Nexera HPLC Troubleshooting Tailing Peaks

Browser lacks capabilities required to play.

Upgrade or switch to another browser.

Loading…

<div class="container"><h1 class="title">Troubleshooting Tailing Peaks</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-tailing-peaks/column.jpg" alt="Troubleshooting Tailing Peaks"></div> <h3>There is always a need to recheck the method.</h3> It is a very good practice to check and recheck the method even if this is a method that is run often. Things to recheck are listed below: <ul> <li>Column type and size</li> <li>Mobile phase composition and gradient conditions</li> <li>Flow rate</li> <li>Wavelength</li> <li>Sample preparation</li> </ul> Re-examine these items and verify that they are appropriate. <div class="ts-question">Is the method being followed properly with respect to the column, mobile phase, flow rate and wavelength?</div><div class="buttons"> <span class="btn-yes">[[Yes->Mobile Phase Preparation]]</span> <span class="btn-no">[[No->Recheck the Method]]</span> </div> </div>

Config.debug = false;

<div class="container"><h1 class="title">Mobile Phase Preparation</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-tailing-peaks/mobile-phase-bottle.jpg" alt="Mobile Phase Preparation"></div> <h3>Sometimes, mobile phase preparation errors or contamination may be the source of the peak tailing.</h3> Follow these precautions during mobile phase preparation: <ul> <li>Use the proper solvents in the proper ratio.</li> <li>Use only HPLC grade solvents or better.</li> <li>Filter and degas all solvents.</li> <li>Ensure that the mobile phase reservoir contains a sufficient supply of mobile phase for the analysis sequence.</li> </ul> <div class="ts-question">Has a freshly prepared and fully degassed mobile phase preparation been tried on the system?</div><div class="buttons"> <span class="btn-yes">[[Yes->Retention Time Change Pattern]]</span> <span class="btn-no">[[No->Preparation of Mobile Phase]]</span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Recheck the Method</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-tailing-peaks/column.jpg" alt="Recheck the Method"></div> <h3>After checking the method, correct any discrepancies.</h3> Some Precautions: <ul> <li>Verify that all flow line connections that are disturbed are replaced properly.</li> <li>Ensure that all mobile phase solvents are thoroughly degassed.</li> <li>Do not allow the maximum flow rate or pressure to exceed the recommendations for the specific column.</li> <li>Allow the lamp(s) to equilibrate fully before proceeding with analysis.</li> <li>Use the mobile phase as the sample diluent if possible</li> </ul> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Retention Time Change Pattern</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-tailing-peaks/retention-time-change.jpg" alt="Retention Time Change Pattern"></div> <h3>Chromatographic problems can manifest themselves as tailing peaks but may actually be another problem.</h3> Several injections must be observed to diagnose a retention time problem. Different patterns in the retention times indicate different diagnoses. <ul> <li>Drifting (shorter retention times) - indicates column degradation.</li> <li>Drifting (longer retention times) - may indicate a steadily decreasing column temperature.</li> <li>Irregular - indicates a random shifting of the flow rate of the analytes through the column matrix.</li> <li>Steady but Different - indicates a change in the volume of the system flow line.</li> </ul> <div class="ts-question">Are the peak retention times consistent with expectations?</div><div class="buttons"> <span class="btn-yes">[[Yes->Dead Volume]]</span> <span class="btn-no"><a href="https://www.ssi.shimadzu.com/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-retention-time/index.html">Troubleshoot Retention Time Problems</a></span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Preparation of Mobile Phase</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-tailing-peaks/dirty-mobile-phase-bottle.jpg" alt="Preparation of Mobile Phase"></div> Either the incorrect mobile phase is being used or the mobile phase was prepared incorrectly. In either case, the mobile phase must be freshly prepared. Ensure that the flow lines are rinsed with the new, freshly prepared and degassed mobile phase. <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Dead Volume</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-tailing-peaks/tubing-connections.jpg" alt="Dead Volume"></div> <h3>Tubing connections can add a significant amount of dead volume to the total system volume that may manifest as tailing peaks.</h3> Regardless of whether stainless steel or resin tubing is being used, it is important to use care when selecting connection parts. Different manufactures have different connection types. It is not advisable to use a ferrule from company A with a union from company B etc. It is necessary to push the tubing all the way into the connector to reduce dead volume that causes band broadening. Proper cutting of the tubing is also critical in performing HPLC connections, as the cut edge will cause dead volume if it is not cut even and square. <div class="ts-question">Is the system tubing properly connected?</div><div class="buttons"> <span class="btn-yes">[[Yes->Column Failure - Causes]]</span> <span class="btn-no">[[No->Tubing - Flow Line Connections]]</span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Column Failure - Causes</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-tailing-peaks/column-parameters.jpg" alt="Column Failure - Causes"></div> By monitoring certain column parameters, it is possible to determine when a column is starting to deteriorate and might need to be replaced. The following column parameters are important to monitor for each column. <ul> <li>Theoretical plates</li> <li>Capacity factor</li> <li>Selectivity</li> <li>Tailing factor</li> <li>Pressure</li> </ul> There are three main reasons for column failure. Select from the items below for more information. <ul> <li>Loss of the bonded phase</li> <li>Formation of voids</li> <li>Build-up of contamination</li> </ul> <span class="btn-link">[[Bonded Phase->Bonded Phase]]</span> <span class="btn-link">[[Void->Void]]</span> <span class="btn-link">[[Contamination->Contamination]]</span> <div class="ts-question">Has a different column of the same type and size been tried on the system?</div><div class="buttons"> <span class="btn-yes">[[Yes->Sample Adsorption]]</span> <span class="btn-no">[[No->Column Replacement]]</span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Tubing - Flow Line Connections</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/tubing-flow-line-connections.jpg" alt="Tubing - Flow Line Connections"></div> <ol> <li>Place a male nut and ferrule on the tubing.</li> <li>Insert the end of the tubing with the ferrule completely into the appropriate fitting.</li> <li>Push the tubing into the fitting until it butts up against the end of the fitting (tubing stop position).</li> <li>While making sure the tubing remains in the tubing stop position, securely tighten the male nut. The ferrule is secured to the tubing.</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: It is important to apply a significant amount of pressure to the tubing while tightening the male nut.