<h3>Two Dimensions, One Solution: Advancing Oligonucleotide Analysis with 2D-LC</h3><h4>Why Consider 2D-LC for Oligonucleotide Analysis?</h4>In oligonucleotide analytics, achieving reliable and precise results is not optional—it is fundamental. These biomolecules are inherently complex, often containing truncated sequences, structural variants, and a wide range of impurities. Such complexity frequently exceeds the separation capabilities of conventional one-dimensional liquid chromatography (1D-LC). As a result, critical components may coelute or remain unresolved, limiting analytical confidence.Two-dimensional liquid chromatography (2D-LC) offers a powerful alternative by significantly enhancing separation performance. By combining two complementary separation mechanisms, 2D-LC improves resolution, sensitivity, and overall analytical reliability. This enables a much deeper understanding of oligonucleotide samples and their compositional intricacies.<blockquote><h4>Previous parts from the Oligos Made Easy series</h4><ul><li>Part 1: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6808">Overview</a></li><li>Part 2: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6846">AEX</a></li><li>Part 3: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6880">IP-RP</a></li><li>Part 4: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6912">HILIC</a></li><li>Part 5: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6944">SEC</a></li><li>Part 6: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6982">Column Navigator</a></li><li>Part 7: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/7012">Large Scale AEX</a></li><li>Part 8: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/7045">Sepapure oliGO</a></li></ul></blockquote><h4>Moving Beyond the Limits of 1D-LC</h4>For many years, 1D-LC has served as a standard tool for biopharmaceutical characterization. However, its inherent limitations become apparent when dealing with highly complex mixtures. The selectivity of a single chromatographic dimension is restricted, and the lack of orthogonality in retention mechanisms often results in insufficient separation. As sample complexity increases, the resolving power of 1D-LC becomes inadequate.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_1_2_D_LC_Principle_Graphic_by_KNAUER_43e04d32a2.jpg" alt="KNAUER: Figure 1 - 2D-LC Principle (Graphic by KNAUER)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_1_2_D_LC_Principle_Graphic_by_KNAUER_43e04d32a2.jpg 245w," sizes="100vw" width="1291" height="391">Two-dimensional LC addresses these limitations by coupling two chromatographic separations, which can be either similar or fundamentally different. Each dimension targets different physicochemical properties of analytes, enabling the resolution of compounds that would otherwise overlap. In essence, 2D-LC introduces an additional level of separation capacity, allowing analysts to disentangle coeluting species that remain unresolved in a single dimension.An important practical consideration is that the overall analysis time is governed by the second dimension. Because fractions from the first dimension must be processed sequentially, the second dimension must operate rapidly and efficiently. High-speed separations in the second dimension are therefore essential to maintain system performance and data quality.<h4>Architecture of a 2D-LC System</h4>At its core, a 2D-LC system relies on precise coordination between its components. The setup typically consists of two chromatographic columns, two pumping systems, and a switching valve that enables fraction transfer between dimensions. The sample is introduced through an autosampler and first separated on the primary column. Fractions of interest are then transferred into a loop via a switching valve and subsequently introduced into the second column for further separation.