Nexera HPLC Troubleshooting Retention Time Problems

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<div class="container"><h1 class="title">Troubleshooting Retention Time Problems</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/retention-time-change.jpg" alt="Troubleshooting Retention Time Problems"></div> <h3><strong>Observe the historical retention time data and determine whether the problem has a pattern or is erratic.</strong></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">Is the retention time shifting from one injection to another?</div><div class="buttons"> <span class="btn-yes">[[Yes->Column Insulation]]</span> <span class="btn-no">[[No->Different But Consistent Retention Times]]</span> </div> </div>

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<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-retention-time-problems/column-temp.jpg" alt="Column Insulation"></div> <h3><strong>Site conditions, such as ambient temperature and exposure to air currents may be causing column temperature fluctuation and the retention time problem.</strong></h3> Maintaining the column temperature at a constant level increases analysis reproducibility and separation performance. There are three different types of thermal insulation devices available for the LC column. <ul> <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> </ul> <div class="ts-question">Is the column temperature stable?</div><div class="buttons"> <span class="btn-yes">[[Yes->Flow Line Leaks]]</span> <span class="btn-no">[[No->Column Oven]]</span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Different But Consistent Retention Times</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/retention-time-different.jpg" alt="Different But Consistent Retention Times"></div> <h3><strong>Observe the historical retention time data and determine whether the problem has a pattern or is erratic.</strong></h3> Different but consistent retention times may be attributable to different factors. <ul> <li>Tubing - changing the inner diameter of the tubing changes the volume of the system. Use care in changing tubing in the HPLC system.</li> <li>Column temperature - temperature changes can affect the analyte retention time.</li> <li>Mobile phase pH - the pH of the mobile phase can affect the analyte retention time.</li> <li>Mobile phase strength - the organic strength of the mobile phase greatly affects the analyte retention time.</li> <li>Flow rate - the flow rate of the mobile phase through the system directly affects the analyte retention time.</li> </ul> Observe the historical retention time data and determine whether the problem has a pattern or is erratic. <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Flow Line Leaks</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/flow-lines.jpg" alt="Flow Line Leaks"></div> <h3><strong>A leak in the flow line could affect the retention time of the analyte peaks.</strong></h3> A leak in the flow line before the detector flow cell could explain the absence of sample peaks. Visually inspect the flow line from needle to flow cell looking for signs of a leak: <ul> <li>Puddles</li> <li>Drips</li> <li>Crystallization of buffer</li> </ul> A leak in the flow line could affect the retention time of the analyte peaks. <div class="ts-question">Does visual examination reveal a leak in the flow line?</div><div class="buttons"> <span class="btn-yes">[[Yes->Tubing - Flow Line Connections]]</span> <span class="btn-no">[[No->Pressure Fluctuation]]</span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Column Oven</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/cto-instrument.jpg" alt="Column Oven"></div> <h3>Site conditions, such as ambient temperature and exposure to air currents may be causing column temperature fluctuation and the retention time problem.</h3> The Shimadzu column oven (CTO-20A/20AC) is an air-circulated thermostatic chamber designed to improve retention time reproducibility and separation performance by maintaining the temperature of the LC column and flow lines. In addition to the column, the oven can also house manual injectors, solvent mixers, column switching valves and other flow line accessories. Installing these items into the column oven isolates them from the influence of ambient temperature fluctuations and allows more stable analysis. Site conditions, such as ambient temperature and exposure to air currents may be causing column temperature fluctuation and the retention time problem. <div class="buttons"> <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> <h3><strong>A leak in the flow line could affect the retention time of the analyte peaks.</strong></h3> A leak in the flow line before the detector flow cell could explain the absence of sample peaks. Visually inspect the flow line from needle to flow cell looking for signs of a leak: <span class="btn-link">[[Cutting Resin->Cutting Resin]]</span> <span class="btn-link">[[Cutting SS->Cutting SS]]</span> <span class="btn-link">[[Tighten SS->Tighten SS]]</span> <div class="buttons"><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Pressure Fluctuation</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/pressure.jpg" alt="Pressure Fluctuation"></div> <h3><strong>Pump pressure can be used to diagnose problems with the flow rate.</strong></h3> When the solvent delivery system is working properly, the pressure indicator will display a very stable value. When the pump displays a large pressure fluctuation, it is an indication that the actual delivery flow rate is changing. For example, a lower pressure or wildly fluctuating pressure indicates a lower or fluctuating flow rate and will result in longer and inconsistent retention times. If the pressure is lower than expected or fluctuating, pump troubleshooting will be required. <div class="ts-question">Is the displayed pump pressure stable?