<h3><strong>A Practical Guide to Oligonucleotide Production – From Concept to Final Product</strong></h3><p><strong>In 2020, an internal innovation at KNAUER significantly reshaped our perspective on nucleic acid technologies. The development of Impingement Jets Mixing (IJM) for lipid nanoparticle (LNP) generation—originally driven by the urgent need for mRNA-based COVID-19 vaccines—sparked a deeper engagement with DNA and RNA-based therapeutics. Although mRNA vaccines were the first application, it soon became clear that the same platform could support a broad range of oligonucleotides, including short DNA and RNA sequences.</strong></p><p>This realization naturally expanded our focus beyond formulation alone. Purification strategies, analytical control, and scalable manufacturing of oligonucleotides became equally compelling challenges. Today, simplifying and optimizing oligonucleotide production workflows is a central pillar of KNAUER’s technology roadmap.</p><h4><strong>Technologies for LNP Manufacturing</strong></h4><ul><li><strong>NanoScaler</strong></li><li><strong>Customized NanoProducer</strong></li></ul><p>These systems provide flexible solutions for research and production-scale LNP generation, supporting nucleic acid delivery applications.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Systems_for_LNP_production_Middle_Nano_Scaler_Right_Customized_Nano_Producer_1cf4727f38.jpg" alt="KNAUER: Systems for LNP production. Middle - NanoScaler, Right - Customized NanoProducer" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Systems_for_LNP_production_Middle_Nano_Scaler_Right_Customized_Nano_Producer_1cf4727f38.jpg 245w," sizes="100vw" width="1547" height="524"></p><h4><strong>What Is Required to Manufacture Oligonucleotides?</strong></h4><p>Whether producing oligonucleotides for internal use or as a CDMO, establishing a dedicated oligo production laboratory requires a sequence of well-defined steps. This introductory article outlines a mid-scale production workflow (from approximately 1 µmol up to several hundred mmol). Each operation is introduced here and will be explored in greater depth in future articles, with particular attention to purification and quality control. But before assessing purity, the oligonucleotide itself must first be synthesized.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Typical_workflow_for_oligonucleotide_production_a52b1aabd0.jpg" alt="KNAUER: Typical workflow for oligonucleotide production" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Typical_workflow_for_oligonucleotide_production_a52b1aabd0.jpg 245w," sizes="100vw" width="1593" height="510"></p><h4><strong>Typical Oligonucleotide Production Workflow</strong></h4><h5><strong>Step 1: Solid-Phase Oligonucleotide Synthesis</strong></h5><p>Oligonucleotide assembly begins on a solid support, most commonly controlled pore glass (CPG) or polystyrene resin. Individual nucleotides are sequentially added through a cyclic series of reactions:</p><ul><li><strong>Detritylation</strong> – removal of temporary protecting groups</li><li><strong>Coupling</strong> – attachment of the next nucleotide</li><li><strong>Oxidation or thiolation</strong> – stabilization of the phosphate backbone</li><li><strong>Capping</strong> – deactivation of unreacted chains</li></ul><p>These repetitive steps involve precise reagent delivery, washing, and activation, and are typically performed on automated synthesizers. KNAUER’s own oligonucleotide synthesizer is currently approaching completion.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Solid_phase_synthesis_5d16092ede.jpg" alt="KNAUER: Solid-phase synthesis" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Solid_phase_synthesis_5d16092ede.jpg 203w," sizes="100vw" width="809" height="622"></p><h5><strong>Step 2: Cleavage and Deprotection</strong></h5><p>Once synthesis is complete, the crude oligonucleotide must be released from the solid support. This is commonly achieved using ammonia-based reagents. Subsequent treatment with heated ammonium hydroxide removes remaining protecting groups. After filtration to remove the solid matrix, the solution is neutralized and prepared for initial analytical evaluation.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Flow_scheme_KNAUER_Synthesizer_a4212a41a2.jpg" alt="KNAUER: Flow scheme KNAUER Synthesizer​" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Flow_scheme_KNAUER_Synthesizer_a4212a41a2.jpg 245w," sizes="100vw" width="834" height="506"></p><h5><strong>Step 3: Chromatographic Analysis and Purification</strong></h5><p>Chromatography plays a central role in both the characterization and purification of oligonucleotides. Commonly used techniques include:</p><ul><li><strong>Anion-Exchange Chromatography (AEX)</strong><br>Separation based on charge; cost-efficient but generates high salt loads requiring downstream removal.</li><li><strong>Ion-Pair Reversed-Phase Chromatography (IP-RP)</strong><br>Offers excellent resolution and typically simplifies downstream processing due to volatile buffers, though at higher operating costs.</li><li><strong>Hydrophilic Interaction Liquid Chromatography (HILIC)</strong><br>An increasingly popular analytical tool, particularly suitable for short or chemically modified oligos, without ion-pairing reagents. Its primary strength lies in analysis rather than large-scale purification.</li></ul><h5><strong>Step 3a: In-Process Quality Control</strong></h5><p>Throughout production, oligonucleotides must be monitored for purity, sequence fidelity, and structural modifications. Liquid chromatography remains the primary analytical technique for these assessments, commonly using IP-RP, AEX, or HILIC methods. This is an area where KNAUER’s analytical expertise and HPLC platforms play a key role.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Representative_KNAUER_analytical_HPLC_system_and_Chromatogram_of_Oligonucleotide_Analysis_26f4619c5b.jpg" alt="KNAUER: Representative KNAUER analytical HPLC system  and Chromatogram of Oligonucleotide Analysis" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Representative_KNAUER_analytical_HPLC_system_and_Chromatogram_of_Oligonucleotide_Analysis_26f4619c5b.jpg 245w," sizes="100vw" width="1342" height="674"></p><h5><strong>Step 3b: Preparative Chromatographic Purification</strong></h5><p>Following analytical assessment, purification removes synthesis-related impurities such as truncated sequences or residual protecting groups. AEX and IP-RP are the most frequently applied methods. Process development teams often evaluate both approaches at small scale before selecting the optimal strategy. Notably, both modes can be implemented on the same preparative system, such as the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=41&amp;manufacturerItems=503&amp;search=lab"><strong>AZURA® Lab </strong></a>or <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=41&amp;manufacturerItems=503&amp;search=pilot"><strong>Pilot Prep HPLC platforms.</strong></a></p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Representative_KNAUER_preparative_LC_System_and_Chromatogram_of_Oligonucleotide_Purification_19415a7614.jpg" alt="KNAUER: Representative KNAUER preparative LC System and  Chromatogram of Oligonucleotide Purification" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Representative_KNAUER_preparative_LC_System_and_Chromatogram_of_Oligonucleotide_Purification_19415a7614.jpg 245w," sizes="100vw" width="1335" height="673"></p><h5><strong>Step 4: Desalting or Solvent Exchange via Tangential Flow Filtration (TFF)</strong></h5><p>After chromatographic purification, oligonucleotides remain in matrices containing salts or organic solvents. Tangential flow filtration enables gentle and scalable buffer exchange. Low-molecular-weight components pass through the membrane, while oligonucleotides are retained. This step prepares the product for formulation or further processing. KNAUER is currently developing a modular TFF solution tailored to this application.</p><h5><strong>Step 5: Endotoxin Reduction</strong></h5><p>For therapeutic applications, endotoxin levels must be rigorously controlled. Lipopolysaccharides from bacterial sources can be efficiently reduced using an additional TFF step. By selecting appropriate membranes, multiple diafiltration cycles can lower endotoxin concentrations by several orders of magnitude, supporting regulatory compliance depending on dose and administration route.</p><h5><strong>Step 6: Lyophilization</strong></h5><p>For preclinical and early-stage batches, freeze-drying is the standard finishing step. The oligonucleotide solution is frozen below –40 °C and subjected to vacuum sublimation, producing a dry, stable product that can be stored at ambient temperature and rapidly reconstituted when needed.</p><h4><strong>Connecting the Workflow with KNAUER Technology</strong></h4><p>KNAUER provides a cohesive portfolio supporting oligonucleotide production from synthesis to final formulation. <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=37&amp;manufacturerItems=503&amp;search=azura"><strong>AZURA® (U)HPLC systems</strong></a> and dedicated columns such as <strong>Sepapure® oliGO</strong> enable robust analytical control and efficient purification. Preparative LC platforms are optimized for oligo workflows, while new solutions—including an in-house oligonucleotide synthesizer and a modular TFF system—are being developed to streamline desalting and endotoxin removal.</p><p>Together, these technologies allow laboratories to build scalable, integrated oligonucleotide production environments without relying on fragmented, multi-vendor solutions.</p>

