Frans_M_Everaerts_Isotachophoresis_378342.pdf

18 PRINCIPLES OF ELECTROPHORETIC TECHNIQUES
by the so-called ‘stacking electrophoresis’*. The importance of this phenomenon is
clear when it is realized that, because the concentrations in the zones are constant, the
length of a zone (the distance between two differential signals) is a direct measure of
the concentration of the sample ionic species.
2.4.3. Concentration adaptation
If zones migrate, they must have a concentration that is fixed by the preceding zones
according to Ohm’s law, and we call them ‘adapted zones’. The effects on changes in
concentration during an isotachophoretic analysis are shown in Figs.2.7a-2.7f for the
separation of a mixture of anionic species A and B, introduced between the leading
electrolyte L and the terminating electrolyte T.
In Fig.2.7a, the original situation is shown. A mixture of A and B (Ao +Bo) is
introduced between the leading ions L and the terminating ions TI. Of course, the
zone A. +Bo is not adapted to L according Ohm’s law, and nor is zone TI. In Fig.2.7b,
the situation is shown after a certain time, where the leading zone L has migrated over a
certain distance, its concentration remaining constant, however. According to all movingboundary
procedures, a zone containing the anionic species A is formed and the concentration
in this zone Al is adapted to zone L. The mixed zone A+B that has passed the
original boundary is also adapted. But behind the original boundary, the originzl mixture
A. +Bo is present, still not adapted. Behind that zone A. +Bo, a zone B1 is formed
that contains only the anionic species B and this zone is adapted to the zone A. + Bo.
Also, the migrated zone Tz is adapted to zone A. +B,. In Fig.2.7c, the original mixture
A. +B, has disappeared, but now there are two zones B, one adapted to the leading
zone L(Bz) and one still adapted to the non-existing original zone A. + Bo. In Fig.2.7d,
the terminator has passed the original boundary and from this time also a zone T3 exists,
already adapted to the leading zone L. At this moment, three T zones exist, viz., a zone
T3 adapted to the leading zone L, a zone T2 adapted to the non-existing zone A. +Bo
and the original zone TI **. In Fig.2.7e, the same situationisshown, the mixed zone A + B
being much smaller. In Fig.2.7f, the mixed-zone A+B has disappeared, ie., anionic species
A and B are separated. Three T zones still exist, marking the spot where the sample was
introduced.
It is important to understand this procedure, although we shall not take this effect into
account, because it is of no importance at the position of detection, as the original
boundaries do not move and never reach the position of detection. In fact, we can never
detect these zones with electrophoretic equipment, as will be discussed later***. These
three zones do not remain sharp, because the ‘self-correcting’ effect, characteristic of
isotachophoresis, does not occur in these zones.
*It should be noted that the proteins in the ‘stack‘ can be easily denatured, because the conditions
are not ideal for proteins, as indicated in Chapter 13 where the separation of proteins is considered.
**In the separation chamber, all zones are now adapted to the composition of the leading zone,
although mixed zones are still present. If a counter flow of electrolyte (as described in section 7.5.5)
is to be applied, it should be applied at this moment, because the zone of the terminating electrolyte,
which has passed the boundary occupied originally by the sample, has already attained its isotacho-
phoretic velocity. Even if 100% counter flow of electrolyte occurs, neither the sample zone nor the
zone of the terminating electrolyte is flushed back.
***No scanning device has yet been constructed.