<h3>Mastering Ion-Pair Reversed-Phase Chromatography for Oligonucleotide Analysis</h3><h4>Why Purity Is Critical in the Oligonucleotide Era</h4>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.<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"><h4>Understanding Ion-Pair Reversed-Phase (IP-RP) Chromatography</h4>For scientists involved in oligonucleotide synthesis and characterization, Ion-Pair Reversed-Phase Chromatography (IP-RP) is one of the most effective analytical tools available.Unlike separation techniques that rely solely on charge differences, IP-RP combines:<ul><li>Ionic interactions</li><li>Hydrophobic interactions</li></ul>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.<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"><h4>The Principle Behind Ion-Pairing</h4>Oligonucleotides possess a highly negatively charged phosphate backbone. In a traditional reversed-phase system, such hydrophilic, charged molecules would show little retention.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.Retention strength depends on:<ul><li>The hydrophobicity of the ion-pair reagent</li><li>The strength of ionic interaction</li><li>The applied organic gradient</li></ul>During gradient elution (commonly using acetonitrile), analytes are released in a controlled manner according to size, sequence, and chemical modifications.<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"><h4>Choosing the Right Ion-Pair Reagent</h4>Successful IP-RP separation depends heavily on selecting an appropriate ion-pair reagent. Two key factors determine retention behavior:<ol><li>Alkyl chain length</li><li>Charge density</li></ol><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"><h4>Strong Cationic Ion-Pair Reagents</h4>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.These reagents are particularly useful when high retention strength is required.<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"><h4>Volatile (Weaker) Ion-Pair Reagents</h4>Short-chain ion-pair reagents generate weaker retention but offer a significant advantage: compatibility with LC–MS detection.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.<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"><h4>Building an IP-RP Method for Oligonucleotides</h4>Developing a robust IP-RP method follows a logical sequence:<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"><h5>1. Characterize the Analyte</h5>Oligonucleotides typically contain 10–50+ nucleotides and exhibit high negative charge density. This necessitates a suitable cationic partner such as TEA⁺ or DIEA⁺.<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"><h5>2. Select the Column</h5>Use a reversed-phase column with:<ul><li>Large pore size (≈300 Å)</li><li>Appropriate internal diameter</li></ul>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.<h5>3. Optimize the Mobile Phase</h5>A typical starting condition includes:<ul><li>10 mM TEA buffer at pH ~7</li><li>Acetonitrile as organic modifier</li></ul>Gradient programs from approximately 5% to 50% organic solvent over 15–30 minutes are commonly effective for complex oligonucleotide mixtures.<h5>4. Adjust Retention Strength if Needed</h5>To increase retention:<ul><li>Raise ion-pair concentration (up to ~100 mM if required)</li><li>Select a more hydrophobic ion-pair reagent</li></ul><h5>5. Validate the Method</h5>Ensure robustness, sensitivity, and reproducibility by carefully evaluating:<ul><li>UV chromatograms</li><li>MS traces</li><li>Peak shape and resolution</li></ul><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"><h4>Why IP-RP Is Considered the Gold Standard</h4>Ion-Pair Reversed-Phase Chromatography uniquely combines the resolving power of reversed-phase LC with the ability to retain highly polar, charged biomolecules.Its advantages include:<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>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.For these reasons, IP-RP is widely regarded as the benchmark technique for oligonucleotide analysis.<h4>Final Thoughts</h4>Mastering IP-RP is less intimidating than it may seem. A successful method comes down to:<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>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.