Oligos Made Easy - Part 1: Overview

<h3><strong>Charged for Action: The Hidden Secrets of AEX Oligonucleotide Analysis</strong></h3><p><strong>Oligonucleotides—short, well-defined fragments of DNA or RNA—have become indispensable tools in modern life sciences. They underpin cutting-edge applications in molecular diagnostics, gene therapy, and pharmaceutical development. Before oligonucleotides can be confidently used in research or clinical settings, however, their quality must be rigorously assessed. Purity, sequence integrity, length distribution, and chemical modifications all play a decisive role in their performance.</strong></p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Oligos_Made_Easy_Part_2_AEX_2_45eee54fa0.jpg" alt="KNAUER: Oligos Made Easy - Part 2: AEX" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Oligos_Made_Easy_Part_2_AEX_2_45eee54fa0.jpg 245w," sizes="100vw" width="1534" height="711"></p><p>Among the analytical techniques available today, <strong>anion exchange chromatography (AEX)</strong> stands out as a robust and highly informative method for characterizing oligonucleotides. By exploiting their intrinsic negative charge, AEX provides detailed insight into product composition and impurity profiles that are difficult to obtain by other approaches.</p><blockquote><p><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6808"><strong>Part 1 of the Oligos series: A Practical Guide to Oligonucleotide Production – From Concept to Final Product</strong></a></p></blockquote><h4><strong>How Scientists Read Oligonucleotides Using Charge</strong></h4><p>At the core of AEX lies a simple but powerful principle: oligonucleotides carry a negatively charged phosphate backbone. This charge increases in a predictable way with strand length and, to a lesser extent, sequence composition. Anion exchange chromatography uses this property to separate oligonucleotides based on differences in charge strength and interaction with a positively charged stationary phase.</p><p>As a result, AEX has become one of the most widely used analytical tools for evaluating oligonucleotide quality. It allows scientists to distinguish full-length products from truncated species, degradation products, or closely related variants—often with single-nucleotide resolution.</p><h4><strong>Why Ion Exchange Works: Binding, Retention, and Release</strong></h4><p>Ion exchange chromatography (IEX) relies on electrostatic interactions between analytes and a charged stationary phase. In the case of oligonucleotides, <strong>anion exchange</strong> is used, where negatively charged molecules interact with positively charged functional groups on the resin—most commonly quaternary amines.</p><p>The system consists of two main components:</p><ul><li><strong>Stationary phase:</strong> A positively charged resin that binds anionic analytes.</li><li><strong>Mobile phase:</strong> An aqueous buffer system, typically combined with a salt gradient, that controls both pH and elution strength.</li></ul><p>Retention in AEX is governed primarily by charge. Molecules with weaker overall charge interact less strongly with the stationary phase and elute earlier, while more highly charged oligonucleotides bind more tightly and require stronger elution conditions.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_1_IEX_overview_including_elution_order_dependency_on_analyte_charge_3419ad3075.jpg" alt="KNAUER: Oligos Made Easy - Part 2 AEX: Figure 1 IEX overview including elution order dependency on analyte charge." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_1_IEX_overview_including_elution_order_dependency_on_analyte_charge_3419ad3075.jpg 245w," sizes="100vw" width="1583" height="654"></p><h4><strong>The Five-Step Workflow of Ion Exchange Chromatography</strong></h4><p>AEX separations generally follow a well-established sequence of steps:</p><ol><li><strong>Column equilibration</strong><br>The column is conditioned with the starting buffer until the baseline and pH are stable. This ensures that all charged sites on the resin are ready for interaction.</li><li><strong>Sample loading</strong><br>Samples, prepared in the starting buffer, are introduced under conditions that promote binding of the target oligonucleotides while minimizing retention of unwanted impurities.</li><li><strong>Washing</strong><br>The column is washed with the starting buffer to remove uncharged or weakly interacting components. The baseline stabilizes as these species are flushed away.</li><li><strong>Elution</strong><br>Bound oligonucleotides are released using a salt or pH gradient. Increasing ionic strength introduces competing ions that displace the analytes from the resin. Weakly bound species elute first; strongly charged oligonucleotides elute later.</li><li><strong>Regeneration and re-equilibration</strong><br>A high-salt buffer removes any remaining bound material. The column is then returned to starting conditions in preparation for the next analysis.</li></ol><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_2_Five_steps_of_IEX_procedure_in_blue_outlined_using_an_example_chromatogram_in_red_38dd70b1b1.jpg" alt="KNAUER: Oligos Made Easy - Part 2 AEX: Figure 2  Five steps of IEX procedure (in blue), outlined using an example chromatogram (in red)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_2_Five_steps_of_IEX_procedure_in_blue_outlined_using_an_example_chromatogram_in_red_38dd70b1b1.jpg 245w," sizes="100vw" width="1583" height="674"></p><h4><strong>Why AEX Is Especially Effective for Oligonucleotides</strong></h4><p>The repeating phosphate backbone of oligonucleotides gives rise to a strong and uniform anionic character across a wide pH range (typically pH 7–9). This makes oligonucleotides exceptionally well suited for anion exchange separations.</p><p>Key factors influencing AEX behavior include:</p><ul><li><strong>Charge density:</strong> Longer oligonucleotides carry more negative charges and therefore exhibit stronger retention.</li><li><strong>Resin chemistry:</strong> Strong and weak anion exchangers differ in functional groups and pH stability, influencing selectivity and robustness.</li><li><strong>Buffer composition:</strong> The choice of buffering ions and salt gradients directly affects elution strength and resolution.</li></ul><p>Salt gradients—commonly based on chloride ions—compete with the oligonucleotide backbone for binding sites on the resin. By carefully tuning gradient slope and composition, analysts can achieve highly reproducible and finely resolved separations.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_3_Ionic_interactions_and_p_H_influence_on_Anion_Exchange_Chromatography_AEX_612e231ba4.jpg" alt="KNAUER: Oligos Made Easy - Part 2 AEX: Figure 3 Ionic interactions and pH influence on Anion Exchange Chromatography (AEX)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_3_Ionic_interactions_and_p_H_influence_on_Anion_Exchange_Chromatography_AEX_612e231ba4.jpg 245w," sizes="100vw" width="1547" height="593"></p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_4_AEX_buffer_selection_and_gradient_strategy_chromatogram_with_red_buffer_gradient_curve_53fd1bb11b.jpg" alt="KNAUER: Oligos Made Easy - Part 2 AEX: Figure 4 AEX buffer selection and gradient strategy (chromatogram with red buffer gradient curve)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_4_AEX_buffer_selection_and_gradient_strategy_chromatogram_with_red_buffer_gradient_curve_53fd1bb11b.jpg 245w," sizes="100vw" width="1600" height="327"></p><h4><strong>AEX as the Gold Standard for Oligonucleotide Separation</strong></h4><p>Anion exchange chromatography has become the method of choice for both analytical and preparative oligonucleotide workflows. Its advantages include:</p><ul><li><strong>High resolving power:</strong> Closely related impurities, including n–1 or n–2 species, can often be separated and quantified.</li><li><strong>Predictable retention behavior:</strong> In many cases, retention time correlates linearly with oligonucleotide length, simplifying identification.</li><li><strong>Compatibility with modified oligonucleotides:</strong> Even chemically modified sequences retain their anionic nature, allowing AEX to resolve both products and related impurities.</li><li><strong>Excellent control via gradient elution:</strong> Salt gradients provide precise tuning of selectivity and resolution.</li></ul><p>Typically, shorter or degraded oligonucleotides elute at lower ionic strength, while full-length products with higher charge density appear later in the chromatogram.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_5_AEX_Advantages_and_Challenges_7835521e34.jpg" alt="KNAUER: Oligos Made Easy - Part 2 AEX: Figure 5 AEX Advantages and Challenges." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Oligos_Made_Easy_Part_2_AEX_Figure_5_AEX_Advantages_and_Challenges_7835521e34.jpg 245w," sizes="100vw" width="1535" height="707"></p><h4><strong>Final Perspective: Mastering AEX with Confidence</strong></h4><p>Anion exchange chromatography may appear complex at first, but with thoughtful optimization of pH, buffer composition, and gradient conditions, it becomes a highly precise and reliable tool for oligonucleotide analysis. When properly applied, AEX delivers sharp peaks, excellent resolution, and robust performance—even for challenging sequences and modifications.</p><p>For laboratories developing or refining oligonucleotide workflows, AEX offers both analytical depth and operational confidence.</p><blockquote><p>If you would like to discuss AEX method development or optimization for oligonucleotides, feel free to contact us at <strong>sales@knauer.net</strong>. More insights into practical oligonucleotide analysis will follow in our <i>“Oligos Made Easy”</i> series.</p><p>For technical questions or in-depth discussion, the author can be reached at <strong>kindler@knauer.net</strong></p></blockquote>

