Frans_M_Everaerts_Isotachophoresis_378342.pdf

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AMINO ACIDS, PEPTIDES AND PROTEINS
Fig.13.6. Isotachopherogram of the separation of a mixture of amino acids obtained in the operational
system listed in Table 13.7. 1 = Chloride; 2 = propionate; 3 = Asp; 4 = I,-Tyr; 5 = His; 6 = Met;
7 = Tyr; 8 = Val; 9 = Ala. All are L-amino acids. A = Increasing UV absorption; R = increasing resistance;
t = time.
these samples are analyzed, one can say that they are being analyzed in isotachophoretic
operational systems, but owing to the presence of the additives isotachophoresis as
strictly defined (see Chapters 2 and 4) does not take place. The proteins are much more
stable if these additives are included, because not only are they present as a single substance
with the counter ion in a specific zone (as is common in isotachophoretic analyses),
but also the density is much lower. Hence the electric current can pass much more easily
and excessive temperatures do not occur. These two effects were verified experimentally.
Ampholines (LKB, Bromma, Sweden) so far seem to be compounds that can be applied
both for the dilution of the various zones (carrier function) and €or spacing the various
zones (spacer function), because they consist of numerous amphiprotic compounds. The
ampholines are mixtures of polyamino polycarboxylic acids of general structure
- CH2 -N-( C Hz )x - N-( CH2 )x-NR2
I I
where x = 2 or 3 and R = H or -CHz -CHz -COOH. These compounds are commonly
applied in isoelectric focusing experiments in order to create a stable pH gradient.
In Fig. 13.7, the space function and the carrier function are shown schematically.
As already said, the ampholyte mixtures will give both characteristics to the separation in