Oligos Made Easy - Part 3: IP-RP

<h4>Why Consider HILIC in Modern Oligonucleotide Analysis?</h4>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.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.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.<h4>How Does HILIC Separate Oligonucleotides?</h4>HILIC essentially reverses conventional reversed-phase logic. It combines:<ul><li>A polar stationary phase</li><li>An organic-rich mobile phase</li></ul>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.This mechanism allows:<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>The key to performance lies in carefully tuning the parameters that govern HILIC selectivity and retention.<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"><h4>What Controls Retention in HILIC?</h4>Several interaction mechanisms contribute to analyte behavior in HILIC:<h5>Hydrophilic Partitioning – The Primary Mechanism</h5>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.<h5>Hydrogen Bonding – Fine-Tuning Effects</h5>Functional groups such as –OH, –NH₂, and –COOH can form hydrogen bonds with the stationary phase, subtly influencing retention time and selectivity.<h5>Electrostatic Contributions – The Role of Charge</h5>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.<h5>Dipole and Ion–Dipole Interactions – Added Selectivity</h5>Highly polar or zwitterionic compounds — including phosphorylated species and sugars — may engage in dipole–dipole or ion–dipole interactions, further refining separation behavior.<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"><h4>How to Develop a HILIC Method for Oligonucleotides</h4>Like any chromatographic method, successful HILIC development follows a structured approach:<h5>Step 1: Define the Analytes</h5>Understand oligonucleotide length, polarity, charge, and modifications.<h5>Step 2: Select a Suitable Column</h5>Choose a polar stationary phase (e.g., amide or zwitterionic) appropriate for the oligonucleotide’s chemical properties.<h5>Step 3: Optimize the Mobile Phase</h5><ul><li>Start with 70–95% acetonitrile</li><li>Use a volatile buffer (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>Small pH shifts can significantly alter selectivity and MS response.<h5>Step 4: Design the Gradient</h5>Employ a gradient that increases water content to modulate retention and separate oligonucleotides by size or chemical modification.<h5>Step 5: Match Injection Conditions</h5>Samples should be injected in a solvent similar to the starting mobile phase (high organic content) to prevent peak distortion.<h5>Step 6: Evaluate Performance</h5>Assess:<ul><li>Resolution</li><li>Retention time stability</li><li>Peak shape</li><li>Reproducibility</li></ul>Balancing separation performance with MS ionization efficiency is essential.<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"><h4>Practical Tips for High-Performance HILIC</h4><h5>Maintain a Stable Water Layer</h5>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.<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"><h5>Use High Organic Content</h5>HILIC typically operates best with 70–95% acetonitrile. Polar analytes are retained more effectively under these conditions.<h5>Include a Volatile Buffer</h5>Use low concentrations (5–20 mM) of ammonium formate or acetate to:<ul><li>Stabilize pH</li><li>Reduce peak tailing</li><li>Maintain MS compatibility</li></ul><h5>Control Injection Solvent</h5>Avoid high-water sample solvents. They disrupt the partitioning mechanism and distort peaks.<h5>Allow Proper Column Equilibration</h5>HILIC columns require longer equilibration times than reversed-phase columns — typically 10–20 column volumes after major gradient or solvent changes.<h5>Consider Temperature</h5>Operating at 30–50 °C can:<ul><li>Improve peak shape</li><li>Reduce mobile phase viscosity</li><li>Stabilize retention behavior</li></ul><h4>From Alternative to Essential? Final Perspective</h4>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.Although HILIC follows different chromatographic principles than reversed-phase separations, once optimized, it can deliver robust and highly selective results.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.If you are developing or refining an oligonucleotide LC-MS method, HILIC may be worth integrating into your analytical toolbox.<blockquote><ul><li>For additional information or discussion, feel free to contact: <a>sales@knauer.net<svg><use></use></svg></a></li><li>For direct scientific inquiries, reach out to the author at: <a>kindler@knauer.net<svg><use></use></svg></a></li></ul></blockquote>Stay tuned for more insights in the “Oligos Made Easy” series.

