Challenges and Solutions in Oligonucleotide Analysis, Part IV – Solvent Mismatch, Denaturing vs. Non-Denaturing Conditions, and Non-Specific Adsorption

The recent expansion of pharmaceutical portfolios to include more therapeutic oligonucleotides (ONs) has led to a dramatic increase in research on chromatographic methods for analysis and purification of these molecules. This has been accompanied by a new set of method development and troubleshooting challenges, particularly for those whose background has been focused on small molecule separations, which have not previously been addressed in any detail in the “LC Troubleshooting” column. Thus, I am thrilled to have Martin Gilar join me for a series of “LC Troubleshooting” installments focused on challenges encountered in LC analyses of ONs. Martin is one of the world’s experts on this topic, and this series of articles should be a rich resource for LC practitioners working in this area.

— Dwight Stoll

In Part IV of this series, we will focus on aspects of oligonucleotide (ON) separations that were only briefly mentioned in previous installments. In Parts I¹ and II², we discussed practical tips for mobile phase selection, the role of mobile phase pH in separation, and the choice of column temperature. In part III, liquid chromatography-mass spectrometry (LC–MS) compatibility, the effect of sorbent pore size on separation, and common problems encountered in ON analysis—including retention drift, bacterial growth in aqueous buffers, and column stability—were discussed.³ In Part IV, we expand the discussion into less well-known problems of oligonucleotide liquid chromatography (ON-LC) separations: (i) peak splitting; (ii) peak distortion due to denaturation of nucleic acids on-column; and (iii) analyte losses due to non-specific adsorption (NSA).

Peak Splitting in LC Analysis of ONs

Practitioners of small-molecule LC analysis who transition to analysis of larger biopolymers quickly learn that the retention behaviors of small and large molecules are very different. For example, the retention of ONs is strongly affected by minor changes of mobile phase strength on the order of 1% of the strong solvent.^4–6 This retention behavior can lead to ON peak splitting or analyte breakthrough when injecting samples dissolved in a “strong” solvent. This behavior is most commonly observed when injecting samples dissolved in acetonitrile in reversed-phase (RP) LC, however, it can also be observed with other separation types. Figure 1 shows peak splitting of ONs separated by hydrophilic interaction liquid chromatography (HILIC), where water acts as a strong solvent. Most HILIC separations of ONs involve about 70% acetonitrile, and injecting large volumes of samples rich in water can lead to severe peak distortion.⁷ For IP RPLC separations of ONs, the preferred sample solvent is water or aqueous ion-pairing buffer. Presence of salts (for example, potassium chloride (KCl) or sodium chloride (NaCl) in the sample can also cause peak splitting in ion-pair (IP) RPLC,⁸ probably due to disruption of ion-pairing equilibria. The recommended sample solvent in anion-exchange LC is water, or the buffer used as the starting solvent in a gradient elution method. In size-exclusion chromatography (SEC), the sample solvent should ideally match the mobile phase. In affinity chromatography (AC) where immobilized oligo-deoxythymidine (dTn) is used to selectively retain messenger ribonucleic acid (mRNA) through hybridization with the polyA tail of the ribonucleic acid (RNA), the sample should be dissolved in the “binding buffer”; in this case, water acts as a strong solvent.

While LC peak splitting due to strong sample solvent affects small molecule separations as well,⁹ biopolymers are much more sensitive to this effect. Figure 1 shows serious splitting resulting from a mere 2-µL injection volume. The peak splitting is typically more prominent for early eluting (less retained) analytes. In addition to correct selection of the sample solvent, the partial loop injection mode also requires appropriate selection of the auxiliary solvent that fills a portion of the injection loop or injection needle to make sure it is not mismatched with the sample. Readers interested in learning more about this solvent mismatch effect, as well as potential solutions, are also referred to last month’s installment of “LC Troubleshooting”.¹⁰

Denaturing vs. Non-Denaturing LC Conditions?

