Nexera HPLC Troubleshooting

Browser lacks capabilities required to play.

Upgrade or switch to another browser.

Loading…

<div class="container"><h1 class="title">Troubleshooting No Peaks</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/SolventPeak.jpg" alt="Troubleshooting No Peaks"></div> <h3>By closely examining the first few minutes of the chromatogram, it can be determined whether an injection is being made.</h3> The injection process typically produces a baseline disturbance caused by the detectors response to: *the sample diluent. *a pressure fluctuation from the rotation of the injection valve. *a change in the refractive index The retention time of this disturbance is indicative of the column volume and is referred to as the void time (time=0). If there is no indication of a successful injection, the problem is most likely with the signal cable, the injection process or the detector. If the solvent peaks are present, it is an indication that something was injected and the detector is responding to it. If there is a chromatography problem, it may be related to the injector, detector, or sample solution. <div class="ts-question">Is there any evidence of an injection, such as the presence solvent peaks?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Sample Vial Position]]</span> <span class="btn-no">[[No->Signal Noise Indication]]</span> </div> </div>

Config.debug = false;

<div class="container"><h1 class="title">Sample Vial Position</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/VialPosition.jpg" alt="Sample Vial Position"></div><h3>Does the autosampler know where to find the sample?</h3> The upper surface of the sample vial rack is marked with numbers at the vial positions. These numbers correspond to the sample vial positions in the chromatography software. Ensure that the sample vial is actually in the position designated in the injection sequence. <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 vial position numbers start with 1. Ensure that the sample vial is actually in the position designated in the injection sequence and that the proper rack type is designated.</strong> <div class="ts-question">Is the sample vial placed in the vial rack according to the injection sequence?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Sample Vial Volume]]</span> <span class="btn-no">[[No->Vial Position in the Autosampler]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Signal Noise Indication</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/SignalNoise.jpg" alt="Mobile Phase Bottle"></div><h3>It is possible that the signal cable is connected and that the injection process is not working is not working properly</h3> By enlarging the baseline and closely examining it for the presence of normal system noise, it can be determined whether the signal is actually reaching the data system. All baselines have an inherent amount of normal system noise. If there is no system noise, it is an indication that the signal is not reaching the data system. If system noise is present, it is an indication that the detector is at least responding minimally, but the injection process or detector may not be working properly. <div class="ts-question">Is the baseline completely flat, without displaying some system noise?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Wire Signal Cable]]</span> <span class="btn-no">[[No->Null Injection]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Wire Signal Cable</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/Wire.jpg" alt="Wire Signal Cable"></div> The detector signal cables must be properly connected to the data system for data collection. It is possible to connect analog signal cables in a way that would produce negative peaks. Use care when connecting these communication cables. <div class="ts-question">Are the signal cables properly connected between the detector and the data system?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Signal Choice]]</span> <span class="btn-no">[[No->Detector Connections]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Null Injection</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/Null_Injection_SIL-30AC.jpg" alt="Null Injection"></div> It is possible to program the autosampler to run a normal analysis process without physically moving the injection mechanisms. Local Control - Examine the SIL display and verify that the vial numbers are not entered as (-1). Remote Control - Examine the data system software to verify that the vial number is not entered as (-1). <div class="ts-question">Is the sample vial programmed as -1 for performing a null injection?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Changing the Injection Parameters]]</span> <span class="btn-no">[[No->Pressure Fluctuation]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Changing the Injection Parameters</h1> <div class="center"><iframe width="900" height="506" src="https://www.youtube.com/embed/XX7kxC-RWV0?rel=0" title="Changing the Injection Parameters" 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: If only one vial is to be injected, use the same number entered in the 'From' field.