Nexera HPLC Troubleshooting - Chromatography Problems

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<div class="container"><h1 class="title">Troubleshooting Chromatography Problems </h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-chromatography-problems/SamplePeaks_a.jpg" alt="Troubleshooting Chromatography Problems "></div> <h3>''Let's evaluate the chromatogram.''</h3> The first step in troubleshooting the chromatogram is to determine whether a sample injection produces a peak in the chromatogram. <ol> <li> Properly equilibrate the HPLC system by flushing it with 15-20 column volumes of mobile phase. </li> <li> Make an injection of a known standard solution according to the appropriate method parameters.</li> <li>Evaluate the chromatogram and determine whether a peak is produced.</li> </ol> <span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-no-peaks/index.html">Troubleshooting No Peaks</a></span> <div class="ts-question">Are the expected peaks present in the chromatogram? </div><div class="buttons"> <span class="btn-yes">[[Yes->HPLC System Stability]]</span> </div> </div>

<div class="container"><h1 class="title">HPLC System Stability </h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-chromatography-problems/RT_TimeDef.jpg" alt="Troubleshooting Chromatography Problems "></div> <h3>''Now evaluate the stability of the HPLC system.''</h3> Retention time is the time interval from the point of injection to the maximum elution of the compound (peak apex).<br>There are a number of factors that affect the retention time: <ul> <li>System equilibration</li> <li>Column equilibration</li> <li>Mobile phase</li> <li>Column age</li> <li>Temperature</li> <li>Pump performance (flow rate)</li> </ul> Now evaluate the stability of the HPLC system. <div class="ts-question">Are the retention times of the peaks reproducible from one injection to another?<br> </div><span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-retention-time/index.html">Troubleshooting Retention Time Problems</a></span><br> <div class="buttons"> <span class="btn-yes">[[Yes->Peak Shape]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

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<div class="container"><h1 class="title">Peak Shape</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-chromatography-problems/PeakShape.jpg" alt="Peak Shape"></div> The most optimal peak shape is a Gaussian curve. A Gaussian curve is a perfectly symmetrical curve that represents a normal distribution pattern. Compare the current analyte peak shape to the established historical peak shape. Determining whether the current chromatogram is equivalent to the historical peak shape allows us to further isolate the problem and find an effective corrective action. The tailing factor is the most common way to determine whether the peak shape is optimal. Most computer-based chromatography software applications can automatically calculate the tailing factor. Consult the software documentation or use the formula above to calculate the tailing factor. Tailing Factor=1.0 is perfect. <div class="ts-question">Is the peak shape normal?</div> <div class="buttons"> <span class="btn-yes">[[Yes->Relative Standard Deviation]]</span> <span class="btn-no">[[No->Loss of Resolution]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Relative Standard Deviation </h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-chromatography-problems/RSD.jpg" alt="Relative Standard Deviation "></div> Determine whether the peak areas are consistent from one injection to another using the following procedure. Make 3-5 injections from a single vial of a known standard solution. Most computer based chromatography software can automatically calculate %RSD. Consult the software documentation or use the calculations above to calculate the mean (average) area, the standard deviation and the %RSD. Passing %RSD is typically less than 1%.<br> <span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-the-autosampler/index.html">Troubleshooting the Autosampler</a></span> <div class="ts-question">Are the peak areas consistent from one injection to another? </div> <div class="buttons"> <span class="btn-yes">[[Yes->Extra (Ghost) Peaks]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Loss of Resolution</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-chromatography-problems/ResolutionLoss.jpg" alt="Loss of Resolution.jpg"></div> Loss of resolution is the decrease in the degree of separation and may ultimately result in a complete loss of separation (co-eluting peaks). Resolution is a measure of the degree of separation between two peaks in a chromatogram. Resolution is commonly monitored as part of a system suitability evaluation. For system suitability, adequate resolution is often defined as a 'resolution greater than' limit. Most computer-based chromatography software applications can automatically calculate resolution. Consult the software documentation or use the calculation to the left to evaluate the resolution. <div class="ts-question">Is the peak shape problem accompanied by a loss of resolution? </div><br><span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-loss-of-resolution/index.html">Troubleshooting the Loss of Resolution</a></span> <div class="buttons"> <span class="btn-no">[[No->Peak Shape Choice]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Extra (Ghost) Peaks</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-chromatography-problems/GhostPeaks.jpg" alt="GhostPeaks"></div> <h3>Evaluate the number of peaks in the chromatogram.</h3> Unwanted peaks in the chromatogram arise from various sources of system contamination. These peaks can occur in every injection, in only a few random injections or in the system blank injection where there should be no peaks present at the retention time of the analyte peak Examine the chromatogram closely and determine whether the peak <ul> <li>is consistent from one injection to another</li> <li>is random throughout the injection sequence.</li> <li>is occurring at the same retention time as the peak of interest.</li> </ul> <div class="ts-question">Are extra peaks present in the chromatogram?</div> <div class="buttons"> <span class="btn-yes">[[Yes->System Peaks or Carryover]]</span> <span class="btn-no">[[No->Small Peaks]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">System Peaks or Carryover</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-chromatography-problems/GhostPeak_BlankSmall.jpg" alt="Ghost Peaks"></div> <h3>Now determine whether the extra peak is a carryover peak or a system peak. </h3> It is possible that the magnitude of the analyte peak may hide any carryover peak. Make an injection of blank sample solvent to verify whether there is a carryover peak eluting at the same time as the analyte peak. If the extra peak has a different retention time from the analyte peak, it is most likely a system peak that could be caused from a variety of system contamination sources. Examine the chromatogram closely and determine whether the peak Some of these sources include: <ul> <li>injector contamination</li> <li>detector contamination</li> <li>column contamination</li> <li>gradient issues</li> </ul> <div class="ts-question">Is the extra peak eluting at the same retention time as the analyte peak?</div> <span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-carryover-peaks/index.html">Troubleshooting Carryover Peaks</a></span><br> <span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-system-peaks/index.html">Troubleshooting System Peaks</a></span> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Small Peaks</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-chromatography-problems/Small Peaks_a.jpg" alt="Small Peaks"></div> <h3>Now compare the chromatogram with historical chromatographic results.</h3> A number of problems can manifest themselves in smaller than normal peaks. Some of these problems include: <ul> <li>major system leak</li> <li>poor injection process</li> <li>software display scale setting</li> <li>detector problems (such as the wrong wavelength)</li> </ul> <div class="ts-question">Are the peaks smaller than expected?</div> <span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-carryover-peaks/index.html">Troubleshooting Carryover Peaks</a></span><br> <span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-system-peaks/index.html">Troubleshooting System Peaks</a></span> <div class="buttons"> <span class="btn-no">[[No->Baseline Stability]]</span> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Peak Shape Choice</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-chromatography-problems/ShapeChoice.jpg" alt="Peak Shape"></div> <h3>Peak shape problems can be manifested in a number of ways.</h3> At this point, we must define the specific peak shape problem being experienced. Examine the abnormal peak shapes below and select the one that best matches the peak problem that you are experiencing. <div class="ts-question">Select the peak shape that most closely matches the abnormal peak shape problem.</div> <span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-tailing-peaks/index.html">Troubleshooting Trailing Peaks</a></span><br> <span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-broad-peaks/index.html">Troubleshooting Broad Peaks</a></span> <br> <span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-split-peaks/index.html">Troubleshooting Split Peaks</a></span> <br> <span class="btn-link">[[Troubleshoot Shark-fin Peaks->Troubleshoot Shark-fin Peaks]]</span> <br> <span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-flat-peaks/index.html">Troubleshooting Flat Peaks</a></span><br> <span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-fronting-peaks/index.html">Troubleshooting Fronting Peaks</a></span><br> <span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-negative-peaks/index.html">Troubleshooting Negative Peaks</a></span> <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>