Oligos Made Easy - Part 4: HILIC

<h3>Size Matters: A Practical Guide to Size Exclusion Chromatography for Oligonucleotides</h3><h4>Why Consider SEC in Modern Oligonucleotide Workflows?</h4>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.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.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.<h4>How SEC Purifies Oligonucleotides</h4>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.When a mixture containing oligonucleotides and impurities passes through:<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>As a result, the target oligonucleotide elutes first, separated from salts, degraded fragments, and other small contaminants.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.<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"><h4>The Core Principle: Size Dictates Elution</h4>In SEC, molecular dimensions determine migration speed:<ul><li>Large oligos cannot penetrate the pores and therefore elute first.</li><li>Small impurities such as salts and solvent residues enter the pores and elute last.</li><li>Intermediate species, including truncated oligos, elute between the two extremes.</li></ul>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.<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"><h4>Step-by-Step Guide to Oligo Purification Using SEC</h4><h5>1. Selecting the Appropriate Column</h5>Typical oligonucleotides range from 10–50 nucleotides (~3–15 kDa).Recommended pore sizes:<ul><li>100–150 Å for short oligos (10–25 nt)</li><li>200–300 Å for longer strands (25–50 nt) or aggregates</li></ul>Columns packed with high-quality SEC media such as AppliChrom® or equivalent materials are well suited for these applications.<h5>2. Preparing the Sample</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>3. Choosing the Mobile Phase</h5>SEC for oligonucleotides typically employs simple aqueous buffers:<ul><li>50 mM phosphate or Tris</li><li>100–150 mM NaCl (to reduce non-specific interactions)</li></ul>Avoid detergents and organic modifiers that may compromise column stability. After buffer use, flush with water (optionally containing sodium azide) to maintain column integrity.<h5>4. Optimizing Flow Rate</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>Always monitor system backpressure.<h5>5. Sample Loading</h5>Avoid overloading. Excess injection volumes compromise resolution. Confirm the resin’s loading capacity before scaling up.<h5>6. Fraction Collection</h5>Monitor UV absorbance at 260 nm. Collect early-eluting peaks corresponding to the target oligo. Later fractions generally contain salts and smaller impurities.<h5>7. Purity Verification</h5>Confirm purity using complementary analytical methods such as:<ul><li>AEX</li><li>IP-RP</li><li>HILIC HPLC</li></ul><h4>Critical Column Parameters That Influence Performance</h4><h5>Pore Size</h5>Pore dimensions determine the effective separation range. Proper pore selection ensures clean size-based discrimination between oligos and contaminants.<h5>Inert Surface Chemistry</h5>The ideal SEC matrix should be non-interactive:<ul><li>No ionic interactions</li><li>No hydrophobic adsorption</li><li>No secondary retention effects</li></ul>Separation should rely solely on molecular size, not chemical affinity.<h5>Mechanical and Chemical Stability</h5>Reliable SEC gels maintain:<ul><li>Structural integrity under pressure</li><li>Stability in various aqueous buffers</li><li>Performance at elevated temperatures</li></ul>Abrupt changes in flow rate, temperature, or solvent composition should be avoided.<h5>Hydrophilicity</h5>SEC media are typically hydrophilic and compatible with aqueous systems. Common materials include:<ul><li>Crosslinked dextran</li><li>Agarose</li><li>Polyacrylamide</li><li>Hydroxylated methacrylate</li></ul>These materials are well suited for oligonucleotide purification.<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"><h4>Practical Optimization Tips</h4><ul><li>Desalting pre-treatment: Avoid redundant desalting if the sample is salt-rich.</li><li>Temperature control: Maintain consistent room temperature or use a column thermostat.</li><li>pH range: Keep between 6.5–8.0 to protect both oligos and gel stability.</li><li>Buffer exchange: Use your final formulation buffer as the mobile phase when possible.</li><li>Injection volume (analytical): &lt;2–5% of column volume.</li><li>Injection volume (preparative): 5–10%, depending on resin capacity.</li><li>Pressure management: Avoid sudden changes to prevent bed collapse.</li><li>Fraction collection: Base cuts on peak shape and symmetry, not retention time alone.</li><li>Column care: Use filtered samples, flush properly, store in preservative (e.g., 20–30% ethanol), and monitor performance routinely.</li></ul>Proper maintenance ensures reproducible, long-term performance.<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"><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"><h4>Strengths and Limitations in Practice</h4>SEC is widely appreciated for its simplicity and gentle operation. It does not require gradients or complex additives and maintains oligonucleotide integrity.However, it is not universally superior.<h5>Where SEC Excels:</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>Where Other Methods May Be Better:</h5><ul><li>High-resolution impurity profiling</li><li>Detailed structural characterization</li><li>Separation of closely related sequence variants</li></ul>Techniques such as AEX, IP-RP, or HILIC frequently provide higher resolution for demanding analytical tasks.<h4>Final Takeaway</h4>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.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.<blockquote>If you're planning to set up or optimize your oligonucleotide workflow, feel free to contact us at sales@knauer.net. Stay tuned for more exciting insights into the Oligonucleotide world in our “Oligos Made Easy” series.</blockquote>