Oligos Made Easy - Part 2: AEX

<h3><strong>Mastering Ion-Pair Reversed-Phase Chromatography for Oligonucleotide Analysis</strong></h3><h4><strong>Why Purity Is Critical in the Oligonucleotide Era</strong></h4><p>Oligonucleotides — short, synthetic strands of DNA or RNA — are central to modern biotechnology. They power gene therapies, advanced diagnostics, and cutting-edge molecular research. However, before these molecules can be used in clinical or research settings, their purity and structural integrity must be thoroughly verified. High-resolution chromatographic analysis plays a crucial role in ensuring quality and safety.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_1_Separation_Example_of_Oligonucleotides_5577514561.jpg" alt="KNAUER: Figure 1 - Separation Example of Oligonucleotides." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_1_Separation_Example_of_Oligonucleotides_5577514561.jpg 245w," sizes="100vw" width="1024" height="368"></p><h4><strong>Understanding Ion-Pair Reversed-Phase (IP-RP) Chromatography</strong></h4><p>For scientists involved in oligonucleotide synthesis and characterization, Ion-Pair Reversed-Phase Chromatography (IP-RP) is one of the most effective analytical tools available.</p><p>Unlike separation techniques that rely solely on charge differences, IP-RP combines:</p><ul><li>Ionic interactions</li><li>Hydrophobic interactions</li></ul><p>This dual retention mechanism enables exceptional resolution — even for oligonucleotides that differ by only a single nucleotide or subtle chemical modification. Whether assessing synthesis efficiency or purifying therapeutic-grade material, IP-RP provides reliable, reproducible separations.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_2_Overview_of_IP_RP_Mode_prinicple_ecd54b6e9c.jpg" alt="KNAUER: Figure 2 - Overview of IP-RP Mode prinicple." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_2_Overview_of_IP_RP_Mode_prinicple_ecd54b6e9c.jpg 245w," sizes="100vw" width="1024" height="456"></p><h4><strong>The Principle Behind Ion-Pairing</strong></h4><p>Oligonucleotides possess a highly negatively charged phosphate backbone. In a traditional reversed-phase system, such hydrophilic, charged molecules would show little retention.</p><p>IP-RP overcomes this limitation by introducing a positively charged ion-pairing reagent into the mobile phase. The cation associates with the negatively charged oligonucleotide, forming a transient ion pair with increased hydrophobic character. This modified complex can now interact with the hydrophobic stationary phase.</p><p>Retention strength depends on:</p><ul><li>The hydrophobicity of the ion-pair reagent</li><li>The strength of ionic interaction</li><li>The applied organic gradient</li></ul><p>During gradient elution (commonly using acetonitrile), analytes are released in a controlled manner according to size, sequence, and chemical modifications.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_3_Ion_Pair_Reversed_Phase_Principle_9a4fcd12f2.jpg" alt="KNAUER: Figure 3 - Ion-Pair Reversed Phase Principle." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_3_Ion_Pair_Reversed_Phase_Principle_9a4fcd12f2.jpg 245w," sizes="100vw" width="1024" height="393"></p><h4><strong>Choosing the Right Ion-Pair Reagent</strong></h4><p>Successful IP-RP separation depends heavily on selecting an appropriate ion-pair reagent. Two key factors determine retention behavior:</p><ol><li>Alkyl chain length</li><li>Charge density</li></ol><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_4_Ion_Pair_Classification_eaad172a4e.jpg" alt="KNAUER: Figure 4 - Ion-Pair Classification." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_4_Ion_Pair_Classification_eaad172a4e.jpg 245w," sizes="100vw" width="1024" height="398"></p><h4><strong>Strong Cationic Ion-Pair Reagents</strong></h4><p>Ion-pair reagents with longer alkyl chains provide stronger hydrophobic interactions and increased retention. A widely used example is tetrabutylammonium (TBA⁺), frequently applied for strongly retained anionic analytes.</p><p>These reagents are particularly useful when high retention strength is required.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Table_1_Strong_Cationic_Ion_Pair_Reagents_8dbf00c57e.jpg" alt="KNAUER: Table 1 - Strong Cationic Ion-Pair Reagents." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Table_1_Strong_Cationic_Ion_Pair_Reagents_8dbf00c57e.jpg 245w," sizes="100vw" width="1024" height="396"></p><h4><strong>Volatile (Weaker) Ion-Pair Reagents</strong></h4><p>Short-chain ion-pair reagents generate weaker retention but offer a significant advantage: compatibility with LC–MS detection.</p><p>Triethylammonium (TEA⁺) is among the most commonly used reagents for oligonucleotide analysis, particularly when mass spectrometric detection is involved. Other options, such as diisopropylethylammonium (DIEA⁺), are also widely applied, often combined with HFIP to enhance MS performance.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Table_2_Weak_Cationic_Ion_Pair_Reagents_6332a4975e.jpg" alt="KNAUER: Table 2 - Weak Cationic Ion-Pair Reagents." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Table_2_Weak_Cationic_Ion_Pair_Reagents_6332a4975e.jpg 245w," sizes="100vw" width="1024" height="283"></p><h4><strong>Building an IP-RP Method for Oligonucleotides</strong></h4><p>Developing a robust IP-RP method follows a logical sequence:</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Table_3_IP_RP_Method_Step_Procedure_for_Oligos_c2216c24a0.jpg" alt="KNAUER: Table 3 - IP-RP Method Step Procedure for Oligos." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Table_3_IP_RP_Method_Step_Procedure_for_Oligos_c2216c24a0.jpg 245w," sizes="100vw" width="1024" height="304"></p><h5><strong>1. Characterize the Analyte</strong></h5><p>Oligonucleotides typically contain 10–50+ nucleotides and exhibit high negative charge density. This necessitates a suitable cationic partner such as TEA⁺ or DIEA⁺.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_5_Cationic_Ion_Pair_Principle_for_Oligonucleotides_cb2dfb7457.jpg" alt="KNAUER: Figure 5 - Cationic Ion-Pair Principle for Oligonucleotides." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_5_Cationic_Ion_Pair_Principle_for_Oligonucleotides_cb2dfb7457.jpg 245w," sizes="100vw" width="512" height="320"></p><h5><strong>2. Select the Column</strong></h5><p>Use a reversed-phase column with:</p><ul><li>Large pore size (≈300 Å)</li><li>Appropriate internal diameter</li></ul><p>These parameters support the so-called “ON–OFF” retention mechanism — a bind-and-elute behavior where analytes are strongly retained until the gradient triggers rapid elution.</p><h5><strong>3. Optimize the Mobile Phase</strong></h5><p>A typical starting condition includes:</p><ul><li>10 mM TEA buffer at pH ~7</li><li>Acetonitrile as organic modifier</li></ul><p>Gradient programs from approximately 5% to 50% organic solvent over 15–30 minutes are commonly effective for complex oligonucleotide mixtures.</p><h5><strong>4. Adjust Retention Strength if Needed</strong></h5><p>To increase retention:</p><ul><li>Raise ion-pair concentration (up to ~100 mM if required)</li><li>Select a more hydrophobic ion-pair reagent</li></ul><h5><strong>5. Validate the Method</strong></h5><p>Ensure robustness, sensitivity, and reproducibility by carefully evaluating:</p><ul><li>UV chromatograms</li><li>MS traces</li><li>Peak shape and resolution</li></ul><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_6_UV_MS_traces_of_IP_RP_Oligonucleotide_Analysis_81faf4e9f0.jpg" alt="KNAUER: Figure 6 - UV-MS traces of IP-RP Oligonucleotide Analysis." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_6_UV_MS_traces_of_IP_RP_Oligonucleotide_Analysis_81faf4e9f0.jpg 245w," sizes="100vw" width="1024" height="365"></p><h4><strong>Why IP-RP Is Considered the Gold Standard</strong></h4><p>Ion-Pair Reversed-Phase Chromatography uniquely combines the resolving power of reversed-phase LC with the ability to retain highly polar, charged biomolecules.</p><p>Its advantages include:</p><ul><li>Tunable selectivity</li><li>Compatibility with UV and MS detection</li><li>Scalability from analytical to preparative workflows</li><li>Applicability from research to pharmaceutical production</li></ul><p>By transforming highly charged oligonucleotides into hydrophobic ion-pair complexes, IP-RP enables precise and reproducible separations that would otherwise be impossible in conventional reversed-phase systems.</p><p>For these reasons, IP-RP is widely regarded as the benchmark technique for oligonucleotide analysis.</p><h4><strong>Final Thoughts</strong></h4><p>Mastering IP-RP is less intimidating than it may seem. A successful method comes down to:</p><ul><li>Selecting the right ion-pair reagent</li><li>Choosing a suitable column</li><li>Optimizing gradient conditions</li><li>Fine-tuning temperature and concentration parameters</li></ul><p>With thoughtful method development, IP-RP delivers sharp peaks, clean separations, and reliable performance — fully compatible with modern mass spectrometry workflows and ready to meet the demands of advanced oligonucleotide analysis.</p>

