Profiling a spectrum (i.e "targeted profiling") can be a subtle and potentially complex task depending on timeframes, research goals, or other requirements. Maintaining a consistent profiling strategy will provide more consistent and more easily-interpreted results.
For more details on specific profiling techniques and how to apply them to your spectra, please see “Tutorial and Sample Files”.
Basic Compound Fitting
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Select a peak-based compound in the Compound Table and select a cluster from the Cluster Navigator to begin. Chenomx recommends selecting an isolated peak for initial profiling.
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Click and drag the cluster to adjust the fit. You can also click and drag the blue triangle on the vertical axis of the graph to change only the compound concentration, or drag a blue triangle on the horizontal axis to change only a cluster's frequency.
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The concentration of the compound will appear in the 'Concentration' column in the Compound Table.
Region-based Compound Fitting
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Select a region-based compound in the Compound Table. Note that there are no region-based compounds in the default Chenomx Reference Library--you will need to create your own and add it to your Compound Library.
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Select a cluster in the Cluster Navigator, click and drag the cluster to adjust the fit. Notice how changing the height of the compound has no effect on which part of the spectrum is fit.
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Concentrations cannot be calculated for region-based compounds. Instead, the otherwise unfit spectrum areas near each cluster will be summed up and displayed in the Area column of the Compound Table. This value is proportional to the true concentration of the compound.
Targeted Profiling
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Open the spectrum in Profiler
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Select either the default, or a custom compound set that corresponds to your sample.
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Identify and profile isolated and prominent signals in the spectrum first(see “First Step: Identify Compounds”). Easily identifiable compounds include CSI compounds like DSS or TSP and pH indicators like imidazole, creatinine or DFTMP.
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Systematically review the remaining signals in the spectrum; identify and profile as needed.
Use information and external links from the Reference Panel to help establish the presence of particular compounds in the samples that you are analyzing.
Match the compound signatures from the Chenomx Library with patterns in your sample spectrum. There are several ways that you can do this.
Identify specific compounds
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Select at least one compound set in the Compound Sets view (see “Compound Sets (Sidebar)”).
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Select a compound in the Compound Table that you believe is present in the sample(see “Compound Table”).
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Review each cluster in the Cluster Navigator and compare the shape of the preview line (light blue) to the spectrum line (black) and decide whether it is reasonable that the preview line could be contributing to the spectrum line in the displayed region.
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If the shape appears reasonable, click the preview line, and adjust the resulting shape by determining appropriate heights and cluster locations for the compound. If the shape does not appear reasonable...
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Select another compound in the Compound Table.
Find possible compounds contributing to a specific peak
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Select a compound set.
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Right-click on the peak in the experiment spectra and select Search for Compounds Near x.xx ppm.
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Select a listed compounds in the Compound Table to display the preview line and review each cluster in the Cluster Navigator.
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Compare the shape of the preview line (light blue) and the spectrum line (black) and decide whether or not it is reasonable that the preview line could be contributing to the spectrum line in the displayed region.
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If the shape appears reasonable, click the preview line, and adjust the resulting shape by determining appropriate heights and cluster locations for the compound. If all clusters appear reasonably well-matched, apply an automatic fit, then adjust the fit for the compound as needed. If the shape does not appear reasonable...
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Select a different suggested compound from the Compound Table.
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Finish with the filtered list and clear the frequency filter to continue your analysis with the full compound list (see “Quick Searches”).
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If no compounds in the list of potential candidates provides a reasonable shape near the peak that you are investigating, consider the possibility that the compound responsible for the peaks is either not in the selected compound library, or not in your currently selected compound set.
To identify all compounds contributing to a particular region
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Select a compound set in the Compound Set Panel.
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Select a region in the Spectrum Graph (see “Select Region”).
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Search the Compound Table for compounds in the selected region (see “Quick Searches”) and select a listed compound.
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Compare the shape of the preview line (light blue) and the spectrum line (black) and decide whether or not it is reasonable that the preview line could be contributing to the spectrum line in the displayed region.
