News from LabRulezLCMS Library - Week 08, 2026

<h3><strong>Size Matters: A Practical Guide to Size Exclusion Chromatography for Oligonucleotides</strong></h3><h4><strong>Why Consider SEC in Modern Oligonucleotide Workflows?</strong></h4><p>Oligonucleotides are now central to advanced therapeutics, molecular diagnostics, and synthetic biology. As their applications expand, so does the need for highly purified and well-characterized products.</p><p>Size Exclusion Chromatography (SEC) offers a valuable solution in this evolving landscape. Unlike separation techniques based on charge or hydrophobicity, SEC separates molecules strictly according to their size. This means oligos remain structurally intact during purification while unwanted components — such as salts, truncated strands, aggregates, and small molecular impurities — are removed.</p><p>SEC can be applied to smaller oligos, but it is particularly powerful for desalting and buffer exchange. For high-resolution separations and detailed characterization, techniques such as AEX or HILIC are often preferred. Understanding the strengths of SEC allows you to use it effectively within your purification strategy.</p><h4><strong>How SEC Purifies Oligonucleotides</strong></h4><p>Imagine a chromatographic column filled with microscopic porous beads. These beads act like selective barriers: molecules are separated based on whether they can enter the pores or not.</p><p>When a mixture containing oligonucleotides and impurities passes through:</p><ul><li>Large oligos are excluded from the pores and move quickly around the beads.</li><li>Smaller molecules enter the pores and travel a longer path.</li><li>Medium-sized species fall somewhere in between.</li></ul><p>As a result, the target oligonucleotide elutes first, separated from salts, degraded fragments, and other small contaminants.</p><p>Importantly, SEC operates under native conditions. There are no strong interactions, no harsh gradients, and no disruption of secondary structures. Separation is governed purely by molecular size.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_1_SEC_Principle_750f59c842.jpg" alt="KNAUER: Figure 1 - SEC Principle." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_1_SEC_Principle_750f59c842.jpg 245w," sizes="100vw" width="951" height="256"></p><h4><strong>The Core Principle: Size Dictates Elution</strong></h4><p>In SEC, molecular dimensions determine migration speed:</p><ul><li><strong>Large oligos</strong> cannot penetrate the pores and therefore elute first.</li><li><strong>Small impurities</strong> such as salts and solvent residues enter the pores and elute last.</li><li><strong>Intermediate species</strong>, including truncated oligos, elute between the two extremes.</li></ul><p>This mechanism makes SEC ideal for allowing your target oligo to “take the fast lane” while smaller contaminants are retained longer within the porous matrix.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KANUER_Figure_2_SEC_Elution_51d86ab0da.jpg" alt="KANUER: Figure 2 - SEC Elution" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KANUER_Figure_2_SEC_Elution_51d86ab0da.jpg 200w," sizes="100vw" width="1075" height="836"></p><h4><strong>Step-by-Step Guide to Oligo Purification Using SEC</strong></h4><h5><strong>1. Selecting the Appropriate Column</strong></h5><p>Typical oligonucleotides range from 10–50 nucleotides (~3–15 kDa).</p><p>Recommended pore sizes:</p><ul><li><strong>100–150 Å</strong> for short oligos (10–25 nt)</li><li><strong>200–300 Å</strong> for longer strands (25–50 nt) or aggregates</li></ul><p>Columns packed with high-quality SEC media such as AppliChrom® or equivalent materials are well suited for these applications.</p><h5><strong>2. Preparing the Sample</strong></h5><ul><li>Allow complete dissolution (overnight if necessary).</li><li>Filter samples to prevent column blockage.</li><li>Maintain moderate concentrations (e.g., 0.1–1 mg/mL) for optimal separation.</li></ul><h5><strong>3. Choosing the Mobile Phase</strong></h5><p>SEC for oligonucleotides typically employs simple aqueous buffers:</p><ul><li>50 mM phosphate or Tris</li><li>100–150 mM NaCl (to reduce non-specific interactions)</li></ul><p>Avoid detergents and organic modifiers that may compromise column stability.<br>After buffer use, flush with water (optionally containing sodium azide) to maintain column integrity.</p><h5><strong>4. Optimizing Flow Rate</strong></h5><ul><li>Analytical columns: 0.2–0.5 mL/min improves resolution.</li><li>Preparative runs: higher flow rates can be used cautiously, balancing speed and separation quality.</li></ul><p>Always monitor system backpressure.</p><h5><strong>5. Sample Loading</strong></h5><p>Avoid overloading. Excess injection volumes compromise resolution.<br>Confirm the resin’s loading capacity before scaling up.</p><h5><strong>6. Fraction Collection</strong></h5><p>Monitor UV absorbance at 260 nm.<br>Collect early-eluting peaks corresponding to the target oligo.