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_2_2_D_LC_System_Configuration_Example_Graphic_by_KNAUER_f5747adf3a.jpg" alt="KNAUER: Figure 2 - 2D LC System Configuration Example (Graphic by KNAUER)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_2_2_D_LC_System_Configuration_Example_Graphic_by_KNAUER_f5747adf3a.jpg 245w," sizes="100vw" width="859" height="517">The valve plays a crucial role in this process, acting as the interface between both chromatographic dimensions. Its function is to alternately collect fractions from the first dimension and inject them into the second dimension under high-pressure conditions. Various valve configurations can be employed, including ten-port or eight-port valves with dual-loop arrangements, or synchronized six-port valves. Regardless of configuration, the underlying principle remains the same: while one loop is being filled, the other is being flushed into the second dimension.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_3_Schematic_figure_of_ten_port_left_and_eight_port_valve_right_2_position_setup_for_2_D_LC_Graphic_by_KNAUER_9209661ea8.jpg" alt="KNAUER: Figure 3 - Schematic figure of ten-port (left) and eight-port valve (right) 2-position setup for 2D-LC (Graphic by KNAUER)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_3_Schematic_figure_of_ten_port_left_and_eight_port_valve_right_2_position_setup_for_2_D_LC_Graphic_by_KNAUER_9209661ea8.jpg 245w," sizes="100vw" width="1287" height="406">This alternating process requires precise timing. The second-dimension separation must be completed before the next fraction is ready for transfer. Consequently, synchronization between flow rates, loop volumes, and switching intervals is essential for maintaining chromatographic integrity.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_4_Schematic_figure_KNAUER_eight_port_2_position_valve_for_2_D_LC_Graphic_by_KNAUER_f9a5f06f5f.jpg" alt="KNAUER: Figure 4 - Schematic figure KNAUER eight-port 2 position valve for 2D-LC (Graphic by KNAUER)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_4_Schematic_figure_KNAUER_eight_port_2_position_valve_for_2_D_LC_Graphic_by_KNAUER_f9a5f06f5f.jpg 245w," sizes="100vw" width="756" height="391"><h4>Applying 2D-LC to Oligonucleotide Analysis</h4>In oligonucleotide workflows, 2D-LC combines two complementary separation strategies to address sample complexity. The first dimension is typically used for bulk separation based on properties such as size, charge, or polarity. Techniques such as size-exclusion chromatography (SEC), anion-exchange chromatography (AEX), ion-pair reversed-phase chromatography (IP-RP), hydrophilic interaction chromatography (HILIC), or mixed-mode chromatography (MMC) are commonly applied. This step reduces sample complexity and can also serve as an online desalting stage, removing salts and buffer components that could interfere with downstream detection, particularly when mass spectrometry is used.Following this initial separation, selected fractions are transferred to the second dimension. This transfer can be performed either selectively or comprehensively, depending on the analytical goal. The second dimension is typically optimized for high-resolution separation, allowing detailed characterization of closely related species such as truncated oligonucleotides, sequence variants, or chemically modified impurities. When coupled with mass spectrometry, this setup enables accurate identification and sequencing.A key advantage of 2D-LC lies in the flexibility of combining different stationary phases and mobile phase conditions.&nbsp;Numerous separation combinations are possible, including:<ul><li>SEC×AEX</li><li>HILIC×IP-RP</li><li>IEC×IP-RP</li><li>IP-RP×IP-RP</li><li>AEX×HIC</li><li>MMC×SEC,</li><li>and even chiral×RP systems.&nbsp;</li></ul>This adaptability allows tailored workflows for specific analytical challenges.<h4>Heart-Cutting vs. Comprehensive 2D-LC</h4>When implementing 2D-LC, two primary transfer strategies are commonly used: heart-cutting and comprehensive transfer. Each approach offers distinct advantages depending on the analytical objective.Heart-cutting 2D-LC focuses on selected regions of interest from the first dimension. Only specific peaks or time windows are transferred to the second dimension for further analysis. This approach allows extended separation time and higher resolution in the second dimension, making it particularly suitable for targeted investigations of known critical compounds. However, because only selected fractions are analyzed, unexpected impurities outside the selected region may remain undetected.