| Fluorescent dye-labeled DNA fragments, including fluorescent labeled oligonucleotides and labeled long double-strand DNA (dsDNA), play an important role in many cutting-edge biological technologies. The analytical method of fluorescent labeled DNA fragments is established by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) and HPLC coupled to negative-ion electrospray ionization mass spectrometry (ESI-MS) in this work. The results show that the best resolution of different length fluorescent labeled oligonucleotides is observed under the HPLC condition of 0.01 mol/L triethylamine-acetic acid (TEAA), pH 7.0, while the optimal condition for labeled long dsDNA is 0.05-0.10 mol/L TEAA. Another study shows that the retention behavior of fluorescent labeled oligonucleotides is significantly different from that of unlabeled isomers in terms of the retention order. The retention time of labeled oligonucleotides is reduced with the increase of the length of the oligonucleotides.The low concentration of TEAA as mobile phase (0.01mol/L) enables this IP-RP-HPLC method to be used in ESI-MS. Thus, it is available for the fluorescent labeled oligonucleotides to be detected easily by LC-MS. In order to obtain the high sensitivity, the parameters of LC-ESI-MS are improved. Moreover, this approach is able to avoid the impact of Na+ and K+ on the detection of molecular weight of oligonucleotides effectively.The fluorescent properties of fluorescent labeled DNA fragments, which are purified by IP-RP-HPLC method, are investigated preliminarily. Interating sphere technique is used to determine fluorescence quantum yields, emission spectra and excitation spectra. The results indicate that the effect of different length of labeled oligonuleotides on fluorescent properties must not be overlooked. It is very useful for the accurate quantitative analysis of fluorescent labeled DNA fragments.
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