</div><div class="buttons"> <span class="btn-yes">[[Yes->Pump Setting]]</span> <span class="btn-no">[[No->Loss of Prime]]</span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Pump Setting</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/LC-20AD-instrument.jpg" alt="Pump Setting"></div> <h3>Examine the pump flow rate setting and determine whether it is appropriate for the specific method.</h3> Verify that the flow rate is entered according to the specified method. The procedure for entering pump parameters from the various data systems is dependent on the data system. Refer to the instruction manual of the data system for the specific directions. Examine the pump flow rate setting and determine whether it is appropriate for the specific method. <div class="ts-question">Is the pump flow rate entered correctly?</div><div class="buttons"> <span class="btn-yes">[[Yes->Measuring the Flow Rate]]</span> <span class="btn-no">[[No->Flow Rate]]</span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Loss of Prime</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/lc-drain-valve.jpg" alt="Loss of Prime"></div> <h3>Air bubbles in the pump can cause a loss of prime that will lead to very unstable pump pressure.</h3> The solvent delivery system (pump) must be properly primed in order to function properly. Improper degassing of the mobile phase or leaks in the flow path can allow air to build up in the pump heads and cause a loss of the primed condition. Air bubbles in the pump can cause a loss of prime that will lead to very unstable pump pressure. <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: Air bubbles in the drain tubing attached to the drain valve are normal and not a cause for concern.</strong> <div class="ts-question">Has the pump been properly primed?</div><div class="buttons"> <span class="btn-yes"><a href="https://www.ssi.shimadzu.com/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-the-pump/index.html">Troubleshoot the Pump</a></span> <span class="btn-no">[[No->Priming the Pump]]</span><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-the-pump/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 ->Cutting SS]]</span> <span class="btn-link">[[Tighten SS ->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-the-pump/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 ->Cutting Resin]]</span> <span class="btn-link">[[Flow Line Connections ->Tubing - Flow Line Connections]]</span> <span class="btn-link">[[Tighten SS ->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-the-pump/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 ->Cutting SS]]</span> <span class="btn-link">[[Cutting Resin ->Cutting Resin]]</span> <div class="buttons"><span class="btn-back"><<back "Back">></span></div> </div>

<div class="container"><h1 class="title">Measuring the Flow Rate</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/lc-measuring-the-flow-rate.jpg" alt="Measuring the Flow Rate"></div> <ol> <li>Remove the column and replace it with a flow line union.</li> <li>Set the flow rate to 2.0 mL per minute.</li> <li>Allow the flow rate to stabilize for 5 minutes.</li> <li>Using a stopwatch and graduated cylinder, collect the flow from the drain tubing in the cylinder for 5 minutes.</li> <li>Calculate the volume of mobile phase collected in the 5 minute period.</li> </ol> <ul> <li>LC-20AB - The collected volume should be 10.0 mL ± 1%.</li> <li>LC-20AD - The collected volume should be 10.0 mL ± 1%.</li> <li>LC-20AT - The collected volume should be 10.0 mL ± 2%</li> </ul> <div class="ts-question">Is the actual flow rate through the system appropriate?</div><div class="buttons"> <span class="btn-yes">[[Yes->Solvent Choice]]</span> <span class="btn-no"><a href="https://www.ssi.shimadzu.com/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-the-pump/index.html">Troubleshoot the Pump</a></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">Solvent Choice</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/solvent-choice.jpg" alt="Solvent Choice"></div> <h3>Mobile phase chemistry could be changing due to mixing, degassing, preparation, etc.</h3> Choosing the correct solvents of suitable quality is the first step in the process. It is also important to use a consistent solvent supplier, because solvents from different suppliers may vary in purity specifications. Always use solvents and reagents (including water) that are specified HPLC grade or better. <span class="btn-link">[[Solvent Preparation->Solvent Preparation]]</span> <span class="btn-link">[[Solvent Delivery->Solvent Delivery]]</span> <span class="btn-link">[[Solvent Mixing->Solvent Mixing]]</span> <div class="ts-question">Is the mobile phase chemistry changing?</div><div class="buttons"> <span class="btn-yes">[[Yes->Mobile Phase Troubleshooting]]</span> <span class="btn-no">[[No->Column Failure - Causes]]</span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Mobile Phase Troubleshooting</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/mixer.jpg" alt="Mobile Phase Troubleshooting"></div> <h3>Mobile phase chemistry could be changing due to mixing, degassing, preparation, etc.</h3> There are a number of ways to solve mobile phase composition problems. If the problem is a mixing issue, install a mixing chamber or premix the mobile phase. If the problem is an air bubble in the solvent delivery system, degas all of the solvents that are included in the mobile phase. Solvent purity may also affect the analyte retention time. Ensure that the solvent used for mobile phase is equivalent to the historical analyses. If the solvent purity is the problem, the improvement options include changing the solvent type or increasing the purity level of the solvents. <div class="buttons"> <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-retention-time-problems/column-param.jpg" alt="Column Failure - Causes"></div> <h3>Monitor the column log book and historical values with this method.</h3> 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 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">Is the column showing signs of degradation?