<div class="container"><h1 class="title">Baseline Stability</h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-chromatography-problems/BaselineStability_0.jpg" alt="Baseline Stability"></div> <h3>The baseline is a final part of the chromatogram that must be evaluated.</h3> Make a sample injection to calculate the signal-to-noise ratio and determine if it is acceptable for your application. <ul> <li>All baselines have an inherent amount of noise. Baseline noise is only a problem if the amount of noise is so excessive as to interfere with integration of the analyte of interest. Data systems often have an auto scale function that can make a normal baseline appear to be very noisy. </li> <li>The signal to noise ratio is a measure of the Peak height with respect to the Noise height. This ratio can be increased by decreasing the noise or by increasing the peak (signal). </li> <li>The sensitivity of the method and the scale of the chromatogram output must be determined before determining whether or not the noise is excessive. </li> </ul> <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: Calculating Signal-to-Noise Ratio</strong> - The signal-to-noise ratio is calculated using the peak height of the noise and the peak height of the sample peak. <br><br> Example: Noise height 2mV - Sample height 10mV - The signal-to-noise ratio is 5. Typically, a signal-to-noise ratio of 3 is required for detection, and 10 for quantitation. <div class="ts-question">Is the baseline stable?</div> <span class="btn-link"><a href="/service-support/virtual-advisor/nexera/advanced-troubleshooting/troubleshooting-baseline-problems/index.html">Troubleshooting Baseline Problems</a></span> <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_start" target="_blank">Yes</a></span> <span class="btn-back"><<back "Back">></span> </div> </div>

5-9-2023 Baseline stability - needs an updated hyperlink to tech support This tween has lots of links outward to other tweens

<div class="container"><h1 class="title">Troubleshooting Shark-fin Peaks </h1> <div class="center"><img src="https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/support/virtual-advisors/nexera/troubleshooting-chromatography-problems/ColumnContamination_0.jpg" alt="Troubleshooting Shark-fin Peaks"></div> The shark fin peak is the classic sign of an overloaded column. • When too much sample is injected onto the column, the column may become overloaded and cause that peak to split or be misshapen. To correct the problem, it may be necessary to use a smaller injection volume or dilute the sample solution. • As the active sites in the stationary phase become saturated (for example, with strongly retained sample components or impurities), column efficiency is reduced leading to misshapen peaks. Consult the column manufacturer's instructions on proper washing techniques for the specific column. <div class="buttons"> <span class="btn-back"><<back "Back">></span> </div> </div>