</strong> <br> <ol> <li>From the Initial screen, press EDIT. The Editing Sample Table screen is displayed briefly.</li> <li>The cursor is active in the 'Rack' field.</li> <li>Using the numeric keypad, enter the rack number and press ENTER.</li> <li>The cursor moves to the 'From' field.</li> <li>Using the numeric keypad, enter the first vial number to inject, and press ENTER.</li> <li>The cursor moves to the 'To' field.</li> <li>Using the numeric keypad, enter the last vial to inject, and press ENTER.</li> <li>The cursor moves to the 'Rep' (repetitions) field.</li> <li>Using the numeric keypad, enter the number of times the autosampler will inject from the specified vial, and press ENTER.</li> <li>The cursor moves to the 'Inj Volume' field.</li> <li>Using the numeric keypad, enter the desired injection volume (µL) and press ENTER.</li> <li>The cursor moves to the 'Runtime' field.</li> <li>Enter the analysis time (min) for each injection, and press ENTER.</li> <li>The next line of the sample table is displayed.</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">Pressure Fluctuation</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/LC_Front_Press.jpg" alt="Waste Discharge "></div> <h3>Monitor the system pressure to determine whether the mobile phase pressure is normal.</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 system pressure equivalent to the historical value?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Waste Discharge]]</span> <span class="btn-no">[[No->Pump Power and Flow Rate]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Pump Power and Flow Rate</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/PowerSwitch.jpg" alt="Waste Discharge "></div> <ol> <li>The power switch is located on the front of the instrument.</li> <li>Press the PUMP key on the pump keypad to start and stop the pumping action</li> <li>Once the power is on and the pump is pumping, the flow rate must be properly set.</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>Press the power switch once to turn the pump ON and again to turn the pump OFF. It is possible for power to the pump to be ON while the pumping action is OFF. The flow rate can be set at the pump (local mode) or via the controller or data system software (remote mode). </strong> <div class="ts-question">Is the pump power ON and is the flow rate set to the proper flow rate?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Mobile Phase Supply]]</span> <span class="btn-no">[[No->Pump Power and Flow Rate Continued]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Waste Discharge</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/Waste.jpg" alt="Waste Discharge "></div> Examine the waste tubing as it enters the waste container. Is the flow of the discharge appropriate for the flow rate? <div class="ts-question">Examine the waste tubing. Is mobile phase flowing from the flow cell outlet? </div><span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-the-autosampler/index.html">Troubleshooting the Autosampler</a></span><br> <div class="buttons"> <span class="btn-no">[[No->Flow Cell Inspection]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Flow Cell Inspection</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/FlowCellInspect.jpg" alt="Flow Cell Inspection"></div> If the flow cell is damaged, it may be leaking and preventing the entire sample from being measured. This could result in small or irregular peaks, or the absence of peaks. Use the following steps to visually inspect the detector and connections for signs of a leak, such as puddles, drips or crystallization of buffers. <ol> <li>Open the front cover of the detector.</li> <li>Loosen the coupling screw and remove the couplings from their holder. Leave the plumbing connected.</li> <li>Unscrew the two flow cell retaining screws and slide the cell out of the detector.</li> <li>With mobile phase flowing through the flow cell, inspect the flow cell for damage.</li> </ol> <div class="ts-question">Is the flow cell damaged?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Flow Cell Disassembly - SPD-M30A]]</span> <span class="btn-no">[[No->Flow Line Leaks]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Pump Power and Flow Rate</h1> <div class="center"><iframe width="900" height="506" src="https://www.youtube.com/embed/CUzqayHkwjs?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: It is possible for power to the pump to be ON while the pumping action is OFF. </strong> <ol> <li>Press the power switch to turn the pump power ON.</li> <li>Press the PUMP key on the pump keypad to start and stop the pumping action.</li> <li> From the Initial screen, press the FUNC key one time. The cursor blinks at flow rate.</li> <li>Press 2 - . - 0 - 0 - 0 - 0 keys to set the flow rate to 2.0000 mL per minute.