<p><strong>This technical tip explores some issues encountered with </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50"><strong>Solid Phase Extraction</strong></a><strong> and in particular to Analyte Recovery. Recovery relates to the percentage of analyte captured and retrieved through SPE.&nbsp;</strong><br><strong>A couple of other commonly used terms relating to recovery, which may be of interest are:</strong></p><p><strong>Relative Recovery:</strong> Indirect determination of analyte capture and retrieval during the extraction process based on the recovery of an internal standard (which is a compound with a structure similar to the target analyte) that is added to the crude sample/matrix prior to the actual extraction procedure.</p><p><strong>Absolute Recovery:</strong> Quantitative method for the determination of the actual amount of analyte captured and retrieved during the extraction process. Analyte recovery is based on an external standard that is added to the eluant at the end of the extraction procedure (rather than being based on an internal standard that is co-extracted with the analyte).</p><h5><strong>Symptom</strong></h5><ul><li>Analyte recovery is less than 100%.</li></ul><h5><strong>Clue</strong></h5><ul><li>Analysis of the unretained fraction (void volume) indicates that the analyte is present in the “flow through”.</li></ul><h5><strong>Problem</strong></h5><ul><li>Analyte binding is not quantitative during the sample loading step.</li></ul><h5><strong>Cause /&nbsp;Solutions / Suggestions</strong></h5><p><strong>A. Column conditioning is improper, incorrect, or not optimized.</strong>:</p><ol><li>Condition column with methanol or isopropanol.</li><li>Use sufficient methanol or isopropanol to wet the entire sorbent bed; allow more than two column volumes to percolate slowly under low vacuum.</li><li>Follow the methanol or isopropanol directly with one column volume of a solution with a composition similar to the actual sample (pH adjusted) but without the analyte(s).</li><li>Do not use too much of this second “conditioning solvent” in (3) above, or allow it to remain in the column for too long.</li><li>Do not over-dry the column during or after conditioning (use low vacuum for ~1 minute).</li></ol><p><strong>B. Sample/matrix is in (or contains) a solvent which is “too strong”.</strong>:</p><ol><li>Dilute the sample in a “weaker” solvent/solution to promote binding (less polar for Normal Phase, more polar for Reversed Phase, less salt, buffered and/or pH adjusted for Ion-Exchange.</li><li>Increase the sample dilution with a “weak” solvent.</li><li>Load less sample.</li><li>Increase the sorbent mass.</li><li>Decrease the flow rate during sample loading.</li><li>Use a “stronger” sorbent with a higher affinity for the analyte.</li><li>Add an organic modifier (or adjust the pH) to enhance binding affinity for the analyte(s).</li><li>Adjust the sample pH so that the analyte is neutral for Reversed Phase or charged for Ion-Exchange.</li><li>Add salt (5 to 10% NaCl) to increase the solvent polarity and to enhance the retention of highly polar analytes in Reversed Phase.</li><li>Add an ion-pair reagent to enhance binding of charged analytes in Reversed Phase.</li></ol><p><strong>C. Column “mass overload” (column is too small and/or the total mass of bound solutes/components is too large).</strong>:</p><ol><li>Decrease the volume of sample loaded.</li><li>Increase the sorbent mass.</li><li>Use a sorbent with higher surface area.</li><li>Use a “stronger” sorbent.</li><li>Decrease the flow rate during loading (to enhance “diffusion”).</li><li>Decrease the column inner diameter but use the same sorbent mass (to increase the pressure drop, reduce flow, and enhance “diffusion”).</li><li>Dilute the sample in a “weaker” solvent to improve capacity.</li></ol><p><strong>D. Flow rate is too high during the sample loading step.</strong>:</p><ol><li>Decrease the flow rate during loading.</li><li>Increase the sorbent mass.</li><li>Decrease the column inner diameter to reduce flow.</li><li>Use a sorbent with a higher surface area.</li></ol><p><strong>E. Sorbent is “too weak” (has poor or low affinity for the analyte relative to the sample/matrix and/or the dilution solvent).</strong>:</p><ol><li>Switch to a “stronger” sorbent, to one with a higher ligand density, from a non-endcapped to an endcapped sorbent (or vice versa).</li><li>See “Solutions/Suggestions” for Cause C (“Column mass overload..”), above.</li><li>See Solutions/Suggestions for Cause B (“Sample/matrix is in (or contains) a solvent which is “too strong””), above.</li></ol><p><strong>F. Conditioning solvent is “washed away” with large sample volumes.</strong>:</p><ol><li>Add 2% methanol or isopropanol to the sample to prevent alkyl chain collapse during the sample loading step.</li></ol>

