Oligonucleotides are diverse in nature, with a wide range of applications. Here, Dr Anastassia Kanavarioti and Dr Sandra Rontree explore the challenges of oligonucleotide analysis in the life sciences sector and outline the different HPLC methods available for the job.
OLIGONUCLEOTIDES are the starting point for many molecular biology and synthetic biology applications. Used in a wide variety of ways, from biopharmaceutical therapeutics to clinical diagnostics and forensic research, the purification and analysis of oligonucleotides is becoming increasingly important across the life sciences sector.1
Are you looking to explore how lipid formulations in softgels can enhance drug absorption and bioavailability. Register for our upcoming webinar to find out!
3 September 2025 | 3:00 PM BST | FREE Webinar
This webinar will delve into the different types of lipid formulations, such as solutions, suspensions, emulsions, and self-(micro)emulsifying systems. Applications span diverse therapeutic areas including HIV therapy, oncology, immunosuppressants, and emerging treatments like medicinal cannabis (eg, CBD).
What You’ll Learn:
Lipid formulation development and screening tools for optimisation
Key steps in scale-up and industrialisation to ensure consistency and efficiency
Impact of lipid-based softgels on drug delivery and patient outcomes.
Can’t attend live? No worries – register to receive the recording post-event.
During the synthesis of oligonucleotides, smaller side chains, failure sequences and impurities can become present, which, depending on the downstream application, can negatively impact results. When choosing a technique to purify and characterise oligonucleotides, the type of solution being eluted and the goals of the analysis are both important. For example, if the downstream application requires only the full-length sequence, using a purification technique to create a purer oligonucleotide, such as high-performance liquid chromatography (HPLC), will be crucial.
However, many traditional analysis techniques can only process shorter chain oligonucleotides in a specific, high-resolution and high-purity manner. As many of the latest technologies, such as CRISPR, require longer scaffold sequences, there is a real need for advanced oligonucleotide purification and analysis.
Fortunately, the technologies available for DNA and RNA analysis are rapidly developing. Advances in HPLC instruments and innovative columns can improve resolution, accuracy and end sample purity. This facilitates precise knowledge of what a sample contains and enables the separation of a solution based on specific characteristics of a particular molecule. However, selecting the most appropriate method for your application is key. Here, we explore the challenges of oligonucleotide analysis and the different HPLC methods available.
Considerations for selecting the most appropriate technique
Within HPLC there are various options for oligonucleotide purification and analysis, including anion exchange (AEX) chromatography and ion‑pair reversed phase (IP-RP) chromatography. So, how do you know which method to select? The first step is to consider the goals of your analysis; for example, those interested in the molecular formula of an HPLC peak will require a different technique to those looking to understand the resolution of components in a mixture.
Optimising the resolution and selectivity of oligonucleotide analysis can be achieved using specifically designed columns for anion exchange (AEX) and ion-pair reversed phase (IP-RP) chromatography
Next, it is important to assess the specific challenges associated with analysing different kinds of oligonucleotides. For instance, synthetic oligonucleotides increasingly have chemical features that improve stability and bioavailability; these include the addition of phosphorothioate linkages, attachment of polyethylene glycol at the terminus and the incorporation of modified nucleosides. Where these chemical features are present, careful column selection and modifications to the purification technique are often required.2,3
The length of the oligonucleotide scaffold can also affect which HPLC technique will be the most appropriate. sgRNAs, for example, which are essential for CRISPR, tend to be 100 nucleotides long making their analysis difficult with traditional HPLC.4 Here, advanced HPLC systems will be required to ensure the accuracy of results and end sample purity. The need for such systems is also seen in diagnostics, where the identification of molecules, including long non-coding RNAs and oligonucleotides 20-300 nucleotides in length, is required.5
AEX and IP-RP chromatography can overcome many of the challenges associated with oligonucleotide analysis and purification – the two chemistries are complementary and each can be used to provide different information.