Oligos Made Easy - Part 5: SEC

<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

<h3>Why Anion Exchange Chromatography Is a Strong Choice for Large-Scale Oligonucleotide Purification</h3>Oligonucleotide therapeutics are becoming increasingly important in modern drug development. Short DNA- and RNA-based medicines are moving through clinical pipelines in growing numbers, and several have already received FDA approval. As this field expands, one question becomes increasingly important: how can these molecules be purified efficiently, reproducibly, and on a manufacturing scale?One of the most reliable answers is anion exchange chromatography (AEX).In earlier discussions, we looked at the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6846">fundamentals of oligonucleotide analysis and the core principles behind AEX separations</a>. Here, the focus shifts to preparative purification at larger scale and to the practical issues that arise when moving from laboratory development toward production. As an example, this article highlights the purification of an oligo(dT) sample on the liter scale, demonstrating how AEX can deliver both high purity and strong recovery.<h4>Why purification is so important</h4>Following chemical synthesis, crude oligonucleotide material contains a mixture of desired product, truncated sequences, and other synthesis-related impurities. For pharmaceutical use, especially in clinical and regulated settings, this crude material must be refined to a much higher purity level.Although multiple HPLC approaches are available for oligonucleotide purification, not every method is equally suitable for scale-up. Some techniques work well in analytical or small-scale formats but become less practical when transferred to larger production environments. What is needed is a purification strategy that combines high performance with straightforward scalability.<h4>What makes AEX attractive?</h4>Anion exchange chromatography has established itself as an effective purification method for oligonucleotides, especially when larger sample volumes are involved. Its main strengths include:<ul><li>High loading capacity, allowing large sample amounts to be processed efficiently</li><li>Strong separation performance, enabling high-purity product recovery</li><li>Robust scalability, from development work through to GMP-oriented production</li><li>More environmentally friendly operation, since aqueous buffer systems can be used instead of harsher organic solvents</li></ul>These features make AEX a practical option for laboratories and manufacturers seeking a reproducible purification platform that can grow with the needs of a project.<h4>Example: large-scale purification of oligo(dT)</h4>To illustrate the approach, an oligo(dT) sample was purified on the liter scale. Oligo(dT) is widely used in molecular biology, for example in mRNA isolation and as a primer in cDNA synthesis.Before chromatography, the sample required the usual preparation steps: cleavage from the solid support, deprotection with ammonia, and subsequent neutralization. The purification itself was performed on the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1621">AZURA® Prep HPLC system</a>, which is designed for high-throughput preparative workflows and offers precise gradient formation, UV detection, and automated fraction collection.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_AZURA_preparative_HPLC_system_b989fe64fb.webp" alt="KNAUER: AZURA preparative HPLC system" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_AZURA_preparative_HPLC_system_b989fe64fb.webp 107w," sizes="100vw" width="653" height="954">The separation used a strong anion exchange resin with gradient elution based on a phosphate buffer and sodium bromide as the counterion. Sodium bromide was selected because it is less aggressive toward stainless steel components than chloride, helping protect instrument hardware during repeated operation. For optimal resolution, anion exchange particles in the 10–15 µm range are typically preferred.<h4>Performance in practice</h4>The results clearly demonstrate the suitability of AEX for oligonucleotide purification at scale.<ul><li>Purity before purification: 89%</li><li>Purity after AEX purification: 95%</li><li>Yield after purification: 90%</li></ul>These values show that the method can effectively improve product quality while maintaining a high recovery, which is a key requirement in preparative and manufacturing settings.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Chromatogram_of_oligonucleotide_purification_from_1_l_sample_a768bb4759.webp" alt="KNAUER: Chromatogram of oligonucleotide purification from 1 l sample" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Chromatogram_of_oligonucleotide_purification_from_1_l_sample_a768bb4759.webp 245w," sizes="100vw" width="1920" height="848"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Analytical_chromatogram_of_purified_and_pooled_oligo_d_T_after_AEX_0d6e5b31c5.webp" alt="KNAUER: Analytical chromatogram of purified and pooled oligo (dT) after AEX." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Analytical_chromatogram_of_purified_and_pooled_oligo_d_T_after_AEX_0d6e5b31c5.webp 240w," sizes="100vw" width="1085" height="705"><h4>Important limitations to keep in mind</h4>Like any chromatographic method, AEX also has its limitations and process considerations.<ul><li>Phosphodiester vs. phosphorothioate separations can be challenging, and resolution may decrease for longer or more heavily modified sequences.</li><li>Aggregation effects caused by secondary structures can reduce purification efficiency.</li><li>Because AEX is typically performed under aqueous conditions, there is also an increased need to monitor and control endotoxin-related process risks.</li></ul>These factors do not diminish the value of AEX, but they do underline the importance of careful method development and process control.<h4>Final thoughts</h4>For oligonucleotide-based therapeutics, purification is not just a supporting step — it is a critical part of the path from crude synthesis to clinical or commercial material. The purification strategy must be scalable, robust, and capable of consistently delivering high-quality product.Anion exchange chromatography meets many of these demands. It offers a compelling balance of purity, yield, scalability, and operational practicality, making it an important tool for both development and production environments.For teams preparing material for research, clinical studies, or GMP manufacturing, AEX on AZURA® Prep systems represents a strong and practical route toward reliable oligonucleotide purification.For additional details, the related application note “<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/VBS_0082_Large_scale_purification_of_oligonucleotides_with_ion_exchange_chromatography_5e622f1914.pdf">Oligonucleotide Purification via Ion Exchange</a>” provides more information on the method.<blockquote><ul><li>If you are planning to build or optimize an oligonucleotide workflow, you can also contact sales@knauer.net</li></ul></blockquote>