Troubleshooting SPE

<p><strong>When performing analyte extraction with </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50"><strong>SPE</strong></a><strong> we expect 100% recovery. If recovery is lower, this would suggest there is an issue with retrieval of the analyte from the sorbent. But what are some of the reasons you may see a recovery greater than what you were expecting (or over 100%)?</strong></p><h3><strong>Common problems</strong></h3><ul><li>Co-eluting interferences</li><li>Contaminants from the solvent or sorbents selected</li><li>Internal standard is compromised making your reference point inaccurate</li><li>Recovery calculations are inaccurate</li></ul><p><strong>Let's examine each of these common problems.</strong></p><h4><strong>Co-eluting interferences</strong></h4><p>These are typically present in the matrix and the goal of the SPE step is to remove them. If you are experiencing recoveries greater than 100%, the first step would be to <strong>check the eluent using an orthogonal analytical technique </strong>to identify that the increased recovery is a result of a co-elution.&nbsp;</p><p><strong>Where a co-elution is identified, there are a few possible solutions.</strong></p><ol><li><strong>Modify the wash steps of the SPE method </strong>to selectively remove the impurity during washing</li><li><strong>Select an alternative SPE sorbent </strong>which will better remove these impurities. This is easier if the analyte / impurity can be selectively ionized, as an ion-exchange sorbent will very often afford cleaner extracts than either reversed or normal phase solutions</li><li><strong>Adjust your analytical method</strong> to resolve the 2 peaks analytically, negating the necessity to remove this impurity during the SPE step.</li><li><strong>Run a blank plasma sample</strong>; this will allow you to ascertain the impurities present and use this blank in the calculations to subtract from your recovery</li></ol><h4><strong>Contaminants from the solvent or sorbent</strong></h4><p>These will typically be impurities present in the starting materials which are similar in nature to your compound of interest, and as a result co-elute with it during SPE and subsequent analytical analysis. You can <strong>follow the steps above to remove co-eluting impurities, or alternatively</strong> as this is not an impurity present in your matrix, you can identify the level present in the sorbent using blank runs and subtract this from your main peak during the calculation. Prior to addition of your sample if you equilibrate your sorbent with the elution solvent this should flush out potential contaminants from the sorbent. For impurities introduced from the solvent, typically switching the solvent should resolve this.</p><h4><strong>Internal standard is compromised</strong></h4><p>If your internal standard is not showing 100% recovery, your subsequent calculations will be inaccurate. It is always good practice to <strong>use an internal standard similar in nature to your compound of interest</strong> as this will give the more consistent reference point for your method.&nbsp;</p><p>If there is concern your internal standard is not showing good recovery from the SPE material using your method, you can use an <strong>external standard</strong> which is added to the elution solvent to calculate absolute recovery.</p><h4><strong>Recovery calculations are inaccurate</strong></h4><p>It is important to follow this step wise to ascertain where the errors are appearing.</p><ol><li>Calculation Errors - Check all calculations and account for all dilution factors</li><li>Integration Errors - Reintegrate all peaks and use manual calculations to confirm</li><li>Analytical method errors – Ensure the analytical method used allows correct integration and calculation. Ensure no strong solvents are used for injection and run a calibration across a number of dilutions to ensure linearity. Troubleshoot your analytical method to determine any factors which may lead to poor reproducibility</li><li>Ensure detections selected are appropriate and working correctly.</li></ol><p>We hope this tip was useful! Check out our resources on <a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/techniques/solid-phase-extraction"><strong>solid phase extraction</strong></a>. See you next month for another tip!</p>

Why are my recoveries greater than 100%?

<h3><strong>Welcome to our new </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50"><strong>SPE </strong></a><strong>technical tip!</strong></h3><p><strong>What is the cause when you encounter interferences post-extraction regardless of the analytical or detection method? Possible sources of the mystery peak(s) are outlined below.</strong></p><h4><strong>Possible Cause/Source &nbsp; / &nbsp; Suggested Remedy</strong></h4><ul><li><strong>Contaminated reagents</strong>: Analyze the purity of reagents and additives or try using these from a different supplier.</li><li><strong>Sample pre-treatment</strong>: Compare chromatograms of pre-treated and non-pre-treated sample.</li><li><strong>Mismatched injection solvent</strong>: Analytes that are dissolved in a strong injection solvent may retain poorly and thus appear as extra peaks. Using the initial mobile phase as the injection solvent is recommended.</li><li><strong>Incorrect SPE method</strong>: The SPE sorbent might not have been properly conditioned and equilibrated. Typically, the strongest solvent used in the method should be used to condition the sorbent. Analyze the conditioning, loading and washing fractions to check if the same interferences appear.</li><li><strong>SPE tube leaching</strong>: Before conditioning, clean the tube with elution solvent. If reagents or the nature of the sample are too harsh for the polypropylene SPE tube, a glass tube or PTFE tube could be used instead.</li><li><strong>Degradation/side reaction</strong>: Check the reactivity and stability of all components in your sample. Degradation can happen if compounds are sensitive to factors such as heat, light, acids, bases, etc. Sample components may be reacting with each other or with reagents/additives.</li></ul><p><strong>We hope this tip was useful. See you soon for another tip!</strong></p>