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_5_2_D_LC_Single_Heart_Cut_Principle_Graphic_by_KNAUER_b1b400784b.jpg" alt="KNAUER: Figure 5 - 2D-LC Single Heart-Cut Principle (Graphic by KNAUER)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_5_2_D_LC_Single_Heart_Cut_Principle_Graphic_by_KNAUER_b1b400784b.jpg 245w," sizes="100vw" width="1276" height="319">In contrast, comprehensive 2D-LC transfers the entire eluate from the first dimension into the second dimension in a continuous manner. This approach provides a complete and detailed representation of the sample, making it ideal for research and development, impurity profiling, and regulatory applications. While more demanding in terms of speed and system performance, comprehensive 2D-LC ensures full sample coverage.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_6_Full_Comprehensive_2_D_LC_Principle_Graphic_by_KNAUER_e13da63ef1.jpg" alt="KNAUER: Figure 6 - Full Comprehensive 2D-LC Principle (Graphic by KNAUER)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_6_Full_Comprehensive_2_D_LC_Principle_Graphic_by_KNAUER_e13da63ef1.jpg 245w," sizes="100vw" width="1327" height="292">In practice, hybrid approaches may also be employed, combining targeted heart-cutting with broader comprehensive analysis to balance resolution and coverage.<h4>Practical Considerations for Method Development</h4>Successful implementation of 2D-LC for oligonucleotide analysis requires careful method design.&nbsp;<h5>1. Column selection&nbsp;</h5>It is a critical starting point, as the combination of stationary phases must provide sufficient orthogonality while maintaining compatibility between mobile phases. Typically, the first dimension is optimized for general separation of oligonucleotide size or class, while the second dimension focuses on sequence-specific resolution and compatibility with mass spectrometry.<h5>2. Mobile phase selection&nbsp;</h5>Differences in pH and organic modifiers can significantly influence separation behavior, but incompatibility between dimensions can compromise performance. Therefore, mobile phases must be designed as a coordinated system. The use of volatile buffers, such as ammonium acetate, is often preferred for MS-based detection.<h5>3. Instrumentation&nbsp;</h5>The switching valve must operate reliably under high pressure, with precise timing to ensure accurate fraction transfer. Loop volumes must be carefully selected to avoid overfilling, as excessive loading can lead to sample loss and distortion due to flow profile effects. As a general guideline, loops should not be filled beyond approximately 70% of their capacity.<h5>4. Method development&nbsp;</h5>Typically begins with optimization of the second dimension, as it dictates overall system timing. Fast separations using short columns and steep gradients are often required to maintain sufficient sampling frequency. Once the second dimension is established, the first dimension can be adjusted to achieve optimal resolution and compatibility.<h4>Final Remarks</h4>Two-dimensional liquid chromatography represents a powerful advancement in oligonucleotide analysis, enabling the resolution of complex mixtures that exceed the capabilities of conventional 1D approaches. By combining orthogonal separation mechanisms, 2D-LC reveals structural details and impurity profiles that would otherwise remain hidden.However, achieving optimal performance requires careful balancing of multiple factors, including column chemistry, mobile phase compatibility, system timing, and data acquisition. When properly implemented, 2D-LC provides a robust and highly informative platform for modern oligonucleotide characterization.<blockquote><ul><li>If you're planning to set up or optimize your oligonucleotide workflow, feel free to contact us at <a href="sales@knauer.net">sales@knauer.net</a>. Stay tuned for more exciting insights into the Oligonucleotide world in our “Oligos Made Easy” series.</li><li>For more in-depth discussion or questions, reach out to the author at <a href="kindler@knauer.net">kindler@knauer.net</a></li></ul></blockquote>