Oligos Made Easy - Part 3: IP-RP

<h4><strong>Why Consider HILIC in Modern Oligonucleotide Analysis?</strong></h4><p>Oligonucleotides play a central role in gene therapies, molecular diagnostics, and cutting-edge biomedical research. Before these molecules reach application, however, they must be carefully characterized to confirm purity, identity, and performance.</p><p>Analyzing therapeutic oligonucleotides is far from straightforward. Their polarity, structural diversity, and modifications demand highly selective and MS-compatible separation methods. Although ion-pair reversed-phase LC (IP-RP-LC) remains the established standard for LC-MS workflows, hydrophilic interaction liquid chromatography (HILIC) is rapidly emerging as a powerful complementary technique.</p><p>When IP-RP approaches reach practical or performance limits, HILIC offers an attractive alternative, delivering strong retention and efficient separation of highly polar oligonucleotides — without relying on ion-pairing reagents.</p><h4><strong>How Does HILIC Separate Oligonucleotides?</strong></h4><p>HILIC essentially reverses conventional reversed-phase logic. It combines:</p><ul><li>A <strong>polar stationary phase</strong></li><li>An <strong>organic-rich mobile phase</strong></li></ul><p>In this configuration, the stationary phase retains a thin, water-enriched layer on its surface. Polar analytes — such as oligonucleotides — partition into this aqueous layer while the acetonitrile-rich mobile phase passes through.</p><p>This mechanism allows:</p><ul><li>Retention of short or highly polar oligonucleotides</li><li>Separation of modified sequences</li><li>LC-MS compatibility without ion-pairing reagents</li></ul><p>The key to performance lies in carefully tuning the parameters that govern HILIC selectivity and retention.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_1_Key_Parameter_of_HILIC_Mode_fb364c9a8b.webp" alt="KNAUER: Figure 1 - Key Parameter of HILIC Mode." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_1_Key_Parameter_of_HILIC_Mode_fb364c9a8b.webp 245w," sizes="100vw" width="945" height="433"></p><h4><strong>What Controls Retention in HILIC?</strong></h4><p>Several interaction mechanisms contribute to analyte behavior in HILIC:</p><h5><strong>Hydrophilic Partitioning – The Primary Mechanism</strong></h5><p>Polar molecules distribute into the thin water layer formed on the stationary phase surface. The greater the polarity of the analyte, the stronger its affinity for this layer, leading to increased retention.</p><h5><strong>Hydrogen Bonding – Fine-Tuning Effects</strong></h5><p>Functional groups such as –OH, –NH₂, and –COOH can form hydrogen bonds with the stationary phase, subtly influencing retention time and selectivity.</p><h5><strong>Electrostatic Contributions – The Role of Charge</strong></h5><p>Some HILIC materials (e.g., bare silica) exhibit surface charges. Depending on mobile phase pH and buffer composition, electrostatic interactions can either enhance or suppress analyte retention.</p><h5><strong>Dipole and Ion–Dipole Interactions – Added Selectivity</strong></h5><p>Highly polar or zwitterionic compounds — including phosphorylated species and sugars — may engage in dipole–dipole or ion–dipole interactions, further refining separation behavior.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_2_Hydrophilic_Partitioning_on_Water_enriched_Layer_41ab9ac902.webp" alt="KNAUER: Figure 2 - Hydrophilic Partitioning on Water-enriched Layer." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_2_Hydrophilic_Partitioning_on_Water_enriched_Layer_41ab9ac902.webp 245w," sizes="100vw" width="945" height="409"></p><h4><strong>How to Develop a HILIC Method for Oligonucleotides</strong></h4><p>Like any chromatographic method, successful HILIC development follows a structured approach:</p><h5><strong>Step 1: Define the Analytes</strong></h5><p>Understand oligonucleotide length, polarity, charge, and modifications.</p><h5><strong>Step 2: Select a Suitable Column</strong></h5><p>Choose a polar stationary phase (e.g., amide or zwitterionic) appropriate for the oligonucleotide’s chemical properties.</p><h5><strong>Step 3: Optimize the Mobile Phase</strong></h5><ul><li>Start with <strong>70–95% acetonitrile</strong></li><li>Use a <strong>volatile buffer</strong> (e.g., ammonium formate or ammonium acetate, typically 5–20 mM)</li><li>Adjust pH (commonly between 6–8, with testing up to pH 9 if column stability allows)</li></ul><p>Small pH shifts can significantly alter selectivity and MS response.</p><h5><strong>Step 4: Design the Gradient</strong></h5><p>Employ a gradient that increases water content to modulate retention and separate oligonucleotides by size or chemical modification.</p><h5><strong>Step 5: Match Injection Conditions</strong></h5><p>Samples should be injected in a solvent similar to the starting mobile phase (high organic content) to prevent peak distortion.</p><h5><strong>Step 6: Evaluate Performance</strong></h5><p>Assess:</p><ul><li>Resolution</li><li>Retention time stability</li><li>Peak shape</li><li>Reproducibility</li></ul><p>Balancing separation performance with MS ionization efficiency is essential.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Table_1_HILIC_Method_Step_Procedure_for_Oligos_f3b9ea6b08.webp" alt="KNAUER: Table 1 - HILIC Method Step Procedure for Oligos." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Table_1_HILIC_Method_Step_Procedure_for_Oligos_f3b9ea6b08.webp 245w," sizes="100vw" width="945" height="357"></p><h4><strong>Practical Tips for High-Performance HILIC</strong></h4><h5><strong>Maintain a Stable Water Layer</strong></h5><p>A minimum of ~5% water is necessary at the start of the run. Too little water can eliminate retention entirely. Adjust water content carefully to fine-tune retention.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_3_Water_Adjustment_Principle_for_HILIC_c0d8616f20.webp" alt="KNAUER: Figure 3 - Water Adjustment Principle for HILIC." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_3_Water_Adjustment_Principle_for_HILIC_c0d8616f20.webp 245w," sizes="100vw" width="951" height="374"></p><h5><strong>Use High Organic Content</strong></h5><p>HILIC typically operates best with 70–95% acetonitrile. Polar analytes are retained more effectively under these conditions.</p><h5><strong>Include a Volatile Buffer</strong></h5><p>Use low concentrations (5–20 mM) of ammonium formate or acetate to:</p><ul><li>Stabilize pH</li><li>Reduce peak tailing</li><li>Maintain MS compatibility</li></ul><h5><strong>Control Injection Solvent</strong></h5><p>Avoid high-water sample solvents. They disrupt the partitioning mechanism and distort peaks.</p><h5><strong>Allow Proper Column Equilibration</strong></h5><p>HILIC columns require longer equilibration times than reversed-phase columns — typically 10–20 column volumes after major gradient or solvent changes.</p><h5><strong>Consider Temperature</strong></h5><p>Operating at 30–50 °C can:</p><ul><li>Improve peak shape</li><li>Reduce mobile phase viscosity</li><li>Stabilize retention behavior</li></ul><h4><strong>From Alternative to Essential? Final Perspective</strong></h4><p>HILIC-MS is steadily advancing as a strong, ion-pair-free option for oligonucleotide analysis. While IP-RP-LC still dominates in sensitivity for many applications, HILIC is closing the gap and offering valuable flexibility — particularly for highly polar or challenging sequences.</p><p>Although HILIC follows different chromatographic principles than reversed-phase separations, once optimized, it can deliver robust and highly selective results.</p><p>Rather than being a niche method, HILIC is becoming a strategic tool in modern oligonucleotide workflows — especially when MS compatibility and method versatility are priorities.</p><p>If you are developing or refining an oligonucleotide LC-MS method, HILIC may be worth integrating into your analytical toolbox.</p><blockquote><ul><li>For additional information or discussion, feel free to contact: <a><strong>sales@knauer.net</strong><span><strong><svg><use></use></svg></strong></span></a></li><li>For direct scientific inquiries, reach out to the author at: <a><strong>kindler@knauer.net</strong><span><strong><svg><use></use></svg></strong></span></a></li></ul></blockquote><p><i><strong>Stay tuned for more insights in the “Oligos Made Easy” series.</strong></i></p>

