IPG gradient to ensure an average buffering power (β_av.) of ca. 3 mequiv./1 · pH and an average conductivity (λ_av.) of 20-30 μS, while in acidic (around pH 4 and below) and alkaline (above pH 9) pH intervals only one third of this concentration is needed, as the carrier ampholytes exhibit substantially higher λ and β values and the hydrolytic products of water begin to contribute to the buffering capacity and conductivity of the system.