Frans_M_Everaerts_Isotachophoresis_378342.pdf

322 AMINO ACIDS, PEPTIDES AND PROTEINS
TABLE 13.8
STEP HEIGHTS (mm) FOUND IN THE LINEAR TRACE OF THE CONDUCTIVITY DETECTOR
SIGNAL IN THE ISOTACHOPHEROGRAMS OBTAINED WITH THE OPERATIONAL SYSTEM
LISTED IN TABLE 13.7
Amino PH
acid
L-cys
L-His
L-Ser
L-Thr
L-Glu
L-Asp
L-Asn
L-Met
L-Ile
Gly
L-Val
L-Trp
L-Tyr
I, -L-T~I
L-Phe
DL-Ala
7.30 7.80
55
67.5
46
43
29.5
24.5
43.5
115
175
170
168
147
135
51
128
190
29.0
60
41.5
39
13.5
13
30
84
128
108
32
103
175
mixed zones has been studied. Pairs of amino acids with comparable effective mobilities,
determined experimentally, were injected simultaneously into the system at pH 7.2. The
results are given in Fig.13.5. This figure, amongst others, shows that histidine can be
separated completely from the other amino acids, which was not possible in the aqueous
systems considered.
The disadvantages of using propionaldehyde are its instability, the extra work involved
in preparing the operational system and its relatively low boiling point (48 C). Because
of the last property, one can work only with relatively mobile components or at low
current densities.
In Fig.13.6, an isotachopherogram is shown of some amino acids obtained in the
operational system specified in Table 13.7. The UV trace is of lower quality than those
with comparable systems in aqueous solutions because propionaldehyde shows a
significant absorption at 256 nm.
13.2. SEPARATION OF PROTEINS IN AMPHOLYTE GRADIENTS
13.2.1. Introduction
While small peptides can usually easily be analyzed by isotachophoresis, the analysis
of proteins is often troublesome. If the proteins are not denatured, they are stacked in
small zones that have a high density, which is not ideal for isotachophoretic separations.