Oligos Made Easy - Part 4: HILIC

News from LabRulezLCMS Library - Week 10, 2026

<h3><strong>Two Dimensions, One Solution: Advancing Oligonucleotide Analysis with 2D-LC</strong></h3><h4><strong>Why Consider 2D-LC for Oligonucleotide Analysis?</strong></h4><p>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.</p><p><strong>Two-dimensional liquid chromatography (2D-LC) </strong>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.</p><blockquote><h4><strong>Previous parts from the Oligos Made Easy</strong> <strong>series</strong></h4><ul><li><strong>Part 1: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6808"><strong>Overview</strong></a></li><li><strong>Part 2: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6846"><strong>AEX</strong></a></li><li><strong>Part 3: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6880"><strong>IP-RP</strong></a></li><li><strong>Part 4: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6912"><strong>HILIC</strong></a></li><li><strong>Part 5: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6944"><strong>SEC</strong></a></li><li><strong>Part 6: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6982"><strong>Column Navigator</strong></a></li><li><strong>Part 7: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/7012"><strong>Large Scale AEX</strong></a></li><li><strong>Part 8: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/7045"><strong>Sepapure oliGO</strong></a></li></ul></blockquote><h4><strong>Moving Beyond the Limits of 1D-LC</strong></h4><p>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.</p><p><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"></p><p>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.</p><p>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.</p><h4><strong>Architecture of a 2D-LC System</strong></h4><p>At its core, a 2D-LC system relies on precise coordination between its components. The <strong>setup typically consists</strong> of <strong>two chromatographic columns</strong>, <strong>two pumping systems</strong>, and a <strong>switching valve</strong> 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.</p><p><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"></p><p>The <strong>valve plays a crucial role</strong> in this process, acting as the interface between both chromatographic dimensions. Its function is to alternately <strong>collect fractions from the first dimension and inject them into the second dimension</strong> 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: <strong>while one loop is being filled, the other is being flushed into the second dimension</strong>.</p><p><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"></p><p>This alternating process requires <strong>precise timing</strong>. 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.</p><p><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"></p><h4><strong>Applying 2D-LC to Oligonucleotide Analysis</strong></h4><p>In oligonucleotide workflows, 2D-LC combines two complementary separation strategies to address sample complexity. The <strong>first dimension</strong> is typically used for <strong>bulk separation</strong> based on properties such as <strong>size, charge, or polarity</strong>. Techniques such as <strong>size-exclusion chromatography</strong> (SEC), <strong>anion-exchange chromatography</strong> (AEX), <strong>ion-pair reversed-phase chromatography </strong>(IP-RP), <strong>hydrophilic interaction chromatography</strong> (HILIC), or <strong>mixed-mode chromatography</strong> (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.</p><p>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.</p><p>A <strong>key advantage of 2D-LC</strong> lies in the <strong>flexibility of combining different stationary phases and mobile phase</strong> conditions.&nbsp;</p><p>Numerous separation combinations are possible, including:</p><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><p>This adaptability allows tailored workflows for specific analytical challenges.</p><h4><strong>Heart-Cutting vs. Comprehensive 2D-LC</strong></h4><p>When implementing 2D-LC, two primary transfer strategies are commonly used: <strong>heart-cutting</strong> and <strong>comprehensive transfer</strong>. Each approach offers distinct advantages depending on the analytical objective.</p><p><strong>Heart-cutting 2D-LC</strong> 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.</p><p><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"></p><p>In contrast, <strong>comprehensive 2D-LC</strong> 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.</p><p><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"></p><p>In practice, <strong>hybrid approaches may also be employed</strong>, combining targeted heart-cutting with broader comprehensive analysis to balance resolution and coverage.</p><h4><strong>Practical Considerations for Method Development</strong></h4><p>Successful implementation of 2D-LC for oligonucleotide analysis requires careful method design.&nbsp;</p><h5><strong>1. Column selection</strong>&nbsp;</h5><p>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.</p><h5><strong>2. Mobile phase selection&nbsp;</strong></h5><p>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.</p><h5><strong>3. Instrumentation&nbsp;</strong></h5><p>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.</p><h5><strong>4. Method development&nbsp;</strong></h5><p>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.</p><h4><strong>Final Remarks</strong></h4><p>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.</p><p>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.</p><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"><strong>sales@knauer.net</strong></a>. Stay tuned for more exciting insights into the Oligonucleotide world in our “<strong>Oligos Made Easy</strong>” series.</li></ul></blockquote>

