Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Toruń, Poland.
Anal Bioanal Chem. 2013 Feb;405(5):1663-72. doi: 10.1007/s00216-012-6590-6. Epub 2012 Dec 14.
The main aim of the present investigation was to study the retention and separation of eight nucleotides with the use of seven stationary phases in RP HPLC mode. Two octadecyl columns were used; however, aminopropyl, alkylamide, cholesterol, alkyl-phosphate, and phenyl were also studied. Special attention was paid to the mobile phase buffer pH, since it appears that this factor is very influential in the case of chromatographic separation of nucleotides. The retention of nucleotides was greater for mobile phase pH 4.0 in comparison with pH 7.0 (except for octadecyl and phenyl packing). This is a consequence of protonization of polar groups present on the stationary phase surface. It was proved that aminopropyl, alkyl phosphate, alkylamide packing materials are not suitable for the resolution of nucleotides. On the other hand, cholesterol and phenyl stationary phases are alternatives for conventional octadecyl phases. Application of cholesterol packing allows separation of small, polar nucleotides, which is impossible to achieve in the case of octadecyl phase. Moreover, a phenyl support allows separation of nucleotides in a shorter time in comparison with octadecyl packing.
本研究的主要目的是研究在反相高效液相色谱（RP HPLC）模式下使用七种固定相保留和分离八种核苷酸。使用了两个十八烷基柱，但也研究了氨基丙基、烷基酰胺、胆固醇、烷基磷酸酯和苯基。特别关注流动相缓冲液 pH 值，因为在核苷酸的色谱分离中，该因素似乎非常有影响。与 pH 值 7.0 相比，核苷酸在流动相 pH 值 4.0 下的保留时间更大（除了十八烷基和苯基包装）。这是固定相表面上存在的极性基团质子化的结果。事实证明，氨基丙基、烷基磷酸酯、烷基酰胺填充材料不适合核苷酸的分辨率。另一方面，胆固醇和苯基固定相是传统十八烷基相的替代品。使用胆固醇包装可以分离小的极性核苷酸，这在十八烷基相的情况下是不可能实现的。此外，与十八烷基包装相比，苯基支撑允许在更短的时间内分离核苷酸。