SPE Troubleshooting - Extra Peak

<p><strong>Solid Phase Extraction (SPE) is one of the most highly selective sample preparation methods, meaning that the targeted analytes and their matrices must be considered when choosing the correct SPE sorbent and format. Drying down the sorbent before elution is an important step.</strong></p><ul><li>Wash solvents are used to flush out unwanted compounds (i.e., contaminants). If the wash solvent is not completely removed, then residual wash solvent could contaminate the final elution.</li><li>If you do not dry the SPE sufficiently, it could make the final solution less concentrated and result in a reduction in sensitivity.</li><li>Drying helps physically break down the interactions between the sorbent and analyte for a more concentrated extract.</li></ul><p>After the wash solvent has been passed through the cartridge, continue to apply negative pressure (vacuum) or positive pressure. Use this pressure to dry the cartridge for 2 to 5 minutes (or until dry). Turn off the pressure and wipe the tips of the manifold needles to remove any residual solvent.</p><h5><strong>Notes</strong></h5><ol><li><strong>Reproducibility:</strong> Drying removes traces of residual sample/matrix and wash solvent from the sorbent. This produces a more concentrated final extract with a consistent volume and composition and yields better reproducibility and higher recoveries.</li><li><strong>Immiscibility:</strong> Drying is especially critical when using elution solvents that are immiscible with water (e.g. hexane, ethyl acetate, and methylene chloride). Residual water in the sorbent bed may prevent dissolution of the analyte in the elution solvent, resulting in low recoveries and irreproducibility.</li><li><strong>Over-drying: </strong>Avoid over-drying the sorbent, since in some cases, analyte recovery may decrease with excessive vacuum or drying times above 5 minutes. Dry cartridges look distinctly different from moist ones. Make a note of the cartridge appearance before and after conditioning. Also, you could touch the cartridge while it is drying; wet cartridges feel colder than dry cartridges.&nbsp;</li></ol>

Why Is It Necessary to Dry the SPE Sorbent Before Elution?

<p><strong>C18 phases do not provide sufficient retention for polar compounds. The result is often coelution and signal suppression in LC–MS. When analyzing polar analytes, C18 is therefore usually not the appropriate choice.</strong></p><p>HILIC (Hydrophilic Interaction Liquid Chromatography) represents a different separation approach.</p><h4><strong>Principle of HILIC</strong></h4><p>The initial mobile phase composition is<strong> 90% acetonitrile </strong>and 1<strong>0% ammonium buffer</strong>. The high organic content creates a thin water-enriched layer on the polar stationary phase. Analytes partition into this layer, and as the water content in the mobile phase increases, they elute.</p><h3><strong>Method Development Essentials</strong></h3><h4><strong>1. Selection of the Stationary Phase</strong></h4><p>Choose an appropriate polar stationary phase, for example:</p><ul><li>silica</li><li>diol</li><li>amino</li><li>zwitterionic</li></ul><h4><strong>2. Initial Mobile Phase Conditions</strong></h4><ul><li>Start at 90% acetonitrile with an ammonium buffer.</li><li>Do not use methanol — it disrupts the water-enriched layer and affects retention.</li></ul><h4><strong>3. Gradient Strategy</strong></h4><p>In HILIC, <strong>water is the strong eluent</strong>. The gradient therefore starts at high organic content and then progressively increases the water content.</p><h4><strong>4. pH Optimization</strong></h4><p><strong>Compare:</strong></p><ul><li>pH 3.2 (ammonium formate)</li><li>pH 5.8 (ammonium acetate)</li></ul><p>Check the differences.</p><h4><strong>5. Equilibration</strong></h4><p>Ensure proper equilibration of the system.</p><h3><strong>Practical Note</strong></h3><p>Excess water in the sample disrupts HILIC retention and leads to poor peak shape.</p>