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Compare the shape of the preview line (light blue) and the spectrum line (black) and decide whether or not it is reasonable that the preview line could be contributing to the spectrum line in the displayed region. If all clusters appear reasonably well-matched, apply an automatic fit, then adjust the fit for the compound as needed. If the shape does not appear reasonable...
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Evaluate the remainder of the compounds in the Compound Table.
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Finish with the filtered list and clear the frequency filter to continue your analysis with the full compound list (see “Quick Searches”).
Tips and Tricks
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Profiler can suggest a fit for a selected compound by clicking Compound > Fit Automatically, or by pressing the space bar.
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It is also possible to create your own compounds that use a region-based fit style, which automatically fit all of the spectrum area in whatever region they are located in. For more information about fit styles, see “Compound Fit Style”.
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Double-click on a peak-based compound's concentration in the Compound Table and type a value directly to set the concentration. Type a number from 0 to 5000 mM (or equivalent concentrations in μM or mg/dL), or type "--" to set the concentration to unknown.
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When you have selected a compound in the Compound Table, you can click its shape in the Spectrum Graph, then use the arrow keys to adjust the compound's concentration or the cluster's position. Hold down Shift to allow finer adjustments.
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To clear all of the fits for the current spectrum, click Edit > Select All and then click Compound > Clear > Fit.
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To clear fits only for selected compounds, select the compounds and click Compound > Clear > Fit. Multiple compounds can be selected by holding down Shift or Ctrl (Cmd in Mac OS X).
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All compound concentrations and areas start out as unknown (--).
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If a compound's concentration is unknown (--) and you drag it up and then back down to the baseline, the concentration is set to 0.00 mM. To return the concentration to unknown, you must clear it.
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You can export a file describing the transform status of all compounds by clicking Tools > Batch Export.
If you have automatically fit a compound, you may need to refine the suggested concentration. Note that it is not possible to determine (or adjust) the concentrations of compounds that make use of the region-based fit style. For those compounds, you must perform your analysis using compound Areas instead. See “Compound Fit Style” for more details. For more details on techniques for determining optimal profiles for compounds in experimental spectra, see “Tutorial and Sample Files”.
Determine the concentration of a compound
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Ensure the subtraction line (green) is visible (see “Legend”).
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Set a concentration for the compound so that you can see the clusters clearly and use the Cluster Navigator to locate a cluster that overlaps clusters as little as possible and approximate the compound concentration.
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Click and drag the height control to adjust the height of the compound until the subtraction line under the cluster approximates a normal spectrum. You may need to move the cluster from side to side somewhat to properly adjust the subtraction line.
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If any of the clusters for the current compound overlap with compounds that you have already profiled, you may need to re-adjust those compounds as well.
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Compound concentrations are shown in the Compound Table (see “Compound Table”).
For all but the simplest spectra, you will need to deal with compounds whose signatures overlap. A consistent strategy for profiling overlapped regions will produce more consistent results.
For more details on this and other profiling strategies, see “Tutorial and Sample Files”.
To profile compounds in an overlapped region of the spectrum
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Identify likely compounds contributing to an overlapped region (see “First Step: Identify Compounds”).
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Evaluate each compound for the presence of additional, non-overlapped peaks elsewhere in the spectrum (see “Cluster Navigator”).
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If they are present:
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Look for clearly visible peaks in the overlapped region. Such clusters occur mostly at the edges of the overlap region, but remember to look throughout the region, as some peaks stand out nicely between the peaks of another cluster.
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Use these partially-exposed peaks to establish the optimal frequency for the cluster.
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If they are not present:
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Look for clusters of peaks in the overlapped region with some clearly visible peaks.
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Use these partially-exposed peaks to establish the optimal frequency for the cluster.
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Once you have approximately profiled most of the groups in the overlap region, fine-tune the heights and frequencies of each cluster in the region for an optimal shape. Use the subtraction line to guide your modifications, as a well-matched cluster will leave a subtraction line that resembles a normal spectrum with the effects of the cluster removed.
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In overlap regions, profile multi-peak clusters before single-peak clusters. If urea is present, you may want to profile it last, to minimize its interference with other compounds in the spectrum.