Challenges and Solutions in Oligonucleotide Analysis, Part II: A Detailed Look at Ion-Pairing Reversed-Phase Separations

Antisense oligonucleotides (ASOs), small interfering ribonucleic acid (siRNA), and messenger RNA (mRNA) are promising classes of therapeutic compounds. Their oligomeric/polymeric nature, complexity, and large size present challenges for analytical methods used to characterize these molecules. In this second installment of a multi-part series, we describe the principles of ion-pair reversed-phase high performance liquid chromatography (IP-RP-HPLC), discuss strategies for method development, and provide practical tips for robust liquid chromatography (LC) analyses of oligonucleotide (ON) mixtures.

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 again for this second installment in a multi-part series of “LC Troubleshooting” articles focused on challenges encountered in LC analyses of oligonucleotides. Gilar 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

Ion-pair reversed-phase liquid chromatography(IP-RP-HPLC) is a flexible and efficient technique for analysis of oligonucleotides (ONs). It is applicable to antisense oligonucleotides (ASOs), short interfering RNA (siRNA), single-guide RNA (sgRNA), transport RNA (tRNA), and messenger RNA (mRNA). The main reason that IP-RP-HPLC separation of ONs gained prominence in recent years is that it enables high resolution of full-length product (FLP) oligonucleotide from closely related molecules, including truncated metabolites, failed products of synthesis, and structurally similar impurities of the same length as the FLP. Another key to success of the IP-RP-HPLC method is its compatibility with mass spectrometric (MS) detection. The LC–MS capability becomes particularly useful in cases when the closely related impurities are not chromatographically resolved from the main FLP peak. In such cases, the resolution and quantitation of ON impurities is accomplished in the MS domain by extracting ion chromatograms for their unique masses.

Other methods, such as hydrophilic interaction chromatography (HILIC), have wide applicability to ONs analysis. HILIC is also compatible with LC–MS, but IP-RP-HPLC remains the method of the choice in many laboratories as a result of its higher chromatographic resolving power and higher LC–MS sensitivity. This is especially the case for analysis of ONs longer than about 30 nucleotides (nt).

In principle, IP-RP-HPLC is a mixed-mode method, where both hydrophobic interactions of ONs with the RPLC sorbent and ionic interactions with ion-pairing reagent contribute to the retention mechanism. The principal optimization parameter available to the analyst during method development is the choice of IP reagent (IPR). IP-RP-HPLC was developed in 1970s, and the retention mechanism was subsequently investigated in 1980s and 1990s, but only for separation of small polar molecules under isocratic conditions. During this period the guidelines for development of methods involving gradient elution were not established in part because in the 1990s the development of therapeutic ONs was in its infancy. In spite of some seminal publications on separations of ONs by IP-RP-HPLC analysis published over the last 30 years (1–11), a gap in knowledge still remains today. Separation scientists new to ON analysis are often surprised with unexpected retention drifts, non-specific sample adsorption, and high carryover compared to their experiences with analysis of small molecules. Therefore, many analysts are looking for guidelines and practical tips for ON method development. In this installment, we will address some questions frequently asked by analysts in this area, with an emphasis on best practices that can help prevent troubleshooting exercises:

• What is IP-RP-HPLC?
• What is the retention mechanism for ONs under IP-RP-HPLC conditions?
• What is the impact of IP reagents on ON separation?
• What are some guidelines that can be used during method development?

History of IP-RP-HPLC and Its Application to ON Analysis

Question #1: What is IP-RP-HPLC?

IP-RP-HPLC was introduced in the 1970s, soon after the commercial introduction of C18 columns for RP-HPLC. While RP-HPLC emerged as the prevalent separation mode in LC for a wide spectrum of compounds, it was less applicable to analysis of highly polar compounds that were poorly retained on C18 columns. Polar analytes are often charged in the mobile phase; Knox and Jurand used an addition of charged IP reagents (IPRs) with amphiphilic properties to enhance the retention of analytes with the opposite charge (for example, amines that exhibit positive charge are used to enhance retention of negatively charged ONs) (12).