Stop Using C18 for Polar Analytes

<p><strong>Traditional protein purification workflows in many laboratories still rely on ultracentrifugation and long incubation steps. These approaches often involve multi-step protocols, extended processing times, and the use of specialized equipment. As a result, workflows can become complex, time-consuming, and prone to variability.</strong></p><p>Magnetic bead-based protein purification offers a faster, simpler, and more efficient alternative by enabling selective capture and rapid magnetic separation.</p><h3><strong>Limitations of Conventional Protein Cleanup Methods</strong></h3><p><strong>In standard laboratory workflows, protein cleanup typically involves:</strong></p><ul><li>Multiple processing steps</li><li>Centrifugation and column equilibration</li><li>Dependence on specialized instrumentation</li><li>Long processing times</li></ul><p><strong>These factors introduce several challenges:</strong></p><ul><li>Increased complexity of workflows</li><li>Variability between runs, reducing reproducibility</li><li>Risk of sample loss during transfers, especially for small sample volumes or scarce targets</li><li>Limited throughput due to manual handling</li></ul><h3><strong>Principle of Magnetic Bead-Based Purification</strong></h3><p>Magnetic beads enable selective protein capture through surface functionalization.</p><p>Each bead is coated with <strong>streptavidin</strong>, allowing the attachment of a <strong>biotinylated ligand</strong>. This ligand can be selected based on the target analyte and may include:</p><ul><li>Antibodies</li><li>Aptamers</li><li>Antigen fragments</li></ul><p>This functionalization step provides the beads with the specificity required to bind the target analyte.</p><h3><strong>Materials and Key Components</strong></h3><h4><strong>Magnetic Beads</strong></h4><ul><li>Streptavidin-coated surface</li><li>Functionalized with biotinylated ligands</li></ul><h3><strong>Ligands</strong></h3><ul><li>Selected for target specificity</li><li>Attached to beads prior to purification</li></ul><h3><strong>Step-by-Step Procedure</strong></h3><h4><strong>1. Bead Activation</strong></h4><p>Load the biotinylated ligand onto the streptavidin-coated magnetic beads to confer specificity.</p><h4><strong>2. Washing</strong></h4><p>Remove impurities after ligand loading to prepare the beads for interaction with the sample.</p><h4><strong>3. Incubation with Sample</strong></h4><p>Incubate the functionalized beads with the sample to allow binding between the ligand and the target analyte.</p><h4><strong>4. Magnetic Separation</strong></h4><p>Place the sample on a magnet:</p><ul><li>Magnetic beads (with bound analyte) are attracted to the vessel wall</li><li>The liquid phase can be separated easily</li></ul><h4><strong>5. Washing</strong></h4><p>Wash the beads while they remain immobilized by the magnetic field to remove:</p><ul><li>Weak interactions</li><li>Non-specific binding</li></ul><h4><strong>6. Elution</strong></h4><p>Add an elution solvent to release the purified analyte from the beads.</p><h3><strong>Downstream Applications</strong></h3><p>After elution, the purified sample is ready for further analysis, including:</p><ul><li>LC-MS</li><li>Western blot</li><li>ELISA</li><li>Activity assays</li></ul><h4><strong>Advantages of Magnetic Bead-Based Workflows</strong></h4><p>Magnetic bead-based purification offers several key benefits:</p><h4><strong>Speed and Simplicity</strong></h4><ul><li>No centrifugation required</li><li>No complex instrumentation needed</li></ul><h4><strong>Improved Reproducibility</strong></h4><ul><li>Consistent magnetic separation</li><li>Reduced variability between runs</li></ul><h4><strong>Reduced Sample Loss</strong></h4><ul><li>Fewer transfer steps</li><li>Suitable for small or scarce samples</li></ul><h4><strong>Scalability and Throughput</strong></h4><ul><li>Compatible with automation</li><li>Supports microplate formats:<ul><li>96-well plates</li><li>384-well plates</li></ul></li></ul><h3><strong>Biozen™ Magnetic Beads Features</strong></h3><p>Phenomenex <strong>Biozen™ magnetic beads</strong> incorporate several design features:</p><ul><li><strong>Hydrophilic co-polymer surface</strong><ul><li>Reduces non-specific hydrophobic interactions</li></ul></li><li><strong>Biotinylated BSA spacer</strong><ul><li>Acts as a linker to streptavidin</li><li>Helps orient capture ligands</li></ul></li><li><strong>Monodispersed capture ligands</strong><ul><li>Optimized binding capacity</li></ul></li></ul><p>These features contribute to:</p><ul><li>Reduced non-specific binding</li><li>Improved capture consistency</li><li>Cleaner eluates</li></ul><h3><strong>When to Use Magnetic Beads</strong></h3><p>Magnetic bead-based purification is particularly suitable when:</p><ul><li>Sample volumes are small</li><li>Fast turnaround is required</li><li>Minimal equipment is preferred</li><li>High-throughput workflows are needed</li></ul><h3><strong>Conclusion</strong></h3><p>Magnetic bead-based protein purification simplifies traditional workflows by eliminating centrifugation and reducing complexity. Through selective capture and efficient magnetic separation, it enables faster processing, improved reproducibility, and compatibility with high-throughput and automated systems.</p>

