Purifying a full-length oligonucleotide product from impurities generated during synthesis is essential to ensure their functionality, safety, and efficacy in pharmaceutical and diagnostic applications. Due to their polyanionic nature, purifying oligonucleotides with high selectivity and resolution is challenging. Ion-pairing reversed-phase chromatography (IP-RP) has emerged as the dominant and preferred technique.
High pH/temperature stable and batch-tested columns with controlled levels of secondary interactions help quickly remove time-consuming bottlenecks. Waters XBridge Oligonucleotide BEH C18 Optimal Bed Density (OBD) Columns are the go-to-choice for preparative columns, delivering assured performance, consistent scalability, and high recovery purification of various oligos with complex chemical modifications.
For preparative columns with smaller aspect ratios, high pressure slurry packing with small particles often fails to achieve the desired bed density found in well-designed analytical columns. Excessive axial compression at the inlet can result in particle breakage, channeling, and reduced bed permeability. Thoughtfully designed OBD columns offer significant benefits, including longer column lifetime, improved efficiency, better peak shape, and lower back pressure.
Nucleic acids, including oligonucleotides are polyanionic (negatively charged) and as such readily adsorb metal oxide surfaces in stainless steel columns. Waters XBridge Premier Oligonucleotide BEH C18OBD Columns are built with hardware components modified to have an inert hybrid organic/inorganic silica-based surface chemistry that greatly reduces non-specific adsorption (NSA) of nucleic acids, thus enabling enhanced recovery and improved reproducibility for certain compounds that need to be isolated from a drug substance for further study. XBridge Premier Oligonucleotide BEH C18 OBD Columns also eliminate the need for column passivation/sample conditioning so you can start your fraction collection runs without delay.
Waters batch-certified Bridged Ethyl Hybrid (BEH) technology for oligonucleotides ensures extra performance, reproducibility, and scale-up for purification. High temperature and high pH are prevalent strategies for minimizing secondary interactions in oligonucleotide separation and purification. BEH particle technology withstands harsh conditions such as high pH, temp, and additives that usually are required for oligonucleotide analysis.
Batch-tested and QC-certified XBridge Oligonucleotide OBD Prep Columns provide extra assurance for reproducibility and method transfer, and support emerging purification needs for standard and new chemically diverse siRNAs, ASOs, and oligonucleotide reagents. Each batch of Oligonucleotide BEH C18 sorbent is QC tested and selected using Waters MassPrep Oligonucleotide Standard and a stringent resolution and peak tailing assessment from a triethylammonium acetate IP-RP separation. These carefully examined sorbents are available in 2.5 and 5 µm particle sizes as well as 130 and 300 Å pore sizes to give oligonucleotide chemists a range of versatile options to purify a variety of synthesis products.
XBridge and XBridge Premier Oligonucleotide OBD Prep Columns are packed with Waters BEH (Bridge Ethyl Hybrid) C18 hybrid silica particles and demonstrate remarkable column longevity under high pH and temperature conditions while maintaining outstanding separation performance. With careful care, Oligonucleotide BEH C18 Columns have been known to exceed 1,0000 injections without loss of efficiency or retention.
In preparative purifications, loading capacity is influenced by multiple parameters. Column efficiency, pore size, particle size, and method parameters play significant roles. Larger pore size columns allow for better mass transfer and sharper peak shapes of larger analytes. With their reduced surface area, they can exhibit lower loading capacity. Waters XBridge Oligonucleotide OBD Prep Columns are available in different pore sizes and a wide range of column I.D.s for predictable scalability and improved loading capacity.
High purity and quality oligonucleotide purification is critical to minimize the safety and efficacy concerns related to unwanted impurities in toxicological studies. The high-efficiency 2.5 µm XBridge Oligonucleotide OBD Prep Columns facilitating isolation of early and co-eluting impurities while maintaining chromatography profile without compromising high recovery.
Oligonucleotides phosphodiester groups can adsorb the metals used in the construction of standard columns. Use of low adsorption column hardware components can improve peak shape by minimizing chelation, leading to more reliable retention and more consistent chromatographic parameters. XBridge Premier Oligonucleotide BEH C18 OBD 130 Å and 300Å 5 µm Prep Columns are manufactured with MaxPeak HPS vapor deposited components to enhance peak shape and improve recovery are available in 10 mm I.D. and 50-, 100-, and 150-mm lengths.
Batch-tested and QC-certified XBridge Oligonucleotide OBD Prep Columns are available in a wide range of diameters and multiple lengths for lab scale purification options that are predictable and flexible for direct scale up. Sizes include:
XBridge Oligonucleotide 130 & 300 Å 5 µm OBD Prep Columns, workhorses for HPLC method development with standard and wide pore options available in stainless steel and MaxPeak hardware, oligo batch tested and QC certified
XBridge Oligonucleotide 130 Å 2.5 µm OBD Prep Columns, offering exceptional sample resolution and superior column life
XBridge Oligonucleotide BEH C18 300 Å 2.5 µm OBD Prep Columns, options are available as custom made products for purifying larger oligos. Please contact sales to request a custom quote
Fraction purity determinations for a 25 mg purification run using a novel XBridge Oligonucleotide BEH C18 130 Å 2.5 µm OBD Prep Column.
(A) Comparison of the chromatograms obtained for hydrocortisone phosphate using MaxPeak Premier (black trace) and standard (gray trace) ACQUITY UPLC BEH C18, 130 Å, 1.7 µm, 2.1 x 50 mm Columns. The mass load was 2 ng. Acetonitrile gradient separations were carried out with a 10 mM ammonium formate (pH 3.0) aqueous mobile phase, a flow rate of 0.5 mL/min and a temperature of 30 °C. The peaks were detected using a PDA detector, with the 246 nm channel displayed. (B) Log-log plot of peak area vs mass load for hydrocortisone phosphate separated using MaxPeak Premier (black symbols) and standard (gray symbols) columns, with mass load from 2–200 ng.