Oligos Made Easy - Part 9: 2D-LC

News from LabRulezLCMS Library - Week 23, 2026

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 15th June 2026? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you application notes by <strong>Agilent Technologies, Shimadzu </strong>and<strong> Thermo Fisher Scientific, </strong>technical note by<strong> KNAUER </strong>and brochure by <strong>Waters Corporation!</strong></p><h3><strong>1. Agilent Technologies: Large-Volume Dilute-and-Shoot Analysis of PFAS in Drinking Water Using the Altura PFAS Column</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_analysis_pfas_drinking_water_altura_5994_9186en_agilent_a66334e5b0.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Per‑ and polyfluoroalkyl substances (PFAS) are synthetic fluorinated compounds valued for their oil‑ and water‑repellent properties, but their environmental persistence and mobility have driven the development of increasingly stringent regulations for drinking water monitoring. Existing dilute-and-shoot methods, such as EPA 8327 and ASTM D7979, were originally designed for screening PFAS in nonpotable water. These methods typically rely on 10 to 30&nbsp;µL injection volumes, setting practical quantitation limits appropriately 10 ng/L.<sup>1,2&nbsp;</sup></p><p>We present a large‑volume injection workflow that extends the capabilities of these methods toward trace PFAS quantitation in drinking water. Using the Agilent 6495D triple quadrupole LC/MS (LC/TQ) coupled with a 1290 Infinity II LC, we achieved injections of 200 µL of methanol‑diluted water on the Altura PFAS column without requiring SPE concentration. The Altura PFAS column maintained peak symmetry and retention stability under these injection conditions. This configuration enabled a working calibration range of 0.25 to 25 ng/L, extending the low-end sensitivity substantially relative to published dilute-and shoot methods.<sup>1,2&nbsp;</sup></p><p>This performance enhancement simplifies operations by removing labor‑intensive extraction steps, while maintaining sensitivity required for regulatory guidelines thereby supporting high‑throughput PFAS workflows suitable for routine drinking water analysis. The PFAS compounds analyzed were PFBS, HFPO-DA, PFHpA, PFHxA, PFHxS, PFOA, PFOS, PFNA, and PFDA. These compounds were selected due to their relevance to ongoing regulatory actions and proposed maximum contaminant level (MCL) considerations for drinking water.&nbsp;</p><p>Specific highlights of this experiment include:&nbsp;</p><ul><li>Minimized peak tailing and solvent effects with 200 µL injections water/methanol (1:1) injections.</li><li>Increased throughput by elimination of solid phase extraction (SPE).&nbsp;</li><li>Enhanced sensitivity of 6495D LC/TQ at low end (0.25&nbsp;ng/L) for the dilute-and-shoot method in drinking&nbsp;water.</li></ul><h4><strong>Experimental&nbsp;</strong></h4><h5><strong>Instrumentation&nbsp;</strong></h5><p><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=+1290+Infinity+II"><strong>Agilent 1290 Infinity II LC</strong></a> coupled to <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6495D"><strong>Agilent 6495D LC/TQ system</strong></a> was utilized for the LC/MS/MS analysis. An Infinity&nbsp;II multisampler with an extended multidraw capillary tube (G7137-68307) was connected to the needle seat and injection valve with a union for large volume injection (200&nbsp;µL). The data were acquired with MassHunter acquisition software version 12.2. All LC/MS parameters are shown in Table 1 and 2. PFAS multiple reaction monitoring (MRM) transition methods were imported from the PFAS MRM database (G1736AA) while labelled homologues were optimized to obtain correct MRM transitions (Table 3).</p><h4><strong>Conclusion&nbsp;</strong></h4><p>This study demonstrates a practical large‑volume dilute-and-shoot workflow for the determination of PFAS in drinking water using the Agilent 1290 Infinity II LC coupled to the 6495D triple quadrupole LC/MS, and Agilent Altura Poroshell PFAS column. With 200 µL water samples diluted in methanol can be injected directly, eliminating the need for SPE concentration and significantly reducing sample preparation time. This method exhibits good sensitivity, accuracy, and reproducibility. The Altura PFAS column maintained consistent peak performance and retention stability even at low sub‑ppt concentrations, supporting long‑term reliability for trace‑level PFAS monitoring. Overall, the findings highlight the method potential to support routine, high‑sensitivity PFAS monitoring in drinking water laboratories.</p><h3><strong>2. KNAUER: Size matters - How to perform narrow calibration using the ClarityChrom® GPC Extension</strong></h3><ul><li>Technical note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/VTN_0049_knauer_size_matters_how_to_perform_narrow_calibration_using_the_claritychrom_gpc_extension_3cfdc7a45b.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Gel Permeation Chromatography (GPC), also known as Size Exclusion Chromatography (SEC), is a separation technique that separates molecules based on their hydrodynamic size rather than chemical interactions. In a GPC column, separation is based on the ability of molecules to access the pores of the stationary phase. Smaller molecules can penetrate these pores and therefore spend more time inside the column, resulting in a delayed elution. Larger molecules, on the other hand, cannot enter the pores and pass through the column more quickly, leading to earlier elution.&nbsp;</p><p>As a result, molecules are separated by size in solution, providing the basis for the determination of molecular weight and molecular weight distributions. Calibration is therefore a fundamental requirement for ensuring the accuracy and reliability of the results. The ClarityChrom® GPC Extension enables users to efficiently create and apply different calibration models for precise analysis. The workflow presented here focuses on narrow calibration using standards with defined molecular weights and low polydispersity (PD ≈ 1).</p><h4><strong>Material and Methods</strong></h4><ul><li><strong>Software&nbsp;</strong><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=503&amp;search=ClarityChrom"><strong>ClarityChrom</strong></a>® 10.1 - station single instrument license one time base</li><li>ClarityChrom® 10.1 - GPC license for GPC data processing &nbsp;</li></ul></li><li><strong>Pump: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2372"><strong>AZURA P 6.1L isocratic HPLC pump</strong></a> with 10 ml pump head for normal phase applications, stainless steel&nbsp;</li><li><strong>Degasser: </strong>Analytical 2 channel GPC degasser&nbsp;</li><li><strong>Autosampler:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2375"><strong>AZURA® AS 6.1L</strong></a>, analytical HPLC autosampler, 862 bar&nbsp;</li><li><strong>Detector:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2429"><strong>AZURA® RID 2.1L</strong></a>, analytical refractive index detector&nbsp;</li><li><strong>Thermostat: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2376"><strong>AZURA® CT 2.1</strong></a> ATC00 Capillaries Start-Up Kit with flexible, precut capillaries for analytical HPLC systems with 1/16" connections</li></ul><h4><strong>Conclusion</strong></h4><p>This technical note demonstrates how to successfully create and apply a narrow calibration using the ClarityChrom® GPC Extension. By following the outlined workflow, users can ensure reliable calibration and determination of molecular weight and molecular weight distributions. The described approach supports consistent and reproducible results, making it a valuable tool for routine GPC/SEC analysis. For further optimization and advanced applications, additional related technical and application notes can be consulted.</p><h3><strong>3. Shimadzu: UHPLC Method for Sensitive Automatic Analysis of Thirty-Seven D/L-Amino Acids and for Liquor Profiling</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_01_01169_en_2dab331398.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>User Benefits</strong></h5><ul><li>Enables high-sensitivity simultaneous fast analysis of thirty-seven D/L-amino acids using simple operations.</li><li>Good reproducibility of analytical results can be achieved by keeping the derivatization reaction time constant.</li><li>Statistical analysis software enables easy multivariate analysis of multiple samples.</li></ul><p>Recent advances in analytical techniques have revealed the presence of D-amino acids in fermented foods and biological samples and clarified their role in the taste, preservation, and aroma of foods , and as potential disease biomarkers. Therefore, the demand for D/L separation of amino acids is increasing. Because D-amino acids are only present in trace amounts in fermented foods and biological systems, unlike L-amino acids, it is necessary to determine the concentration of each D-amino acid by separating them from the high concentrations of Lamino acids.