Magnetic Bead-Based Protein Purification: A Faster and More Efficient Workflow

<p><strong>This technical note explains how to improve recovery of oligonucleotides and oligonucleotide conjugates using the </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2216"><strong>Clarity™ XRO</strong></a><strong> extraction kit. The workflow focuses on optimizing sample preparation and solid-phase extraction (SPE) conditions to achieve higher recovery, reproducibility, and efficiency when working with complex biological matrices.</strong></p><p><strong>Clarity XRO</strong> is a next-generation extraction kit designed for isolating oligonucleotides and their conjugates. The Pro version specifically addresses variability in daily workflows and reduces method optimization time.</p><h3><strong>Materials and Tools</strong></h3><ul><li><strong>Clarity™ XRO extraction kit</strong></li><li><strong>Lysis loading buffer</strong></li><li><strong>Proteinase K </strong>(included in kit)</li><li><strong>Optional antioxidants</strong> (e.g., cyanide-based additives mentioned in video)</li><li><strong>Ceramic beads</strong> (for tissue homogenization)</li><li><strong>SPE tubes or plates</strong></li><li><strong>Equilibration buffer</strong></li><li><strong>Wash buffer solution</strong></li><li><strong>Elution buffer</strong> (MS-compatible)</li><li><strong>Vacuum system</strong> (for SPE plates)</li><li><strong>Lyophilizer</strong> (optional for concentration)</li></ul><h3><strong>Principle of the Method</strong></h3><p><strong>The workflow consists of three main stages:</strong></p><ol><li><strong>Sample lysis</strong> using a specialized buffer</li><li><strong>Protein digestion </strong>using Proteinase K</li><li><strong>Solid-phase extraction </strong>(SPE) based on weak ionic interactions</li></ol><p>This approach enhances oligonucleotide stability by inhibiting nuclease activity and enables efficient enrichment from complex biological samples.</p><h3><strong>Step-by-Step Procedure</strong></h3><h4><strong>1. Sample Pre-Treatment (Lysis)</strong></h4><p>Sample pre-treatment is a critical step for recovery.</p><ul><li>Use a <strong>lysis loading buffer-to-sample ratio of 3:1</strong> as a starting condition</li><li>For complex samples, higher ratios may be required</li><li>For specific cases (e.g., siRNA), a <strong>1:1 ratio may be optimal</strong></li><li>Incubate samples for <strong>5–15 minutes</strong> to ensure complete lysis</li><li>Add antioxidants if oxidation is an issue</li><li>For tissue samples:<ul><li>Perform <strong>homogenization using ceramic beads</strong></li><li>Use the <strong>Clarity Protein Cleanup Kit</strong> to improve protein digestion</li></ul></li></ul><h4><strong>2. Solid-Phase Extraction (SPE)</strong></h4><h5><strong>Conditioning and Equilibration</strong></h5><ul><li>Condition and equilibrate SPE material before loading</li><li>Skipping this step reduces recovery</li></ul><h5><strong>Sample Loading</strong></h5><ul><li>Adjust sample <strong>pH to ~5.5</strong> for optimal binding</li><li>Load sample slowly, especially in small formats (e.g., 2 mg plates)</li><li>Apply sample near the top frit</li><li>Use <strong>low flow rates</strong> to improve:<ul><li>Recovery</li><li>Accuracy</li><li>Reproducibility</li></ul></li></ul><h4><strong>3. Washing Steps</strong></h4><ul><li>Perform <strong>two wash steps</strong>:<ol><li>Equilibration buffer</li><li>Stronger wash buffer</li></ol></li><li>Optimization may be required depending on format</li><li>For small plate formats (≤25 mg):<ul><li>Start with <strong>low vacuum</strong></li><li>Gradually increase vacuum to fully remove wash solution</li></ul></li></ul><h4><strong>4. Elution</strong></h4><ul><li>Use the <strong>new MS-compatible elution buffer</strong></li><li>Dilute <strong>1:1 with water</strong> for direct injection</li></ul><p>Optional concentration step:</p><ul><li>Use a lyophilizer if lower detection limits are required</li><li>Avoid drying to complete dryness (difficult reconstitution)</li></ul><h4><strong>5. Troubleshooting Recovery Issues</strong></h4><p>If recovery is lower than expected:</p><ul><li>Perform <strong>two elution steps</strong></li><li>Increase organic content in the elution solvent</li></ul><h3><strong>Discussion and Key Considerations</strong></h3><ul><li>Sample pre-treatment is the most critical factor for recovery</li><li>Buffer ratios and incubation conditions may require optimization</li><li>Proper SPE conditioning and controlled flow rates significantly affect performance</li><li>Washing and elution conditions must be adapted to sample complexity and format</li></ul><p>The method emphasizes reproducibility and robustness while minimizing variability in routine workflows.</p><h3><strong>Conclusion</strong></h3><p><strong>Clarity™ XRO </strong>provides a streamlined and effective workflow for extracting oligonucleotides from complex biological samples. By carefully optimizing lysis conditions, SPE parameters, and elution strategies, users can significantly improve recovery, reproducibility, and overall analytical performance.</p>

