News from LabRulezLCMS Library - Week 07, 2026

<h3>How to Choose the Right Column for Oligonucleotide Analysis</h3><h4>Finding the Best Column for Your Oligonucleotides</h4>Selecting an appropriate chromatographic column for oligonucleotide analysis can be challenging. These molecules are long, highly charged, and often require specialized separation strategies. With multiple chromatographic techniques available, choosing the most suitable approach for your application is not always straightforward.However, understanding the characteristics of oligonucleotides and the analytical requirements of your workflow can significantly simplify the process.<h4>Why Are Oligonucleotides Difficult to Separate?</h4>Over the past several years, oligonucleotides have become increasingly important in molecular biology, biotechnology, and therapeutic research. Their growing role in regulated environments, including pharmaceutical development, means that precise and reliable analytical methods are essential.From a chromatographic perspective, oligonucleotides present several challenges.These molecules are typically highly polar and strongly charged, consisting of phosphodiester or phosphorothioate backbones. During synthesis, numerous closely related impurities can be generated. These include fragments that differ slightly in chain length (shortmers or longmers), as well as molecules with minor structural variations such as single-base substitutions or functional group modifications.In addition, oligonucleotide samples frequently contain residual reagents, salts, and by-products from synthesis. These components can overload chromatographic columns and negatively affect separation efficiency.For analytical laboratories, this means that a suitable column must deliver:<ul><li>High resolution between closely related structures</li><li>Symmetrical and well-defined peaks</li><li>Fast analysis times</li><li>Compatibility with mass spectrometry (MS) detection</li></ul>At the same time, the method must provide reliable reproducibility and minimize sample loss during elution or desalting.Understanding these analytical requirements is the first step toward selecting the most appropriate chromatographic solution.<h4>Which Chromatographic Approach Should You Choose?</h4>Separating oligonucleotides is inherently complex because many compounds differ only slightly in structure, sometimes by a single nucleotide or modification.Different chromatographic techniques are therefore used depending on the analytical objective.For purification of synthesized strands, preparative liquid chromatography or size exclusion chromatography (SEC) is typically employed.For analytical characterization, UHPLC coupled with mass spectrometry is widely used because it enables both qualitative and quantitative analysis of products and impurities.Before purification or analysis, samples may also require desalting, which can be achieved efficiently using dedicated SEC desalting cartridges to remove interfering salts.Even when the general purpose—purification or analysis—is known, it is still necessary to decide how detailed the separation needs to be and whether robustness or analytical depth should take priority.<blockquote>“Clearly defining your chromatographic goals is the first step toward selecting the right separation method.”Dr. Ulrike Krop, Head of Applications &amp; Academy, KNAUER</blockquote><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Knauer_Figure_1_Decision_tree_Which_separation_technology_for_your_purification_task_5564a4be93.jpg" alt="KNAUER: Figure 1: Decision tree - Which separation technology for your purification task?" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Knauer_Figure_1_Decision_tree_Which_separation_technology_for_your_purification_task_5564a4be93.jpg 178w," sizes="100vw" width="1920" height="1683"><h4>Choosing a Method for Oligonucleotide Purification</h4>When the goal is purification, the amount of material to be processed plays a key role.If only small quantities of oligonucleotides need to be purified, columns with smaller internal diameters and lower flow rates are typically sufficient.For applications where high resolution is required, ion-pair reversed-phase chromatography (IP-RP) can be an effective option.Alternatively, laboratories that prioritize environmentally friendly eluents, cost efficiency, and high loading capacity may prefer anion exchange chromatography (AEX) for preparative separations.If the objective is mainly to separate smaller fragments or remove residual synthesis reagents, size exclusion chromatography (SEC) provides a simple and environmentally friendly solution.