Frans_M_Everaerts_Isotachophoresis_378342.pdf

316 AMINO ACIDS, PEPTIDES AND PROTEINS
TABLE 13.4
STEP HEIGHTS (mm) FOUND IN THE LINEAR TRACE OF THE CONDUCTIVITY DETECTOR
SIGNAL IN THE ISOTACHOPHEROGRAMS OBTAINED WITH THE OPERATIONAL SYSTEM
LISTED IN TABLE 13.2.
Amino PH
acid
L-Asp
L-cys
L-Glu
I, -L-Tyr
L-Ser
L-Thr
DL-Tyr
DL-Met
Gly
L-His
L-Phe
L-Ma
L-Val
L-Trp
3-L-Hyp
L-Ile
L-Leu
P-Ala
9.00 9.20 9.36 9.55
40.5
51.5
49.5
76
112.5
113
135
133
138.5
145
147.5
180
184
191
185
203.5
205
239
33
39.5
40
63
88
98.5
114.5
116
119
124
127.5
154
159
161.5
162
172.5
172.5
210
30.5
35.5
34
59
84
90
107
112.5
106
118
121
147
151
157
151
162.5
164.5
20 1
38.5
34.5
35.5
58
85
94.5
105
117.5
110.5
123
126.5
149
155
161
154
167
170
190
The results obtained when using the operational systems specified in Tables 13.2
and 13.3 are given in Tables 13.4 and 13.5.
Differences in step heights of about 15-20 mm are sufficient for a complete separation
of the various amino acids. The differences found in the two operational systems
considered must be ascribed mainly to the difference in effective mobility of the counter
ion used. While in the operational system specified in Table 13.2 about eight amino
acids can be separated in a single run, in that specified in Table 13.3 about ten amino
acids can be separated simultaneously. L-Lysine has a very small effective mobility at the
pH of the leading electrolyte chosen, while the effective mobility of ethanolamine is
considerably greater.
The idea that pure water, adjusted to a high pH by adding barium hydroxide, can be
used as an optimal terminating electrolyte in operational systems at high pH is nearly
always wrong. If double-distilled water adjusted to a high pH is applied as the terminating
electrolyte*, one can expect the buffer capacity of the counter ion to be insufficient. If,
instead, a suitable terminator, e.g., p-alanine, is added to the water, also adjusted to a high
pH, the pH of the zone of the terminating electrolyte does not need to be increased so
*OH- may carry the electricity because the pH in the zone is increased sufficiently as water is a weak
acid in this electrolytic system. This, in combination with the high absolute mobility, will give OH-
a sufficient high effective mobility.