Abstract
There is a growing demand for the separation and identification of short nucleic acid fragments, such as oligonucleotides. There were two main goals of the present investigation, namely, evaluation of the impact of stationary phase type and the influence of various ion-pair reagents on the retention behavior of oligonucleotides in ion-pair liquid chromatography. Three types of ion-pair reagents were studied: triethylammonium acetate, dimethylbuthylammonium acetate and mixtures of 1,1,1,3,3,3-hexafluoro-2-propanol and triethylamine. Two novel types of packing materials, namely, cholesterol and alkylamide were used for this purpose for the first time. The results indicate that the mechanism of oligonucleotides retention is determined by the hydrophobicity of ion-pair reagents and polar ligands localized on the surface of stationary phases. Oligonucleotides were most effectively separated with the use of alkylamide and cholesterol packings. These two stationary phases reduce the time of analysis in comparison with the octadecyl packing material. Moreover, separation was achieved under non-denaturating conditions.
Highlights
Oligonucleotides are short (3–200 nucleotides), singlestranded fragments of nucleic acids
Packing materials containing hydrophobic and hydrophilic moieties bonded to a silica support have been used: alkylamide (SG-AP) and cholesterol (SG-CHOL) stationary phases (Table 2)
In the second step of synthesis process, alkylamide groups are chemically bonded to the SG-AP surface, while cholesterol molecule is attached in case of SG-CHOL
Summary
Oligonucleotides are short (3–200 nucleotides), singlestranded fragments of nucleic acids. The study of Apffel et al [15] is of great importance, because he introduced volatile triethylamine with 1,1,1,3,3,3-hexafluoro-2-propanol as an IP reagent for the analysis of nucleic acid components. This novel additive resulted in good HPLC separation and efficient negative ion mode electrospray ionization. The authors observed small suppression of mass spectrometry signal suppression. They achieved acceptable separation of native and synthetic oligonucleotides [15]. The HFIP/TEA buffer has become the most widely used for liquid chromatography mass spectrometry (LC-MS) determination of these compounds [20, 21]
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