Question #2: What is the Retention Mechanism for ONs Under IP-RP-HPLC Conditions?

The retention mechanism of IP-RP-HPLC is still the subject of debate in the chromatography community. Readers interested in learning more about the arguments supporting different views are referred to the references in the following list. The classic hypotheses presented in reports from the 1970’s through the 1990s are:

1. Ion-pair model (IPM), where an IPR and charged analyte of interest form a neutral (hydrophobic) ion-pair in the mobile phase that is subsequently adsorbed on the RP-HPLC sorbent (13);
2. Dynamic ion-exchange model (DIEM), where an IPR present in the mobile phase is first adsorbed on the stationary phase, dynamically modifies it with charge, and the analyte is subsequently retained by charge-to-charge interactions as in ion-exchange chromatography (13,14);
3. Dynamic complex model (DCM), where both processes take place simultaneously. Ion-pairs coexist with free IPR and analyte in the mobile phase. Ion-pairs can displace the ion-pairing reagents adsorbed on the stationary phase (15,16). An illustration of the processes involved in this model is shown in Figure 1b.

Question #3: What is the Impact of IP Reagents on ON Separation?

In the absence of IPRs, unmodified oligonucleotides elute from C18 columns with less than 5% acetonitrile in the mobile phase and are generally not resolved by length. The retention and resolution of ONs improves with addition of any of a variety of alkylamine IPRs (see examples in Figure 1a) to the mobile phase. The concentration and hydrophobicity of these IPRs have strong effects on separation selectivity. The selection of IPR is an important optimization parameter in IP-RP-HPLC and will be discussed later in this article (3,4,11,17).

The three models described above (IPM, DIEM, DCM), which were developed to describe retention of small molecules under IP-RP-HPLC conditions, are also applicable to IP-RP-HPLC separation of ONs. However, the models assume isocratic elution conditions, where the free IPR and ion-pairs are equilibrated between the mobile and stationary phases. ONs are typically separated with organic solvent gradient elution conditions, and gradients disrupt the established pre-run equilibration of the IPR between the mobile and stationary phases. At the beginning of a gradient where the concentration of organic solvent in the mobile phase is low, the hydrophobic IPR is strongly adsorbed on the stationary phase, but at the end of the gradient the IPRs are effectively desorbed. To better understand the adsorption of IPRs on a C18 sorbent, we measured the retention factor k for several amines (Figure 2) in 10 mM ammonium acetate buffer at 60 °C in various mobile phase concentrations of acetonitrile. Under these conditions the IPRs are almost entirely protonated and positively charged because the pK_as of the protonated species are well above 9. The data in Figure 2 are useful to sort the IP reagents into the classes of hydrophilic, hydrophobic and very hydrophobic alkylamines as presented in Figure 1. Figure 2 shows that in mobile phases typically used at the start of solvent gradients (that is, below about 10%), the IPRs are adsorbed on the stationary phase with k >1-10. In other words, the majority of the IPR present in the column is adsorbed to the stationary phase.

Sorting the IPRs into the classes (Figure 1) is somewhat arbitrary, but practically useful. For example, we see that triethylamine (TEA) is a rather hydrophilic IP reagent; it requires low mobile phase organic solvent concentrations for it to be functional as an IPR in ON separations. Tripropylamine (TPA), dibutylamine (DBA), and hexylamine (HA) have comparable hydrophobicity; with these IPRs one can use similar gradient elution methods. The very hydrophobic IPRs octylamine (OA), tributylamine (TBA), and dihexylamine (HA) require more than 50% of acetonitrile to elute ONs from C18 columns. Gilar and associates confirmed that alkylamine IPR hydrophobicity correlates with ON retention and n/n-x resolution (that is, the resolution of a given ON [n] and the same sequence but with one less base [n-1]). The authors suggested that hydrophobic IPRs are better suited for challenging separations of long ONs (18).

