Nexera HPLC Troubleshooting Negative Peaks

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<div class="container"><h1 class="title">Troubleshooting Negative Peaks</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-negative-peaks/negative-peaks.jpg" alt="Troubleshooting Negative Peaks"></div> <h3>To further diagnose the problem, it is important to determine the extent of the negative peak problem.</h3> When only a few of the peaks present in a negative manner, it is an indication that some of the sample components have a lower absorbance than the mobile phase. When all of the peaks are presenting in a negative manner, it is an indication that there is a problem with the polarity or physical connection determining the polarity of the detector signal. <div class="ts-question">Are all of the peaks presenting in a negative direction?</div><div class="buttons"> <span class="btn-yes">[[Yes->Scaling]]</span> <span class="btn-no">[[No->Background Subtraction]]</span> </div> </div>

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<div class="container"><h1 class="title">Scaling</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-negative-peaks/scaling.jpg" alt="Scaling"></div> <h3>Verify that you are looking at the chromatogram in the correct orientation and at the proper scale.</h3> Most data systems give the user the ability to change the Y-axis of the display chromatogram. It is also often possible to invert the chromatographic display. Refer to the instruction manual for the particular data system to determine how to examine these parameters. <div class="ts-question">Is the Y-axis scale of the chromatogram display set properly, and not inverted?</div><div class="buttons"> <span class="btn-yes">[[Yes->Signal Parameters]]</span> <span class="btn-no">[[No->Correcting Chromatogram Display Settings]]<span class="btn-back"><<back "Back">></span></span> </div> </div>

<div class="container"><h1 class="title">Background Subtraction</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-negative-peaks/subtraction.jpg" alt="Background Subtraction"></div> Most data systems allow for the user to run a blank injection and subtract the background scan from all subsequent injections. This parameter delivers a chromatographic output that does not include any peak responses from the system background irregularities. <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 a sample chromatogram is inadvertently used as the background scan, it is possible to have negative peaks appear in the final chromatographic output.</strong> <div class="ts-question">Is a sample chromatogram being subtracted from each injection?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Background Subtraction and Chromatogram Selection]]</span> <span class="btn-no">[[No->Solvent Absorption]]</span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Signal Parameters</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-negative-peaks/signal.jpg" alt="Signal Parameters"></div> Digital signals are generally preferred because they require fewer parameters to manipulate them, making the digital signal easier to use than analog signals. However, more types of instrumentation can be accessed via the analog signal than the digital signal. <div class="ts-question">Is the detector polarity parameter set correctly?</div><div class="buttons"> <span class="btn-yes">[[Yes->Detector Connection]]</span> <span class="btn-no">[[No->Detector Polarity]]<span class="btn-back"><<back "Back">></span></span> </div> </div>

<div class="container"><h1 class="title">Correcting Chromatogram Display Settings</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-negative-peaks/scaling.jpg" alt="Correcting Chromatogram Display Settings"></div> Many different types of data systems are available. It is not possible to describe the procedures for all of these data systems completely. Therefore, please refer to your data system instruction manual to learn how to examine and change these parameters. <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Background Subtraction and Chromatogram Selection</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-negative-peaks/subtraction.jpg" alt="Background Subtraction and Chromatogram Selection"></div> Refer to your data system instruction manual to determine how to select and use the background subtraction parameter. <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 a sample chromatogram is inadvertently used as the background scan, it is possible to have negative peaks appear in the final chromatographic output.</strong> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Solvent Absorption</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-negative-peaks/absorbance.jpg" alt="Solvent Absorption"></div> Absorbance is a measure of the amount of light that is absorbed (removed) as the light beam passes through the flow cell and any solution that it contains. Usually when the flow cell is filled with the sample matrix, more light is absorbed than when the flow cell contains only mobile phase. Occasionally, less light is absorbed by the sample matrix than the mobile phase. When this phenomenon occurs, the baseline will dip to below zero causing a negative peak. All of the other peaks will still present in the normal (positive direction). <div class="ts-question">Is the absorption of the sample matrix lower than the mobile phase absorbance?</div> <div class="buttons"> <span class="btn-yes">[[Yes->UV Cutoff Values]]</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">UV Cutoff Values</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-negative-peaks/solvent-uv.jpg" alt="UV Cutoff Values"></div> Use this table to select solvents with appropriate UV cutoff values. When selecting mobile phase solvents, remember that the inclusion of mobile phase modifiers usually elevates the UV cutoff of the resulting mobile phase. <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 values in this table are representative of solvents that do not include additives.</strong> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Detector Connection</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-negative-peaks/detector-connection.jpg" alt="Detector Connection"></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">Is the detector properly connected to the data system?</div><div class="buttons"> <span class="btn-yes"><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-no">[[No->Detector Connections]]<span class="btn-back"><<back "Back">></span></span> </div> </div>

<div class="container"><h1 class="title">Detector Polarity</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-negative-peaks/polarity.jpg" alt="Detector Polarity"></div> The detector polarity is easily changed. By pressing the Display key on the detector keypad, the operation keys become active. Press the POL key to change the detector output polarity. The POL indicator is illuminated when the detector polarity is set to the negative (-) setting. <div class="buttons"> <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-negative-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> <span class="btn-link">[[Analog Connection to a Strip-Chart Recorder->Analog Connection to a Strip-Chart Recorder]]</span> <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 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-negative-peaks/recorder-connect.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> <span class="btn-link">[[SPD Connections->Detector Connections]]</span> <span class="btn-link">[[Analog Connection to an Integrator->Analog Connection to an Integrator]]</span> <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-negative-peaks/fiber-optic-connect.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; margin-bottom:20px" 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> <span class="btn-link">[[SPD Connections->Detector Connections]]</span> <span class="btn-link">[[Analog Connection to an Integrator->Analog Connection to an Integrator]]</span> <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">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-negative-peaks/chromatopac-connect.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">[[SPD Connections->Detector Connections]]</span> <span class="btn-link">[[Analog Connection to a Strip-Chart Recorder->Analog Connection to a Strip-Chart Recorder]]</span> <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>