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Knauer_Figure_2_Decision_tree_What_are_your_priorities_for_your_analysis_2e26d9955e.jpg" alt="KNAUER: Figure 2: Decision tree – What are your priorities for your analysis?" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Knauer_Figure_2_Decision_tree_What_are_your_priorities_for_your_analysis_2e26d9955e.jpg 163w," sizes="100vw" width="1920" height="1840"><h4>Selecting an Analytical Strategy</h4>In analytical workflows, the decision often comes down to a balance between maximum resolution and operational robustness.High-throughput laboratories that routinely perform similar analyses and require stable long-term operation typically choose between IP-RP and AEX methods.Among these approaches, ion-pair reversed-phase chromatography has become widely established due to its excellent efficiency and resolution. In addition, IP-RP methods generally offer good compatibility with mass spectrometry, making them suitable for detailed impurity profiling.Columns specifically designed for oligonucleotide separations, such as Sepapure oliGO columns, are optimized for this application and provide reliable performance across a wide range of workflows.<h4>Methods for Advanced Research Applications</h4>In research and development settings, analytical flexibility and sensitivity may be more important than routine efficiency.For example, if the goal is to identify minor differences in side-chain modifications between two otherwise identical oligonucleotides, techniques such as HILIC (hydrophilic interaction chromatography) or mixed-mode chromatography can be particularly useful.These methods provide additional separation mechanisms and allow extensive method optimization. Depending on the chosen eluents, they can also remain compatible with mass spectrometric detection.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Benefits_and_Limitations_at_a_Glance_34c0cc3066.jpg" alt="KNAUER: Benefits and Limitations at a Glance" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Benefits_and_Limitations_at_a_Glance_34c0cc3066.jpg 138w," sizes="100vw" width="959" height="1082"><h4>Do You Need Bioinert Hardware?</h4>Bioinert chromatographic hardware is commonly associated with protein purification, where metal surfaces can interact with amino acid residues such as histidine or cysteine and alter protein structure.To prevent these interactions, chromatographic systems may use passivated surfaces, titanium components, or glass-lined flow paths. However, these solutions can significantly increase costs.For oligonucleotide separations, the benefits of bioinert hardware are less clear.<blockquote>“I have performed many analyses with different oligonucleotides and have not observed significant differences between bioinert and stainless steel hardware. Only in extremely fast UHPLC runs—around three minutes—might this become relevant.”Marvin Schaek, Product Manager Oligosynthesizer, KNAUER &amp; XNAPharma GmbH</blockquote>Since oligonucleotide structure is generally less sensitive to conformational changes than proteins, stainless steel hardware often performs adequately.However, in cases where phosphorothioate backbones are present, interactions with metal surfaces may occur. Some studies suggest that column passivation can improve peak shape in very fast UHPLC separations, although the impact in typical 10-minute methods appears minimal.<h4>Final Thoughts</h4>Anyone working with oligonucleotides will inevitably encounter the need for purification and high-quality chromatographic analysis.Fortunately, a variety of chromatographic techniques are available, allowing scientists to tailor their workflows to specific analytical goals. By carefully evaluating sample characteristics, desired resolution, and operational priorities, laboratories can select the most appropriate column and separation method.For IP-RP workflows ranging from UHPLC to semi-preparative scale, columns such as Sepapure oliGO provide a reliable and cost-effective option specifically designed for oligonucleotide applications.<blockquote>If you are planning to develop or improve your oligonucleotide workflow, feel free to <a href="sales@knauer.net">contact the KNAUER team</a> for further guidance.</blockquote>