Oligos Made Easy - Part 7: Large Scale AEX

<h3>A Dedicated Solution for Oligonucleotide Analysis</h3>Oligonucleotides have become key players in modern pharmaceutical and biological research, particularly in the development of highly targeted and personalized therapies. As increasing numbers of oligonucleotide-based drugs enter clinical trials or receive regulatory approval, analytical laboratories face growing demands for both throughput and data quality. These molecules present unique chromatographic challenges due to their long, highly polar structures and subtle variations in nucleobase sequence or functionalization.<h3>Sepapure oliGO – Designed for Oligonucleotide Separations</h3>Selecting the optimal chromatographic approach for oligonucleotide analysis can be complex. Many laboratories rely on ion-pair reversed-phase (IP-RP) chromatography because of its speed, robustness, and compatibility with mass spectrometry. However, commonly used ion-pair reagents can degrade conventional silica-based stationary phases.<a target="_blank" rel="noopener noreferrer" href="https://www.knauer.net/columns-oligonucleotide-columns">Sepapure oliGO</a> addresses this limitation through a hybrid silica matrix combined with specialized surface end-capping, ensuring resistance to aggressive IP-RP reagents. Its functionalized surface enables both strong reversed-phase interactions and controlled ionic contributions, effectively creating a mixed-mode separation mechanism. This design results in improved durability compared with standard mixed-mode columns, making Sepapure oliGO a robust choice for routine oligonucleotide workflows.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_1_Sepapure_oli_GO_columns_with_laser_etched_column_information_23f545be81.jpg" alt="KNAUER: Figure 1 - Sepapure oliGO columns with laser-etched column information." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_1_Sepapure_oli_GO_columns_with_laser_etched_column_information_23f545be81.jpg 223w," sizes="100vw" width="1186" height="828"><h3>Method Transfer and Comparative Performance</h3>In collaboration with BianoGMP GmbH, KNAUER’s application team evaluated Sepapure oliGO against a widely used reference column. The study compared oligonucleotides of varying lengths and purities, including a fully thiolated 43-mer DNA. Particular attention was paid to potential interactions between thiolated oligonucleotides and stainless-steel hardware, which have been reported to cause peak tailing.The results demonstrated that direct method transfer between the reference column and Sepapure oliGO was generally feasible without significant adjustment. Chromatographic resolution remained comparable, while retention times were slightly shorter on Sepapure oliGO. Furthermore, the anticipated benefits of expensive bioinert hardware were not confirmed under the tested conditions, suggesting that such effects may primarily influence very fast (U)HPLC separations.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Figure_2_Overlayed_chromatogram_of_crude_product_of_a_43mer_thiolate_oligonucleotide_7ae9613efd.jpg" alt="KNAUER: Figure 2: Overlayed chromatogram of crude product of a 43mer thiolate oligonucleotide. Light blue - reference column and dark blue – Sepapure oliGO." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Figure_2_Overlayed_chromatogram_of_crude_product_of_a_43mer_thiolate_oligonucleotide_7ae9613efd.jpg 245w," sizes="100vw" width="1223" height="453"><h3>Low Backpressure for Reliable Operation</h3>Sepapure oliGO can be applied effectively in both HPLC and (U)HPLC workflows. Its low operating backpressure minimizes risks associated with clogging, excessive system wear, and performance deterioration over time. Typical IP-RP separations using 3 µm particles generated pressures around 200 bar, enabling laboratories without ultra-high-pressure systems to perform high-quality oligonucleotide analysis.<h3>Best Practices for Column Performance</h3>To maintain optimal performance, users should observe recommended operating conditions, including a broad pH range of 1.5–10 and temperatures up to 60 °C. Employing a guard column can prevent coarse synthesis by-products from reaching the analytical column. If pressure increases are observed, backflushing at low flow rates can restore performance.When the system is idle, thorough flushing to remove salts is essential. For storage, the column should be kept in a 1:1 mixture of methanol–water or acetonitrile–water. Gradient transitions are recommended when switching between standard RP eluents and IP-RP reagents. Regeneration procedures developed for RP columns are also suitable for Sepapure oliGO.<h3>Beyond Analytical Separations</h3>For large-scale purification, ion-exchange chromatography (AEX) may offer advantages. Scalable preparative systems are available to support purification workflows, ranging from compact laboratory setups to pilot-scale platforms with flow rates up to 800 mL/min. Bioinert configurations can be integrated where required, allowing laboratories to tailor purification solutions to their specific oligonucleotide applications.<blockquote><ul><li>If you're planning to set up or optimize your oligonucleotide workflow, feel free to contact us at sales@knauer.net. Stay tuned for more exciting insights into the Oligonucleotide world in our “Oligos Made Easy” series.</li></ul></blockquote>