Donegan and coauthors (8) and Gilar and associates (11) propose that IP-RP-HPLC is a mixed-mode separation. Hydrophilic IP systems permit greater contribution of hydrophobic forces to separation, whereas the use of hydrophobic IP amines lead to the predominantly charge-based ON separations with n/n-x resolution independent of the ON sequence or modifications.

The data in Figure 2 were used to establish the log k=log k₀-S• relationships for different IPRs. Once the S and k₀ values are known for a given IPR, we can calculate the local retention factor of the IPR in the mobile phase present at the column exit when a given ON elutes from the column. Doing so, we found that regardless of the IPR chemistry (hydrophilic, hydrophobic, or very hydrophobic), the ONs always eluted after the IPR retention factor fell below about 0.2 or 0.07 for 20 nt and 40 nt ONs, respectively. In other words, ONs are retained under IP-RP-HPLC conditions until the charged IPR is stripped from the C18 sorbent surface. In the light of this observation, we believe that the dynamic ion-exchange model best describes the retention mechanism for ONs under IP-RP-HPLC conditions. It is possible that analyte-IPR ion-pairs also exist in the mobile phase (15,19,20), but the fact that the IPR is strongly adsorbed on the C18 sorbent at the start of a gradient elution program, and nearly fully desorbed at the moment when ONs exit the column, supports the DIEM hypothesis. The scheme illustrated in Figure 1b depicts the equilibrium between a negatively charged ON, positively charged IPR, and the neutral ion-pair [ON:IP] in the mobile phase. This equilibrium should not be strongly affected by the organic content per se. Each component in solution — IP, ON, or [ON:IP] pair — can be adsorbed on the C18 stationary phase. The bold arrows in Figure 1b highlight the processes that we believe dominate the IP-RP-HPLC retention. First, the hydrophobic IPR is adsorbed on stationary phase surface, followed by electrostatic interaction of the ON with the charge-loaded C18 sorbent. The result is illustrated in Figure 1c. When solvent gradient elution is used with IP-RP-HPLC, retention of the ON is decreased dramatically as the IPR is desorbed from the stationary phase surface. Secondary hydrophobic interactions can contribute to the IP-RP-HPLC separation of ONs when using hydrophilic IP reagents, especially when ONs are conjugated with hydrophobic labels such as dyes or lipids (21).

Question #4: What Are Some Guidelines that Can Be Used During Method Development?

Efficiency vs. Selectivity

In the first installment in this series (22), we stated that all chromatographic methods have principal limitations for separation of ONs closely related to the target product. N/n-x separation becomes progressively more difficult for longer oligonucleotides, as shown for example in the IP-RP-HPLC separation in Figure 3a. In such scenarios, where it is difficult to manipulate the separation selectivity of closely eluting species, highly efficient columns provide the only means to further improve resolution. Figure 3a illustrates that when the separation selectivity is identical, resolution improves when the column length is held constant, but the column is packed with smaller particles. This explains why UHPLC columns packed with sub-2 μm particles are generally preferred for high resolution separations of ONs. One notable exception is the analysis of fully phosphorothioated (PS) ONs. As a result of the many diastereomers present in a sample like this, the peak widths for PS ONs are wider compared to phosphorodiester ONs (23–25). The analysis for PS ONs will be discussed in more detail in a future installment in this series.

IP-RP-HPLC offers more flexibility than other LC modes because it is a mixed-mode technique. The spacing between closely eluting ON species depends not only on their charges (lengths), but also their hydrophobicities. Figure 3b illustrates that for heterooligonucleotide species the spacing between the n/n-x peaks is affected by the specific sequence of bases. Sequence can either enhance or diminish the resolution of critical pairs. In extreme cases the retention order may even switch; this was observed for the 24 and 25 nt peaks in Figure 3b, where the 25 nt ON elutes before the 24 nt one, which coeluted with the 26 and 27 nt species.

