Nexera HPLC Troubleshooting

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<div class="container"><h1 class="title">Troubleshooting Broad Peaks</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-broad-peaks/ColumnEquilibrium.jpg" alt="Troubleshooting Broad Peaks"></div> Column equilibration is the time required for the mobile phase and stationary phase of the column to stabilize. Typically, when the mobile phase first starts flowing through the column, the baseline will drift dramatically and begin to stabilize when equilibration is reached. The time required for column equilibration is dependant on the column dimensions. Typically, allowing 15-20 column volumes of mobile phase to flow through the column is necessary to achieve equilibration. The table above displays some average times required for column equilibration. <div class="ts-question">Has the column been allowed to properly equilibrate to the mobile phase?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Following the Method?]]</span> <span class="btn-no">[[No->Longer Column Equilibration]]</span> </div> </div>

<div class="container"><h1 class="title">Following the Method?</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-broad-peaks/Column.jpg" alt="Following the Method"></div> <h3>It is a very good practice to check and recheck the method even if this is a method that is run often. </h3> 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->Column Pressure]]</span> <span class="btn-no">[[No->Following the Method]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Longer Column Equilibration</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-broad-peaks/ColumnEquilibrium.jpg" alt="Troubleshooting Broad Peaks"></div> The time required for column equilibration is dependent on the column dimensions. Typically, flushing 15-20 column volumes through the column is necessary to achieve equilibration. Sometime the column may become 'contaminated' with sample matrix etc. On these occasions, it may be necessary to flush the column for a longer period than the average recommendations. Use the table above to determine the average time required for column equilibration. <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Column Pressure</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-broad-peaks/ColumnDiameter.jpg" alt="Column Diameter"></div> <h3>If the mobile phase flow rate is not appropriate for the column, the column may become damaged.</h3> The back-pressure limits for the column differ according to the manufacture's recommendation. Generally, these limits are based on the column length, diameter and size of the packing material. (See table to the left.) It is important to record the initial column back-pressure as a baseline for the increase that is expected with extended use. Record this information in a column log book for future reference and for monitoring column performance. Refer to documentation provided by the column vendor for the pressure limits and expected initial back-pressures for that column. <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: Conversion</strong> 1kgf/cm2=0.098MPa=0.98bar=14.2psi <div class="ts-question">Is the mobile phase flow rate appropriate for the column?</div> <div class="buttons"> <span class="btn-yes">[[Yes->System Volume]]</span> <span class="btn-no">[[No->Changing the Flow Rate]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Following the Method</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-broad-peaks/Column.jpg" alt="Following 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">System Volume</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-broad-peaks/BandBroadening.jpg" alt="System Volume"></div> <h3>System volume plays a large role in band broadening.</h3> The sample is injected in a concise band within the flow line tubing. All areas in the pathway where there is more space than before allows the sample band to spread out or broaden. The wider the sample band, the longer it takes to move through the column and the broader the sample peak will become. Examine the system and determine whether the tubing length or inner diameter can be reduced, number of connections can be reduced or quality of the tubing connections can be improved. <div class="ts-question">Is the total system volume excessive to the extent of causing band broadening?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Total System Volume]]</span> <span class="btn-no">[[No->Tubing Connections]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Changing the Flow Rate</h1> <div class="center"> <iframe width="900" height="506" src="https://www.youtube.com/embed/Dfbl50LIxuU?rel=0" title="YouTube video player" frameborder="0" allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture; web-share" allowfullscreen></iframe></div> <ol> <li>From the Initial screen, press the FUNC key once. The cursor becomes active at the flow rate setting.</li> <li>Enter the desired flow rate using the keypad.</li> <li>Press ENTER to save the new flow rate settings.</li> <li> Press CE to return to the Initial screen.</li> </ol> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Total System Volume</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-broad-peaks/TotalSystemVolume.jpg" alt="Total System Volume"></div> <strong>The total volume of the system is affected by:</strong> <ul> <li>Length of the tubing </li> <li>Inner diameter of the tubing </li> <li>Number of connections </li> <li>Quality of the connections </li> <li>Diameter of the column </li> <li>Length of the column </li> <li>Size of the flow cell </li> </ul> Take efforts to minimize all of these items to reduce band broadening. <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Tubing Connections</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-broad-peaks/Tubing_Conn.jpg" alt="Tubing Connections"></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->Autosampler Tubing]]</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">Autosampler Tubing</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-broad-peaks/Flow.jpg" alt="Autosampler Tubing"></div> <h3>The INLET and OUTLET tubing to the autosampler can be easily mistaken for one another.</h3> The tubing into and out of the autosampler may cause some confusion. The inlet tubing brings mobile phase from the pump or mixer into port 6 of the high-pressure valve.This inlet tubing IS marked with a blue band. The outlet tubing carries the mobile phase and sample from port 5 of the high-pressure valve to the column inlet. This outlet tubing IS NOT marked with a blue band. <div class="ts-question">Is the autosampler plumbed properly?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Column Performance]]</span> <span class="btn-no">[[No->Autosampler Flow Line]]</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-broad-peaks/Tubing_FlowLineConnections.jpg" alt="Tubing - Flow Line Connections"></div> List of Steps: <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><br> <span class="btn-link">[[Cutting Resin->Cutting Resin]]</span><br> <span class="btn-link">[[Cutting SS->Cutting SS]]</span><br> <span class="btn-link">[[Tighten SS->Tighten SS]]</span><br> <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-broad-peaks/TubingKnife.jpg" alt="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">[[Tubing-Flow Line Connections->Tubing-Flow Line Connections]]</span><br> <span class="btn-link">[[Cutting SS->Cutting SS]]</span><br> <span class="btn-link">[[Tighten SS->Tighten SS]]</span><br> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div>