<br>Later fractions generally contain salts and smaller impurities.</p><h5><strong>7. Purity Verification</strong></h5><p>Confirm purity using complementary analytical methods such as:</p><ul><li>AEX</li><li>IP-RP</li><li>HILIC HPLC</li></ul><h4><strong>Critical Column Parameters That Influence Performance</strong></h4><h5><strong>Pore Size</strong></h5><p>Pore dimensions determine the effective separation range. Proper pore selection ensures clean size-based discrimination between oligos and contaminants.</p><h5><strong>Inert Surface Chemistry</strong></h5><p>The ideal SEC matrix should be non-interactive:</p><ul><li>No ionic interactions</li><li>No hydrophobic adsorption</li><li>No secondary retention effects</li></ul><p>Separation should rely solely on molecular size, not chemical affinity.</p><h5><strong>Mechanical and Chemical Stability</strong></h5><p>Reliable SEC gels maintain:</p><ul><li>Structural integrity under pressure</li><li>Stability in various aqueous buffers</li><li>Performance at elevated temperatures</li></ul><p>Abrupt changes in flow rate, temperature, or solvent composition should be avoided.</p><h5><strong>Hydrophilicity</strong></h5><p>SEC media are typically hydrophilic and compatible with aqueous systems. Common materials include:</p><ul><li>Crosslinked dextran</li><li>Agarose</li><li>Polyacrylamide</li><li>Hydroxylated methacrylate</li></ul><p>These materials are well suited for oligonucleotide purification.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_3_SEC_Column_Gel_Parameter_Characterization_1f8fa93d39.jpg" alt="KNAUER: Figure 3 - SEC Column Gel Parameter Characterization" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_3_SEC_Column_Gel_Parameter_Characterization_1f8fa93d39.jpg 245w," sizes="100vw" width="951" height="541"></p><h4><strong>Practical Optimization Tips</strong></h4><ul><li><strong>Desalting pre-treatment:</strong> Avoid redundant desalting if the sample is salt-rich.</li><li><strong>Temperature control:</strong> Maintain consistent room temperature or use a column thermostat.</li><li><strong>pH range:</strong> Keep between 6.5–8.0 to protect both oligos and gel stability.</li><li><strong>Buffer exchange:</strong> Use your final formulation buffer as the mobile phase when possible.</li><li><strong>Injection volume (analytical):</strong> &lt;2–5% of column volume.</li><li><strong>Injection volume (preparative):</strong> 5–10%, depending on resin capacity.</li><li><strong>Pressure management:</strong> Avoid sudden changes to prevent bed collapse.</li><li><strong>Fraction collection:</strong> Base cuts on peak shape and symmetry, not retention time alone.</li><li><strong>Column care:</strong> Use filtered samples, flush properly, store in preservative (e.g., 20–30% ethanol), and monitor performance routinely.</li></ul><p>Proper maintenance ensures reproducible, long-term performance.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_4_Preparative_SEC_with_Fraction_Collection_ff1251708d.jpg" alt="KNAUER: Figure 4 - Preparative SEC with Fraction Collection" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_4_Preparative_SEC_with_Fraction_Collection_ff1251708d.jpg 245w," sizes="100vw" width="951" height="585"></p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KANUER_Figure_5_SEC_Column_Maintenance_92b48ca7e7.jpg" alt="KANUER: Figure 5 - SEC Column Maintenance" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KANUER_Figure_5_SEC_Column_Maintenance_92b48ca7e7.jpg 159w," sizes="100vw" width="798" height="781"></p><h4><strong>Strengths and Limitations in Practice</strong></h4><p>SEC is widely appreciated for its simplicity and gentle operation. It does not require gradients or complex additives and maintains oligonucleotide integrity.</p><p>However, it is not universally superior.</p><h5><strong>Where SEC Excels:</strong></h5><ul><li>Desalting</li><li>Buffer exchange</li><li>Removal of small molecules</li><li>Rapid quality control checks</li><li>Aggregate analysis</li></ul><h5><strong>Where Other Methods May Be Better:</strong></h5><ul><li>High-resolution impurity profiling</li><li>Detailed structural characterization</li><li>Separation of closely related sequence variants</li></ul><p>Techniques such as AEX, IP-RP, or HILIC frequently provide higher resolution for demanding analytical tasks.</p><h4><strong>Final Takeaway</strong></h4><p>Size Exclusion Chromatography is a dependable and versatile tool in oligonucleotide purification. While it may not replace higher-resolution chromatographic methods, it remains an essential component of efficient purification workflows.</p><p>The key is not choosing one method exclusively — but knowing when SEC is the right solution and when to complement it with alternative techniques for optimal results.</p><blockquote><p>If you're planning to set up or optimize your oligonucleotide workflow, feel free to contact us at <strong>sales@knauer.net</strong>. Stay tuned for more exciting insights into the Oligonucleotide world in our “Oligos Made Easy” series.</p></blockquote>