Improving Oligonucleotide Conjugate Recovery with Clarity XRO

<p><strong>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.</strong></p><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><span><strong>Principle of SEC Separation</strong></span></h3><p>The separation mechanism is based on molecular size:</p><p><strong>Large molecules</strong><br>→ do not enter the pores<br>→ travel through the interstitial spaces between particles<br>→ elute earlier</p><p><strong>Small molecules</strong><br>→ enter the pores<br>→ follow a longer path<br>→ elute later</p><h3><span><strong>Key Parameters for Column Selection</strong></span></h3><h4><span><strong>1. Molecule Nature</strong></span></h4><p><strong>Start by evaluating:</strong></p><ul><li>hydrodynamic radius</li><li>molecular shape</li></ul><p><strong>Examples:</strong></p><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><span><strong>2. Pore Size Selection</strong></span></h4><p>The pore size of the stationary phase should be:</p><ul><li><strong>3–4 times larger than the hydrodynamic radius of the target molecule</strong></li></ul><h4><span><strong>3. Particle Size</strong></span></h4><ul><li>≤ 3 µm</li><li>ensures high separation efficiency</li></ul><h4><span><strong>4. Stationary Phase Material</strong></span></h4><ul><li>Prefer <strong>mechanically robust media</strong>, such as silica</li><li>Capable of withstanding <strong>high back pressure</strong></li></ul><h4><span><strong>5. Surface Chemistry</strong></span></h4><ul><li><strong>Chemical surface inertness is critical</strong></li><li><strong>Diol chemistry is preferred</strong>, because:<ul><li>it minimizes interactions</li><li>it provides a <strong>highly hydrophilic surface</strong></li></ul></li></ul><h4><span><strong>6. Column Hardware</strong></span></h4><ul><li>Use <strong>bio-inert hardware</strong></li><li>ensures:<ul><li>minimal analyte loss</li><li>high reproducibility</li></ul></li></ul><h3><span><strong>Available SEC Column Options (</strong></span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=353&amp;manufacturerItems=50&amp;search=dsec"><span><strong>BioZen dSEC Family</strong></span></a><span><strong>)</strong></span></h3><h4><span><strong>1. </strong></span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2192"><span><strong>90 Å (9 nm)</strong></span></a></h4><ul><li>peptides</li><li>small proteins</li><li>aggregates up to ~50 kDa</li></ul><h4><span><strong>2. </strong></span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1534"><span><strong>200 Å</strong></span></a></h4><ul><li>antibodies</li><li>antibody–drug conjugates</li><li>short oligonucleotides</li></ul><h4><span><strong>3. </strong></span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1535"><span><strong>700 Å (70 nm)</strong></span></a></h4><ul><li>AAVs</li><li>mRNA</li><li>large protein complexes (e.g., IgM)</li></ul><h3><span><strong>Column Characteristics</strong></span></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><span><strong>Performance and Calibration</strong></span></h3><h4><span><strong>Proteins and AAVs</strong></span></h4><ul><li><strong>90 Å</strong> → low molecular weight (up to ~50 kDa)</li><li><strong>200 Å</strong> → medium range (up to ~450–500 kDa)</li><li><strong>700 Å</strong> → large molecules (including AAVs)</li></ul><h4><span><strong>RNA and DNA</strong></span></h4><ul><li>separation of molecules with non-globular shapes</li><li><strong>200 Å</strong> → smaller oligonucleotides</li><li><strong>700 Å</strong> → larger nucleic acid chains (up to ~3000–4000 nucleotides)</li></ul><h3><span><strong>Conclusion</strong></span></h3><p>Selecting the correct SEC column requires:</p><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><p>This enables optimal separation of a wide range of biologics.</p>

