Frans_M_Everaerts_Isotachophoresis_378342.pdf

170 DETECTION SYSTEMS
to pH 4.2. The electric current was stabilised at 70 PA and W detection was carried out
at a wavelength of 256 nm. The drop in the height of the UV trace is clearly visible. The
trace from the linear conductivity detector does not resolve these two acids in the mixture.
The step height in the UV trace for salicylic acid can be plotted against the concentra-
tion ratio of phosphate to salicylic acid, as shown in Fig.6.33’. A 50-fold dilution of
salicylic acid could easily be determined, which means that the resolution improves by a
factor of 50. The step height in the W trace may be influenced by the following factors:
the pH of the ‘mixed zone’ may vary as a function of the composition, which may
influence the step height if there is a large difference between the acidic and basic forms
of the molar absorptivity; and the molar extinction coefficient of the W-absorbing
component. It does not need to be explained that a larger improvement in the resolution
may be expected if the molar absorptivity increases. Moreover, the use of a En-Log
converter will further improve this method.
Anticipating on analyses discussed later, Fig.6.34 demonstrates that salicylic acid and
phosphate can be separated at another pH, the so-called separation according to pK values.
Thee isotachopherograms are shown, demonstrating the separation of phosphate and
salicylic acid (1 : 1) at pH values of 3.2,4.0 and 7.0. It can clearly be seen that these two
acids can be separated at pH values both below and above 4.0. At a high pH, the
influence of carbonate can be seen, because no precautions were taken.
w-
4 3 2
I-
7L
i l 1
I
~
a -
-7
2+3
L;@
, - 1
-L
Fig.6.34. Isotachopherograms of the separation of phosphate and salicylic acid, demonstrating that
the separation is possible at both a ‘high‘ pH (7) and a lower pH (3). Because no precautions were
taken during the preparation of the operational system at pH 7, the influence of carbonate can be
seen. This aspect is considered further in Chapter 12. (a) Separation at pH 3; (b) separation at pH 4.2:
(c) separation at pH 7. Glutamate was used as terminator. 1 = Chloride; 2 = phosphate; 3 = salicylate;
4 = glutamate. A = Increasing UV absorption; R = increasing electric resistance; t = time.
*The signal-to-noise ratio of the W detector is such that an amplification of at least 1000-fold is
possible.
R