Nexera HPLC Troubleshooting Fronting Peaks

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<div class="container"><h1 class="title">Troubleshooting Fronting Peaks</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-fronting-peaks/shark-fin-fronting-peaks.jpg" alt="Troubleshooting Fronting Peaks"></div> <h3>Sample insolubility in mobile phase causes a special kind of peak shape that is often referred to as fronting.</h3> Fronting is the phenomenon in which the top of the peak is delayed due to inadequate solubility of the sample. When the sample is injected into the mobile phase for transport to the column, the sample mixes with some portion of the mobile phase. If the sample is not quite soluble in the mobile phase, these types of fronting peaks may be produced. <div class="ts-question">Is the sample insolubility causing the shark-fin shaped fronting peaks?</div><div class="buttons"> <span class="btn-yes">[[Yes->Sample Solubility Problems - Decreasing the Effect]]</span> <span class="btn-no">[[No->Sample Solvent - Strong]]</span> </div> </div>

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<div class="container"><h1 class="title">Sample Solubility Problems - Decreasing the Effect</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-fronting-peaks/shark-fix.jpg" alt="Sample Solubility Problems - Decreasing the Effect"></div> There are three different ways to reduce the effect of sample solubility problems. <ul> <li>Dilution - Dilute the sample solution until the peak shape returns to a more appropriate shape.</li> <li>Change the Diluent - Use mobile phase as the sample solvent or change the diluent to match the mobile phase composition.</li> <li>Inject Less - Reduce the injection volume.</li> </ul> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Sample Solvent - Strong</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-fronting-peaks/fronting-peak.jpg" alt="Sample Solvent - Strong"></div> Often when the sample is dissolved in a stronger solvent (i.e. 100% MeOH) than the mobile phase (i.e. 50:50 MeOH:Water), the peak shape will be affected. In this situation, fronting shoulders are more noticeable in the earlier eluting peaks than in the later peaks. Decrease the injection volume by 1/2 and make 3-5 repeat injections. If decreasing the injection volume improves the peak shape, it is an indication that the sample solvent is stronger than the mobile phase. If decreasing the injection volume does not improve the peak shape, it is an indication of a probable mixing problem. Add a mobile phase mixing chamber or increase the mixing volume. <div class="ts-question">Is the sample solvent too strong for the mobile phase?</div><div class="buttons"> <span class="btn-yes">[[Yes->Strong Sample Solvents - Remedial Action]]</span> <span class="btn-no">[[No->Column Performance]]</span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Strong Sample Solvents - Remedial Action</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-fronting-peaks/fronting-peak.jpg" alt="Sample Solvent - Strong"></div> BEST - The optimal choice for sample diluent is mobile phase at the initial conditions. NEXT - For typical reverse-phase systems where the BEST choice is not applicable, use less organic solvent or more water in the sample diluent. LAST - If the BEST and NEXT choices will not work with the application, a reduction in the injection volume may reduce the fronting problem. <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Column Performance</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-fronting-peaks/column-param.jpg" alt="Column Performance"></div> It is necessary to use a column logbook to record the performance of the column. Record the initial column parameters such as pressure, analyte retention time, Resolution (R), Plate count (N) and Tailing Factor (T). By periodically recording this information, it is possible to determine when a column needs to be flushed, regenerated or retired. <div class="ts-question">Has the column performance degraded?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Column Regeneration]]</span> <span class="btn-no"><a href="https://www.ssi.shimadzu.com/forms/tech-support/index.html?product_category=VirtualAdvisor&product_name=Nexera&page=webform_start">Contact Shimadzu</a></span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Column Regeneration</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-fronting-peaks/column.jpg" alt="Column Regeneration"></div> <strong>Reversed-Phase Column Regeneration</strong> <ol> <li>Disconnect the column and reconnect it to the pump in the reverse direction. Do not connect it to the detector.</li> <li>Flush out the salts and or buffers with 25 mL of mobile phase (without buffer) at 1mL/min.</li> <li>Flush the column with 25 mL of HPLC grade water.</li> <li>Flush the column with 25 mL of isopropanol.</li> <li>Flush the column with 25 mL of methylene chloride.</li> <li>Flush the column with 25 mL of hexane.</li> <li>Remove the hexane by flushing again with 25 mL of methylene chloride.</li> <li>Remove the methylene chloride by flushing again with 25 mL of isopropanol.</li> <li>Reconnect the column in the proper direction and flush the column with the mobile phase (without buffer), then reintroduce the buffer.</li> <li>Equilibrate the column with 25-50 mL of mobile phase.</li> <li>Inject a standard solution to re-evaluate column performance.</li> </ol> <strong>Normal-Phase Column Regeneration</strong> <ol> <li>Disconnect the column and reconnect it to the pump in the reverse direction. Do not connect it to the detector.</li> <li>Flush the column with 50 mL of 50:50 methanol:chloroform.</li> <li>Flush the column with 50 mL of ethyl acetate.</li> <li>Reconnect the column in the proper direction.</li> <li>Equilibrate the column with 50 mL of mobile phase.</li> <li>Inject a standard solution to re-evaluate column performance.</li> </ol> <img class="warning" style="float: left;" src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/virtual-advisor-warning-icon.png" alt="warning-icon"><strong>NOTE: In some cases, dimethyl-formamide might be a better choice of 'cleaning' solvent than the methylene chloride and hexane. Always refer to the column vendor for the recommended washing technique for that column type.</strong> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>