Frans_M_Everaerts_Isotachophoresis_378342.pdf

Chapter I4
Separation of nucleotides in aqueous systems
SUMMARY
Experiments were carried out in order to separate nucleotides comprising the mono-,
di- and triphosphates of adenosine, cytidine, guanosine and uridine with water as solvent.
The time of analysis is approximately 30-45 min for the thermometric detector and
approximately 15 min for the high-resolution detectors, from the start of the experiment
to the detection of the last zone.
14.1. INTRODUCTION
The nucleotides are amphiprotic substances and at intermediate pH values they are
negatively charged and show a behaviour similar to that of acids. As examples, the
structures of the 5-monophosphates of the nucleotides adenosine, cytidine, guanosine
and uridine are given in Fig. 14.1. This group of substances form the basis of the nucleic
acids and play an important role in carbohydrate, lipid and vitamin metabolisms. The
adenosine and guanosine phosphates are derived from the purine bases adenine and
guanine, and the cytidine and uridine phosphates are derived from the pyrimidine bases
cytosine and uracil.
Exact data on the pK values and mobilities of these nucleotides are not known but it
would be expected that a separation according to pK values would be the most successful.
The pH of the electrolyte system regulates the extent of dissociation of the nucleotides
and is therefore an important factor affecting the effective mobilities.
In the first section some operational systems and data are given for the separation of
nucleotides, using thermometric detection, and in the second section data are given for
separations using a conductivity detector and a UV absorption detector. In this chapter,
the abbreviations A, C, G and U are used for adenosine, cytidine, guanosine and uridine,
respectively, and MP, DP and TP for mono-, di- and triphosphate, respectively.
14.2. SEPARATION USING A THERMOMETRIC DETECTOR
The experiments for the determination of the optimal pH of the operational system at
which the analyses are performed gave a series of operational systems as specified in
Tables 14.1-14.7. These systems were used only with thermometric recording of the
various zones. Later a UV absorption detector became available and this precludes the use
of strongly W-absorbing counter ions. For the experiments in which a thermometric
detector was used, the equipment described in section 7.4.2 was applied.
In Table 14.8, all of the step heights measured for the different systems are given.
They were all obtained with the same thermocouple. In Fig.14.2, the step heights for the
different systems are shown graphically.
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