Oligos Made Easy - Part 9: 2D-LC

News from LabRulezLCMS Library - Week 14, 2026

<h3>We're excited to announce that <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/999">Clarity Chromatography Software</a> version 10.1.2 is now available!</h3>This minor update expands Clarity’s portfolio with new control modules.<h4>Newly added Instrument control modules</h4><ul><li>Ecom auxiliary modules</li><li>HTA ICF control modules</li><li>Spark pump</li></ul><h4>Updated Instrument control modules</h4><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/companies/168">Ecom</a> HPLC</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/companies/328">Knauer</a> HPLC</li><li>Spark ICF control modules</li><li>YoungIn Chromass HPLC</li></ul><blockquote>For a complete list of module changes, see the <a target="_blank" rel="noopener noreferrer" href="https://www.dataapex.com/version_history?product=1">Changelog</a>. For more info about exciting new features directly in Clarity, explore what was introduced in the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/5622">Clarity 10.1 release highlights</a>.Clarity can control 1000+ instruments; the <a target="_blank" rel="noopener noreferrer" href="https://www.dataapex.com/controls">list can be previewed here</a>.</blockquote><h4>Supported Instruments in Clarity (DataApex) — Quick Guide</h4>Clarity Chromatography Software supports a wide range of chromatographs and peripherals via digital control (DD) and analog acquisition (A/D) using the Colibrick module.<h5>Gas Chromatographs (GC)</h5>Digital integration over LAN/RS232 (many via ICF):<ul><li>Agilent: 5890, 6890, 7890, 8860, 8890, Intuvo 9000</li><li>Shimadzu: GC-2010/2014, Nexis GC-2030</li><li>Thermo Fisher: TRACE 1300/1310</li><li>PerkinElmer: Clarus 400–690</li><li>Also supported: Dani, Ellutia, Young In Chromass, Apix, Fuli, GOW-MAC (selected models)</li></ul><blockquote>Note: Most Agilent 6890–8890 and Intuvo use LAN – ICF.</blockquote><h5>HPLC Systems</h5>Full system/module control:<ul><li>Agilent 1100–1290 Infinity II (ICF)</li><li>Shimadzu LC-10/20 and Nexera LC-40</li><li>PerkinElmer Flexar / NexSAR</li><li>Knauer Azura / BlueShadow / Smartline</li><li>Hitachi Chromaster / Primaide</li><li>Young In Chromass ChroZen series</li><li>Sykam S500–S600, Watrex Streamline, Hanon LC7000–7300</li></ul><h5>HPLC Modules (Pumps, Detectors, Ovens, Fraction Collectors)</h5><ul><li>Pumps: Agilent 1100–1290 families, Knauer, Shimadzu, PerkinElmer, Sykam, SSI, Ecom</li><li>Detectors: VWD/DAD(PDA)/RID/FLD/ELSD across Agilent, Shimadzu, Knauer, PerkinElmer, Young In Chromass, Ecom, etc.</li><li>Column Ovens: Agilent G1316x / G7116x, Shimadzu CTO-20/30/40, Knauer CT2.1, PerkinElmer Flexar</li><li>Fraction Collectors: Knauer FC 6.1, Teledyne FOXY series, Ecom ECF 2096, Waters Fraction Collector II</li></ul><h5>Autosamplers</h5><ul><li>Agilent GC ALS (7673/7683/7693/8697), HPLC ALS modules (G1313A–G7167C)</li><li>Shimadzu AOC-20 / AOC-5000+, SIL-10 / SIL-20 / SIL-40 series</li><li>CTC Analytics PAL, HTA autosamplers, Spark Alias/UHPLC, Knauer AS 6.1L</li><li>PerkinElmer Flexar / NexSAR AS</li></ul><h5>Other Devices</h5><ul><li>GC×GC modulators (e.g., Charles University GC×GC Modulator)</li><li>Valves &amp; switch modules (Agilent, Knauer, Shimadzu, VICI-Valco)</li><li>Degassers, flow monitors, managers, interfaces</li><li>LC-MS / GC-MS: Advion Expression CMS, Microsaic 4500 MiD, Markes BenchTOF (and others)</li><li>Balances (EA extension): Mettler Toledo, Sartorius</li></ul><h5>Analog Acquisition (Colibrick)</h5>A/D support for legacy or analog-output devices:<ul><li>GC: Agilent 5880/4890A, Varian 3xxx/3800/3900, PerkinElmer Clarus series, Thermo TRACE, Shimadzu GC-14/9A, Scion 43x/45x/83xx/85xx, etc.</li><li>HPLC Detectors: Waters 2487/2489, ESA Corona, Hitachi L-series, JASCO FP/UV, Ecom LCD series, many others</li><li>Elemental &amp; Amino Acid Analyzers: Carlo Erba/Costech ECS, Biochrom 20/30+</li></ul>If you are interested in a DEMO VERSION, contact us!

DataApex Clarity Chromatography software 10.1.2 just released!

Article

Product

Application

Academy

Software

Since 1991 DataApex develops and manufactures chromatography data stations. DataApex is constantly striving to provide users with efficient and up-to-date tools for chromatography data processing.