</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-retention-time-problems/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">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-retention-time-problems/bonded-phase.jpg" alt="Column Failure - Bonded Phase"></div> <h3>Monitor the column log book and historical values with this method.</h3> 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="ts-question">Is the column showing signs of degradation?</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 Failure - Void</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/column-void.jpg" alt="Column Failure - Void"></div> <h3>Monitor the column log book and historical values with this method.</h3> 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="ts-question">Is the column showing signs of degradation?</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 Failure - Contamination</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/column-contamination.jpg" alt="Column Failure - Contamination"></div> <h3>Monitor the column log book and historical values with this method.</h3> 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. Monitor the column log book and historical values with this method. <img class="warning" style="float: left; margin-bottom:30px" 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="ts-question">Is the column showing signs of degradation?</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">Priming the Pump</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/purge.jpg" alt="Priming the Pump"></div> <ol> <li>Fill the mobile phase reservoir with degassed mobile phase.</li> <li>Place the suction filter in the reservoir.</li> <li>Place the end of the drain tubing into the waste container, which has been placed on the floor.</li> <li>Turn the drain valve knob 90° counterclockwise to open the drain valve.</li> <li>Press CE to return to the Initial screen.</li> <li>Verify that the pressure for the displayed valve is in the range of [-0.3 to 0.3 MPa].</li> <li>Press the PURGE key. Pumping begins at the preset flow rate (P-FLOW).</li> <li>Allow the purge to run until the mobile phase flows from the drain tubing in a continuous and air free stream.</li> <li>Press the PURGE key to stop the pump.</li> </ol> <img class="warning" style="float: left; margin-bottom:30px" src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/virtual-advisor-warning-icon.png" alt="warning-icon"><strong>NOTE: Air bubbles in the drain tubing attached to the drain valve are normal and not a cause for concern. NOTE: If no mobile phase comes from the drain tubing, insert the tip of the supplied needle into the end of the drain tubing and using an attached syringe, draw the mobile phase through the tubing.</strong> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Solvent Preparation</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/solvent-delivery.jpg" alt="Solvent Preparation"></div> <h3>Mobile phase chemistry could be changing due to mixing, degassing, preparation, etc.</h3> Whether preparing premixed isocratic mobile phases or non-mixed gradient solvents, care should be taken in the preparation process. Use caution to ensure that clean containers are used for solvent reservoirs. Employ a methodical process for buffer preparation to ensure repeatable preparations. All solvents should be filtered through a 0.45 µm (or smaller) pore size. Incorporate a thorough degassing process in the preparation process. <span class="btn-link">[[Solvent Choice->Solvent Choice]]</span> <span class="btn-link">[[Solvent Delivery->Solvent Delivery]]</span> <span class="btn-link">[[Solvent Mixing->Solvent Mixing]]</span> <div class="ts-question">Is the mobile phase chemistry changing?</div><div class="buttons"> <span class="btn-yes">[[Yes->Mobile Phase Troubleshooting]]</span> <span class="btn-no">[[No->Column Failure - Causes]]</span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Solvent Delivery</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/solvent-delivery.jpg" alt="Solvent Delivery"></div> <h3>Mobile phase chemistry could be changing due to mixing, degassing, preparation, etc.</h3> It is important to use the designated solvent delivery type. Results may vary if results are compared from systems that incorporate different pump types. Differences in high-pressure and low-pressure gradient elution processes make it impossible to interchange them. Be sure to use the correct pump type for the application as specified in the method. <span class="btn-link">[[Solvent Choice->Solvent Choice]]</span> <span class="btn-link">[[Solvent Preparation->Solvent Preparation]]</span> <span class="btn-link">[[Solvent Mixing->Solvent Mixing]]</span> <div class="ts-question">Is the mobile phase chemistry changing?</div><div class="buttons"> <span class="btn-yes">[[Yes->Mobile Phase Troubleshooting]]</span> <span class="btn-no">[[No->Column Failure - Causes]]</span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Solvent Mixing</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-retention-time-problems/solvent-mix.jpg" alt="Solvent Mixing"></div> <h3>Mobile phase chemistry could be changing due to mixing, degassing, preparation, etc.</h3> It is important to use the designated solvent delivery type. Results may vary if results are compared from systems that incorporate different pump types. Differences in high-pressure and low-pressure gradient elution processes make it impossible to interchange them. Be sure to use the correct pump type for the application as specified in the method. <span class="btn-link">[[Solvent Choice->Solvent Choice]]</span> <span class="btn-link">[[Solvent Preparation->Solvent Preparation]]</span> <span class="btn-link">[[Solvent Delivery->Solvent Delivery]]</span> <div class="ts-question">Is the mobile phase chemistry changing?</div><div class="buttons"> <span class="btn-yes">[[Yes->Mobile Phase Troubleshooting]]</span> <span class="btn-no">[[No->Column Failure - Causes]]</span><span class="btn-back"><<back "Back">></span> </div> </div>