Oligos Made Easy - Part 5: SEC

News from LabRulezLCMS Library - Week 11, 2026

<p><strong>Even in chromatography, appearances can be misleading. If you have ever performed a Liquid Chromatography (LC) or Gel Permeation Chromatography/Size Exclusion Chromatography (GPC/SEC) analysis and noticed unexpected signals appearing in your chromatogram, you may have encountered so-called chromatographic “ghosts.” These unanticipated peaks can complicate data interpretation and challenge even experienced analysts. Fortunately, these ghost peaks are not supernatural phenomena. In this Halloween-themed overview, we explore their origin, discuss strategies to minimize their occurrence, and describe practical approaches to identify them.</strong></p><h4><strong>What are ghost peaks?</strong></h4><p>When a chromatogram displays additional peaks that cannot be directly attributed to analytes of interest, these signals are commonly referred to as ghost peaks or system peaks [1]. They arise when the equilibrium between the mobile phase and stationary phase is temporarily disturbed [1]. Such disturbances often occur during sample injection, when the system experiences a transient compositional or physical imbalance [1][2].</p><p>Ghost peaks are particularly evident when using refractive index (RI) detection, where even injections of pure mobile phase can generate detectable signals (see Fig. 1). In GPC/SEC applications, these peaks frequently appear toward the end of the chromatogram, corresponding to the transition between size-based separation and interaction-driven effects [2]. Their appearance can closely resemble genuine analyte peaks and, in some cases, they may partially overlap with them, further complicating interpretation.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Fig_1_Chromatogram_of_a_20_l_blank_injection_mobile_phase_with_RI_detection_Graphic_by_KNAUER_72b81051f9.jpg" alt="KNAUER: Fig. 1 Chromatogram of a 20 µl blank injection (= mobile phase) with RI detection. Graphic by KNAUER." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Fig_1_Chromatogram_of_a_20_l_blank_injection_mobile_phase_with_RI_detection_Graphic_by_KNAUER_72b81051f9.jpg 245w," sizes="100vw" width="1024" height="611"></p><h4><strong>How to minimize ghost peaks?</strong></h4><p>Ghost peaks may manifest as either positive or negative signals. While their retention time typically remains consistent, their intensity, shape, and area can vary depending on injection conditions, sample composition, and injection volume (see Fig. 2). Due to this variability, they are not suitable as reference markers, for example in GPC/SEC analyses.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Fig_2_Overlay_chromatograms_measured_with_RID_and_20_l_injection_volume_c5b4b8f3f6.jpg" alt="KNAUER: Fig. 2 Overlay chromatograms measured with RID and 20 µl injection volume. Dotted black: citric acid; solid black: ethylene glycol. Graphic by KNAUER." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Fig_2_Overlay_chromatograms_measured_with_RID_and_20_l_injection_volume_c5b4b8f3f6.jpg 245w," sizes="100vw" width="1024" height="611"></p><p>To reduce their impact, several practical measures can be implemented:</p><ul><li>Ensure that samples are dissolved in the same mobile phase used in the chromatographic system</li><li>Use smaller injection volumes to limit system disturbance</li><li>Allow sufficient equilibration time for the system before analysis</li></ul><p>Despite these precautions, ghost peaks cannot be completely eliminated. Therefore, recognizing and correctly assigning them is essential for reliable data interpretation. Failure to do so may lead to misinterpretation of chromatographic results.</p><h3><strong>How to unmask ghost peaks?</strong></h3><p>A systematic approach can help identify the origin of ghost peaks. One effective strategy involves preparing each component of the mobile phase individually, spiking them into the mobile phase, and injecting them separately.