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

<p><strong>This technical note presents a practical troubleshooting strategy for one of the most common issues in liquid chromatography (LC): shifting retention times. The approach is based on a systematic distinction between system-related (physical) and chromatographic (chemical) causes.</strong></p><p>A key diagnostic parameter used throughout the process is the <strong>T₀ (dead time)</strong>, representing an <strong>unretained peak</strong> that passes through the column without interaction.</p><h3><span><strong>Key Concept: T₀ (Dead Time / Unretained Peak)</strong></span></h3><p>T₀ is defined as the retention time of a compound that does not interact with the stationary phase and therefore elutes directly with the mobile phase.</p><h4><span><strong>How to Determine T₀</strong></span></h4><p>T₀ can be determined experimentally by adding a marker compound to the system:</p><ul><li><strong>Uracil</strong></li><li><strong>Potassium nitrate</strong></li></ul><p>These compounds are added to a blank or method and produce a measurable unretained peak used for troubleshooting.</p><h3><span><strong>Step 1: Evaluate T₀ Behavior</strong></span></h3><p>The first and most important step is to determine whether <strong>T₀ is constant or variable</strong>.</p><ul><li><span><strong>Case A: T₀ is Variable (Shifting)</strong></span><br>→ Indicates a <strong>system-related (physical) problem</strong><br>&nbsp;</li><li><span><strong>Case B: T₀ is Constant</strong></span><br>→ Indicates a <strong>chromatographic or chemical problem</strong></li></ul><h3><span><strong>System-Related Issues (T₀ is Variable)</strong></span></h3><p>If T₀ is shifting, the problem is likely related to the <strong>LC system hardware or physical parameters</strong>. This is especially true when <strong>all peaks shift together</strong> in the chromatogram.</p><h3><span><strong>Possible Causes</strong></span></h3><ul><li><span><strong>Incorrect Flow Rate</strong></span></li><li><span><strong>Pump Malfunction</strong></span><ul><li>Inconsistent or unstable flow delivery can cause retention time variability.</li></ul></li><li><span><strong>Incorrect Column Length</strong></span><ul><li>Using a column with different dimensions than expected.</li></ul></li><li><span><strong>Solvent Delivery Issues</strong></span><ul><li>Empty solvent reservoir (system running dry)</li><li>Blockages or restrictions (e.g., frits, solvent lines)</li></ul></li><li><span><strong>Tubing or System Volume Changes</strong></span><ul><li>Changes in tubing configuration</li><li>Differences in system dwell (swell) volume</li></ul></li></ul><h3><span><strong>Chromatographic / Chemical Issues (T₀ is Constant)</strong></span></h3><p>If T₀ remains stable but <strong>only specific peaks shift</strong>, the issue is likely related to <strong>chemistry or column conditions</strong>.</p><h4><span><strong>Possible Causes</strong></span></h4><ul><li><span><strong>Incorrect Stationary Phase</strong></span><ul><li>Column with same dimensions but different chemistry (e.g., different silica or bonded phase)</li></ul></li><li><p><span><strong>Ionizable Analytes</strong></span><br>Ionizable compounds exist in:</p><ul><li><strong>Neutral form - </strong>More <strong>hydrophobic</strong>, retained longer on a <strong>reversed-phase column</strong></li><li><strong>Ionized form</strong></li></ul><p><span><strong>Impact of pH</strong></span><br>Small pH changes in the mobile phase can significantly affect:</p><ul><li>Ionization state</li><li>Retention time of specific analytes</li></ul></li><li><span><strong>Mobile Phase Issues</strong></span><ul><li>Old solvents</li><li>Evaporation of volatile components</li><li>Changes in composition</li></ul></li><li><span><strong>4. Temperature Variations</strong></span><ul><li>Temperature affects analyte–stationary phase interactions</li><li>Even small fluctuations can shift retention times</li></ul></li></ul><h3><span><strong>Step-by-Step Troubleshooting Workflow</strong></span></h3><ol><li><span><strong>Add T₀ Marker</strong></span><ol><li>Use uracil or potassium nitrate</li></ol></li><li><span><strong>Monitor T₀</strong></span><ol><li>Determine whether it is constant or shifting</li></ol></li><li><span><strong>If T₀ is Variable → Check System</strong></span><ol><li>Flow rate settings</li><li>Pump performance</li><li>Solvent levels and lines</li><li>Column dimensions</li><li>Tubing and system volume</li></ol></li><li><span><strong>If T₀ is Constant → Check Chemistry</strong></span><ol><li>Column phase</li><li>Mobile phase composition and pH</li><li>Solvent quality</li><li>Temperature stability</li><li>Nature of analytes (ionizable vs. non-ionizable)</li></ol></li></ol><h3><span><strong>Discussion and Practical Insight</strong></span></h3><p>This troubleshooting approach simplifies a complex problem by dividing it into two main categories:</p><ul><li><strong>Physical/system-related</strong></li><li><strong>Chemical/chromatographic</strong></li></ul><p>The use of T₀ as a diagnostic tool allows rapid identification of the problem origin, reducing unnecessary trial-and-error steps.</p><p>Particular attention should be paid to <strong>ionizable compounds</strong>, as they are highly sensitive to <strong>pH variations</strong>, which can lead to significant and selective retention time shifts.</p><h3><span><strong>Conclusion</strong></span></h3><p>Retention time shifts in LC can be efficiently diagnosed by evaluating the behavior of the <strong>T₀ (unretained peak)</strong>:</p><ul><li><strong>Variable T₀ → System issue</strong></li><li><strong>Constant T₀ → Chromatographic/chemical issue</strong></li></ul><p>By following this structured approach, analysts can quickly narrow down the root cause and implement targeted corrective actions.</p>

