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chromatography (IP-RP) [4,5] IP-RP utilises ionic interaction between analytes and the ion-pair reagent and the hydrophobic interaction between the stationary phase and the ion-pair reagent. IP-RP provides high-resolution separation of failure sequences and can be directly coupled to mass spectrometry for identification of target oligonucleotides and any related impurities. Here, we look at the HPLC-UV [6] analysis of oligonucleotides using the Thermo Scientific™ DNAPac™ RP column.
The DNAPac RP column [6] is a reversed- phase column designed for the high resolution separation of oligonucleotides and their failure sequences. These include oligonucleotides with modifications designed to improve stability in biological fluids, such as ribose modification, phosphorothioate (PS) substitution and linkage isomers. This column also supports gel-like separation of dsDNA fragments generated by restriction enzymes for cloning and next generation or high throughput sequencing (NGS, HTS). The column is based on a hydrophobic, polymer resin, so it is stable under high pH and/or high temperature conditions which often provide higher resolution for challenging oligonucleotide samples. In addition, the particles harbour a wide pore size (1000~2000 Å). The wide pores on the DNAPac RP column have been found to deliver excellent separation of large, double-stranded nucleic acids, up to 10k base pairs, with low carry-over. The hydrophobic nature of the resin comes from phenyl groups within the Poly(styrene- divinylbenzene) particles. Therefore this column provides alternative selectivity to the conventional silica based C18 columns.
Fast, High Resolution Separation of Single Stranded DNA
It is generally accepted that the ion- pair reagent, such as trimethylamine, interacts with the non-polar stationary phase via hydrophobic interaction which in turn acts as an ion-exchange site for the negatively charged oligonucleotides [1]. At pH values between 6 and 8, standard oligonucleotides have one negative charge for each phosphodiester bond. Therefore, a terminally non-phosphorylated oligonucleotide will harbour a charge equal to one less than the number of bases. Since the number of phosphodiesters,
Figure 1. Fast Separation of 12-40mer Deoxythymidines (dTs).
Figure 2. High Resolution Separation of 12-60mer dTs.
Figure 3. Separation of failure sequences.
and thus the number of IP reagent molecule interactions is proportional to the length of the oligonucleotide, longer oligonucleotides elute later at neutral pH values. As the oligonucleotides get longer, the percent charge difference between oligonucleotides decreases, so resolution
between oligonucleotides decreases with oligonucleotide length. The separation of single stranded poly-deoxythymidine (dT) using IP-RP is illustrated in Figure 1 and Figure 2. Fast Separation of 12-40mer Deoxythymidines (dTs).
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