Oligos Made Easy - Part 6: Column Navigator

News from LabRulezLCMS Library - Week 15, 2026

News from LabRulezLCMS Library - Week 16, 2026

<h4>The science behind gummy candies</h4>Who can resist colourful, fruity gummy candies 🍬 with their signature chewy and elastic texture? While they may appear simple, their formulation is actually quite sophisticated. Each gummy is produced using a precisely balanced combination of gelling agents, sugars, acids, flavourings, and colourants, all working together to deliver the desired taste and texture.For instance, the characteristic chewiness of gummy sweets results from the interaction between gelatin and sugars. Even minor adjustments—such as changing the type or concentration of the gelling agent—can significantly alter the final product. In addition, manufacturers must meet regulatory and labeling requirements, including certifications like Halal compliance. This makes accurate and consistent monitoring of ingredients essential throughout production.<h4>The importance of SEC fingerprinting</h4>Size Exclusion Chromatography (SEC), also known as Gel Permeation Chromatography (GPC), offers a fast and reliable way to analyze gummy formulations. By separating molecules according to their hydrodynamic size, SEC generates a unique molecular “fingerprint” for each product.This fingerprint enables manufacturers to:<ul><li>confirm the type and origin of gelling agents (e.g., for Halal verification)</li><li>ensure batch-to-batch consistency and detect production variations</li><li>monitor changes during storage to assess shelf life</li><li>evaluate low-molecular-weight compounds such as sugars in a semi-quantitative manner</li></ul>Overall, SEC fingerprinting supports consistent product quality and regulatory compliance, ensuring that every gummy maintains the same expected characteristics.<h4>Influence of column selection on SEC performance</h4>Column selection plays a crucial role in achieving high-quality separations and detailed molecular fingerprints. To illustrate this, porcine and bovine gelatin standards were analyzed using two different configurations:<ul><li>a single Multipore <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1655">SuperOH-P column</a></li><li>a coupled system of SuperOH-P 350 and SuperOH-P 150 columns</li></ul>Although both setups cover the same molecular weight range (100–1,000,000 Da), the coupled configuration effectively doubles the separation length. This results in improved resolution, which was confirmed experimentally. The coupled columns provided clearer separation between porcine and bovine gelatin, producing more distinct and informative fingerprints.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Knauer_Fig_1_Overlay_chromatograms_measured_with_DAD_at_230_nm_20_L_d81181cfb4.jpg" alt="KNAUER: Fig. 1 Overlay chromatograms measured with DAD at 230 nm, 20 µL. Dark blue: porcine reference standard; red: bovine reference standard; solid line: measured with column A; dotted line: measured with column set B. Graphic by KNAUER." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Knauer_Fig_1_Overlay_chromatograms_measured_with_DAD_at_230_nm_20_L_d81181cfb4.jpg 245w," sizes="100vw" width="1024" height="611"><h4>Achieving reliable SEC fingerprints</h4>Reproducibility is key when using SEC for quality control. The process begins with consistent sample preparation—maintaining identical dissolution procedures, concentrations, and filtration steps is essential, as even small variations can influence peak shape and overall results.Beyond the column, the entire system setup is equally important. For example, combining a Refractive Index Detector (RID) with a Diode Array Detector (DAD) allows simultaneous detection of sugars, acids, and polymers within a single analysis.Accurate peak identification further enhances reliability. By analyzing reference standards based on known ingredient compositions, chromatographic peaks can be correctly assigned, enabling detailed interpretation of both high- and low-molecular-weight components.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Knauer_Fig_2_Zoom_overlay_chromatograms_measured_with_RID_20_L_and_column_set_B_37caf5ac59.jpg" alt="KNAUER: Fig. 2 Zoom overlay chromatograms measured with RID, 20 µL and column set B. Black: Katjes® Yoghurt Gums; turquoise: apple pectin diluted 1:10; dark green: preserving sugar 1:1 (sugar; citric acid; pectin; fully hydrogenated sunflower oil); dark blue: maltodextrin; pink: sodium potassium tartrate; yellow: potassium citrate; red: citric acid; purple: lactic acid; beige: lactose; orange: sucrose; green: glucose; grey: blank. Graphic by KNAUER. " srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Knauer_Fig_2_Zoom_overlay_chromatograms_measured_with_RID_20_L_and_column_set_B_37caf5ac59.jpg 245w," sizes="100vw" width="1024" height="611">Additionally, incorporating a flow marker helps correct for system-related variations, such as fluctuations in flow rate, thereby improving retention time stability and peak assignment accuracy. More details on the flow marker and its use in SEC are described in <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/VTN_0043_knauer_gpc_sec_data_reproducibility_with_flow_marker_96ee6b3cd9.pdf">this technical note</a>.