&nbsp;</p><p>A comprehensive method for quantitating the thirty-seven D/Lamino acids was previously reported1), in which two chiral derivatizing reagents were employed to provide different diastereomers for each amino acid, with the instrument automatically switching between two separation methods to analyze each sample twice.&nbsp;</p><p>This article introduces an improved method that employs a single chiral derivatizing reagent to enable simultaneous analysis of D/L-amino acidsin a shorter time.</p><h4><strong>D/L-Amino Acid Analysis by UHPLC&nbsp;</strong></h4><p>In general, LC/MS or multi-dimensional LC is used to analyze D/L-amino acids by HPLC, because it is difficult to separate proteinogenic D/L-amino acids with a single separation mode. However, it is known that LC/MS is susceptible to matrix effects and less quantitative than HPLC. It is also known that the multidimensional LC method requires a long analysis time and very complicated HPLC setup. Therefore, a simple operational method that provides good separation for D/L-amino acids in a short time is needed.&nbsp;</p><p>For food analysis, a small particle column is used to achieve appropriate separation of the small amounts of D-amino acids from the large amounts of L-amino acids and co-existing contaminants. A <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=X3"><strong>UHPLC system (Nexera X3</strong></a>) was employed in this study due to the increased system pressure caused by the use of a small particle column.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>A method was developed for simultaneous separation of OPA/NIBC diastereomers for thirty-seven D/L-amino acids using a simple UHPLC system in approximately half the time of the conventional methods. By optimizing the derivatization reaction, all compounds were determined sensitively, accurately, and precisely. Separation of the target compounds from the contaminants was good in the liquor samples when evaluated using real liquor samples. It is expected to be easily applicable to D/L-amino acid profiling in food and beverage samples without using expensive MS detection or complicated multidimensional HPLC configurations as long as samples are prepared properly. Moreover, D/L-amino acid profiling could be used to research and develop foods containing D-amino acids intended to help to maintain and improve the intestinal environment, and could help in the early detection of diseases and determining optimal treatment strategies through biomarker discovery</p><h3><strong>4. Thermo Fisher Scientific: Screening of per- and polyfluoroalkyl substances (PFAS) in cosmetics: Utilizing a new combustion-ion chromatography system for total organic fluorine (TOF) analysis</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ap_004098_ic_systems_combustion_ic_environmental_ap004098_na_en_a3a1d33a24.pdf"><strong>Full PDF for download</strong></a></li></ul><p>This application note describes a method for <strong>screening per- and polyfluoroalkyl substances (PFAS) in textiles</strong> using total organic fluorine (TOF) analysis by combustion-ion chromatography (C-IC). PFAS are widely used in textiles to provide water, oil, and stain resistance, but increasing concerns about their environmental persistence and potential health effects have led to stricter regulations worldwide. California’s AB 1817 legislation, for example, limits TOF concentrations in textiles to 100 ppm beginning in 2025 and 50 ppm by 2027, creating a need for reliable analytical methods capable of assessing PFAS content in textile products.</p><p>The study evaluates a new <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2150"><strong>Thermo Scientific Cindion Combustion Ion Chromatography System</strong></a>, which combines a Dionex Inuvion IC system with a Cindion combustion/absorption module. The system features an optimized Z-fold combustion tube design that improves combustion efficiency, shortens analysis time, reduces instrument footprint, and allows complete control through Chromeleon software. Unlike surface-sensitive techniques such as PIGE, the combustion-IC approach analyzes the entire sample, providing results that are independent of sample thickness. The method measures total fluorine (TF) after combustion and total inorganic fluorine (TIF) by direct IC analysis, with TOF calculated as the difference between TF and TIF.</p><p>To validate the approach, six textile samples representing waterproof and stain-resistant consumer products were analyzed and compared with results previously obtained using a Nittoseiko combustion-IC system. TOF concentrations ranged from approximately 1 ppm in one nylon fabric to nearly 375 ppm in a polyester baby bib. The values generated by the new Cindion system showed excellent agreement with the reference system, achieving between 100% and 105% of the previously reported results. Reproducibility was also strong, with relative standard deviations below 8% for all samples.</p><p>The authors conclude that the new Cindion C-IC platform provides an accurate, reliable, and efficient solution for PFAS screening in textiles. In addition to matching the performance of the established combustion-IC method, the enhanced system offers a flexible “2-in-1” configuration that allows seamless switching between combustion-IC and standalone IC operation. This improves workflow efficiency, reduces manual intervention, and provides manufacturers and testing laboratories with a practical tool for monitoring compliance with evolving PFAS regulations in textile products.</p><h3><strong>5. Waters Corporation: Advanced Acquisition Modes for the </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2433"><strong>Xevo MRT P10 Mass Spectrometer</strong></a></h3><ul><li>Brochure</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009362_9baa39e7de.pdf"><strong>Full PDF for download</strong></a></li></ul><div><div><section><div><div><div><div><div><div><p>The Waters solution sheet introduces the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2433"><strong>Xevo MRT P10 Mass Spectrometer</strong>,</a> a next-generation high-resolution mass spectrometer designed to provide significantly greater analytical depth, sensitivity, and flexibility for modern omics workflows. Compared with the previous Xevo MRT platform, the instrument delivers a reported <strong>20-fold increase in MS/MS sensitivity</strong>, incorporates Wideband Enhanced Duty Cycle (WB EDC) technology, and supports ultrafast acquisition rates of up to <strong>100 Hz for MS data</strong> and <strong>200 Hz for MS/MS data</strong>. These performance improvements are intended to enhance the detection of low-abundance compounds while maintaining high-quality spectral information across complex biological samples.</p><p>A major focus of the document is the introduction of the new <strong>SONAR Pulse Acquisition Mode</strong>, an advanced data-independent acquisition (DIA) approach. SONAR Pulse improves selectivity by rapidly stepping a quadrupole isolation window across a defined precursor mass range, allowing fragment ions to be associated with narrower precursor windows than in conventional DIA experiments. This strategy reduces spectral complexity, improves confidence in compound identification, and increases the likelihood of detecting low-level analytes that might be missed by traditional data-dependent acquisition (DDA) methods. The method supports precursor and product ion scans up to m/z 2000, multiple quadrupole isolation window sizes, acquisition speeds up to 200 Hz, and full compatibility with WB EDC technology.</p><p>The second major innovation described is <strong>Mixed Mode Acquisition</strong>, a hybrid workflow that combines targeted and untargeted analysis within a single injection. In this approach, high-sensitivity <strong>Tof MRM</strong> quantification is performed simultaneously with untargeted DIA (MSE) acquisition. This enables users to obtain quantitative data for predefined targets while also collecting comprehensive full-scan information for discovery and screening purposes. The workflow integrates seamlessly with Waters software tools, allowing targeted quantification through the MS Quan application and untargeted analysis through UNIFI or export to open data formats such as mzML for processing in third-party software packages including MZmine, MS-DIAL, DIA-NN, PEAKS, and Progenesis QI.</p><p>According to Waters, both acquisition strategies are designed to maximize information obtained from each sample injection. SONAR Pulse emphasizes deeper molecular coverage, improved selectivity, and simplified interpretation of complex DIA datasets, while Mixed Mode provides a unified quantitative–qualitative workflow that combines targeted measurement and discovery screening without compromising sensitivity or acquisition speed. Together, these capabilities position the Xevo MRT P10 as a versatile platform for metabolomics, lipidomics, proteomics, and other multiomics applications where both comprehensive characterization and accurate quantification are required.</p></div></div></div></div></div></div></section></div></div>