In a recent study, we proposed some empirical rules that dictate IP-RP-HPLC selectivity (11):

• Peak spacing between truncated ON species depends on the hydrophobicity of nucleotides;
• The relative hydrophobicity of nucleotides is U<C<G<A<T; 2’-O-methyl or 2’-Fluoro modifications increase the hydrophobicity to a similar degree;
• Truncation (loss) of hydrophobic nucleotides in the sequence results in enhanced resolution of n/n-1 peaks. Loss of hydrophilic nucleotides decreases the resolution of n/n-1 peaks;
• The uneven spacing between peaks is amplified with hydrophilic IP reagents. This could be used as optimization tool to improve the separation of critical pairs of interest. HILIC separations exhibit selectivity that is generally the opposite of what is observed with IP-RP-HPLC and can be used in scenarios where an IP-RPLC method does not provide enough selectivity;
• Hydrophobic IPRs minimize the impact of oligonucleotide sequence on selectivity. In this case, retention is driven primarily by the charge on the analyte (and therefore, the length of the ON), and separation is largely sequence independent.

The mixed-mode nature of IP-RP-HPLC becomes particularly apparent in the analysis of ONs conjugated with fluorescent dyes, dimethoxytrityl (DMT) protecting groups, or lipids. Figure 4a illustrates the IP-RP-HPLC separation of 25 nt ONs of the same sequence but labeled with different dyes. Native (unlabeled) ONs elute first, followed by other ONs retained in the order of the dye hydrophobicity. This property of IP-RP-HPLC is used for purification of dually labeled qPCR probes from singly-labeled and unlabeled products of synthesis (21). Please note that if the ONs are conjugated with a very hydrophobic moiety (DMT group or lipid), the retention order may be reversed and shorter oligonucleotides elute after the FLP (Figure 4b), at least with hydrophilic ion-pairing systems (for example, TEA). In these cases, the expected elution order (short ONs eluting before the FLP) can still be achieved with hydrophobic IP reagents (27).

Selection of Column Temperature

ONs are most often separated at 60 °C. The rationale for this selection is simple: analyte diffusivity improves as temperature increases, which improves mass transfer and leads to narrower peaks. Second, the high separation temperature denatures potential secondary structures of ONs and minimizes the impacts of these structures on LC retention. Naturally, there are certain sequence motifs that have highly stable secondary structures, such as C/G-rich oligonucleotides, G-quadruplexes, stable hairpins, and oligo G sequences (28). In some cases, temperatures of 90 °C or even higher are required to achieve acceptable peak shapes. However, such high temperatures generally reduce column lifetime, so these conditions should be used only when necessary. Thus, the 60 °C column temperature appears to be a good compromise that balances reasonable column lifetime with improved peak efficiency. This column temperature selection was further endorsed by Sturm and colleagues, who investigated the impact of temperature on retention prediction for ONs. The authors concluded that 60 °C is the lowest temperature at which the retention of ONs was accurately predicted. Temperatures below 50 °C yielded many aberrant results, which the authors assert were due to ONs secondary structures giving rise to unexpected retention shifts.

On the other hand, duplex DNA and siRNA separations are performed at temperatures below 40 °C to preserve duplex stability. Low mobile phase buffer concentration and high organic modifier content can also destabilize the duplexes and induce on-column melting. The impact of mobile phase composition and LC separation mode on siRNA duplex melting temperature was investigated by Gilar and associates (29). IP-RP-HPLC is suitable for siRNA duplex analysis and provides excellent resolution of single-strand RNA species from siRNA duplex, when performed at low column temperature.

Guidelines for Gradient Elution

We mentioned earlier in this installment that separation selectivity for structurally similar ONs is limited. To achieve the desirable n/n-x resolution the analysis is performed with shallow gradients. When using hydrophilic IPRs such as TEA, the gradient could be as shallow as 0.1% acetonitrile per minute. When using hydrophobic IPRs, slightly steeper gradients of 0.25–0.5% acetonitrile per minute are often used. These gradients are significantly shallower than those used for small molecules, which can be challenging for LC pumps. To improve retention time repeatability under these conditions, solvents A and B may be pre-mixed (3). For example, when using 100 mM hexylamine acetate as the IPR, we prepare 100 mM hexylamine acetate in 25/75 acetonitrile/water as mobile phase A, and 100 mM hexylamine acetate dissolved in 75/25 acetonitrile/water as mobile phase B. Addition of organic solvent to both the A and B solutions both improves solubility of hexylamine and inhibits the growth of bacteria in the mobile phase.

