Once you've finished processing your spectrum, you should calibrate it so that it can be analyzed in Profiler or used to create compound signatures and simulations in Compound Builder and Spin Simulator.
The chemical shape indicator (CSI) settings form the basis for all
fits performed in Profiler and Compound Builder. The accuracy of these
settings also have a direct impact on the accuracy of profiled spectra
(.cnx), exported data
(.txt), compound files (.xcpd)
and spin simulations (.xss). Calibrate CSI mode lets
you view, define and change these settings for the current
spectrum.
When you import a spectrum, Processor attempts to automatically locate the CSI, and determine its width and height. You can change these automatically determined parameters using Calibrate CSI mode if necessary.
| Menu Location | Tools > Calibrate CSI... |
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How do I modify the CSI settings?
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Switch to Calibrate CSI mode.
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Type the concentration of the CSI in your sample in the Concentration (mM) text entry box under Calibration Peak.
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Select the CSI present in your sample using the drop-down box under Chemical Shape Indicator (CSI). Chenomx NMR Suite includes CSI definitions for DSS, TSP and formate. A custom CSI can also be defined.
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Automatic: Click the Calibrate Automatically button.
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Manual:
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Click and drag the calibration peak to match it with the spectrum line (black).
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Adjust the width by dragging the width control (red triangles to either side of the peak) or typing a number directly into the Width (Hz) text entry box.
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Use the up and down arrow keys to adjust peak height, and the left and right arrow keys to adjust peak position.
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How do I handle samples that have no CSI?
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Switch to Calibrate CSI mode.
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Using the drop-down box under Chemical Shape Indicator (CSI), select None.
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Click and drag the horizontal axis to set a chemical shift reference.
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Set an appropriate line width for the spectrum by typing a value in Hz under Calibration Peak.
Tips and Tricks
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Calibrate the CSI only after you have finished applying all necessary processing layers. If you change a spectrum's processing, you will need to recalibrate its CSI to ensure accurate quantification and generally good fits.
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Processor can only automatically calibrate the CSI if its signal has a chemical shift less than 0 or greater than 10. This includes DSS, TSP, and some Custom CSIs, but excludes formate.
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If Processor cannot calibrate the CSI automatically when you use DSS or TSP, check the phase correction and baseline correction of your spectrum and try again. If Processor still cannot calibrate the CSI, you need to use the manual tools to locate it instead.
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If you have selected DSS as the CSI, then the indicated center position always reads 0 ppm. If you use TSP or formate instead, the center position varies based on the current pH value for the spectrum.
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If you would like to adjust the height or width of the calibration peak while keeping its total area constant, check the Preserve Peak Area box.
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When you select None as the CSI, the red calibration peak in the Spectrum Graph disappears. You can only adjust the chemical shift and width settings as described above.
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To make adjustments to the CSI in more than one spectrum at a time, use the “Batch Process” tool.
If you have spectra that were acquired with a CSI other than the three supported by Chenomx NMR Suite (DSS, TSP or formate), you can define a custom CSI for use with Chenomx NMR Suite.
| Menu Location | Tools > Define Custom CSI... |
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How do I define the custom CSI?
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Open a spectrum of a sample that contains both your custom CSI and a Chenomx-supported CSI (DSS, TSP or formate). This spectrum should already be calibrated using its Chenomx-supported CSI.
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Open the Define Custom CSI dialog.
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Adjust the custom CSI peak (red) to match the appropriate peak in the calibrated spectrum.
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Enter a name for your custom CSI.
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Click Accept.
Tips and Tricks
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You may only have one custom CSI defined at a time.
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The spectrum that you use to define the custom CSI should also contain a Chenomx-supported CSI like DSS, TSP or formate. This reference spectrum must be calibrated using the Chenomx CSI before you use it to define the custom CSI.
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Once you have defined the custom CSI, you may not delete it. You can only edit its parameters.
The signal position and shape of compounds in aqueous solution can be very sensitive to pH. The accuracy of the pH that you set for a spectrum will have an impact on the quality and accuracy of fits that you perform on that spectrum in Profiler, Compound Builder or Spin Simulator. Calibrate pH mode lets you calculate or set the pH for the current spectrum.
If your sample contains any combination of creatinine, imidazole or DFTMP, Processor may be able to calculate the spectrum pH. If your sample does not contain these compounds, or if you prefer to use an externally measured pH, you may enter a pH value manually.
While importing a spectrum, Processor attempts to calculate the pH of the sample automatically using the frequencies of the imidazole, creatinine and DFTMP peaks. You can refine or change this calculated pH in Calibrate pH mode. If Processor cannot calculate the pH of your sample, you need to set it manually.
Whether you use automatic or manual methods to set the spectrum pH, you can also set an uncertainty for the pH value. The pH uncertainty helps to determine the size of the transform windows for pH-sensitive compound signatures in Profiler.
The predicted frequencies of the imidazole, creatinine and DFTMP peaks, based on the pH value that you currently have set, appear in the Spectrum Graph as a pair of blue (imidazole) and purple lines (histidine) at low frequencies (high ppm value)s, a pair of green lines(creatinine) at mid frequency, and a single orange line (DFTMP) at high frequency. You can use these lines during manual pH determination by aligning them with the corresponding peaks for creatinine, imidazole, histidine and DFTMP, when these compounds are present.
| Menu Location | Tools > Calibrate pH... |
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How do I set the spectrum pH?
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Switch to Calibrate pH mode.
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Set the desired pH using any one of the following methods:
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Click the Find Automatically button.
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Click and drag the pH slider bar to set the determined frequencies of imidazole, creatinine and DFTMP as close as possible to the actual frequencies of the peaks in the spectrum (if known). The resulting pH appears in the text box beside the slider bar.
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Select the pH slider control, and use the arrow keys to make fine adjustments to the pH (with the Fine Tune checkbox selected).
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Type the desired numeric pH into the text entry box to the right of the slider bar.
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Tips and Tricks
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Setting an accurate spectrum pH helps Profiler choose more accurate starting positions and transform windows for clusters.
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Once you have made coarse adjustments to the pH, you can zoom in to particular indicator lines to make finer adjustments. Select the Fine Tune check box to let you make more detailed adjustments over a smaller pH range.
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When aligning the displayed lines to peaks in your spectrum, it is not necessary to have the lines match the peak positions precisely. A common situation is to arrive at two pH values separated by 0.01, one of which places the indicator lines on one side of the peak, while the other places them on the other side of the peak. In such cases, you may use either value.
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If you have selected TSP or formate as the CSI, then changing the pH changes the position of the horizontal axis, since the frequencies of the TSP and formate peaks are pH-sensitive.
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To make adjustments to the pH in more than one spectrum at a time, use the “Batch Process” tool.