Anion exchange chromatography (AEX)
AEX is an example of an HPLC system suited to analyse short to long oligos as well as plasmid DNAs.4,6 Depending on the requirements of the analysis, different specifications of AEX column can be employed. For example, there are many columns available that vary in terms of resin type; pore size; and the temperature and pressure the system allows, providing great flexibility.
Traditionally, resin is functionalised using a quaternary or tertiary amine, which is positively charged. When the negatively charged oligonucleotide binds to the stationary phase, it elutes as the salt concentration increases. As oligonucleotide length increases, the more negative charge it has; therefore, longer oligonucleotides usually elute later. Changes are also seen in the elution, depending on the sequence and structure of the oligonucleotide.
The technologies available for DNA and RNA analysis are rapidly developing”
Variations in pH levels, particle size and the ability to operate at different temperature and pressure ranges also affect what can be achieved with a given AEX system. Particle size typically varies from 4-13μm, with 4μm giving ultra-high resolution, for instance, while high-throughput oligonucleotide separation can be achieved with high pressures of 10,000psi. Temperature and pH tolerances also vary widely, ranging from <35°C to 85°C and <8.5 to 12.5 respectively. By modulating pH, temperature, solvent and eluent salt, oligonucleotide retention and secondary structures can be controlled.
The ability to control these parameters makes AEX suitable for analysing both single- and double-stranded oligonucleotides. Notably, AEX is particularly effective for ultra high-resolution separation of components in a mixture.
Ion-pair reversed phase chromatography (IP-RP)
IP-RP is an alternative analytical strategy for RNA separation and is often used with organic solvents at high temperature. IP-RP can be used in conjunction with AEX with each method providing different information. Oligonucleotides have unique features that suit IP-RP. Each molecule contains one negative charge for each phosphodiester linkage when in aqueous solution above pH 4; this means traditional reversed phase chromatography is not suitable, while ion-exchange techniques such as IP-RP work well.7
It is important to assess the specific challenges associated with analysing different kinds of oligonucleotides”
However, retention and elution order are affected by the charge of the oligonucleotides, length of alkyl chain in the ion-pairing reagent and the proportion of organic solvent in the mobile phase.2 For example, the number of charges in the oligonucleotides increases the retention time, as does increased hydrophobicity in the ion-pairing agent long alkyl chain.8
Optimising the resolution and selectivity of the column can be achieved by altering the mobile phase.9 The most commonly used ion-pair reagent is triethylammine (TEAA) but other ion-pair reagents could provide alternative selectivity. For example, tetrabutylammonium bromide (TBAB) as an ion-pairing agent can improve separation,2 while using hexafluoro-2-propanol (HFIP) in the mobile phase makes the system compatible with mass spectrometry, enabling molecular weight determination.
IP-RP systems can often be modulated to allow separation of different molecules; for example, different ion-pair reagents could be used to gain optimal separation for specific samples. Additionally, some IP-RP systems use polymer resin, as opposed to silica. This gives excellent stability at high temperatures (up to 100°C) and supports high pH (0-14 pH range), giving the freedom to operate in conditions required for high selectivity and resolution. Large pore IP-RP systems can also be used, allowing long oligonucleotides to diffuse in and out to generate sharper peaks than a small-pore system.
In short, novel IP-RP technology can resolve both very small and very large oligonucleotides and can be compatible with mass spectrometry, a great benefit if determining the compound identity.
Better results with better tools
Effective and reliable oligonucleotide analysis and purification is critical for many life science applications. Traditional analytical methods are unreliable for processing the longer scaffold sequences often required by the latest technologies such as CRISPR. Making use of the latest analytical systems that can work with longer sequences, such as AEX and IP-RP, is key to avoid these downstream applications being compromised.
By considering the goals and requirements of your specific analysis needs and selecting the most appropriate method, you will be able to generate accurate and reproducible results and ensure end sample purity.
About the authors
Dr Anastassia Kanavarioti has 40 years’ experience using HPLC analysis with a wide range of chemical entities. She has used HPLC analysis to elucidate mechanisms of reactions, to assess purity and stability of pharmaceutical preparations and in support of formulation development. Her primary focus is oligonucleotide separations and profiling of oligonucleotide mixtures. She founded Yenos Analytical LLC in 2013 and is currently using HPLC to validate nanopore devices as analytical sensors for microRNAs in biological fluids. Tessi has over 60 peer‑reviewed publications showcasing HPLC methods that successfully address separation and mechanistic questions.
