Frans_M_Everaerts_Isotachophoresis_378342.pdf

Chapter 13
Amino acids, peptides and proteins
SUMMARY
The separation of amino acids in aqueous solutions at low pH, at high pH and at
‘neutral’ pH when propionaldehyde is added to the electrolytes is discussed. Experimental
data for the amino acids in several operational systems are given. The separation of
proteins in an operational system at neutral pH is discussed. The addition of a mixture of
amphiprotic substances, by which the proteins are diluted in their zone, stabilizes
proteins of high moleculer weight, although this technique deviates from the originiil
principle of isotachophoresis as discussed in Chapter 4. For small peptides, the addition
of amphiprotic substances is not necessary.
The time of analysis is approximately 15 min from the start of the experiment to the
detection of the last zone.
13.1. AMINO ACIDS
13.1.1. Introduction
The analysis of amino acids is extremely important and nearly all separation techniques
have been applied to them. Good results have been obtained by various research workers
who analyzed these substances by liquid chromatography [ 1,2] , gas chromatography and
electrophoresis. Many references can easily be found and they are not cited here because
only an incomplete list could be given. So far, little attention has been paid to the
separation of amino acids by isotachophoresis [3-51.
In this section, we discuss various systems in which amino acids can be analyzed by
isotachophoresis. The application of ths technique to amino acids is particularly
interesting because in theory they can be separated both as cations and as anions. The
possibility of achieving a complete separation according to pK values (Chapter 5) is, of
course, considered first. It is clear that no systems at a neutral pH can be chosen,
because most amino acids have their pZ values at neutral pH and hence will have a
negligible migration in an electric field. It is also well known that amino acids form
stable complexes, e.g, with metals and aldehydes. If such a complex is formed, not only
the molecular size and solvation change, but also the pK values, and the effective mobility
therefore changes in an operational system chosen.
Several operational systems are considered below in order to show complex formation
and the variations in the effective mobilities. Much more research, however, needs to be
carried out. In particular, solvents or a combination of solvents in which the amino acids
are more soluble than they are in aqueous systems must be sought. Unusual combinations
of systems may be obtained e.g., a combination of urea and water to increase the
solubility of the amino acids, to which an aldehyde must be added to decrease the pl
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