Nexera HPLC Troubleshooting Small Peaks

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<div class="container"><h1 class="title">Troubleshooting Small Peaks</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-small-peaks/full-scale-display.jpg" alt="Troubleshooting Small Peaks"></div> <h3>The full scale display parameter affects the appearance of the peaks in the chromatogram.</h3> The display Y-axis display parameter affects the displayed peak height and size. Absorbance units or volts are displayed on the Y-axis. Changing the Y-axis full scale display will change the size of the displayed peaks. If the displayed peaks are all small, it may be due to an inappropriate full scale Y-axis setting. <div class="ts-question">Are the area values similar to the historical results?</div><div class="buttons"> <span class="btn-yes">[[Yes->Full Scale Display]]</span> <span class="btn-no">[[No->Adequate Reproducibility]]</span> </div> </div>

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<div class="container"><h1 class="title">Full Scale Display</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-small-peaks/full-scale-display.jpg" alt="Full Scale Display"></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 the display parameters. <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Adequate Reproducibility</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-small-peaks/reproducibility.jpg" alt="Adequate Reproducibility"></div> Make five injections of a known standard solution and evaluate the reproducibility of the peak areas. If the reproducibility is adequate, it indicates that the problem is not related to the autosampler. If the reproducibility is not adequate, it indicates that the problem is most likely related to the autosampler. <div class="ts-question">Is the reproducibility adequate?</div><div class="buttons"> <span class="btn-yes">[[Yes->Sample Preparation]]</span> <span class="btn-no">[[No->Sample Vial Volume]]</span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Sample Preparation</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-small-peaks/sample-preparation.jpg" alt="Sample Preparation"></div> <h3>Sample preparation procedures can affect the amount of sample that actually reaches the sample vial.</h3> Depending on the complexity of the sample preparation procedure, including the extraction process, there are a various stages at which preparation errors can be made. Some common things to re-examine are listed below: <ul> <li>Clean glassware - glassware may be contaminated with substances that can absorb the analyte or keep it from being fully dissolved.</li> <li>Appropriate solvents - inappropriate solvents may not fully dissolve the sample.</li> <li>Correct measurements - ensure that the appropriate amount of sample was added to the sample vial.</li> <li>Proper filtration - the wrong filter could absorb the sample before it reaches the sample vial.</li> <li>Vial and septa type - the wrong vial type or septa type may absorb the sample from the sample solution.</li> </ul> Carefully examine the sample preparation and review the process for accuracy. <div class="ts-question">Was the sample solution prepared properly?</div><div class="buttons"> <span class="btn-yes">[[Yes->Injection Volume]]</span> <span class="btn-no">[[No->Re-prepare the Sample Solution]]</span><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-small-peaks/vial-choice.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 there an adequate volume of sample solution in the sample vial?</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">Injection Volume</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-small-peaks/injection-volume.jpg" alt="Injection Volume"></div> The Injection Volume parameter has a direct affect on the amount of sample that is introduced onto the column. Increasing the sample volume increases the area of each of the peaks in the sample chromatogram. <div class="ts-question">Is the injection volume set appropriately for the method?</div><div class="buttons"> <span class="btn-yes">[[Yes->Wavelength Determination]]</span> <span class="btn-no">[[No->Changing the Injection Parameters]]</span><span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Re-prepare the Sample Solution</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-small-peaks/sample-preparation.jpg" alt="Re-prepare the Sample Solution"></div> The reason that all of the peaks are smaller than expected may be due to the sample preparation process. Discard the erroneously prepared sample and prepare the sample solution again, while strictly adhering to the method specifications. It may be necessary to examine the method to ensure that the sample preparation (extraction) procedure is effective for the sample type. The Injection Volume parameter has a direct affect on the amount of sample that is introduced onto the column. Increasing the sample volume increases the area of each of the peaks in the sample chromatogram. <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-small-peaks/filling-sample-vial.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">Wavelength Determination</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-small-peaks/wavelength-determination.jpg" alt="Wavelength Determination"></div> Each analyte in the sample solution has a wavelength at which maximum absorbance occurs (Lambda Max). When a sample is analyzed using the lambda max wavelength the absorbance values are maximized. Analyzing a sample at a wavelength other than lambda max may result in a smaller analyte peak. A multi wavelength scan of the sample solution will show the best wavelength to use for analysis. <div class="ts-question">Is the proper wavelength being monitored?</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->Setting the Wavelength]]</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="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 only one vial is to be injected, use the same number entered in the 'From' field.</strong> <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">Needle Stroke Depth</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-small-peaks/needle-stroke-depth.jpg" alt="Needle Stroke Depth"></div> <h3>Now determine if the needle is actually reaching the sample.</h3> 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. Now determine if the needle is actually reaching the sample. <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 needle stroke set appropriately for the sample vial type?</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->NEEDLE STROKE Parameter]]</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="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: 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> 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>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">Setting the Wavelength</h1> <div class="center"><iframe width="900" height="506" src="https://www.youtube.com/embed/aEtSrv_5vXU?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. The PARAMETER screen is displayed.</li> <li>Press ENTER. The Channel 1 wavelength-setting screen (LAMBDA 1) is displayed.</li> <li>Press the numeric keys to enter the desired wavelength (220 nm in our example) and then press ENTER</li> <li>The wavelength is set to 220 nm.</li> <li>Press the CE key twice to return to the Initial screen.</li> </ol> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>