Dr Sandra Rontree is a product marketing manager for chromatography columns and consumables in the Chromatography and Mass Spectrometry group at Thermo Fisher Scientific. Sandra has more than 20 years’ experience with analytical instrumentation and has a strong interest in UHPLC, mass spectrometry and the life sciences.
References
Sun H, Zhu X, Lu PY, et al. Oligonucleotide aptamers: New tools for targeted cancer therapy. Mol Ther Nucleic Acids 2014;3:182.
Gilar M. Analysis and purification of synthetic oligonucleotides by reversed-phase high-performance liquid chromatography with photodiode array and mass spectrometry detection. Biochem 2001;298:196–206
Abeydeera ND, Egli M, Cox N, Yang YB. Evoking picomolar binding in RNA by a single phosphorodithioate linkage. Nucleic Acids Res 2016;44:8052–8064
Kanavarioti A. HPLC methods for purity evaluation of man-made single-stranded RNAs. Scientific Reports 2019;9(1019).
Kang ASW, Bernasconi JG, Jack W, Kanavarioti A. Ready-to-use nanopore platform for the detection of any DNA/RNA oligo at attomole range using an Osmium tagging complementary probe. Scientific Reports 2020;10(19790).
Thayer JR, McCormick RM, Avdalovic N. High-resolution nucleic acid separations by high-performance liquid chromatography. Methods Enzymol 1996;271:147-74.
Biba M, Mao B, Welch CJ. Liquid chromatography methods for the separation of short RNA oligonucleotides. LCGC N Am. 2014;32:42–50
Zhang Q, Lv H, Wang L, et al. Recent methods for purification and structure determination of oligonucleotides. International Journal of Molecular Sciences; 2016:17(2134)
Andrus A, Bloch W. HPLC of oligonucleotides and polynucleotides. HPLC Macromol 1998; 872:141
This website uses cookies to enable, optimise and analyse site operations, as well as to provide personalised content and allow you to connect to social media. By clicking "I agree" you consent to the use of cookies for non-essential functions and the related processing of personal data. You can adjust your cookie and associated data processing preferences at any time via our "Cookie Settings". Please view our Cookie Policy to learn more about the use of cookies on our website.
This website uses cookies to improve your experience while you navigate through the website. Out of these cookies, the cookies that are categorised as ”Necessary” are stored on your browser as they are as essential for the working of basic functionalities of the website. For our other types of cookies “Advertising & Targeting”, “Analytics” and “Performance”, these help us analyse and understand how you use this website. These cookies will be stored in your browser only with your consent. You also have the option to opt-out of these different types of cookies. But opting out of some of these cookies may have an effect on your browsing experience. You can adjust the available sliders to ‘Enabled’ or ‘Disabled’, then click ‘Save and Accept’. View our Cookie Policy page.
Necessary cookies are absolutely essential for the website to function properly. This category only includes cookies that ensures basic functionalities and security features of the website. These cookies do not store any personal information.
Cookie
Description
cookielawinfo-checkbox-advertising-targeting
The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Advertising & Targeting".
cookielawinfo-checkbox-analytics
This cookie is set by GDPR Cookie Consent WordPress Plugin. The cookie is used to remember the user consent for the cookies under the category "Analytics".
cookielawinfo-checkbox-necessary
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Necessary".
cookielawinfo-checkbox-performance
This cookie is set by GDPR Cookie Consent WordPress Plugin. The cookie is used to remember the user consent for the cookies under the category "Performance".
PHPSESSID
This cookie is native to PHP applications. The cookie is used to store and identify a users' unique session ID for the purpose of managing user session on the website. The cookie is a session cookies and is deleted when all the browser windows are closed.
viewed_cookie_policy
The cookie is set by the GDPR Cookie Consent plugin and is used to store whether or not user has consented to the use of cookies. It does not store any personal data.
zmember_logged
This session cookie is served by our membership/subscription system and controls whether you are able to see content which is only available to logged in users.