News from LabRulezLCMS Library - Week 25, 2026

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 13th July 2026? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you posters by <strong>Agilent Technologies / ASMS, Shimadzu / ASMS, Thermo Fisher Scientific / HPLC Symposium </strong>and<strong> Waters Corporation / HPLC Symposium </strong>and technical note by<strong> KNAUER!</strong></p><h3><strong>1. Agilent Technologies / ASMS: Intact Protein Analysis in Plasma Using Automated 2D-LC/TOF with Multiple Heart-Cutting</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_intact_protein_mhc_2d_lc_tof_wp_679_asms_2026_en_agilent_edb54f4854.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Direct intact and top-down protein analysis enables comprehensive characterization of proteoforms, post-translational modifications, and fragments that are often lost in bottom-up workflows.1 However, intact mass analysis in complex biological matrices such as whole plasma is challenged by extreme dynamic range and ion suppression, commonly necessitating affinity-based enrichment.&nbsp;</p><p>Here, we demonstrate a non-affinity, orthogonal separation strategy using a 2D-LC/TOF workflow with multi-heart-cutting for native intact protein analysis directly in plasma. Therapeutic monoclonal antibodies (mAbs) trastuzumab and antibody-drug conjugate T-DM1, were used as model systems to evaluate performance.&nbsp;</p><p>The workflow employs an Agilent 1290 Infinity III 2D-LC system for orthogonal separations and precise multiple heart-cutting, coupled to a high-resolution, high-sensitivity Agilent 6230C Accurate Mass TOF LC/MS System.</p><h4><strong>Experimental</strong></h4><h5><strong>Instrumentation&nbsp;</strong></h5><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+III+2D-LC"><strong>Agilent 1290 Infinity III 2D-LC system </strong></a>comprised the following modules:&nbsp;<ul><li>1290 Infinity III Bio Multisampler with Thermostat (G7137A)&nbsp;</li><li>1290 Infinity III Multicolumn Thermostat (G7116B)&nbsp;</li><li>1290 Infinity III Diode Array Detector (G7117B) with Max-Light cartridge cell (G7117-60020)&nbsp;</li><li>1260 Infinity III Bio Flexible Pump (G7131C)&nbsp;</li><li>1290 Infinity III Bio High-speed Pump (G7132A)&nbsp;</li><li>1290 Infinity Valve Drive (G1170A) with 2D-LC ASM Valve (G5643B)&nbsp;</li><li>Two 1290 Infinity III Valve Drives (G1170A) with multiple heart-cutting valves (G5642-64000) equipped with 40 µL Bio loops&nbsp;</li><li>1290 Infinity III Valve Drive (G1170A) with a 2- position/6-port valve (G4231A) (used as a diverter valve)&nbsp;</li></ul></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6230C"><strong>Agilent 6230C Accurate Mass TOF LC/MS System</strong></a></li></ul><h5><strong>LC/MS Analysis&nbsp;</strong></h5><p>Data acquisition and analysis were carried out using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=MassHunter+acquisition"><strong>MassHunter acquisition</strong></a> (12.2), <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=BioConfirm"><strong>BioConfirm</strong></a> (12.1) and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=MassHunter+Qualitative+Analysis"><strong>MassHunter Qualitative Analysis </strong></a>(13.0) software.</p><h4><strong>Conclusions</strong></h4><ul><li>Agilent 2D-LC enables direct native MS analysis of intact proteins from plasma without affinity-based enrichment.&nbsp;</li><li>UV-guided and multiple heart-cutting simplify 2D-LC method development and provide high-resolution isolation of native proteins from complex biological matrices such as plasma.&nbsp;</li><li>The Agilent 6230C Accurate Mass TOF delivers high sensitivity and excellent mass accuracy, enabling confident intact protein characterization under native conditions.</li></ul><h3><strong>2. KNAUER: Mind the pressure in preparative up-scaling – effects of particle size and solvent composition on back pressure</strong></h3><ul><li>Technical note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/VTN_0051_knauer_mind_the_pressure_in_preparative_up_scaling_effects_of_particle_size_and_solvent_comp_12e49884d3.pdf"><strong>Full PDF for download</strong></a></li></ul><p>The back pressure of a chromatographic system is generated by the system components, the capillaries, the column, the solvent, and the applied flow rate. Each system has a maximum pressure which it can sustain, and therefore the pressure must be monitored as it is a limiting factor during method development. The back pressure generated by a column is dependent on the particle size and its length. The development of a preparative method is ideally performed on an analytical scale and after optimization the method is then scaled up to preparative scale.</p><p>As preparative HPLC systems normally have lower maximum pressures, it is important to keep the generated pressure in the range of the later preparative system.&nbsp;</p><p>The larger the particles of the column material are, the lower will be the generated back pressure. With increasing particle size, the resolution is decreasing at the same time. Depending on the separation task, the resolution can be a critical point, and it is important to find the right compromise between resolution and backpressure. Additionally, larger particles are significantly less price intensive, which is another factor to be considered especially for large scale purifications. Often solvent mixtures of water with acetonitrile, methanol or ethanol are used for reverse phased methods. Depending on the organic solvent composition, the generated back pressure differs significantly which could be reduced by changing the solvent composition slightly or the organic solvent if possible. The back pressure generated by the solvent will not or only slightly change during the up-scaling process.&nbsp;</p><p>Here, the influence of particle size, flow rate and solvent composition on the back pressure was evaluated. Further, a practical scale-up was performed to evaluate the values obtained on analytical scale to those in larger scale.</p><h4><strong>User Tips:&nbsp;</strong></h4><ul><li>What is the finale scale you want to operate later?&nbsp;</li><li>What are the pressure limitations of the preparative system?&nbsp;</li><li>Be aware of the solvent back pressure&nbsp;</li><li>Consider particle size not only for best separation&nbsp;</li><li>Consider the flow rate</li></ul><h4><strong>MATERIAL AND METHODS&nbsp;</strong></h4><ul><li><strong>Pump: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2372"><strong>AZURA® P 6.1LHPG</strong></a>, 10 ml, sst&nbsp;</li><li><strong>Autosampler: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2375"><strong>AZURA AS 6.1L</strong></a></li><li><strong>Detector:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2423"><strong>UV AZURA DAD 2.1L&nbsp;</strong></a></li><li><strong>Flow cell: </strong>Pressure proof 10 mm, 10 µl, 300 bar&nbsp;</li><li><strong>Thermostat:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2376"><strong>CT2.1&nbsp;</strong></a></li><li><strong>Columns:&nbsp;</strong><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1633"><strong>Eurospher II 100-5 C18, 150 x 4.6 mm&nbsp;</strong></a></li><li>Eurospher II 100-10 C18, 150 x 4.6 mm&nbsp;</li><li>Eurospher II 100-15 C18, 150 x 4.6 mm&nbsp;</li><li>Eurospher II 100-20/45 C18, 150 x 4.6 mm &nbsp;</li></ul></li><li><strong>Software&nbsp;</strong><ul><li>ClarityChrom 10.1&nbsp;</li><li>ClarityChrom 10.1 PDA extension</li></ul></li></ul><h4><strong>CONCLUSION&nbsp;</strong></h4><p>The back pressures of four C18 150 x 4.6 mm columns, which differed only in particle size were measured and compared at different flow rates and solvent compositions. As expected, the 5 µm column showed the highest and the 20/45 µm column the lowest back pressure at all tested conditions. The pressure curves of ethanol and methanol gradients were similar, with a maximum pressure at approximately 50% solvent. The pressure curve of the acetonitrile gradient differed from the other two, showing the highest pressure at a lower organic solvent content of approximately 35%, after which the pressure dropped. Ethanol showed the highest pressure, followed by methanol and then acetonitrile. Determining the column back pressure at an analytical scale is an important tool during preparative method development. Ideally, if a linear scale up from the analytical to the preparative scale is conducted, the column back pressure should stay the same. This helps to predict if the method can be scaled up to the desired size in terms of back pressure.</p><h3><strong>3. Shimadzu / ASMS: Enhanced Quantification of Emerging UV Filter Contaminants in Drinking Water Using Optimized LC-MS/MS Technique</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/TP_121_Enhanced_Quantification_of_Emerging_UV_Filter_Contaminants_in_Drinking_Water_Using_Optimized_LC_MSMS_Technique_e733c64828.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Ultraviolet (UV) filter chemicals from sunscreens and personal care products are increasingly recognized as emerging contaminants of concern in drinking water1 . Their persistence and reported biological effects highlight the need for sensitive and reliable monitoring approaches2 . The objectives were to optimize quantitation to achieve ppt level detection without offline extraction and assess the occurrence of these compounds in multiple drinking water matrices. In this study, 20 UV filters were analyzed using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-8060RX"><strong>Shimadzu LCMS8060RX triple quadrupole mass spectrometer.</strong></a></p><h4><strong>Conclusion</strong></h4><p>A highly sensitive, precise, and robust direct injection LC–MS/MS method enables sub-ppt to high-ppb quantification of 20 UV filters in drinking water. Overall, the highly sensitive and robust Shimadzu LCMS-8060RX Triple Quadrupole Mass Spectrometer combined with this extraction-free workflow provides a simple, reliable, and high-throughput approach for quantifying emerging UV filters at ng/L concentrations in drinking water.</p><h3><strong>4. Thermo Fisher Scientific / HPLC Symposium: Comparison of biocompatible and inert UHPLC systems for LC-UV quantitation of large RNA</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_004752_hplc_vanquish_amplify_metal_sensitive_analyte_analysis_po004752_na_en_ff99cfde49.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Accurate LC-UV analysis of large RNA and oligonucleotide therapeutics requires highly inert chromatographic flow paths because interactions between the negatively charged phosphodiester backbone and metallic surfaces can cause analyte loss, peak broadening, and reduced quantitative sensitivity, particularly at low concentration.&nbsp;</p><p>Here, an inert UHPLC system – Vanquish Amplify system, incorporating inert-coated flow path components was evaluated against a biocompatible UHPLC configuration using MP35N alloy capillaries and additionally against a commercially available inert UHPLC platform. Large RNA fragments of 500 and 5000 nucleotides were analyzed by IP-RPLC to assess the difference between these systems. To further investigate flow path interactions, adenosine 5′- (α,β-methylene) diphosphate (AMPcP), a metal-sensitive analyte, was analyzed alongside adenosine, a noninteracting reference compound.</p><h5><strong>Material and Methods</strong></h5><ul><li><strong>Instrumentation: </strong>Thermo Scientific Vanquish Amplify UHPLC system</li><li><strong>Column:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=3&amp;search=SurePac+Oligo"><strong>Thermo Scientific SurePac Oligo RP MDi HPLC column</strong></a>,<br>2.5 µm 2.1 X 50 mm (P/N: 43712-052132</li><li><strong>Data Analysis: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/76"><strong>The Thermo Scientific Chromeleon Software 7.3.2 MUc Chromatography Data System (CDS)</strong></a> was used for all data acquisition and analysis.</li></ul><h4><strong>Conclusions&nbsp;</strong></h4><p>Using AMPcP with the Vanquish Amplify system demonstrated up to a twofold increase in sensitivity compared to systems with MP35N fluidics under conditions where metal interactions are most pronounced&nbsp;</p><p>For large RNA, the Vanquish Amplify UHPLC system delivered 1.25–1.35× higher signal intensity compared to systems with MP35N fluidics, enabling reduced sample consumption and improved quantitative reliability.&nbsp;</p><p>The Vanquish Amplify UHPLC system demonstrated comparable system inertness under the tested conditions for larger RNA fragments (5,000 nt) and slightly better system inertness for smaller fragments (500 nt) in this study than the MaxPeak HPS containing system, based on peak height and area comparisons using an external UV detector</p><h3><strong>5. Waters Corporation / HPLC Symposium: Development and Transfer of Quantitative Analytical Methods using Universal Detectors</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009493_8cbe4c3cc2.pdf"><strong>Full PDF for download</strong></a></li></ul><div><div><section><div><div><div><div><div><div><p>Universal detectors such as <strong>Charged Aerosol Detection (CAD)</strong> and <strong>Evaporative Light Scattering Detection (ELSD)</strong> provide valuable alternatives to traditional LC detectors including UV, fluorescence, and mass spectrometry, particularly for analytes lacking chromophores. Although both detectors are suitable for quantitative analysis, they rely on different detection principles—CAD measures the electrical charge transferred to aerosol particles, whereas ELSD detects light scattered by dried analyte particles. Because both detectors exhibit non-linear responses, quantitative methods typically require calibration models such as log-log linear or quadratic regression rather than conventional linear calibration. The poster discusses key considerations for developing robust quantitative methods and highlights the challenges associated with transferring methods between different universal detection platforms.</p><p>To demonstrate detector transferability, the authors migrated a <strong>HILIC method for sugar analysis in fruit juice</strong> from ELSD to CAD. The separation was performed on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/306"><strong>Waters Arc HPLC System</strong></a> equipped with an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=5&amp;search=XBridge+BEH+Amide"><strong>XBridge BEH Amide</strong></a><strong> (4.6 × 150 mm, 2.5 μm)</strong> column using a water/acetonitrile gradient containing triethylamine. While the chromatographic conditions remained unchanged, CAD detector parameters were optimized. Both detectors produced highly comparable quantitative performance across a calibration range of <strong>50–1000 μg/mL</strong>, achieving calibration coefficients (R²) above <strong>0.995</strong>, area RSD values below <strong>1.5%</strong>, and nearly identical concentrations of fructose, sorbitol, glucose, and sucrose in an apple juice sample. These results demonstrate that quantitative HILIC methods can be successfully transferred between ELSD and CAD with only detector-specific optimization.</p><p>The poster also evaluates method transfer between different CAD platforms using the <strong>USP Deoxycholic Acid assay</strong>. The method was implemented on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/198"><strong>Waters ACQUITY UPLC H-Class PLUS System</strong></a> with an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1823"><strong>XBridge BEH C18</strong></a><strong> (4.6 × 150 mm, 3.5 μm)</strong> column and compared against a competing LC/CAD system. After scaling the method according to <strong>USP Chapter &lt;621&gt;</strong>, both systems met all USP system suitability requirements, including repeatability and signal-to-noise criteria. Quantitative results for deoxycholic acid and related impurities were virtually identical, confirming that validated CAD methods can be reliably transferred between different vendors' LC/CAD platforms without compromising analytical performance.</p><p>Overall, the study demonstrates that both ELSD and CAD are robust universal detection techniques for quantitative liquid chromatography. While method transfer between identical detector types is generally straightforward, successful migration between ELSD and CAD—or between different CAD systems—requires careful optimization of detector parameters while preserving chromatographic conditions. The presented workflows illustrate that with appropriate calibration and detector tuning, laboratories can achieve equivalent sensitivity, precision, and quantitative accuracy, facilitating method standardization and transfer across analytical platforms.</p></div></div></div></div></div></div></section></div></div>

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