</li> <li>Press ENTER to save the 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">Mobile Phase Supply</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/MobilePhase.jpg" alt="Mobile Phase Supply"></div> A sufficient supply of mobile phase is required to perform LC measurement. If the suction filter is blocked or not submerged in the mobile phase solution, air will be pulled into the flow line and could cause all flow to stop. Examine the mobile phase reservoir to ensure that the suction filter is fully submerged in the mobile phase solution. <div class="ts-question">Is there sufficient solvent in the mobile phase reservoir?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Drain Valve Closed]]</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">Drain Valve Closed</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/DrainValve.jpg" alt="Drain Valve Closed"></div> During normal pump operation, the drain valve is closed. However, the valve can be opened to replace the mobile phase in the flow lines or to bleed air from the flow lines. If the valve is not fully closed after these operations, the flow line remains open and mobile phase does not flow to the column. <div class="ts-question">Is the drain valve closed?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Flow Line Leaks]]</span> <span class="btn-no">[[No->Drain Valve Replacement]]</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-no-peaks/Bottle.jpg" alt="Mobile Phase Preparation"></div> 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="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Drain Valve Replacement</h1> <div class="center"><iframe width="900" height="506" src="https://www.youtube.com/embed/sk-GCHXTzSw?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: If the drain valve ASSY is forced in at an angle, the seal may be damaged. Any damage can compromise the seal.</strong> <ol> <li>Open the door</li> <li>Unscrew the drain valve ASSY by turning counterclockwise and pull straight out of the drain valve</li> <li>Wet the seal of the new drain valve ASSY by using the 2-propanol.</li> <li>Insert the drain valve ASSY into the hole of the housing in a straight line.</li> <li>Screw the new drain valve ASSY as far as it will go to close</li> <li>Screw the knob to the drain valve ASSY and insert the cap into the knob while the knob position is vertical..</li> <li>Close the door.</li> </ol> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Flow Cell Disassembly - SPD-M30A </h1> <div class="center"><iframe width="900" height="506" src="https://www.youtube.com/embed/nc_SYQNxK44?rel=0" title="Flow Cell Disassembly - SPD-M30A" 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: Clean the inner surface of the cell with a cotton swab moistened with 2-propanol. Sonicate the lenses for 5 minutes in 2-propanol. NOTE: The convex lens should be on the notch side with the convex side facing outward. </strong> <ol> <li>Open the door.</li> <li>Loosen the clamp securing the flow line couplings and disconnect the flow lines.</li> <li>Disconnect the heater assembly connector.</li> <li>Loosen screws and remove the flow cell.</li> <li>Remove the cell window screws, flat lens/convex lens and gaskets.</li> <li>Install new gaskets and flat lens/convex lens, and secure with the cell window screws.</li> <li>Check the flow cell for leaks.</li> <li>Reinsert the flow cell and finger tighten the screws.</li> <li>Reconnect the heater assembly connector.</li> <li>Reconnect the flow lines and secure the couplings in the clamp.</li> <li>Close the door.</li> </ol> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Signal Choice</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/SystemChoice.jpg" alt="Mobile Phase Bottle"></div> <h3>Since the connections are properly connected, we need to further troubleshoot the signal.</h3> Most often, multiple HPLC systems are connected to the data system. It is important to ensure that you are monitoring the correct instrument, channel, and detector wavelength. <div class="ts-question">Is the data system set up to monitor the proper signal?</div> <div class="buttons"> <span class="btn-yes">[[Yes->LAMP On]]</span> <span class="btn-no">[[No->System Signal Selection]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Detector Connections</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/SPD_Front.jpg" alt="Detector Connections"></div> Connections between the detector and data system vary depending on the type of data system being used. Some data systems connect digitally to the detector through the fiber optic cable, while others connect via analog signal cables. If the detector and data system are properly connected and the signal is either absent or unsuitable, check the data system parameters to ensure that the proper channels are being monitored. <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>WARNING: Turn OFF all of the system units and unplug the power cables before performing any wiring procedures. Do not use cables or perform any wiring connections other than those specified in the detector User Manual. Failure to follow these warnings could result in fire, electric shock or instrument malfunction.