Best Practices for Oligonucleotide Analysis Using Ion-Pair Reversed-Phase (IP-RP) Liquid Chromatography – Columns and Chemistries

Application Note

Best Practices for Oligonucleotide Analysis 
Using Ion-Pair Reversed-Phase (IP-RP) 
Liquid Chromatography – Columns and 
Chemistries

Martin Gilar,

 Nicholas J. Zampa

Waters Corporation

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This is an Application Brief and does not contain a detailed Experimental section.

Abstract

Waters presents some critical column and chemistry best practices for performing successful 

oligonucleotide analysis by ion-pair reversed-phase (IP-RP) liquid chromatography. Ensuring high 

quality oligonucleotides, either in diagnostic applications or as therapeutic entities, relies on robust 

analytical methods. For example, in response to the global COVID-19 pandemic, PCR-based 

diagnostic kits have been developed to detect SARS-CoV-2 genetic code in novel coronavirus 

patients. Accurate viral detection via PCR requires high-quality oligonucleotide primers and probes. 

Additionally, oligonucleotides are being investigated as therapeutics (including mRNA-based 

vaccines) for the treatment and prevention of COVID-19.

Benefits

Waters BEH-based C18 particles enable high pH and high temperature oligonucleotide 

separations in LC or LC-MS compatible buffers

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Batches are QC tested with MassPREP Oligo Standard to ensure consistency

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Different particle sizes and column dimensions for preparative or analytical applications

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Three step method development of <5-minute IP-RP oligonucleotide separations

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Introduction

In this application brief, Waters provides a short list of best practices to characterize 

oligonucleotides by ion-pair reversed-phase (IP-RP) liquid chromatography. These best practices 

cover separation temperature, pH optimization, pore size selection, mobile phase choice, rapid 

method development considerations, purification guidelines, and popular buffer recipes. Following 

these best practices for oligonucleotide analysis helps ensure robust methods, enabling the delivery 

of consistently high quality oligonucleotides for therapeutic or diagnostic applications.

The COVID-19 pandemic serves as an example of the importance of oligonucleotides in diagnostic 

applications. Timely diagnosis of SARS-CoV-2 viral infection remains paramount to successfully 

managing the novel coronavirus pandemic. As a result, many firms have developed in vitro 

diagnostic tests to detect the presence of SARS-CoV-2 genetic material. Of the emergency use 

authorizations (EUA) for in vitro diagnostic tests in the United States, most of them are PCR-based 

(e.g. qPCR, RT-PCR) tests which detect SARS-CoV-2 viral genetic code.1 Diagnostic kits indicated 

for the molecular detection of viral genetic code rely on oligonucleotides as primers and probes 

during PCR amplification and detection. As a result, analytically assessing the quality and ensuring 

appropriate purity is of principle importance for a successful diagnostic test. 

Results and Discussion

Waters created a short list of best practices that have emerged as a result of reviewing method and 

product development resources for IP-RP oligonucleotide separations. We discuss 6 key practices 

below.

1)   Perform oligonucleotide separations at elevated pH and temperature for best results.

i.  Elevated temperature prevents the oligonucleotide secondary structure from impacting retention 

(60 °C). Elevated temperature ensures that DNA/RNA secondary structure does not affect the 

separation. For CG rich or G-rich oligonucleotides with high degree of secondary structure, it may 

be necessary to increase column temperature to 80 or 90 °C.

ii.  High pH buffers (pH ≥ 7) are commonly used in oligonucleotide separations (e.g. TEAA).

iii. TEA-HFIP was found to be a robust mobile phase offering superior LC-MS sensitivity and 

resolution across various sized single stranded oligonucleotides.2

iv.  The high pH used for IP-RP oligonucleotide separations renders most common silica-based 

stationary phases unsuitable.

v.  Waters BEH sorbent technology lends itself well for oligonucleotide separation due to its high pH 

stability and temperature tolerance.