Oligos Made Easy - Part 8: Sepapure oliGO

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

Oligos Made Easy - Part 9: 2D-LC

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.<h4>What are ghost peaks?</h4>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].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.<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"><h4>How to minimize ghost peaks?</h4>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.<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">To reduce their impact, several practical measures can be implemented:<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>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.<h3>How to unmask ghost peaks?</h3>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.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.<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">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.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.<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">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.<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"><h4>Conclusion</h4>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.<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">Technical Note VTN0046</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 sales@knauer.net.&nbsp;</li></ul></blockquote>

GPC/SEC Made Easy – Unmask Ghost Peaks

In German, there is a well-known saying: “Der April macht, was er will!” — loosely translated as “April does whatever it wants.” While this usually refers to unpredictable weather, it can sometimes describe other situations just as well.As an analytical application specialist at KNAUER, I was genuinely excited when, at the end of 2023, my colleague and product manager Mareike Myrach and I were assigned the project of the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1454">Liquid Handler LH 8.1</a>. At that time, we had no prior hands-on experience with the system, but we quickly got up to speed — largely because learning by doing turned out to be surprisingly intuitive.Following an intensive phase of testing, method development, and optimization, the Liquid Handler LH 8.1 was officially introduced by KNAUER in April 2025. Returning to the April proverb — this time, April behaved quite differently. Rather than being unpredictable, everything came together smoothly (even if not weather-wise!). All documentation was finalized, product configurations defined, and specifications locked in. It was a very satisfying milestone.&nbsp;<h3>What makes the Liquid Handler LH 8.1 stand out?</h3>Let’s take a closer look at some of its key features.<h4>Flexible configuration tailored to your workflow</h4>The KNAUER LH 8.1 is a highly customizable XYZ autosampler designed to adapt to a wide range of laboratory requirements. It supports numerous modules and accessories, allowing users to build a system that fits their specific applications.Core components include the injection valve, the Robotic Cooler, and the fast wash station — all essential for efficient operation. Beyond these, we are continuously exploring additional enhancements.&nbsp;For example, how about seasonal upgrade kits?<ul><li>LH 8.1 Easter edition</li><li>LH 8.1 Christmas edition</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Fig_1_Left_LH_8_1_Upgrade_Kit_7b9a711bab.jpg" alt="KNAUER: Fig. 1: Left: LH 8.1 Upgrade Kit “Easter”, right: LH 8.1 Upgrade Kit “Christmas” (limited availability — only 1 in stock!)" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Fig_1_Left_LH_8_1_Upgrade_Kit_7b9a711bab.jpg 234w," sizes="100vw" width="1263" height="843"><h4>Flexible sample storage and high-throughput capability</h4>For laboratories focused on high-throughput analysis, the Robotic Cooler module is indispensable. Each unit can accommodate up to three sample racks, with 130 vials per rack — totaling 390 vials per module.Depending on the rail length, multiple Robotic Coolers can be installed and operated independently. On the longest rail configuration, up to three modules can be used simultaneously, increasing the total capacity of a single LH 8.1 system to as many as 1,170 vials (1.5 mL each).Naturally, the system is not limited to standard vials. It can store a variety of sample types within a temperature range of 4–40 °C.