In Part II of this series, we discussed the importance of column temperature for ON analysis.² Although the basic pH and high temperatures generally shorten column lifetime, denaturing conditions of 60 °C or higher are typically used for IP RPLC. Elevated column temperature improves peak shape and minimizes the impact of ON secondary structure on their retention. A recent publication discussed LC conditions suitable for analysis of small interfering ribonucleic acid (siRNA).¹¹ Non-denaturing conditions are useful for analysis of intact duplexes, while denaturing conditions are used for analysis of single-stranded ONs. The transition from duplex (or inter/intramolecular secondary structures such as hairpins or aggregates) into linearized single-stranded ONs depends on mobile composition and LC stationary phase type. The stability of duplexes/secondary structures increases with increasing mobile phase ionic strength. The type of cation has a strong effect; duplexes are more stable in sodium chloride or ammonium acetate solutions than in triethylammonium acetate. Acetonitrile, hexafluoropropanol, and other organic mobile phase additives further lower the melting temperature (T_m; a measure of duplex stability). A general recommendation is to operate above T_m for ON analysis or well below T_m if the goal is to investigate ON secondary structure or duplexes. When working near T_m, on-column analyte melting will result in peak smearing.¹¹ This scenario is illustrated in Figure 2. IP RPLC analysis at 40 °C preserves the intact siRNA duplex; small amounts of single-stranded species are also observed, and they elute prior to the duplex. The retention time of single-stranded ONs is highlighted by overlaying chromatograms for 21 nt and 23 nt complementary ONs (red and blue chromatograms, respectively). Analyses at 50 °C (and 60 °C) show wider peaks as a result of on-column duplex melting. However, moving to 70 °C yields two clear peaks for single-stranded ONs; the siRNA duplex melts rapidly when it enters the column. Figure 2b shows SEC analysis of 102 nt guide RNA (gRNA); the second earlier eluting peak is probably aggregated molecules. A multi-angle light scattering (MALS) detector (Figure 2c) was used to confirm the identity of the peaks. The first peak appears to be a dimer (64,400 g/mol) of the guide ribonucleic acid (gRNA; 34,230 g/mol). Because RNA has a propensity to form stronger secondary structures than deoxyribonucleic acid (DNA) molecules, denaturing LC conditions are preferred for RNA analysis.

Non-Specific Adsorption of ONs on LC Hardware

One of the unusual behaviors LC practitioners observe in ONs analysis is that the very first injection into a new column is not consistent with subsequent injections. Low recovery or missing peaks can be observed in these chromatograms. Additional injections gradually improve the recovery. Such behavior is illustrated in Figure 3 for IP RPLC ONs analysis with hexylammonium acetate as the ion-pairing system. However, similar behavior is often observed with anion-exchange LC, HILIC, or SEC analyses of ONs. Some analysts routinely practice system “conditioning”, which involves repeated injections of a sacrificial ON sample to condition the system (Figure 3a). This NSA phenomenon^12–15 is caused by the adsorption of acidic molecules (ONs) on metal-oxide surfaces of LC hardware.¹⁶ Figure 3c illustrates the mechanism of NSA: metal surfaces are covered with a metal-oxide (MO) layer that has an amphoteric nature. MO surfaces are positively charged under neutral or acidic conditions and strongly adsorb negatively charged ONs, leading to analyte loss and poor recovery. During system conditioning, the MO surfaces are saturated with an excess of ONs, leading to improved sample recovery (Figure 3a).

A recent study used LC column frits to investigate the NSA phenomenon in detail.¹⁶ The frits have the largest surface area in an LC system, and therefore strongly affect the observed NSA. Figure 3b shows that a single 2.1-mm-i.d. stainless-steel frit placed between injector and detector (no column in the flow path) causes significant loss of a 25 nucleotide (nt) ON in early injections. The ON signal gradually increases and reaches 95–100% recovery after 40 injections. The study confirmed that ON loss due to NSA is more pronounced at acidic pH, and when injecting small amounts (that is, mass, or moles) of analyte.

Various approaches have been explored to minimize ON losses due to NSA, including the use of non-metallic LC surfaces, system washing with phosphoric acid, and analyte conditioning as discussed above. Recently, surface modification of LC hardware with hybrid organic-inorganic silica surface technology (HST) was shown to be an effective solution to the NSA problem in ON analysis. Columns with this technology have been introduced commercially for separations of nucleic acids by RP LC, IP RPLC, HILIC, and SEC.

Another common nuisance in ON analysis is sample carryover (often 0.5–1% or greater), which can be directly related to NSA. ONs adsorbed on metallic surfaces slowly bleed from the LC system following elution of the main peak, and can show up as peaks or elevated baselines in subsequent blank analyses. The use of LC hardware with HST technology has potential to reduce the carryover problem as well.¹⁷

Summary

In this installment of “LC Troubleshooting” we have discussed three more practical aspects of therapeutic oligonucleotide analysis that can be confusing and frustrating, particularly for those coming from a background of small molecule analysis. These include: i) peak splitting and distortion due to the effects of mismatch between the sample and mobile phase solvents; ii) the effects of column temperature and mobile phase composition on the stability and melting of oligonucleotide secondary structures (for example, double-stranded duplexes); and iii) analyte loss and poor recovery of oligonucleotides due to non-specific adsorption on metal oxide surfaces. In each case, we’ve briefly discussed the fundamental issue at hand and provided tips and recommendations for avoiding pitfalls in method development. We’re hopeful that these insights will help users avoid problems before they occur, and/or help identify potential problems during troubleshooting when unfavorable results and behaviors are observed.

Acquity, UPLC and BEH are trademarks of Waters Technologies Corporation.