</strong> <span class="btn-link">[[Cutting Resin->Tubing - Cutting Resin]]</span> <span class="btn-link">[[Cutting SS->Tubing - Cutting SS]]</span> <span class="btn-link">[[Tighten SS->Tubing - Tighten SS]]</span> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Column Failure - Bonded Phase</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-tailing-peaks/bonded-phase.jpg" alt="Column Failure - Bonded Phase"></div> Consistently decreasing retention times may be caused by a loss of bonded phase from the column's silica support. Hydrolysis of the bonded phase is most severe in systems with a mobile phase pH of less than 3. As a column deteriorates, retention times and resolution decrease; peak tailing may also become evident. <span class="btn-link">[[Causes->Column Failure - Causes]]</span> <span class="btn-link">[[Void->Void]]</span> <span class="btn-link">[[Contamination->Contamination]]</span> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Column Failure - Void</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-tailing-peaks/column-void.jpg" alt="Column Failure - Void"></div> The most common causes of voids at the head of the column are violent pressure changes, gradual settling in poorly packed columns and high pH dissolving the silica packing. As a void forms at the head of the column, efficiency (N) drops, resolution decreases and peaks broaden, split or begin to tail. <span class="btn-link">[[Causes->Column Failure - Causes]]</span> <span class="btn-link">[[Bonded Phase->Bonded Phase]]</span> <span class="btn-link">[[Contamination->Contamination]]</span> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Sample Adsorption</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-tailing-peaks/adsorbtion-active-sites.jpg" alt="Sample Adsorption"></div> Active sites in the column and sample flow line may cause band spreading that may look like tailing peaks. Sometimes, the sample components may adhere to the surface of the chromatographic system components such as stainless steel tubing polymeric seal surfaces or the column. These sample components may also adhere to material that has been deposited in the system from previous dirty sample matrixes. This phenomenon may be manifested in missing peaks, tailing peaks, carryover and other chromatographic problems. Some suggestions for remedial action include, flushing the system, washing the column and replacing SS tubing with PEEK tubing. Run a test mix made for your system to test for sample adsorption. <div class="ts-question">Are sample components adhering to active sites in the system?</div><div class="buttons"> <span class="btn-yes">[[Yes->HPLC System Flush]]</span> <span class="btn-no">[[No->Test Mix]]</span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Column Replacement</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-tailing-peaks/column-replacement.jpg" alt="Column Replacement"></div> Sometimes it is necessary for the purpose of troubleshooting to remove the current column and replace it with one of the same type and size. This technique allows the user to determine whether the initial column is the source of the chromatography problem. It is important to use another column of known adequate performance. Using a brand new column is recommended. Columns are a consumable item and eventually 'wear out' causing tailing, band broadening, and other chromatographic abnormalities. <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>IMPORTANT: If replacing the column does not correct the problem, rinse the new column for storage and replace the initial column into the flow path.</strong> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Column Failure - Contamination</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-tailing-peaks/column-contamination.jpg" alt="Column Failure - Contamination"></div> Column contamination is caused by a build-up of retained material on the stationary phase or by plugging of the column frit and packing by particulate matter, precipitated sample or buffer. Contamination can lead to shifting retention times, loss of resolution and poor peak shape. <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: Filtering of all sample solution is highly recommended. Avoid injecting a cloudy sample solution.</strong> <span class="btn-link">[[Causes->Column Failure - Causes]]</span> <span class="btn-link">[[Bonded Phase->Bonded Phase]]</span> <span class="btn-link">[[Void->Void]]</span> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">HPLC System Flush</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-tailing-peaks/column-tubing-union.jpg" alt="HPLC System Flush"></div> Contaminants in the system tubing may create active sites to which sample components adhere, causing peak tailing and other chromatographic problems. In these instances, it is necessary to flush (clean) the system tubing to remove these contaminants and active sites. <ol> <li>Remove the column and replace it with a tubing union.</li> <li>Depending on the composition of the current mobile phase, flush the system with 100% filtered degassed water for 10 minutes at a flow rate of 3mL/min.</li> <li>Exchange the water for an organic solvent in which the suspected contaminant is soluble.</li> <li>Flush the system with 100% filtered degassed organic solvent for 10 minutes at a flow rate of 3mL/min.</li> <li>Replace the solvent with water and flush for 10 more minutes at a flow rate of 3mL/min.</li> <li>Purge the system with fully degassed and filtered mobile phase.</li> <li>Reinstall the column and equilibrate the system at the appropriate method parameters (flow rate, etc.).</li> </ol> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Test Mix</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-tailing-peaks/test-mix.jpg" alt="Test Mix"></div> The Shimadzu HPLC Installation Test Kit (PN/ 220-91394-00) is used to verify the hardware performance after installation of a new Prominence HPLC system. This kit may also be used to evaluate the performance of the system hardware at regular maintenance intervals and before contacting a service provider. If the Test Kit performance is acceptable, it indicates that the system hardware is working adequately and that the column, mobile phase or sample solution is responsible for the abnormal peak shape. <ol> <li>Obtain a Shimadzu HPLC Installation Test Kit (PN/ 220-91394-00) from your local service provider.</li> <li>Flush the system (union - no column) with the mobile phase provided in the plastic bag at 2 mL/min for 10 minutes.</li> <li>Turn off the flow and install the column.</li> <li>Equilibrate the column at 1.0 mL/min for about 30 minutes.</li> <li>Open an ampoule of the test mix and transfer the contents to a clean sample vial.</li> <li>Set the detector wavelength to 254 nm.</li> <li>Inject 5 µl of the test mix onto the column.</li> <li>Record the results and compare them to the chromatogram show in the Certificate of Composition provided with the Test Kit.</li> </ol> <div class="ts-question">Run a test mix solution on the system, is the chromatogram normal?</div><div class="buttons"> <span class="btn-yes">[[Yes->HPLC System Flush]]</span> <span class="btn-no">[[No->Column Regeneration]]</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-tailing-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>