</strong> <span class="btn-link">[[Analog Connection to an Integrator->Analog Connection to an Integrator]]</span><br> <span class="btn-link">[[Analog Connection to a Strip-Chart Recorder->Analog Connection to a Strip-Chart Recorder]]</span><br> <span class="btn-link">[[Digital Connection to a System Controller->Digital Connection to a System Controller]]</span><br> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Analog Connection to a Strip-Chart Recorder</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/RecorderConnect.jpg" alt="Analog Connection to a Strip-Chart Recorder"></div> The detector can be connected to a recorder via the analog signal cable. <ol> <li>Attach the supplied signal cable to the RECORDER connection and recorder terminals as shown.</li> <li>Reconnect the power cables to the system units and turn ON the power switches of all the system units.</li> <li>Set the recorder RANGE.</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>CAUTION: If the signal cable is reversed at the recorder terminals, the chromatogram may be represented in a negative direction.</strong> <br><br> <span class="btn-link">[[Detector Connections->Detector Connections]]</span> <br> <span class="btn-link">[[Analog Connection to an Integrator->Analog Connection to an Integrator]]</span> <br> <span class="btn-link">[[Digital Connection to a System Controller->Digital Connection to a System Controller]]</span> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Analog Connection to an Integrator</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/ChromatopacConnect.jpg" alt="Analog Connection to an Integrator"></div> The detector can be connected to an integrator to monitor 1 or 2 channels. The optional Channel 2 accessory board is required to monitor the second channel. <ol> <li>Attach the detector signal cable to the INTEGRATOR connector and to the Chromatopac as shown. </li> <li>Reconnect the power cables to the system units and turn ON the power switches of all the system units.</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>CAUTION: If the signal cables are reversed, the chromatogram may be represented in a negative direction. NOTE: If dual wavelength mode is used, the AUX RANGE and REC MODE parameters must be set properly.</strong> <span class="btn-link">[[Detector Connections->Detector Connections]]</span> <br> <span class="btn-link">[[Analog Connection to a Strip-Chart Recorder->Analog Connection to a Strip-Chart Recorder]]</span> <br> <span class="btn-link">[[Digital Connection to a System Controller->Digital Connection to a System Controller]]</span> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Digital Connection to a System Controller</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/FiberOpticConnect.jpg" alt="Digital Connection to a System Controller"></div> The fiber optic cable supplied with the unit provides two-way communication for both transmission and reception of the signals, and is connected to the REMOTE connector. <ol> <li>Before connecting the fiber optic cable, remove the cap from the REMOTE connection channel to be used.</li> <li>Insert the fiber optic cable plug into the REMOTE connector until it clicks into place.</li> <ul> <li>Do not bend the fiber optic cable to less than a 35 mm radius.</li> <li>Grasp the plug, not the cable, when inserting or removing the fiber optic cable.</li> <li>Do not bend the cable where it joins the plug.</li> </ul> <li>Connect the other end of the cable to the system controller REMOTE connector.</li> <li>Insert the power plug into the outlet, and turn the power switch on.</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>CAUTION: Make sure the connector is not connected across two different channels. NOTE: Keep the removed cap in a safe place to use if the fiber optic cable is unplugged. Channels 3-8 of the system controller REMOTE connector are typically used for this purpose. If the fiber optic cable is not properly connected, 'Detector not linked' will be displayed.</strong> <br><br> <span class="btn-link">[[Detector Connections->Detector Connections]]</span> <br> <span class="btn-link">[[Analog Connection to an Integrator->Analog Connection to an Integrator]]</span> <br> <span class="btn-link">[[Analog Connection to a Strip-Chart Recorder->Analog Connection to a Strip-Chart Recorder]]</span> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Sample Vial Volume</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/VialChoice.jpg" alt="Sample Vial Volume"></div> Although the autosampler uses a direct volume injection with no sample loss, the minimum required sample volume is dependent on a number of factors. Consider all of these parameters when selecting the type of sample vial to be used and the final volume of sample in each vial. <ul> <li>Sample vial type</li> <li>Injection volume</li> <li>Needle stroke</li> </ul> It is also important to consider the maximum volume of sample in the sample vial. If the sample volume is too large, the sample may not cool (SIL-20AC only) properly. It is also possible that if the sample volume is too large, it could impede the sample aspiration process. <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>WARNING: The amount of sample introduced into the vial should not exceed the corresponding heights displayed in the diagram.