Ion pairing reversed-phase (IPRP) chromatography is the method of choice for synthetic oligonucleotide characterization and impurities analysis. The method helps scientists resolve synthesis and process-related impurities better than any other chromatographic technique, including anion exchange (AEX), HILIC, and SEC. The technique is not without its challenges. Multiple factors can directly affect separation performance and reproducibility, including the purity and stability of ion-pairing reagents, non-specific adsorption to chromatographic hardware, and batch-to-batch variations in sorbent. IPRP relies on high pH and temperature conditions for optimal separations, requiring column materials to have high pH and temperature tolerance.
Gain confidence in your results using Waters Oligonucleotide Reversed Phase Columns that offer:
Nucleic acids, including oligonucleotides, are polyanionic (negatively charged) and readily adsorb to metal oxide surfaces in stainless steel columns. Waters MaxPeak Premier Oligonucleotide Columns have an inert hybrid organic/inorganic silica-based surface chemistry that nearly eliminates NSA of nucleic acids, enabling enhanced sensitivity, reproducibility and productivity. With Waters, your lab can eliminate time-consuming column passivation and sample conditioning steps prior to analysis.
IP-RP chromatography of synthetic oligonucleotides resolves sample components based on minor changes in size, charge, and hydrophobicity. Waters ACQUITY (UPLC: 1.7 µm particle size) and XBridge (HPLC/UHPLC: 2.5 µm particle size) Premier Oligonucleotide BEH C18 Columns deliver exceptional oligonucleotide sample resolution, sensitivity, and reproducibility.
Waters ACQUITY and XBridge Premier Oligonucleotide Columns are packed with Waters BEH (Ethylene Bridged Hybrid) C18 hybrid silica particles and demonstrate remarkable column longevity under high pH and temperature conditions while maintaining outstanding separation performance.
Oligonucleotides and longer nucleic acids exhibit a strong on/off bind and elute mechanism, which makes them amenable to “ballistic gradients” and ultra-fast separations that can improve productivity in high-throughput testing labs. Waters ACQUITY Premier Oligonucleotide BEH C18 2.1 x 20 mm Columns in 130 Å and 300 Å pore sizes enable ultra-fast separations of short-mer (10–40 nt) and long-mer (> 40 nt) oligonucleotides, respectively.
Both ACQUITY (1.7 µm) and XBridge (2.5 µm) Premier Oligonucleotide Columns are available in 130 Å and 300 Å pore sizes that demonstrate differences in retentivity and peak sharpness depending on the length of the oligonucleotide. The increased surface area of 130 Å pore size particles enables greater retention and resolution of short-mer oligonucleotides (< ~40 mer), while 300 Å pore size particles enable greater diffusion and enhanced resolution of longer oligonucleotides (> ~40 mer).
Four consecutive injections of 3 µL of Duplex C (0.40 mg/mL) using the ACQUITY Premier Oligonucleotide BEH C18, 300 Å Column.
Fraction purity determinations for a 25 mg purification run.
Complete (100%) sequence coverage, presented in a dot-map format, produced by high energy MSE untargeted fragmentation.
Experimental chromatogram observed with a 1.33-minute concave (logarithmic) gradient on an ACQUITY Premier Oligonucleotide 2.1 x 20 mm ultra-short column and operated at F = 1.5 mL/min and T = 70 °C.