<div class="container"><h1 class="title">Tubing - Cutting Resin</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/tubing-cutting-resin.jpg" alt="Tubing - Cutting Resin"></div> When using PEEK tubing or equivalent resin tubing, use a straight edge razor knife to cut the tubing. To avoid the creation of void volumes, verify that the cut edges are clean right-angled cuts. <span class="btn-link">[[Flow Line Connections->Tubing - Flow Line Connections]]</span> <span class="btn-link">[[Cutting SS->Tubing - Cutting SS]]</span> <span class="btn-link">[[Tighten SS->Tubing - Tighten SS]]</span> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Tubing - Cutting SS</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/tubing-cutting-ss.jpg" alt="Tubing - Cutting SS"></div> When using HPLC stainless steel tubing, it is best to use precut stainless steel tubing or employ a cutting technique that produces a smooth and even surface to the cut edge. If the cut edge of the tubing is not smooth and square, a void volume is created in the connection. <span class="btn-link">[[Cutting Resin->Tubing - Cutting Resin]]</span> <span class="btn-link">[[Flow Line Connections->Tubing - Flow Line Connections]]</span> <span class="btn-link">[[Tighten SS->Tubing - Tighten SS]]</span> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Tubing - Tighten SS</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/tubing-tighten-ss.jpg" alt="Tubing - Tighten SS"></div> Verify that the stainless steel unions are securely connected. Use two wrenches to tighten stainless steel couplings. <span class="btn-link">[[Flow Line Connections->Tubing - Flow Line Connections]]</span> <span class="btn-link">[[Cutting SS->Tubing - Cutting SS]]</span> <span class="btn-link">[[Cutting Resin->Tubing - Cutting Resin]]</span> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

Combined two Column failure - causes https://nexerava.ssi.shimadzu.com/node/1926 https://nexerava.ssi.shimadzu.com/node/2336