</strong> <div class="ts-question">Is the sample volume in the sample vial adequate for the analysis?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Needle Stroke Depth]]</span> <span class="btn-no">[[No->Filling the Sample Vial]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Vial Position in the Autosampler</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/VialPosition.jpg" alt="Sample Vial Position"></div> <ol> <li>Open the door of the autosampler.</li> <li>Place the vial in the rack with the cap facing up.</li> <li>Slide the sample vial rack along the guides all the way to the back of the instrument. The sample vial rack clicks into place when it is inserted correctly.</li> <li>Close the door.</li> <li>Enter the proper sample vial positions into the injection sequence</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: The vial position numbers start with 1. Ensure that the sample vial is actually in the position designated in the injection sequence and that the proper rack type is designated.</strong> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Filling the Sample Vial</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/FillingSampleVial.jpg" alt="Filling the Sample Vial"></div> Ensure that the final volume of sample in the vial is sufficient for the injection volume and the number of repeat injections. Verify that the sample solution does not contain particulate matter. Filter the sample or adjust the sample preparation procedure to ensure adequate sample quality and volume. Ensure that the sample vial is properly covered and sealed to avoid sample evaporation. The table above displays the residual volume left in the bottom 2 mm of the vial. <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Needle Stroke Depth</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/NeedleStrokeDepth.jpg" alt="Needle Stroke Depth"></div> The distance that the needle descends into the sample vial is determined by the NEEDLE STROKE parameter. The parameter resets to the default position (2 mm from the bottom of the vial) when the sample vial rack is changed. This parameter must be adjusted for each of the different rack types. <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 table above displays the default values for needle stroke.</strong> <div class="ts-question">Is the sample needle depth adequate for the sample type? </div> <div class="buttons"> <span class="btn-yes">[[Yes->Purging the Autosampler]]</span> <span class="btn-no">[[No->NEEDLE STROKE Parameter]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Purging the Autosampler</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/ManPurge.jpg" alt="Purging the Autosampler"></div> Although the injection process may still function if the rinse solution runs dry, the reproducibility will suffer greatly. When the rinse solution is used up, air may be drawn into the injection tubing. Air in the injection tubing interferes with the reproducibility of the injection process. In such cases, the autosampler must be manually purged. <div class="ts-question">Has the autosampler been purged recently?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Column in Flow Path]]</span> <span class="btn-no">[[No->MANUAL PURGE]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Needle Stroke Parameter</h1> <div class="center"><iframe width="900" height="506" src="https://www.youtube.com/embed/IapCWjZ0UFk?rel=0" title="Needle Stroke Parameter" 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: When the rack is changed, this parameter is automatically set to the default value. The default setting causes the needle to descend to 2 mm from the bottom of the sample vial.</strong><br><br> The NEEDLE STROKE parameter sets the distance that the needle descends into the sample vial. <ol> <li>From the Initial screen, press the FUNC key</li> <li>The Parameter setting screen is displayed. Press the ENTER key.</li> <li> From the Initial screen, press the FUNC key one time. The cursor blinks at flow rate.</li> <li>Then press the FUNC key seven times. The NEEDLE STROKE parameter screen is displayed.</li> <li>Enter the numeric value for the parameter and press the ENTER key.</li> </ol> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Column in Flow Path</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/ColumnFlowPath.jpg" alt="Column in Flow Path"></div> <h3>It is possible to have a column connected to the system and not have the flow going through it.</h3> In many multiple-column configurations, one of the pathways incorporates a bypass or a direct flow to the waste. In these situations, it is possible to program the valve position in a mistaken position. If the sample does not pass through the column or passes directly to a waste bottle, peaks will not be present in the chromatogram <div class="ts-question">Is there a column in the flow path? </div> <div class="buttons"> <span class="btn-yes"><a href="https://www.ssi.shimadzu.com/forms/tech-support/index.html?product_category=Virtual-Advisor&product_name=Nexera&page=webform_startContact Shimadzu" target="_blank">Contact Shimadzu</a></span> <span class="btn-no">[[No->Valve Position - FCV-20AH6]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">MANUAL PURGE</h1> <div class="center"><iframe width="900" height="506" src="https://www.youtube.com/embed/l_BJzmGimOk?rel=0" title="Needle Stroke Parameter" frameborder="0" allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture; web-share" allowfullscreen></iframe></div> <br> It is a good practice to follow all manual purges with an automatic purge. <ol> <li> From the Initial screen, press the FUNC key twice. The CONTROL screen is displayed.