Most ONs separations are performed with simple linear solvent gradients. Separation selectivity is typically sufficient for short ONs, but resolution of longer species is more difficult. Non-linear gradients can be used to achieve simultaneous separation of short and long ONs. Figure 5 illustrates the benefit of a segmented concave gradient for separation of long ONs.

Suggested optimization guidelines for mobile phase gradients are:

• Select the type of ion-pairing system based on the desired performance, MS compatibility (see the next installment in this series for more on MS compatibility), and type of application.
• Select the initial mobile phase strength (that is, organic solvent concentration) to ensure sufficient retention of the ONs of interest. Explore the effects of different gradient slopes. Steep gradients may lead to partial loss of resolution; shallow gradients may improve separation, but at the cost of long analysis times.

When a suitable gradient slope is identified for the required resolution, one can optimize the starting point in the gradient to reduce analysis time. If the starting and ending concentrations of organic solvent in the mobile phase are increased by the same amount, the gradient slope (that is the change in % organic solvent per minute) will be unchanged, but all peaks will shift to earlier times, resulting in a shorter method without loss of n/n-x resolution.

Initial method development work is best performed with short columns (for example, 50 mm length) to save time. If the achieved resolution is unsatisfactory, the method can be transferred to longer columns. Transferring from a 50 mm to a 100 mm column will improve the resolution 1.4-fold (square root of two), at the cost of a doubling of the analysis time. Similarly, transferring from a 50 mm to a 150 mm column will improve resolution 1.7-fold.

Tips for ONs Analysis by IP-RP-HPLC

Analysts new to the field of ONs often find that IP-RP-HPLC separation requires different expertise than analysis of small molecules. In this section, we highlight practical tips to address some of the challenges encountered in IP-RP-HPLC analyses.

IP-RP-HPLC uses volatile mobile phase constituents. The volatility of commonly used IPRs is a pre-requisite for LC–MS applications. However, evaporation of mobile phase components may cause practically significant intra-day and inter-day retention time shifts. Batch-to-batch variability in mobile phase preparation may arise due to difficulty in measuring the volatile mobile phase constituents (for example, HFIP). Solubility of hydrophobic IPRs in aqueous mobile phases is limited; long periods of mixing required to homogenize these solvents will result in evaporative loss of alkylamines if the container is not closed. Please note that both amines and HFIP have an intense and unpleasant odor; these solutions should be prepared in a fume hood.

Aqueous solutions buffered around pH 7 are susceptible to bacterial growth. Bacteria in the mobile phases are primary sources of column failures due to occlusion of the inlet frit. We recommend adding organic solvent to both A and B solvents to improve IPR solubility and inhibit bacteria growth.

pH adjustment of alkylamine-containing mobile phases to around pH 7 with acetic acid is problematic since the pK_as of amines and acetic acid (about 10 and 5, respectively) are far from the target pH of 7. Preparing these solutions in the pH range of 8.0 to 8.5 provides slightly more buffer capacity, and also has the benefit of reducing non-specific adsorption of ONs on LC hardware (30).

Non-specific adsorption (NSA; that is, ON losses on LC and MS system components) has been observed in IP-RP-HPLC (30,31). Use of “inert” columns is recommended over column conditioning that is often practiced by analysts (32,33). Relatedly, NSA can also lead to analyte carryover, which can exceed levels of 0.5–1%. This carryover is caused by ONs leaching from LC hardware during the column equilibration prior to an analysis where solvent gradient elution is used. The sources of NSA and its mitigation will be discussed in more detail in a future installment in this series.