Troubleshooting Retention Time Shifts in Liquid Chromatography 

<p><strong>Size exclusion chromatography (SEC) columns are highly sensitive to operating conditions, including the composition of the mobile phase and the nature of analyzed samples. Improper handling and unsuitable method conditions can significantly reduce column lifetime. This guide summarizes common causes of column degradation and provides practical recommendations for extending column lifespan.</strong></p><h3><span><strong>Common Causes of Reduced Column Lifetime</strong></span></h3><p>Several factors can negatively affect the performance and durability of SEC columns:</p><h4><span><strong>1. Contamination of the Stationary Phase</strong></span></h4><ul><li>Accumulation of impurities from samples or mobile phases</li><li>Microbial growth due to infrequent replacement of mobile phase</li></ul><h4><span><strong>2. Stationary Phase Degradation</strong></span></h4><ul><li>Caused by unsuitable pH conditions</li><li>Exposure to inappropriate temperatures</li><li>Use of harsh chemical agents</li></ul><h4><span><strong>3. Improper Column Storage</strong></span></h4><ul><li>Incorrect storage conditions may reduce column performance over time</li></ul><h4><span><strong>4. Column Inlet Blockage</strong></span></h4><ul><li>Particulates from insufficiently prepared samples can clog the column inlet</li></ul><h3><span><strong>Preventive Measures and Best Practices</strong></span></h3><h4><span><strong>1. Preventing Contamination</strong></span></h4><ul><li>Use high-quality solvents</li><li>Prepare buffer solutions daily</li><li>Filter both mobile phases and samples before use</li><li>Perform regular column cleaning</li></ul><p><strong>Important note: </strong>Not all SEC columns can be reverse flushed. For example:</p><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1534"><strong>Biozen dSEC-2</strong></a> and Yarra SEC-X7 columns <strong>must not be backflushed</strong></li></ul><h4><span><strong>2. Protecting the Stationary Phase</strong></span></h4><ul><li>Operate within recommended pH and temperature limits</li><li>When using strong denaturants (e.g., urea or SDS):<ul><li>Perform immediate and thorough water flushing after use</li></ul></li></ul><h4><span><strong>3. Proper Column Storage</strong></span></h4><p><strong>Before storage </strong>always clean the column thoroughly</p><ul><li><strong>Two storage options:</strong><ul><li><strong>Buffer with bacteriostatic agent</strong> (e.g., sodium azide)</li><li><strong>No buffer:</strong> water with 20% methanol</li></ul></li></ul><p><strong>When introducing methanol:</strong></p><ul><li>Use <strong>low flow rates</strong></li><li>Prevent pressure spikes that could damage the column</li></ul><p><strong>When restarting the column:</strong></p><ul><li>Consider that the column contains 20% methanol</li><li>Adjust flow conditions accordingly</li></ul><h4><span><strong>4. Preventing Column Inlet Clogging</strong></span></h4><p>Use protective components:</p><ul><li><strong>Guard column</strong></li><li><strong>Precolumn</strong></li></ul><p>These act as barriers to prevent contamination of the analytical column.</p><h5><span><strong>Types of Protection:</strong></span></h5><ul><li><strong>Security guard cartridges</strong><ul><li>Minimal added length</li></ul></li><li><strong>Short precolumns</strong><ul><li>Same stationary phase as analytical column</li></ul></li></ul><h5><span><strong>Key Advantage:</strong></span></h5><ul><li>Essential for columns that <strong>cannot be backflushed</strong></li><li>Protects against:<ul><li>Poorly centrifuged samples</li><li>Particulate contamination</li><li>Dirty sample matrices</li></ul></li></ul><h3><span><strong>Conclusion</strong></span></h3><p>Extending the lifetime of SEC columns requires careful attention to:</p><ul><li>Sample preparation</li><li>Mobile phase quality</li><li>Operating conditions</li><li>Storage procedures</li><li>Use of protective accessories</li></ul><p>Implementing these practices helps prevent contamination, degradation, and mechanical damage, ensuring consistent column performance and longer service life.</p>

Extending the Lifetime of Size Exclusion Columns

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