Oligonucleotide Reversed-Phase Columns

Oligonucleotide Reversed-Phase Columns

Achieve industry-leading pH & temperature tolerance and exceptional resolution with Waters oligonucleotide reversed-phase columns for oligonucleotide characterization and impurity analyses. Each batch of sorbent is quality control (QC) tested with oligonucleotide standards to ensure lot-to-lot performance and reproducibility.

Achieve industry-leading pH & temperature tolerance and exceptional resolution with Waters oligonucleotide reversed-phase columns for oligonucleotide characterization and impurity analyses. Each batch of sorbent is quality control (QC) tested with oligonucleotide standards to ensure lot-to-lot performance and reproducibility.
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Overview

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: 

  • Industry-leading pH and temperature stability with BEH C18 hybrid silica particle technology for exceptional resolution and column life.
  • A dramatic reduction of non-specific adsorption (NSA) with MaxPeak Premier High-Performance Surfaces (HPS) Technology for maximum sensitivity, reproducibility, and productivity.
  • Oligonucleotide-based QC testing to ensure lot-to-lot separations performance and reproducibility.

Enhanced sensitivity & reproducibility without column conditioning

Enhanced sensitivity & reproducibility without column conditioning

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.

Exceptional resolution of synthetic oligonucleotides

Exceptional resolution of synthetic oligonucleotides

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.

Outstanding column lifetime

Outstanding column lifetime

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.

Solutions

2.1 x 20 mm columns for ultra-fast separations

2.1 x 20 mm columns for ultra-fast separations
ACQUITY Premier 2.1 x 20 mm Oligonucleotide BEH C18 Columns

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.

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  • Ultrafast Oligonucleotide Separations
  • Reversed-Phase Analysis

MaxPeak Premier Wide-Pore (300 Å) Columns for long-mer oligonucleotides

MaxPeak Premier Wide-Pore (300 Å) Columns for long-mer oligonucleotides
MaxPeak Premier Wide-Pore (300 Å) Columns

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).

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  • Long-mer Oligonucleotide Separations
  • Reversed-Phase Analysis

Oligonucleotide / nucleic acid standards for benchmarking performance

Oligonucleotide / nucleic acid standards for benchmarking performance
Oligonucleotide & Nucleic Acid Standards

Waters provides a range of high-quality lot-traceable analytical standards that complement our oligonucleotide reversed-phase columns to facilitate system suitability testing, method development, and troubleshooting. These include Waters MassPREP Oligonucleotide Standard containing a 15-35mer oligodT ladder to QC verify all of Waters oligonucleotide branded columns, a 20mer ssDNA standard for benchmarking LC-MS/MS fragmentation and sequence analysis, a lipid-conjugated heavily modified antisense oligonucleotide (ASO), ssDNA and dsDNA ladders, and more.

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  • Reversed-Phase Analysis
  • Oligonucleotide Separations

VanGuard FIT Columns for extended column lifetimes

VanGuard FIT Columns for extended column lifetimes
VanGuard FIT Column

All ACQUITY and XBridge Premier Oligonucleotide BEH C18 columns are available in the VanGuard FIT Column format, which includes a fully integrated 5 mm pre-column cartridge packed with the same sorbent and acts as a guard to prevent fouling of the analytical column. The fully integrated design has zero impact on sample resolution. The cartridge can be easily replaced should fouling occur, extending the life of your analytical column.

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  • Reversed-Phase Analysis
  • Oligonucleotide Separations

Applications

Waters has produced more than 30 application notes addressing oligonucleotide characterization and impurities analysis using our technology-leading liquid chromatography and mass spectrometry tools for therapeutic and diagnostic purposes. The following recent examples highlight our deep applications expertise and newest, most advanced tools.

Waters has produced more than 30 application notes addressing oligonucleotide characterization and impurities analysis using our technology-leading liquid chromatography and mass spectrometry tools for therapeutic and diagnostic purposes. The following recent examples highlight our deep applications expertise and newest, most advanced tools.

Application Notes

Application Notes
Colorful 3D rendering of an oligonucleotide

As a double-stranded RNA, small interfering RNA (siRNA) oligonucleotide therapeutics present unique analytical challenges. These application notes demonstrate both non-denaturing and denaturing chromatographic separations.

