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
Need Help? To learn more about how Waters can help you in your efforts against
COVID-19, please contact the COVID-19 Innovation Response Team
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
■
Batches are QC tested with MassPREP Oligo Standard to ensure consistency
■
Different particle sizes and column dimensions for preparative or analytical applications
■
Three step method development of <5-minute IP-RP oligonucleotide separations
■
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.
■
Evaluation of Alternative Ion-pairing Reagents in the Analysis of Oligonucleotides with the
ACQUITY QDa Detector. 720005830EN.
■
Hexylammonium Acetate as an Ion-Pairing Agent for IP-RP LC Analysis of Oligonucleotides.
■
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).
■
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).
■
For more detailed method development considerations for XBridge OST Columns (e.g. starting
gradient slopes, buffer formulations, and column considerations) see Waters 715001476.
■
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.
■
UPLC Separation of Oligonucleotides: Method Development. 720002383EN.
■
XBridge OST C18 Method Guidelines. 715001476.
■
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.
■
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.
■
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.
■
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-
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.
Featured Products
720006948, July 2020
© 2020 Waters Corporation. All Rights Reserved.