Summary

IP-RP-HPLC separations are flexible and commonly used for analysis of therapeutic ONs. Despite the widespread use of this technique, the mechanism of IP-RP-HPLC separation is not completely understood. In this installment we described our understanding of how IP-RP-HPLC separations of ONs work, based on the published literature and our own experiences, and provided practical method development guidance and troubleshooting tips. The choices of LC column, ion-pairing reagent, and buffering acid on separation selectivity were highlighted in Figures 1–5. We hope this advice helps readers develop highly effective and rugged methods for their ON analyses. The mobile phase MS compatibility will be discussed in the next part of this series.

BEH is a trademark of Waters Technologies Corporation. TaqMan is a trademark of Roche Molecular Systems, Inc.

References

1. Huber, C. G.; Oefner, P. J.; Bonn, G. K. High-Resolution Liquid Chromatography of Oligonucleotides on Nonporous Alkylated Styrene-Divinylbenzene Copolymers. Anal. Biochem. 1993, 212 (2), 351–358. https://doi.org/10.1006/abio.1993.1340.
2. Apffel, A.; Chakel, J. A.; Fischer, S.; Lichtenwalter, K.; Hancock, W. S. Analysis of Oligonucleotides by HPLC−Electrospray Ionization Mass Spectrometry. Anal. Chem. 1997, 69 (7), 1320–1325. https://doi.org/10.1021/ac960916h.
3. Gilar, M.; Fountain, K. J.; Budman, Y.; Neue, U. D.; Yardley, K. R.; Rainville, P. D.; Russell Ii, R. J.; Gebler, J. C. Ion-Pair Reversed-Phase High-Performance Liquid Chromatography Analysis of Oligonucleotides: J. Chromatogr. A 2002, 958 (1–2), 167–182. https://doi.org/10.1016/S0021-9673(02)00306-0.
4. Levin, D. S.; Shepperd, B. T.; Gruenloh, C. J. Combining Ion Pairing Agents for Enhanced Analysis of Oligonucleotide Therapeutics by Reversed Phase-Ion Pairing Ultra Performance Liquid Chromatography (UPLC). J. Chromatogr. B 2011, 879 (19), 1587–1595. https://doi.org/10.1016/j.jchromb.2011.03.051.
5. Enmark, M.; Rova, M.; Samuelsson, J.; Örnskov, E.; Schweikart, F.; Fornstedt, T. Investigation of Factors Influencing the Separation of Diastereomers of Phosphorothioated Oligonucleotides. Anal Bioanal Chem. 2019, 411 (15), 3383–3394. https://doi.org/10.1007/s00216-019-01813-2.
6. Roussis, S. G.; Pearce, M.; Rentel, C. Small Alkyl Amines as Ion-Pair Reagents for the Separation of Positional Isomers of Impurities in Phosphate Diester Oligonucleotides. J. Chromatogr. A 2019, 1594, 105–111. https://doi.org/10.1016/j.chroma.2019.02.026.
7. Goyon, A.; Yehl, P.; Zhang, K. Characterization of Therapeutic Oligonucleotides by Liquid Chromatography. J. Pharm. Biomed. Anal. 2020, 182, 113105. https://doi.org/10.1016/j.jpba.2020.113105.
8. Donegan, M.; Nguyen, J. M.; Gilar, M. Effect of Ion-Pairing Reagent Hydrophobicity on Liquid Chromatography and Mass Spectrometry Analysis of Oligonucleotides. J. Chromatogr. A 2022, 1666, 462860. https://doi.org/10.1016/j.chroma.2022.462860.
9. Rentel, C.; Gaus, H.; Bradley, K.; Luu, N.; Kolkey, K.; Mai, B.; Madsen, M.; Pearce, M.; Bock, B.; Capaldi, D. Assay, Purity, and Impurity Profile of Phosphorothioate Oligonucleotide Therapeutics by Ion Pair–HPLC–MS. Nucleic Acid Therapeutics 2022, 32 (3), 206–220. https://doi.org/10.1089/nat.2021.0056.