Performance cookies are includes cookies that deliver enhanced functionalities of the website, such as caching. These cookies do not store any personal information.
Cookie
Description
cf_ob_info
This cookie is set by Cloudflare content delivery network and, in conjunction with the cookie 'cf_use_ob', is used to determine whether it should continue serving “Always Online” until the cookie expires.
cf_use_ob
This cookie is set by Cloudflare content delivery network and is used to determine whether it should continue serving “Always Online” until the cookie expires.
free_subscription_only
This session cookie is served by our membership/subscription system and controls which types of content you are able to access.
ls_smartpush
This cookie is set by Litespeed Server and allows the server to store settings to help improve performance of the site.
one_signal_sdk_db
This cookie is set by OneSignal push notifications and is used for storing user preferences in connection with their notification permission status.
YSC
This cookie is set by Youtube and is used to track the views of embedded videos.
Analytics cookies collect information about your use of the content, and in combination with previously collected information, are used to measure, understand, and report on your usage of this website.
Cookie
Description
bcookie
This cookie is set by LinkedIn. The purpose of the cookie is to enable LinkedIn functionalities on the page.
GPS
This cookie is set by YouTube and registers a unique ID for tracking users based on their geographical location
lang
This cookie is set by LinkedIn and is used to store the language preferences of a user to serve up content in that stored language the next time user visit the website.
lidc
This cookie is set by LinkedIn and used for routing.
lissc
This cookie is set by LinkedIn share Buttons and ad tags.
vuid
We embed videos from our official Vimeo channel. When you press play, Vimeo will drop third party cookies to enable the video to play and to see how long a viewer has watched the video. This cookie does not track individuals.
wow.anonymousId
This cookie is set by Spotler and tracks an anonymous visitor ID.
wow.schedule
This cookie is set by Spotler and enables it to track the Load Balance Session Queue.
wow.session
This cookie is set by Spotler to track the Internet Information Services (IIS) session state.
wow.utmvalues
This cookie is set by Spotler and stores the UTM values for the session. UTM values are specific text strings that are appended to URLs that allow Communigator to track the URLs and the UTM values when they get clicked on.
_ga
This cookie is set by Google Analytics and is used to calculate visitor, session, campaign data and keep track of site usage for the site's analytics report. It stores information anonymously and assign a randomly generated number to identify unique visitors.
_gat
This cookies is set by Google Universal Analytics to throttle the request rate to limit the collection of data on high traffic sites.
_gid
This cookie is set by Google Analytics and is used to store information of how visitors use a website and helps in creating an analytics report of how the website is doing. The data collected including the number visitors, the source where they have come from, and the pages visited in an anonymous form.
Advertising and targeting cookies help us provide our visitors with relevant ads and marketing campaigns.
Cookie
Description
advanced_ads_browser_width
This cookie is set by Advanced Ads and measures the browser width.
advanced_ads_page_impressions
This cookie is set by Advanced Ads and measures the number of previous page impressions.
advanced_ads_pro_server_info
This cookie is set by Advanced Ads and sets geo-location, user role and user capabilities. It is used by cache busting in Advanced Ads Pro when the appropriate visitor conditions are used.
advanced_ads_pro_visitor_referrer
This cookie is set by Advanced Ads and sets the referrer URL.
bscookie
This cookie is a browser ID cookie set by LinkedIn share Buttons and ad tags.
IDE
This cookie is set by Google DoubleClick and stores information about how the user uses the website and any other advertisement before visiting the website. This is used to present users with ads that are relevant to them according to the user profile.
li_sugr
This cookie is set by LinkedIn and is used for tracking.
UserMatchHistory
This cookie is set by Linkedin and is used to track visitors on multiple websites, in order to present relevant advertisement based on the visitor's preferences.
VISITOR_INFO1_LIVE
This cookie is set by YouTube. Used to track the information of the embedded YouTube videos on a website.