The Natural pH-Gradient

Svensson [24,25] regarded the natural pH gradient lEF as a true steady-state process and expected its unlimited stability. However, as noted by Svensson, a true steady state was never reached in gel lEF [27]. Instabilities of pH gradient, such as the plateau phenomenon, cathodic drift, anodic drift, or symmetric drift, were commonly observed [1,27-34]. There have been hypotheses to explain the instability of the pH gradient in lEF by isotachophoresis (ITP) mechanism [35,36] or stationary neutralization reaction boundary equilibriums (SNRBEs) [37]. Hjerten et al. [38] suggested, as he proposed the mechanism for chemical mobilization in cIEF, that pH instability is inherent in natural pH gradient lEF due to the need for electroneutral conditions. 

Natural pH-gradients are located and maintained by the electric current itself. The following is a simplified explanation of the natural pH-gradient. 

Earlier, before the modern development of the natural pH-gradient, muUichamber systems were used, as was filter paper technique. 

The natural pH-gradient is now well established and has shown most encouraging possibilities and properties. Although improvements will occur, the natural pH-gradient will probably exist principally in its present form for a long time. 

As previously mentioned, resolution in CIEF strongly depends on the ampholyte composition. The estimated maximum resolving power of IEF is 0.02 pH units when carrier ampholytes are used to create the pH gradient.99 The Law of Monotony" formulated by Svensson in 1967 states that a natural pH gradient increases continually and monotonically from the anode to the cathode that the steady state does not allow for reversal of pH at any position along the gradient and that two ampholytes (in stationary electrolysis) cannot be completely separated from each other unless the system contains a third ampholyte of intermediate pH (or pi). The latter explains why better resolution is obtained when mixing ampholytes from different vendors and production batches as the number of ampholytes species increases, the chance that one or more ampholytes have intermediate pi relative to those of the sample components also increases. 

Svensson H (1961), Isoelectric fractionation, analysis, and characterization of ampholytes in natural pH gradients, the differential equation of solute concentrations at a steady state and its solution for simple cases, Acta Chem. Scand. 15 325-341. 

Boltz et al. (1978) worked out the mathematics of a stable natural pH gradient for a simple citrate buffer system. A stable pH gradient from 3.08 to 7.21 was formed in 0.0015M citrate, that most likely did not form... 

The law of pH monotony was formulated by Svensson in 1967. It states that when a natural pH-gradient is created by an electric current, it is positive throughout. In other words, the pH increases all the way from the anode to the cathode. This law can be exemplified by considering a pair of ampholytes in the same system, e.g., aspartic acid (pi = 2.77) and glutamic acid (pi = 3.22). Both acids carry considerable proportions of electric charged species even in the neighborhood of their isoelectric points. However, they can never become completely separated by the electric current. A zone of pure water cannot arise between them. Each of them always contains a certain proportion of the other. 

The law of pH monotony can be expressed in another way An electrolytic vacuum is not permissible in natural pH-gradients. This explains why complete separation of proteins could not be achieved before introduction of suitable carrier ampholytes. 

Ampholytes (zwitterions) include an extensive range of substances that could be used as self buffers. Table 2.6 gives a list of isoionic ampholytes proposed for use as buffers in protein fractionation in a natural pH gradient (Svensson, 1962). Solutions of these substances in water have pH values close to the listed value of pi and when p7 - pA i is less than 1.5 they can be considered to be self buffers. 

Loss of nitrogen compounds from soils is also a major pathway into the atmosphere for some compounds (e.g., N2O, NO, and NH3). As in the aquatic systems, parameters that play an important role in this process include the nature of the compound soil temperature, water content, pH, aeration of the soil and a concentration gradient of the gas in question. 

Also in this case the calculated (predicted) retention values showed good agreement with the experimental results. It has been concluded that pH gradient elution may enhance the separation efficacy of RP-HPLC systems when one or more analyses contain dissociable molecular parts [81]. As numerous natural pigments and synthetic dyes contain ioniz-able groups, the calculations and theories presented in [80] and [81] and discussed above may facilitate the prediction of the effect of mobile phase pH on their retention, and consequently may promote the rapid selection of optimal chromatographic conditions for their separation. 

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