Improved Peak Recovery and Peak Shape of Oligonucleotides Using Waters Premier Columns

Oligonucleotide separation using XBridge Premier Oligonucleotide BEH C₁₈ Columns yielded higher peak areas and sharper peaks compared to a competitor column based on conventional column hardware. An average of 40% peak area loss was observed on the competitor column for the five analyte separations.

Benefits

• Improved analyte recovery
• Improved peak shape
• Out-of-box performance

Adsorptive losses to column hardware, commonly referred to as non-specific adsorption, is a common issue in many workflows and is most problematic for large molecule applications, like oligonucleotide separations, where multiple residues on the analyte can interact with the various surfaces. Stainless-steel and other metallic surfaces are particularly problematic in LC systems and column hardware as they introduce ionic secondary interactions and the potential for metal chelation. This can lead to reduced peak area, peak shape distortions, and in some extreme cases total peak loss. Work-arounds exist, including the use of passivation agents—like medronic acid—or even saturating the system with a similar analyte to occupy the active sites on the metal surfaces. However, these techniques are time consuming, and are often inconsistent or temporary fixes.

By using Waters MaxPeak Premier Columns, which utilize hardware based on High Performance Surface (HPS) Technology, the undesirable interaction of oligonucleotides with column hardware can be drastically reduced. To demonstrate this, a standard LC instrument and two separate columns were used to analyze a simple mixture of oligonucleotides. The first column to be tested was a fully porous XBridge Premier Oligonucleotide BEH C₁₈, 130 Å, 2.5 µm Column while the other was a superficially porous 2.7 µm competitor C₁₈ column constructed from stainless steel hardware. Both stationary phases were pH resilient and designed for use with ion pairing RPLC conditions. The analytes examined were five chains of oligodeoxythymidine (15, 20, 25, 30, and 35 nt) contained in the Waters MassPREP OST Standard (p/n: 186004135). Mobile phases containing 25 mM of hexylammonium acetate (pH 7.0) as an ion pairing agent were used along with ACN as an eluent and a column temperature of 60 °C.

As shown in the figure, the competitor column showed decreased peak areas for all five of the oligos. On average, the competitor column yielded a peak area 40% lower than the XBridge Premier Oligonucleotide BEH C₁₈ Column. Additionally, peak tailing for most of the oligos was worse on the competitor column. Particularly of interest is the 35 nt which co-elutes with an impurity on the competitor column but resolves on the MaxPeak Premier Column. With its HPS column hardware, the MaxPeak Premier Column addresses undesired secondary interactions. A MaxPeak Premier System was not employed for this separation, but a significant level of performance is still gained by employing the MaxPeak Premier Column. Additional performance and robustness gains are likely to be found by pairing a MaxPeak Premier Column with a MaxPeak Premier System.

The Waters MaxPeak Premier brand of columns reduces the adsorptive interactions between analytes and column hardware surfaces. While problematic secondary interactions can occur across a wide range of applications, separation of oligonucleotides is an important and ever relevant area of separation science. Oligonucleotides are generally very sensitive to metallic surfaces as a result of their structure which can lead to significant challenges when it comes to recovery (peak area) and achieving good peak shape. In this work, an XBridge Premier Oligonucleotide BEH C₁₈ Column was compared to a competitor column based on conventional stainless-steel hardware. The MaxPeak Premier Column yielded higher peak areas and sharper peaks compared to the competitor column. Switching to MaxPeak Premier Columns will provide better separations and more accurate results. Even more benefits can be found by using MaxPeak Premier Columns on a MaxPeak Premier LC System.

1. Waters MassPREP OST Standard Care and Use Manual, 715001677EN.