As a double-stranded RNA, small interfering RNA (siRNA) oligonucleotide therapeutics present unique analytical challenges. These application notes demonstrate both non-denaturing and denaturing chromatographic separations.

Application Notes

Application Notes
stock-viral-genome-close-up-novel-virus-RNA-sequence.jpg

The nucleotide sequence for diagnostic and therapeutic oligonucleotides is critical to their specificity and function. It is therefore essential to accurately confirm their sequence. LC-MS workflows enable 100% sequence confirmation of oligonucleotides and offer the unique ability to confirm the identity and location of modified nucleotides in the sequence, whether for synthetic oligonucleotides or those generated from a nuclease digest of longer chain nucleic acids (e.g., mRNA or ssDNA oligo mapping workflow).

The nucleotide sequence for diagnostic and therapeutic oligonucleotides is critical to their specificity and function. It is therefore essential to accurately confirm their sequence. LC-MS workflows enable 100% sequence confirmation of oligonucleotides and offer the unique ability to confirm the identity and location of modified nucleotides in the sequence, whether for synthetic oligonucleotides or those generated from a nuclease digest of longer chain nucleic acids (e.g., mRNA or ssDNA oligo mapping workflow).

Application Notes

Application Notes
stock-DNA-sequence-in-digital-matrix.jpg

Key critical quality attributes for mRNA-based therapeutics and vaccines include confirming the length and polydispersity of the polyA tail as well as the identity of the 5’ Cap and any possible low-level variants present. These application notes demonstrate these analyses via ion-pair reversed-phase chromatography.

Key critical quality attributes for mRNA-based therapeutics and vaccines include confirming the length and polydispersity of the polyA tail as well as the identity of the 5’ Cap and any possible low-level variants present. These application notes demonstrate these analyses via ion-pair reversed-phase chromatography.

Application Notes

Application Notes

During drug development, from early discovery through to late-stage development, adequate purified oligonucleotide material is required to facilitate a range of different studies and improve purification schemes. This application note presents a cost-effective, high throughput, and systematic approach for both analytical separations and a preparative purification scheme for a 20-mer oligonucleotide.

During drug development, from early discovery through to late-stage development, adequate purified oligonucleotide material is required to facilitate a range of different studies and improve purification schemes. This application note presents a cost-effective, high throughput, and systematic approach for both analytical separations and a preparative purification scheme for a 20-mer oligonucleotide.

Application Notes

Application Notes

The data speaks for itself

The data speaks for itself
Four consecutive injections of 3 µL of Duplex C (0.40 mg/mL) using the ACQUITY Premier Oligonucleotide BEH C18, 300 Å Column.
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.
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.
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
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.

Webinars and Resources

  • Wall Chart

LC and LC-MS Consumables for Bio-Separation – Wall Chart

LC and LC-MS Consumables for Bio-Separation – Wall Chart
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  • Infographic

ACQUITY Premier Columns for Oligonucleotide Analysis Infographic

ACQUITY Premier Columns for Oligonucleotide Analysis Infographic
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  • User Manuals

ACQUITY UPLC and ACQUITY Premier Oligonucleotide BEH C18 Columns Care and Use Manual

ACQUITY UPLC and ACQUITY Premier Oligonucleotide BEH C18 Columns Care and Use Manual
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  • User Manuals

Oligonucleotide Separation Technology Standard Care and Use Manual

Oligonucleotide Separation Technology Standard Care and Use Manual
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  • User Manuals

XBridge and XBridge Premier Oligonucleotide BEH C18 Columns Care and Use Manual

XBridge and XBridge Premier Oligonucleotide BEH C18 Columns Care and Use Manual
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  • eBook

Purifying Oligonucleotides eBook

Purifying Oligonucleotides eBook
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  • eBook

Quantifying Oligonucleotides Guidebook

Quantifying Oligonucleotides Guidebook
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Learn more about Oligonucleotide Reversed-Phase Columns.

Learn more about Oligonucleotide Reversed-Phase Columns.
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