</p><p>For example, in an aqueous SEC method using phosphate-buffered saline (PBS), individual injections of buffer components such as NaCl, KCl, KH₂PO₄, and Na₂HPO₄ can be compared with a blank injection (see Fig. 3). By overlaying these chromatograms, it becomes possible to assign specific ghost peaks to individual buffer constituents. This not only clarifies their origin but also provides an opportunity to optimize the method by adjusting buffer composition or concentration.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Fig_3_Overlay_chromatograms_measured_with_RID_and_20_l_injection_volume_c5f57e44f6.jpg" alt="KNAUER: Fig. 3 Overlay chromatograms measured with RID and 20 µl injection volume. Black: blank (mobile phase = PBS); light green: sodium chloride; dark green: potassium chloride; light blue: potassium dihydrogen phosphate; dark blue: disodium hydrogen phosphate. Graphic by KNAUER." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Fig_3_Overlay_chromatograms_measured_with_RID_and_20_l_injection_volume_c5f57e44f6.jpg 245w," sizes="100vw" width="1024" height="611"></p><p>Another practical approach involves comparing a blank chromatogram (mobile phase only) with that of the sample. Peaks that appear at identical retention times in both chromatograms can be confidently identified as ghost peaks.</p><p>This concept is illustrated in Fig. 4 for an aqueous SEC system using PBS. Blank injections reveal two small peaks detected by the RI detector. In the case of citric acid, only two distinct peaks are observed instead of three, as one ghost peak coelutes with the analyte, forming a subtle shoulder (Fig. 4A). This demonstrates how ghost peaks can distort peak shapes and affect interpretation.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Fig_4_Left_4a_Overlay_of_chromatograms_recorded_with_the_RID_using_a_20_L_injection_volume_1732ef3a18.jpg" alt="KNAUER: Fig. 4 Left (4a): Overlay of chromatograms recorded with the RID using a 20 µL injection volume. Orange: citric acid; black: blank (mobile phase = PBS)" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Fig_4_Left_4a_Overlay_of_chromatograms_recorded_with_the_RID_using_a_20_L_injection_volume_1732ef3a18.jpg 245w," sizes="100vw" width="1024" height="611"></p><p>In contrast, ethylene glycol produces three clearly resolved peaks (Fig. 4B), where ghost peaks do not interfere directly with the analyte signal. Nevertheless, their identification remains crucial to ensure accurate evaluation of the chromatographic data.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Fig_4_Left_4b_Overlay_of_chromatograms_recorded_with_the_RID_using_a_20_L_injection_volume_149ed84d87.jpg" alt="KNAUER: Fig. 4b - Overlay of chromatograms recorded with the RID using a 20 µL injection volume. Orange: ethylene glycol; black: blank (mobile phase = PBS). Graphic by KNAUER." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Fig_4_Left_4b_Overlay_of_chromatograms_recorded_with_the_RID_using_a_20_L_injection_volume_149ed84d87.jpg 245w," sizes="100vw" width="1024" height="611"></p><h4><strong>Conclusion</strong></h4><p>Ghost peaks are a common yet often misunderstood phenomenon in LC and GPC/SEC analyses. Although they may initially appear mysterious, they are the result of predictable physicochemical processes within the chromatographic system. By understanding their origin, applying appropriate experimental strategies, and using systematic identification approaches, analysts can effectively distinguish them from true analyte signals. Recognizing these “ghosts” ensures more accurate data interpretation and helps prevent misleading conclusions in chromatographic analysis.</p><blockquote><ul><li>If you’d like to learn more about detecting and minimizing system or ghost peaks in practice, take a look at our <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/VTN_0046_knauer_understanding_system_peaks_in_gpc_sec_for_accurate_and_reliable_analysis_f134154935.pdf"><strong>Technical Note VTN0046</strong></a> on identifying and reducing system peaks in GPC/SEC.</li><li>If you're planning to set up or optimize your GPC/SEC workflow, feel free to contact us at <strong>sales@knauer.net</strong>.&nbsp;</li></ul></blockquote>