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Fig_2_Left_Tray_with_common_samples_middle_tray_with_special_samples_right_very_very_special_samples_b1d2e3daea.jpg" alt="KNAUER: Fig. 2: Left: tray with standard samples, middle: tray with specialized samples, right: very special samples" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Fig_2_Left_Tray_with_common_samples_middle_tray_with_special_samples_right_very_very_special_samples_b1d2e3daea.jpg 245w," sizes="100vw" width="1176" height="402"><h4>True zero sample loss</h4>Have you ever encountered the concept of “sandwich injections” in liquid chromatography? It may sound unusual at first. Replace the bread with standard HPLC solvents and the filling with your sample, separated by small air gaps — and you have a liquid “sandwich.”The LH 8.1 takes this concept even further. It allows sampling from virtually any container type. Whether it’s a standard vial or something more unconventional — like a filled chocolate egg — the system can handle it. The result? A rather creative “egg sandwich.”<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Fig_3_Left_Exemplary_sandwich_build_up_right_aspiration_from_double_coated_chocolate_sample_container_8e9f9fa243.jpg" alt="KNAUER: Fig. 3: Left: example of sandwich configuration, right: aspiration from a chocolate-based sample container" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Fig_3_Left_Exemplary_sandwich_build_up_right_aspiration_from_double_coated_chocolate_sample_container_8e9f9fa243.jpg 245w," sizes="100vw" width="1218" height="751"><blockquote>Please note: using such unconventional containers may significantly increase both carryover and... enjoyment.</blockquote><h4>Carryover and injection precision</h4>When evaluating HPLC injectors, injection precision is one of the most critical parameters. After optimizing the injection conditions, we achieved excellent performance.To ensure ease of use, all optimized parameters have been integrated into the software drivers. This allows users to benefit from high precision without complex programming.<h5>Injection precision (RSD):</h5><ul><li>Full loop injection: ≤ 0.10%</li><li>Sandwich injection (&gt;5 µL): ≤ 0.15%</li></ul>Another key parameter is sample carryover, evaluated using recommended washing protocols. During testing, we measured carryover using chlorhexidine as a standard compound to allow comparison with other autosamplers. Additionally, caffeine was tested due to its widespread use and accessibility in laboratories.Taking advantage of the LH 8.1’s flexibility, we also explored sampling from unconventional containers.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Fig_4_Exemplary_sample_take_up_for_the_determination_of_carry_over_47706172c7.jpg" alt="KNAUER: Fig. 4: Left: injection of diluted caffeine sample for quantification, right: injection of concentrated sample for carryover assessment" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Fig_4_Exemplary_sample_take_up_for_the_determination_of_carry_over_47706172c7.jpg 224w," sizes="100vw" width="1248" height="870"><h5>Measured carryover values:</h5><ul><li>Chlorhexidine: &lt; 0.005%</li><li>Caffeine: &lt; 0.002%</li></ul>Returning to the beginning of this article — you may have noticed that not only “April does whatever it wants,” but sometimes KNAUER blog authors do as well.Despite the seasonal theme, we hope you discovered a few hidden “Easter eggs” throughout the text — and didn’t take everything too seriously.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Fig_5_KNAUER_blog_authors_72de1a0c72.jpg" alt="KNAUER: Fig. 5: KNAUER blog authors" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Fig_5_KNAUER_blog_authors_72de1a0c72.jpg 237w," sizes="100vw" width="1150" height="757"><blockquote><ul><li>More reputable information: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1454">KNAUER Liquid Handler LH 8.1</a></li></ul></blockquote>

Seasonal update on liquid handling

<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></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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