The influence of experimental conditions on chromatographic behaviour of promising oligodeoxynucleotide double‐labelled molecular probes containing an azaphthalocyanine macrocycle as a perspective dark quencher was studied. A recently introduced new stationary phase based on styrene‐divinylbenzene copolymer was tested. The planar and hydrophobic structure of the azaphthalocyanine is considerably different from those of currently used fluorophores and quenchers. Thus, the most challenging issue was the separation of the double‐labelled probe from its main impurity represented by a mono‐labelled probe, containing only the azaphthalocyanine macrocycle. The absorbance measurement cannot simply determine this impurity, and its presence fundamentally compromises the biological assay. The commonly used gradient elution was not suitable and isocratic conditions seemed to be more appropriate. The azaphthalocyanine moiety influences the properties of the modified oligodeoxynucleotides substantially, and thus their chromatographic behaviour was determined predominantly by this quencher. Acetonitrile was the preferred organic solvent for the analysis of probes containing the azaphthalocyanine quencher and the effect of ion‐pairing reagents was dependent on the probe structure. The temperature seemed to be an effective parameter for fine‐tuning of the separation and mass transfer improvement. Generally, our findings could be helpful in method development for purity evaluation of double‐labelled oligodeoxynucleotide probes and semipreparative methods.

中文翻译：

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含氮酞菁染料作为猝灭剂的新型双标记寡脱氧核苷酸探针的色谱行为，以评估其纯度

研究了实验条件对有前景的以氮杂酞菁大环作为暗猝灭剂的寡脱氧核苷酸双标记分子探针色谱行为的影响。测试了最近推出的基于苯乙烯-二乙烯基苯共聚物的新型固定相。氮杂酞菁的平面和疏水结构与目前使用的荧光团和淬灭剂有很大不同。因此，最具挑战性的问题是将双标记探针与其主要杂质分离，该杂质由仅含氮酞菁大环的单标记探针代表。吸光度测量不能简单地确定这种杂质，并且它的存在从根本上损害了生物测定。常用的梯度洗脱不合适，等度条件似乎更合适。氮杂酞菁部分基本上影响修饰的寡脱氧核苷酸的性质，因此，其色谱行为主要由该猝灭剂确定。乙腈是分析含有氮酞菁猝灭剂的探针的首选有机溶剂，离子对试剂的作用取决于探针的结构。温度似乎是微调分离和改善传质的有效参数。通常，我们的发现可能有助于双标记寡聚脱氧核苷酸探针和半制备方法纯度评估的方法开发。氮杂酞菁部分基本上影响修饰的寡脱氧核苷酸的性质，因此，其色谱行为主要由该猝灭剂确定。乙腈是分析含有氮酞菁猝灭剂的探针的首选有机溶剂，离子对试剂的作用取决于探针的结构。温度似乎是微调分离和改善传质的有效参数。通常，我们的发现可能有助于双标记寡聚脱氧核苷酸探针和半制备方法纯度评估的方法开发。氮杂酞菁部分基本上影响修饰的寡脱氧核苷酸的性质，因此，其色谱行为主要由该猝灭剂确定。乙腈是分析含有氮酞菁猝灭剂的探针的首选有机溶剂，离子对试剂的作用取决于探针的结构。温度似乎是微调分离和改善传质的有效参数。通常，我们的发现可能有助于双标记寡聚脱氧核苷酸探针和半制备方法纯度评估的方法开发。乙腈是分析含有氮酞菁猝灭剂的探针的首选有机溶剂，离子对试剂的作用取决于探针的结构。温度似乎是微调分离和改善传质的有效参数。通常，我们的发现可能有助于双标记寡聚脱氧核苷酸探针和半制备方法纯度评估的方法开发。乙腈是分析含有氮酞菁猝灭剂的探针的首选有机溶剂，离子对试剂的作用取决于探针的结构。温度似乎是微调分离和改善传质的有效参数。通常，我们的发现可能有助于双标记寡聚脱氧核苷酸探针和半制备方法纯度评估的方法开发。