<h4>Verification of gelatin origin using SEC</h4>Determining the source of gelatin is critical for proper labeling and compliance, particularly for Halal-certified products where porcine gelatin is not permitted.To demonstrate SEC’s capability, two gummy samples with identical formulations but different gelatin sources were analyzed. One sample contained porcine gelatin, while the other used bovine gelatin. The resulting SEC fingerprints showed clear differences in the gelatin region, allowing unambiguous identification. The standard Haribo® Goldbears corresponded to the porcine reference, whereas the Halal version matched the bovine standard.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Knauer_Fig_3_Overlay_chromatograms_measured_with_DAD_at_230_nm_20_L_and_column_set_B_12e99a43e7.jpg" alt="KNAUER: Fig. 3 Overlay chromatograms measured with DAD at 230 nm, 20 µL and column set B. Solid dark blue line: Haribo® Goldbears; solid red line: Halal Haribo® Goldbears; dotted dark blue line: porcine reference standard; dotted red line: bovine reference standard. Graphic by KNAUER. " srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Knauer_Fig_3_Overlay_chromatograms_measured_with_DAD_at_230_nm_20_L_and_column_set_B_12e99a43e7.jpg 245w," sizes="100vw" width="1024" height="611"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Fig_4_Overlay_chromatograms_measured_with_RID_20_L_and_column_set_B_2_c29ea59bc4.jpg" alt="KNAUER: Fig. 4 Overlay chromatograms measured with RID, 20 µL and column set B. Solid dark blue line: Haribo® Goldbears; solid red line: Halal Haribo® Goldbears; dotted dark blue line: porcine reference standard; dotted red line: bovine reference standard. Graphic by KNAUER." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Fig_4_Overlay_chromatograms_measured_with_RID_20_L_and_column_set_B_2_c29ea59bc4.jpg 245w," sizes="100vw" width="1368" height="432">This confirms that SEC is a reliable method for distinguishing gelatin origin.<h4>Role of low-molecular-weight components</h4>In addition to polymers such as gelatin, SEC can also reveal smaller molecules, including sugars and organic acids. While SEC is not a replacement for fully validated HPLC methods, it serves as a rapid screening tool for comparing samples and supporting quality control.Using RID detection, differences in sugar composition become visible. For example, chromatograms showed a significantly higher sucrose signal in preserving sugar compared to gummy candies, reflecting formulation differences.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Knauer_Fig_5_Zoom_overlay_chromatograms_measured_with_RID_20_L_and_column_set_B_06c072e069.jpg" alt="KNAUER: Fig. 5 Zoom overlay chromatograms measured with RID, 20 µL and column set B. Black: sucrose 1mg/ml; blue: Haribo® Goldbears; green: preserving sugar 1:1 (sugar; citric acid; pectin; fully hydrogenated sunflower oil). Graphic by KNAUER." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Knauer_Fig_5_Zoom_overlay_chromatograms_measured_with_RID_20_L_and_column_set_B_06c072e069.jpg 245w," sizes="100vw" width="1024" height="611"><h4>Conclusion</h4>SEC is a powerful analytical technique for monitoring product quality and ensuring process consistency in gummy candy production. By generating detailed molecular fingerprints, it enables manufacturers to verify ingredients, maintain consistency, and comply with regulatory requirements.Importantly, this approach is not limited to gummy candies—it can be adapted to a wide range of products where molecular characterization is essential.<blockquote><ul><li>If you're planning to set up or optimize your GPC/SEC workflow, feel free to contact us at sales@knauer.net. Stay tuned for more exciting insights into the GPC/SEC world in our “GPC/SEC Made Easy” series.</li><li>For more information on the technical details, you can refer to <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/VFD_0195_sec_for_fingerprint_analysis_of_gummy_candies_ce5349cd95.pdf">this application note.</a></li><li>For further information on this topic, please contact our author: wesolowski@knauer.net</li></ul></blockquote>

GPC/SEC Made Easy – Fingerprint analysis of Gummy Candies

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

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