Nucleosides retention time

It was found that the polymer exhibited selectivity towards phosphomonoester dianions. Less polar compounds were found to bind non-specifically to the polymer. The polymer was then used as a stationary phase for a HPLC column. A mixture containing dA, 5 -dAMP and 3, 5 -cAMP was thus separated. As expected, the retention time of 5 -AMP was larger than those for dA and 3, 5 -cAMP. The same was tme for other nucleotides compared to the corresponding nucleosides. When the Zn2+-free control polymer was used, all compounds were immediately eluted. The possibility to use polymer-anchored recognition units to separate biologically important phosphates was thus proved. 

Retention times vary for different nucleotides, but as a very rough guide, they are approximately nucleoside 5 min, monophosphate 9 min, S -bisphosphate 17 min and triphosphate 19 min. 

Set up a gradient of 0-30% of solution B over a period of 30 min, and then back down to 0% in 10 min can be used to achieve separation. The retention times of triphsophates are between 20 and 30 min but these can vary according to the nucleoside with mono- and diphosphates eluting earlier (note the order of elution on reversed-phase HPLC using the conditions above is mono-, di-and then triphosphate last). 

Set up an isocratic gradient of 15% solution B (Protocol 20). Retention times of (l-thio)triphosphates are normally between 10 and 20 min but can vary according to the nucleoside (Note the 5p diastereoisomer always elutes first). Atypical HPLC trace is shown in Figure 9.11. 

The use of the enzyme purine nucleoside phosphorjiase for enzymatic peak identification is illustrated by the RPLC serum profile from a patient with severe depression. Based upon the retention time, the peak eluting at approximately 15 min was tentatively identified as inosine (Fig. 14A). Cochromatography with an inosine reference compound resulted in a subsequent increase in peak area for the compound of interest (Fig. 14B). Fur-fiiermore, stopped-flow UV spectra indicated a similarity between the inosine standard and the peak tentatively identified as inosine (Fig. 15). 

Finally, the serum sample was incubated with the enzyme purine nucleoside phosphorylase and rechromatographed (Fig. 14C). From the disappearance of the inosine peak and the appearance of a peak with the retention time of hypoxanthine, it can be concluded that the peak under investigation was indeed inosine. 

The three HPLC modes commonly used for the separation of these compounds include ion-exchange, reversed phase and reversed phase ion-pair. The p T values of the bases and nucleosides play a major role in determining their retention times in all chromatographic modes and a list is presented in Table 11.1.1 for reference (pAT b and pifaa refer to the first gain and to the first loss of a proton). 

The classical methods for the identification of nucleoside eluent peaks is to use either retention time or spiking (co-chromatography) with reference compoimds. In addition, chemical treatment with periodate can be used to identify ribonucleosides by observing the lost peaks from a second chromatogram (Hartwick et al., 1979b). Similarly, enzymatic modification can be useful in a number of specific cases (Table 11.1.2). 

The separation of nucleosides and their corresponding bases has also been achieved on unmodified siUca. The retention times of at least fifty biologically important compounds, including nucleoside derivatives, have been reported (Ryba and Beranek, 1981). Several... 

The retention time of nucleosides decreases as a linear function of the increase in temperature in the range 25 to 55 °C. The column efficiency significantly increases with increasing temperatures. 

A number of papers on the reversed-phase h.p.l.c. analysis of nucleosides and related compounds have appeared. The analysis of both the major and modified ribonucleosides present in nucleic acids and biological fluids using photodiode array detection has been critically reviewed. Retention times under standardised chromatographic conditions and u.v.-data, along with the preparation of samples from various RNA s, are dealt with in detail. Useful increases in the retention of... 

Figure 15. Retention times of nucleoside with water mobile phase. Flow rate 1.0 mL/min. Temperature 20 C. Detector 254 nm. Samples (A), adenosine (C), cytidine (U), uridine (G), guanosine.

A number of papers on the reversed-phase h.p.l.c. analysis of nucleosides and related compounds have appeared. Correlations between structure and retention time have been drawn for a series... 

In early studies the kinetics appeared to be sequential because of the convergence of double-reciprocal plots (72, 77). It was not entirely clear at the time that the phosphatase activity was an intrinsic part of the phosphotransferase activity, and the kinetics was not pursued further. Efforts to isolate an active phosphoenzyme failed, presumably due to its hydrolysis in the absence of a nucleoside 73, 77). The stereochemical analysis showing retention of configuration at phosphorus reopened the mechanistic question and stimulated further studies that... 

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