DataApex

Everything from the world of analytical chemistry in one place. We connect people in solving their problems. At Labrulez you will find all the necessary information easily, quickly and clearly. Stop searching and start finding.

LabRulez

Based in Berlin, KNAUER is a medium-sized, owner-managed company that has been serving the sciences since 1962. We develop and manufacture scientific instruments of superior quality for liquid chromatography, including: Analytical HPLC/UHPLC, Preparative HPLC, Fast protein liquid chromatography (FPLC), Multi-column chromatography/Simulated moving bed (SMB) chromatography. High pressure dosing, in-line detection systems, and osmometry are additional business areas.

KNAUER

Determination of nitrosamine content (Bioanalytic)

Our offer includes comprehensive development and validation of methods for determination of nitrosamines in pharmaceutical products. We perform the work according to the criteria and needs of the client based on the EMA note.

Determination of nitrosamines is one of the pharmaceutical industry's top priorities. After the presence of N-nitrosodimethylamine (NDMA) was first reported in an active pharmaceutical ingredient (API), the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) mandated the identification and control of all APIs and finished products in which there is a risk of nitrosamines.The content of nitrosamines in drug products can be controlled primarily by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. More than that, a reliable analysis result in our offer consists of a well-chosen sample preparation procedure, a selective and sensitive analytical method, and the experience of the analyst, who selects the best method parameters at the optimization stage.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Determination_of_nitrosamine_content_Bioanalytic_2_ce4f440837.jpg" alt="Bioanalytic: Determination of nitrosamine content." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Determination_of_nitrosamine_content_Bioanalytic_2_ce4f440837.jpg 234w," sizes="100vw" width="1536" height="1024">We have gained experience working with leading pharmaceutical companies. As part of our ongoing work, we have developed analytical methods for small-molecule nitrosamines of 70-150 g/mol. These compounds are mainly formed during manufacturing processes and depend on the presence and type of excipients (e.g. NDBA - N-nitroso-dibutylamine). We have also developed methods for determining molecules with higher nitrosamine drug substance related impurities (NDSRIs). In our work, we focus on achieving the highest possible sensitivity, selectivity and precision. To this end, we individually select the parameters of the methods depending on the composition of the drug and the expected nitrosamines. Due to the above criteria and the required detection limits, we perform nitrosamine impurity determinations using LC-MS/MS kits based on <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=52&amp;search=QTRAP+5500%2B">QTRAP 5500+</a> and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=52&amp;search=QTRAP+6500%2B">QTRAP 6500+</a> mass spectrometers (<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=55">SCIEX</a>).<blockquote>Our offerOur offer includes comprehensive development and validation of methods for determination of nitrosamines in pharmaceutical products. We perform the work according to the criteria and needs of the client based on the EMA note.At the stage of sample preparation, we focus on:<ul><li>obtaining the highest possible recovery of nitrosamines (matrix purification);</li><li>limiting additional synthesis of impurities (selection of appropriate equipment and reagents);</li><li>ensuring the stability of the sample prepared for LC-MS/MS analysis.</li></ul>During the optimization of the LC-MS/MS method, we solve such problems as:<ul><li>selection of an appropriate internal standard;</li><li>separation of the impurity from the API;</li><li>obtaining the expected sensitivity of the analytical method.</li></ul>Quantitative analysis of nitrosamines requires sensitivity, precision and accuracy, which we provide to our customers.</blockquote><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Determination_of_nitrosamine_content_Bioanalytic_1_d629e00e62.jpg" alt="Bioanalytic: Determination of nitrosamine content." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Determination_of_nitrosamine_content_Bioanalytic_1_d629e00e62.jpg 103w," sizes="100vw" width="1130" height="1710">

Products from category Pharmaceutical analyses distributed by Bioanalytic - page 1

Related content

DataApex Clarity Chromatography software 10.1.2 just released!

News from LabRulezLCMS Library - Week 14, 2026

Oligos Made Easy - Part 9: 2D-LC