This guide explains how to select an appropriate size exclusion chromatography (SEC) column for the analysis of biologics. SEC separates molecules based on their hydrodynamic radius, reflecting their size and shape rather than molecular weight.<ul><li>Molecules penetrate the pores of the stationary phase</li><li>There are no chemical interactions with the stationary phase</li><li>Larger molecules elute faster</li><li>Smaller molecules elute later</li></ul><h3>Principle of SEC Separation</h3>The separation mechanism is based on molecular size:Large molecules → do not enter the pores → travel through the interstitial spaces between particles → elute earlierSmall molecules → enter the pores → follow a longer path → elute later<h3>Key Parameters for Column Selection</h3><h4>1. Molecule Nature</h4>Start by evaluating:<ul><li>hydrodynamic radius</li><li>molecular shape</li></ul>Examples:<ul><li>Peptides: ~0.7 nm</li><li>Large biomolecules (e.g., AAVs): ~25 nm</li><li>mRNA: elongated, non-globular structure</li></ul><h4>2. Pore Size Selection</h4>The pore size of the stationary phase should be:<ul><li>3–4 times larger than the hydrodynamic radius of the target molecule</li></ul><h4>3. Particle Size</h4><ul><li>≤ 3 µm</li><li>ensures high separation efficiency</li></ul><h4>4. Stationary Phase Material</h4><ul><li>Prefer mechanically robust media, such as silica</li><li>Capable of withstanding high back pressure</li></ul><h4>5. Surface Chemistry</h4><ul><li>Chemical surface inertness is critical</li><li>Diol chemistry is preferred, because:<ul><li>it minimizes interactions</li><li>it provides a highly hydrophilic surface</li></ul></li></ul><h4>6. Column Hardware</h4><ul><li>Use bio-inert hardware</li><li>ensures:<ul><li>minimal analyte loss</li><li>high reproducibility</li></ul></li></ul><h3>Available SEC Column Options (<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=353&amp;manufacturerItems=50&amp;search=dsec">BioZen dSEC Family</a>)</h3><h4>1. <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2192">90 Å (9 nm)</a></h4><ul><li>peptides</li><li>small proteins</li><li>aggregates up to ~50 kDa</li></ul><h4>2. <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1534">200 Å</a></h4><ul><li>antibodies</li><li>antibody–drug conjugates</li><li>short oligonucleotides</li></ul><h4>3. <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1535">700 Å (70 nm)</a></h4><ul><li>AAVs</li><li>mRNA</li><li>large protein complexes (e.g., IgM)</li></ul><h3>Column Characteristics</h3><ul><li>silica-based particles</li><li>diol surface chemistry (hydrophilic, inert)</li><li>bio-inert hardware</li><li>designed for:<ul><li>high recovery</li><li>high reproducibility</li></ul></li></ul><h3>Performance and Calibration</h3><h4>Proteins and AAVs</h4><ul><li>90 Å → low molecular weight (up to ~50 kDa)</li><li>200 Å → medium range (up to ~450–500 kDa)</li><li>700 Å → large molecules (including AAVs)</li></ul><h4>RNA and DNA</h4><ul><li>separation of molecules with non-globular shapes</li><li>200 Å → smaller oligonucleotides</li><li>700 Å → larger nucleic acid chains (up to ~3000–4000 nucleotides)</li></ul><h3>Conclusion</h3>Selecting the correct SEC column requires:<ul><li>understanding molecule size and shape</li><li>choosing an appropriate pore size</li><li>selecting suitable particle size</li><li>ensuring inert surface chemistry and bio-inert hardware</li></ul>This enables optimal separation of a wide range of biologics.

How to Select the Right SEC Column for the Analysis of Biologics

<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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GPC/SEC Made Easy – Fingerprint analysis of Gummy Candies

How to Select the Right SEC Column for the Analysis of Biologics