Figure 1. Schematic structure of 

BEH sorbent. Hydrolytic stability 

is achieved by bridging ethyl 

groups. For oligonucleotide 

analysis, the surface of sorbent is 

alkylated by C18 functional groups.

For more detailed information, see Waters Application Note

HPLC and UPLC Columns for the Analysis of Oligonucleotides. 720002376EN.

2)   Choose a relevant pore size for your oligonucleotide separation. Choosing the correct pore size 

enables appropriate analyte diffusivity resulting in the best interaction between the oligonucleotide 

and the ligand. Improved ligand interactions improve peak shape.

 i.   130 Å pore size is ideally suited for single stranded oligonucleotides (2-100 mers).

 ii.  300 Å pore size allows for efficient separation of both single stranded oligonucleotides and 

longer dsDNA fragments.

3)  Choose an appropriate mobile phase. See section 6 for buffer recipes.

i.   Triethylamine/hexafluoroisopropanol (TEA/HFIP) is MS compatible and has impressive resolving 

power. Higher TEA/HFIP buffer concentrations improve separation performance. Lower 

concentrations improve MS sensitivity.

ii.   Hexylammonium acetate (HAA) also offers exceptional resolution and MS compatibility, 

however, the MS compatibility of HAA is less than that of TEA/HFIP. Use of HAA may result in 

better separation of labeled oligonucleotides and longer oligonucleotides (>35-mer) compared to 

TEA/HFIP. This may be relevant if performing only LC analysis.

iii.   Fresh TEA/HFIP and HAA/HFIP mobile phases are critical to good separations. These semi-

volatile mobile phases can gradually lose their separation strength and MS spectra become 

contaminated with alkali ion adducts. For robust day-to-day results, make mobile phases daily or in 

limited quantities. Upper limit of mobile phase usability is one week.

iv.   Both TEA and HFIP should be prepared in a fume hood, use Waters APC solvent bottle caps to 

prevent gassing out, and if possible, use a snorkel above the system.

Figure 2. Separation of 

heteromeric oligonucleotides. The 

type of ion-pairing mobile phase 

influences the selectivity of 

separation. With “weak” IP 

systems such as TEAA both 

hydrophobic and ion-pairing 

interactions participate in 

separation. With “strong” ion-

pairing systems (TEA/HFIP, HAA) 

the oligonucleotide separation is 

mostly driven by their 

charge/length.

For more detailed information, see the below Waters Application Notes

UPLC/MS Separation of Oligonucleotides in Less than Five Minutes: Method Development. 

720002387EN.

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Evaluation of Alternative Ion-pairing Reagents in the Analysis of Oligonucleotides with the 

ACQUITY QDa Detector. 720005830EN.

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Hexylammonium Acetate as an Ion-Pairing Agent for IP-RP LC Analysis of Oligonucleotides. 

720003361EN.

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4)   Quick three step method development for <5-minute IP-RP oligonucleotide separations.

i.   Identify suitable initial gradient strength or start with a scouting gradient. With 15 mM TEA/400 

mM HFIP ion pairing system, an example scouting gradient may be:

Start at 20% MeOH, perform at 1%/min MeOH gradient with 0.2 mL/min flow rate (0.2 mL/min 

is useful to enhance column efficiency for macromolecules).

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ii.   Adjust gradient slope to achieve desired separation (shallower gradients increase resolution, but 

increase time needed for analysis). Adjust the starting percentage of MeOH to reduce the time of 

analysis as needed. Extend the MeOH gradient time as needed until the target oligonucleotide and 

impurities are eluted. High organic flush is then inserted to elute highly retained components (often 

non-oligonucleotide components) and minimize carryover. Target oligonucleotides should elute 

during the gradient and not in the high organic flush.

iii.   If speed is important, speed up the separation by increasing the flow rate while proportionally 

reducing gradient time (constant gradient column volume). Selectivity of separation should not 

change while minimal loss in resolution can be observed. When driving towards sensitivity or a need 

for optimal resolution, use lower flow rates.