10. 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. https://doi.org/10.1016/j.chroma.2023.464184.
11. Gilar, M.; Schomann, N.; Schott, S.; Rühl, M. Impact of Nucleotide Hydrophobicity on Oligonucleotides Separation in Liquid Chromatography. J. Chromatogr. A 2025, 1753, 465968. https://doi.org/10.1016/j.chroma.2025.465968.
12. Knox, J. H.; Jurand, J. Determination of Paracetamol and Its Metabolites in Urine by High-Performance Liquid Chromatography Using Ion-Pair Systems. J. Chromatogr. A 1978, 149, 297–312. https://doi.org/10.1016/S0021-9673(00)80994-2.
13. Bidlingmeyer, B. A.; Deming, S. N.; Price, W. P.; Sachok, B.; Petrusek, M. Retention Mechamism for Reversed-Phase Ion-Pair Liquid Chromatography. J. Chromatogr. A 1979, 186, 419–434. https://doi.org/10.1016/S0021-9673(00)95264-6.
14. Bartha, A.; Vigh, G.; Varga-Puchony, Z. Basis of the Rational Selection of the Hydrophobicity and Concentration of the Ion-Pairing Reagent in Reversed-Phase Ion-Pair High-Performance Liquid Chromatography. J. Chromatogr. A 1990, 499, 423–434. https://doi.org/10.1016/S0021-9673(00)96989-9.
15. Melander, W. R.; Horváth, C. Mechanistic Study on Ion-Pair Reversed-Phase Chromatography. J. Chromatogr. A 1980, 201, 211–224. https://doi.org/10.1016/S0021-9673(00)83876-5.
16. Horváth, C.; Melander, W.; Molnar, I.; Molnar, P. Enhancement of Retention by Ion-Pair Formation in Liquid Chromatography with Nonpolar Stationary Phases. Anal. Chem. 1977, 49 (14), 2295–2305. https://doi.org/10.1021/ac50022a048.
17. Enmark, M.; Furlan, I.; Habibollahi, P.; Manz, C.; Fornstedt, T.; Samuelsson, J.; Örnskov, E.; Jora, M. Expanding the Analytical Toolbox for the Nondenaturing Analysis of siRNAs with Salt-Mediated Ion-Pair Reversed-Phase Liquid Chromatography. Anal. Chem. 2024, 96 (47), 18590–18595. https://doi.org/10.1021/acs.analchem.4c05248.
18. Gilar, M.; Doneanu, C.; Gaye, M. M. Liquid Chromatography Methods for Analysis of mRNA Poly(A) Tail Length and Heterogeneity. Anal. Chem. 2023, 95 (38), 14308–14316. https://doi.org/10.1021/acs.analchem.3c02552.
19. Shibue, M.; Mant, C. T.; Hodges, R. S. The Perchlorate Anion Is More Effective than the Trifluoroacetate Anion as an Ion-Pairing Reagent for Reversed-Phase Chromatography of Peptides. J. Chromatogr. A 2005, 1080 (1), 49–57. https://doi.org/10.1016/j.chroma.2005.02.063.
20. Tutiš, L.; Ferguson, P. D.; Benstead, D.; Clarke, A.; Heatherington, C.; Gripton, C.; Vanhinsbergh, C. J.; Somsen, G. W.; Gargano, A. F. Ion-Pairing Hydrophilic Interaction Chromatography for Impurity Profiling of Therapeutic Phosphorothioated Oligonucleotides. Anal. Chem. 2025, 97 (29), 15717–15726. https://doi.org/10.1021/acs.analchem.5c01407.
21. Fountain, K. J.; Gilar, M.; Budman, Y.; Gebler, J. C. Purification of Dye-Labeled Oligonucleotides by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography. J. Chromatogr. B 2003, 783 (1), 61–72. https://doi.org/10.1016/S1570-0232(02)00490-7.