<div class="container"><h1 class="title">Tubing - Cutting Stainless Steel</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-broad-peaks/TubingSS.jpg" alt="Cutting Stainless Steel"></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->Cutting Resin]]</span><br> <span class="btn-link">[[Tubing-Flow Line Connections->Tubing-Flow Line Connections]]</span><br> <span class="btn-link">[[Tighten SS->Tighten SS]]</span><br> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div>

<div class="container"><h1 class="title">Tubing - Tighten Stainless Steel</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-broad-peaks/TubingTighten.jpg" alt="Tighten Stainless Steel"></div> Verify that the stainless steel unions are securely connected. Use two wrenches to tighten stainless steel couplings. <span class="btn-link">[[Tubing-Flow Line Connections->Tubing-Flow Line Connections]]</span><br> <span class="btn-link">[[Cutting SS->Cutting SS]]</span><br> <span class="btn-link">[[Cutting Resin->Cutting Resin]]</span><br> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </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-broad-peaks/ColumnParam.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 deteriorated to cause broad peaks?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Column Regeneration]]</span> <span class="btn-no"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-the-autosampler/index.html">Troubleshooting the Autosampler</a></span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Autosampler Flow Line</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-broad-peaks/Flow.jpg" alt="Autosampler Flow Line"></div> <h3>The autosampler flow line is detailed above. Ensure that the flow lines are plumbed correctly. Please examine these flow lines and connections for blockage or leaks.</h3> The tubing into and out of the autosampler may cause some confusion. The inlet tubing brings mobile phase from the pump or mixer into port 6 of the high-pressure valve. This inlet tubing is marked with a blue band. The outlet tubing carries the mobile phase and sample from port 5 of the high-pressure valve to the column inlet. This outlet tubing is NOT marked with a blue band. <div class="buttons"> <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-broad-peaks/Column.jpg" alt="Column Regeneration"></div> <h3>Reversed-Phase Column Regeneration</h3> <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> <h3>Normal-Phase Column Regeneration</h3> <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. </strong>Always refer to the column vendor for the recommended washing technique for that column type. <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>