Frans_M_Everaerts_Isotachophoresis_378342.pdf

SEPARATION OF PROTEINS IN AMPHOLYTE GRADIENTS 325
I I I
Fig.13.7. [llustration of carrier and spacer functions. If an ionic species S is added to a sample
consisting of ions A and B such that mA > ms > mg, and the differences in mobility are sufficient
for a complete separation, S acts as a ‘Spacer’ for ions A and B. If a component C is added to a
sample consisting of ions A and B such that the effective mobility of C is equal to the effective
mobility of B in the operational system chosen, component C acts as a ‘carrier’ for ion B. In specific
instances it is possible for a component to be added such that a mixed zone is formed between the
ions A, B and the component added, although generally an enrichment of A in front and an
enrichment of B at the rear can be expected.
question. One has to bear in mind that although the differences in effective mobility
between the compounds of interest remain constant, the separation capacity decreases
because compounds are added that have effective mobilities between those of the
compounds of interest. The final result of the detection of the various zones, which really
move with equal speed, will be less sharp than under ideal isotachophoretic conditions
because the self-sharpening effect is much lower.
Apart from the addition of, e.g., ampholytes to the leading electrolyte, the terminating
electrolyte can also be doped with a suitable ion with an effective mobility higher than
that of the most mobile protein. However, in some instances the elution effect due to the
substance added may play a dominant role (i.e., isotachophoresis will gradually become
zone electrophoresis). Experiments along these lines will not be discussed in this book,
because they lie far outside its scope.
13.2.2. Experimental
All experiments described in this section were performed in the operational system
specified in Table 13.9. Various operational systems can be used, depending mainly on the
particular proteins to be separated.
Only a general discussion is presented here but we hope it will be sufficient for
scientists interested in the separation of proteins.
Glutamic acid was chosen as the leading ion because it is commercially available in a
very pure form (‘isotachophoretically pure’) and its mobility is sufficiently high in
comparison with that of the most mobile protein at a pH of the leading electrolyte of 7.2.
As already indicated in the analyses discussed in section 13.1, the influence of carbonate
(hydrogen carbonate) on both the qualitative and quantitative results are negligible,