0.4 mL/min is a good compromise between speed and analytical performance. For high-

throughput separations a 0.8 mL/min flow rate is recommended (1-3 min separation time, 2.1 

mm column I.D). 

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For more detailed method development considerations for XBridge OST Columns (e.g. starting 

gradient slopes, buffer formulations, and column considerations) see Waters 715001476.

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Figure 3. Separation of 30 to 60 nt 

oligodeoxythymidines using 2.1 x 50 mm, 1.7 

µm ACQUITY UPLC OST C18 Column.

Figure 4. Separation of 15 to 35 nt 

oligodeoxythymidines.

For more detailed information, see te below Waters Application Notes

UPLC/MS Separation of Oligonucleotides in Less than Five Minutes: Method Development. 

720002387EN.

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UPLC Separation of Oligonucleotides: Method Development. 720002383EN.

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XBridge OST C18 Method Guidelines. 715001476.

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5)   Guidelines for oligonucleotide purification using XBridge Oligonucleotide BEH C18 Columns.

i.   XBridge Oligonucleotide BEH C18, 130 Å Columns are the preferred offering for detritylated 

oligonucleotide purifications due to the availability of column sizes designed to meet lab-scale 

isolation requirements.  

ii.   The choice of XBridge Oligonucleotide C18 Column dimension and operating flow rate depends 

primarily on the scale of the synthesis reaction mixture.

 For example, a 4.6 × 50 mm column containing XBridge Oligonucleotide BEH C18, 130 Å, 2.5 μm 

material is an excellent selection when oligonucleotide mass loads are less than or equal to 0.2 

μmol.

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 Selection of the appropriate column size for oligonucleotide sample load is recommended to 

maximize component resolution and recovery of the target product from non-desired failure 

sequences. See Table 1 for mass loading guidelines.

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Figure 5. HPLC purification of a synthetic 

21-mer oligonucleotide. Sample 

concentration was 2.8 nmol/µL, with on-

column loading ranging from 1.4 to 140 

nmol.

*Values are only approximates and vary depending on 

oligonucleotide length, base composition, and “heart-

cutting” fraction collection method used.

Table 1. XBridge OST C18 Column selection guide for 

detritylated oligonucleotide purification.

6)   How to make select IP-RP buffers (1 liter)

i.   Perform all work in a fume hood.

ii.   Filter all mobile phases through a solvent compatible, 0.45 µm membrane filter and store in 

bottles that are clean and particulate free.

These are all recipes for mobile phase A. Mobile phase B is generally a mixture of organic (e.g. 

ACN, MeOH) mixed with mobile phase A at a suitable percentage (e.g. 20-50%). The higher 

concentration buffer (15 mM TEA/400 mM HFIP) can be used for separations involving G-rich 

oligonucleotides. The lower strength buffer (8.6 mM TEA/100 mM HFIP) is often enough for 

routine detriylated oligonucleotide LC-MS applications.

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Conclusion

In support of customers working on oligonucleotides, Waters offers these critical best practices to 

ensure consistent high-performance IP-RP liquid chromatographic separations for oligonucleotide 

analysis. Robust methods are critical to delivering high-quality oligonucleotides in diagnostic or 

therapeutic applications. For example, high-quality oligonucleotide primers and probes are essential 

for accurate PCR-based diagnostic assays, including those for COVID-19.

References

In Vitro Diagnostic EUAs - Test Kit Manufacturers and Commercial Laboratories Table 

[Internet]. Washington (DC). FDA. [cited 2020 Jun 1]. Available from: 

https://www.fda.gov/medical-devices/emergency-situations-medical-devices/emergency-use-

authorizations#covid19ivd

1. 

Gilar M, Fountain KJ, Budman Y, Holyoke JL, Davoudi H, Gebler JC. Characterization of 

Therapeutic Oligonucleotides Using Liquid Chromatography with On-line Mass Spectrometry 

Detection. Oligonucleotides. 2003 13(4):229-243.

2. 

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720006948, July 2020

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