22. Gilar, M.; Stoll, D. R. Challenges and Solutions in Oligonucleotide Analysis, Part I: An Overview of Liquid Chromatography Methods and Applications. LCGC International 2025, 2 (7), 8–15. https://www.chromatographyonline.com/view/oligonucleotide-analysis-chromatography-methods-therapeutic-nucleic-acids
23. Gilar, M.; Koshel, B. M.; Birdsall, R. E. Ion-Pair Reversed-Phase and Hydrophilic Interaction Chromatography Methods for Analysis of Phosphorothioate Oligonucleotides. J. Chromatogr. A 2023, 1712, 464475. https://doi.org/10.1016/j.chroma.2023.464475.
24. Kadlecová, Z.; Kalíková, K.; Tesařová, E.; Gilar, M. Phosphorothioate Oligonucleotides Separation in Ion-Pairing Reversed-Phase Liquid Chromatography: Effect of Ion-Pairing System. J. Chromatogr. A 2022, 1676, 463201. https://doi.org/10.1016/j.chroma.2022.463201.
25. Kadlecová, Z.; Kalíková, K.; Tesařová, E.; Gilar, M. Phosphorothioate Oligonucleotides Separation in Ion-Pairing Reversed-Phase Liquid Chromatography: Effect of Temperature. J. Chromatogr. A 2022, 1681, 463473. https://doi.org/10.1016/j.chroma.2022.463473.
26. Fekete, S.; Imiolek, M.; Lauber, M. A. Platform Ion Pairing RPLC Method for Oligonucleotides Using High Throughput 20 Mm Length Columns; 720008460-pt; Waters Corporation. https://www.waters.com/content/dam/waters/en/app-notes/2024/720008460/720008460-pt.pdf?srsltid=AfmBOop1ybKf5NI0ZSp75YAmvIMFSa61CqXsfpeWQcgtKuimzlHcINEX (accessed 2025-09-25).
27. McCarthy, S. M.; Gilar, M. Hexylammonium Acetate as an Ion-Pairing Agent for IP-RP LC Analysis of Oligonucleotides; 720003361EN; Waters Corporation, 2010. https://www.waters.com/content/dam/waters/en/app-notes/2016/720003361/720003361-en.pdf (accessed 2025-09-25).
28. Oefner, P. J. Allelic Discrimination by Denaturing High-Performance Liquid Chromatography. J. Chromatogr. B: Biomed. Sci. Appl. 2000, 739 (2), 345–355. https://doi.org/10.1016/S0378-4347(99)00571-X.
29. 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. https://doi.org/10.1016/j.chroma.2024.465285.
30. Gilar, M.; DeLano, M.; Gritti, F. Mitigation of Analyte Loss on Metal Surfaces in Liquid Chromatography. J. Chromatogr. A 2021, 1650, 462247. https://doi.org/10.1016/j.chroma.2021.462247.
31. Tuytten, R.; Lemière, F.; Witters, E.; Dongen, W. V.; Slegers, H.; Newton, R. P.; Onckelen, H. V.; Esmans, E. L. Stainless Steel Electrospray Probe: A Dead End for Phosphorylated Organic Compounds? J. Chromatogr. A 2006, 1104 (1–2), 209–221. https://doi.org/10.1016/j.chroma.2005.12.004.
32. DeLano, M.; Walter, T. H.; Lauber, M. A.; Gilar, M.; Jung, M. C.; Nguyen, J. M.; Boissel, C.; Patel, A. V.; Bates-Harrison, A.; Wyndham, K. D. Using Hybrid Organic–Inorganic Surface Technology to Mitigate Analyte Interactions with Metal Surfaces in UHPLC. Anal. Chem. 2021, 93 (14), 5773–5781. https://doi.org/10.1021/acs.analchem.0c05203.
33. Nguyen, J. M.; Gilar, M.; Koshel, B.; Donegan, M.; MacLean, J.; Li, Z.; Lauber, M. A. Assessing the Impact of Nonspecific Binding on Oligonucleotide Bioanalysis. Bioanalysis 2021, 13 (16), 1233–1244. https://doi.org/10.4155/bio-2021-0115.

About the Authors