</li> <li>Press ENTER. The Clear SMPTBL (clear sample table) screen is displayed.</li> <li>Press the FUNC key four times. The MANUAL PURGE screen is displayed.</li> <li>Insert the manual syringe into the tubing that is connected to port 3 of the low-pressure valve.</li> <li>Press 1 on the numeric keypad.</li> <li>Draw in the rinse solution with the manual syringe.</li> <li>Press 0 on the numeric keypad. The MANUAL PURGE process is finalized.</li> </ol> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Valve Position - FCV-20AH6</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/ValvePosition.jpg" alt="Valve Position - FCV-20AH6"></div> Set up the correct column switching methods using the table above to program the contact inputs for the FCV-20AH6. Be sure to set the same position for the column inlet side and outlet side valve positions. The flow line will be non-continuous if set otherwise. <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 FCV-AH2 is a dual channel valve that can be used to switch between two columns or between a column and a bypass pathway. Verify that the valve is programmed properly and that the sample is flowing through the column correctly.</strong> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">LAMP On</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/LampOnOff.jpg" alt="LAMP On"></div> <h3>Now determine whether the detector is emitting a signal. The lamp power must be ON.<br> Click the appropriate detector type to review the procedure for checking the LAMP power. </h3> <div class="ts-question">Is the lamp power turned ON?</div> <span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-pda-detector/index.html">Troubleshooting The PDA Detector</a></span> <div class="buttons"> <span class="btn-no">[[No->Turning the LAMP Power ON and OFF - SPD-M20A]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">System Signal Selection</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/SystemChoice.jpg" alt="System Signal Selection"></div> Most often, multiple HPLC systems are connected to the data system. Each of the HPLC systems has multiple components such as controllers, autosamplers, pumps, and detectors as well as other individual units. It is important to ensure that you are monitoring the correct instrument, channel and detector wavelength. Refer to the appropriate instruction manual (data system, controller, detector, etc.) to ensure that the displayed signal is the desired one. <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Turning the LAMP Power ON and OFF - SPD-M20A</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-no-peaks/LampOnOff.jpg" alt="Turning the LAMP Power ON and OFF - SPD-M20A"></div> <ol> <li>Turn ON the PC that controls the instrument and launch the web browser on the PC.</li> <li>With the instrument running, enter the IP address of the instrument in the 'Address' field. When a connection with the instrument is established, the Login screen is displayed.</li> <li>Log in to the system as a 'guest'. The Analysis Parameters screen is displayed. The current operating conditions are displayed. They can be changed or edited as needed.</li> <li>Press the UPDATE button. The parameters are updated and the Analysis Setting and Execution screen is displayed. The parameters are saved in the unit's EEPROM and are kept even after the unit is turned OFF.</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: When parameters are changed via the system controller or data system, they are updated accordingly.</strong> <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-no-peaks/FlowLine.jpg" alt="FlowLine"></div> 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> <div class="ts-question">Is there a leak in the flow path before the head of the column?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Tubing - Flow Line Connections]]</span> <span class="btn-no"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-the-pump/index.html">Troubleshooting The Pump</a></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-no-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-no-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-no-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">[[Tubing - Flow Line Connections->Tubing - Flow Line Connections]]</span><br> <span class="btn-link">[[Tighten SS->Tighten 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">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-no-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>

Flow line leaks - merged two nodes