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<div class="container"><h1 class="title">Troubleshooting the Loss of Resolution </h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-loss-resolution/Gradient.jpg" alt="Troubleshooting Chromatography Problems "></div> <h3>''Re-examine the gradient programming.''</h3> Typically, shallower gradients (smaller change in concentration per minute) produce greater resolution. Sharper gradients (larger changes in concentration per minute) may provide adequate resolution while increasing sample throughput. Ensure that the gradient time is long enough for adequate resolution or is as the method specifies. <div class="ts-question">Are the gradient parameters entered properly for the desired gradient changes?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Mobile Phase Preparation]]</span> <span class="btn-no">[[No->Entering the Gradient]]</span> </div> </div>

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<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-loss-resolution/Bottle.jpg" alt="Mobile Phase Bottle"></div><h3>Re-examine the mobile phase preparation.</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">Was the mobile phase prepared properly?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Flow Rate Effect on Resolution]]</span> <span class="btn-no">[[No->Mobile Phase - Preparation]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Entering the Gradient</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-loss-resolution/GradientEnter.jpg" alt="Entering the Gradient"></div> Follow these precautions during mobile phase preparation: Often, errors are made when entering gradient parameters. To ensure the use of a linear or step gradient, it is important that the parameters be entered correctly. Using the example above, re-examine the method and the gradient parameters to ensure that the gradient parameters have been entered properly. <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Flow Rate Effect on Resolution</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-loss-resolution/FlowResolution.jpg" alt="Flow Rate Effect on Resolution"></div> The flow rate can have a moderate effect on resolution. Refer to the column information to select the appropriate flow rate. <div class="ts-question">Is the flow rate parameter entered properly? </div> <div class="buttons"> <span class="btn-yes">[[Yes->Column Insulation]]</span> <span class="btn-no">[[No->Flow Rate]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<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-loss-resolution/DirtyBottle.jpg" alt="Mobile Phase Preparation"></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">Column Insulation</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-loss-resolution/ColumnTemp.jpg" alt="Column Insulation"></div> <h3>Maintaining the column temperature at a constant level increases analysis reproducibility and separation performance. </h3> There are three different types of thermal insulation devices available for the LC column. <ol> <li>CTO-30A and the CTO-30AS with the SIL-30AC MP Column Oven - Uses a heating block to control the temperature of the LC column.</li> <li>CTO-20A/20AC Column Oven - Uses an air-circulated thermostatic chamber to maintain the temperature of the LC column and flow lines.</li> <li>Jacket-type Devices - Uses plumbing insulation or heat tape to a lesser degree of effectiveness.</li> </ol> <div class="ts-question">Is the column temperature parameter entered properly? </div> <div class="buttons"> <span class="btn-yes">[[Yes->Check and Recheck the Method]]</span> <span class="btn-no">[[No->Setting the Temperature]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Flow Rate</h1> <div class="center"> <iframe width="900" height="506" src="https://www.youtube.com/embed/49GhQ7HaOAI?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">Check and Recheck the Method</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-loss-resolution/Column.jpg" alt="Column Check and ReCheck"></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 column in use one of the correct type and size?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Column Performance]]</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">Setting the Temperature</h1> <div class="center"> <iframe width="900" height="506" src="https://www.youtube.com/embed/lLyenJrQ--U?rel=0" title="YouTube video player" frameborder="0" allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture; web-share" allowfullscreen></iframe></div> <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: The SET TEMP (operating temperature setting) screen can also be displayed by pressing the TEMP key. NOTE: The temperature set here is displayed as the set temperature on the Initial screen and remains in memory when the power is turned off.</strong> <ol> <li>From the Initial screen, press the FUNC key. The PARAMETER screen is displayed.</li> <li>Press ENTER. This displays the SET TEMP screen.</li> <li>Enter the desired operating temperature (4-85°). 40°C is used here for this example.</li> <li>Press 4 - 0 and the ENTER key.</li> <li>Press CE twice 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">Column Performance</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-loss-resolution/ColumnParam.jpg" alt="Column Check and ReCheck"></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?</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">Following the Method</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-loss-resolution/Column.jpg" alt="Column Check and ReCheck"></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">Column Regeneration</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-loss-resolution/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. 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>

Troubleshooting the Loss of Resolution

Re-examine the gradient programming.

Typically, shallower gradients (smaller change in concentration per minute) produce greater resolution.

Sharper gradients (larger changes in concentration per minute) may provide adequate resolution while increasing sample throughput.

Ensure that the gradient time is long enough for adequate resolution or is as the method specifies.

Are the gradient parameters entered properly for the desired gradient changes?

Yes No

Nexera Virtual Advisor

Troubleshooting the Loss of Resolution

Re-examine the gradient programming.