References

1. Gilar, M.; Stoll, D. Challenges and Solutions in Oligonucleotide Analysis, Part I: An Overview of Liquid Chromatography Methods and Applications. LCGC International 2025, 2 (7), 8–15. DOI: https://doi.org/10.56530/lcgc.int.aw2283b9

2. Gilar, M.; Stoll, D. Challenges and Solutions in Oligonucleotide Analysis, Part II: A Detailed Look at Ion-Pairing Reversed-Phase Separations. LCGC International 2026, 3 (2), 8-15. DOI: 10.56530/lcgc.int.mh1387e9

3. Gilar, M.; Stoll, D. Challenges and Solutions in Oligonucleotide Analysis, Part III: LC-MS Methods. LCGC International 2026, 3 (3), 10–14. DOI: 10.56530/lcgc.int.dv6581j4

4. Gilar, M.; Fountain, K. J.; Budman, Y.; et al. Ion Pair Reversed-Phase High-Performance Liquid Chromatography Analysis of Oligonucleotides: Retention Prediction. J. Chromatogr. A 2002, 958 (1-2), 167–182. DOI: 10.1016/s0021-9673(02)00306-0

5. Gilar, M.; Neue, U. D. Peak Capacity in Gradient Reversed-Phase Liquid Chromatography of Biopolymers. Theoretical and Practical Implications for the Separation of Oigonucleotides. J. Chromatogr. A 2007, 1169 (1-2), 139–150. DOI: 10.1016/j.chroma.2007.09.005

6. Sorensen, M. J.; Paulines, M. J.; Maloney, T. D. Evaluating Orthogonality Between Ion-Pair Reversed Phase, Anion Exchange, and Hydrophilic Interaction Liquid Chromatography for the Separation of Synthetic Oligonucleotides. J. Chromatogr. A 2023, 1705, 464184. DOI: 10.1016/j.chroma.2023.464184

7. Verduin, J.; Tutis, L.; Kritsima, A.; et al. Enabling Large-Volume Injections in Hydrophilic Interaction Chromatography of Oligonucleotides Through In-Line Mmixing. J. Sep. Sci. 2026, 49 (2), e70372. DOI: 10.1002/jssc.70372

8. Birdsall, R. E.; Gilar, M.; Shion, H.; et al. Reduction of Metal Adducts in Oligonucleotide Mass Spectra in Ion-Pair Reversed-Phase Chromatography/Mass Spectrometry Analysis. Rapid Commun. Mass Spectrom. 2016, 30 (14), 1667–1679. DOI: 10.1002/rcm.7596

9. Gritti, F.; Gilar, M.; Hill, J. Mismatch Between Sample Diluent and Eluent: Maintaining Integrity of Gradient PeaksUsing in silico Approaches. J. Chromatogr. A 2019, 1608, 460414. DOI: 10.1016/j.chroma.2019.460414

10. Stoll, D. More Tools for Sample Injection when Faced with Mobile Phase/Sample Solvent Mismatch. LCGC International 2026, Online in May.

11. Gilar, M.; Redstone, S.; Gomes, A. Impact of Mobile and Stationary Phases on siRNA Duplex Stability in Liquid Chromatography. J. Chromatogr. A 2024, 1733, 465285. DOI: 10.1016/j.chroma.2024.465285

12. Tuytten, R.; Lemière, F.; Witters, E.; et al. Stainless Steel Electrospray Probe: A Dead End for Phosphorylated Organic Compounds? J. Chromatogr. A 2006, 1104 (1-2), 209–221. DOI: 10.1016/j.chroma.2005.12.004

13. Iqubal, M. A.; Sharma, R.; Kamaluddin. Surface Interaction of Ribonucleic Acid Constituents with Spinel Ferrite Nanoparticles: A Prebiotic Chemistry Experiment. RSC Adv. 2016, 6, 68574–68583. DOI: 10.1039/C6RA12247G

14. Lardeux, H.; Goyon, A.; Zhang, K.; et al. The Impact of Low Adsorption Surfaces for the Analysis of DNA and RNA Ooligonucleotides. J. Chromatogr. A 2022, 1677, 463324. DOI: 10.1016/j.chroma.2022.463324

15. Nguyen, J. M.; Gilar, M.; Koshel, B.; et al. Assessing the Impact of Nonspecific Binding on Oligonucleotide Bioanalysis. Bioanalysis 2021, 13(16), 1233–1244. DOI: 10.4155/bio-2021-0115

16. Gilar, M.; DeLano, M.; Gritti, F. Mitigation of Analyte Loss on Metal Surfaces in Liquid Chromatography. J. Chromatogr. A 2021, 1650, 462247. DOI: 10.1016/j.chroma.2021.462247

17. Yogendrarajah, P.; Suarez Marina, I.; Verluyten, W.; et al. Analysis of siRNA with Denaturing and Non-Denaturing Ion-Pair Reversed-Phase Liquid Chromatography Methods. LCGC North America 2023, 41 (2), 60–66.