GPC/SEC Made Easy – Unmask Ghost Peaks

<h4><span><strong>The science behind gummy candies</strong></span></h4><p><strong>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.</strong></p><p>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.</p><h4><span><strong>The importance of SEC fingerprinting</strong></span></h4><p>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.</p><p>This fingerprint enables manufacturers to:</p><ul><li><strong>confirm the type and origin</strong> of <strong>gelling agents</strong> (e.g., for Halal verification)</li><li>ensure <strong>batch-to-batch consistency</strong> and detect production variations</li><li><strong>monitor changes during storage</strong> to assess shelf life</li><li>evaluate <strong>low-molecular-weight compounds</strong> such as sugars in a semi-quantitative manner</li></ul><p>Overall, SEC fingerprinting supports consistent product quality and regulatory compliance, ensuring that every gummy maintains the same expected characteristics.</p><h4><span><strong>Influence of column selection on SEC performance</strong></span></h4><p>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:</p><ul><li>a single Multipore <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1655"><strong>SuperOH-P column</strong></a></li><li>a coupled system of SuperOH-P 350 and SuperOH-P 150 columns</li></ul><p>Although both setups cover the same molecular <strong>weight range (100–1,000,000 Da)</strong>, 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.</p><p><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"></p><h4><span><strong>Achieving reliable SEC fingerprints</strong></span></h4><p>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.</p><p>Beyond the column, the entire system setup is equally important. For example, <strong>combining</strong> a <strong>Refractive Index Detector</strong> (RID) with a <strong>Diode Array Detector (DAD)</strong> allows simultaneous detection of sugars, acids, and polymers within a single analysis.</p><p>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.</p><p><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"></p><p>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"><strong>this technical note</strong></a>.</p><h4><span><strong>Verification of gelatin origin using SEC</strong></span></h4><p>Determining the source of gelatin is critical for proper labeling and compliance, particularly for Halal-certified products where porcine gelatin is not permitted.</p><p>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.</p><p><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"></p><p><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"></p><p>This confirms that SEC is a reliable method for distinguishing gelatin origin.</p><h4><span><strong>Role of low-molecular-weight components</strong></span></h4><p>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.</p><p>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.</p><p><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"></p><h4><span><strong>Conclusion</strong></span></h4><p>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.</p><p>Importantly, this approach is not limited to gummy candies—it can be adapted to a wide range of products where molecular characterization is essential.</p><blockquote><ul><li>If you're planning to set up or optimize your GPC/SEC workflow, feel free to contact us at <strong>sales@knauer.net</strong>. 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"><strong>this application note.</strong></a></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.

KNAUER

TIDES USA 2026

Eurachem/CITAC Workshop on Quality in Analytical Measurements 2026

Waters Informatics Users Meeting 2026

Uncovering Microplastics: Identification, Quantification, and Exposure Assessment

Size variant analysis of antibodies and ADCs using SEC, SEC-MALS, SEC-MS, and 2D Protein A-SEC Methods

Beyond C18 and Phenyl: Expanding Selectivity with Biphenyl Columns Built for UV and pH Robustness