Quantitative Analysis

There are three different approaches for quantitative analysis with ChemAdder: 1) Quantitative quantum mechanical spectral analysis (qQMSA), 2) Quantitative 1D NMR integration and 3) Deconvolution. Deconvolution can also be combined with the QMSA (see the serum example: C:\ChemAdder\Examples\Serum).

Quantitative Quantum Mechanical Spectral Analysis

The basic idea behind quantitative quantum mechanical analysis (qQMSA) is to merge all the parameter files of the mixture components into one parameter file. ChemAdder contains a metabolite libraries called Adaptive Spectral Libraries which are designed for qQMSA.

The following contains a basic protocol for qQMSA using adaptive spectral libraries.

  1. Write the name of the metabolite to the search field and select the metabolite from the table. Press Shift + X to cut the regions of the chemical shifts from the spectrum. If necessary, adjust the positions of the shifts by dragging them with left mouse. Press the [upward arrow ⇧ on the green background] button to add the selected metabolite to the spectrum.

    Search and select a metabolite. The green upward arrow button adds the selected metabolite to the spectrum.
  2. Repeat the step 1 until all the desired metabolites are added. Simulate spectrum. It is sensible to save the parameter file now. Select File → Save “filename” As…

  3. Remove/cover solvent and other extra signals via Ignore Region Between Cursors button, as shown in the image below.

    Signals can be removed via Ignore Region Between Cursors button.
  4. Make sure all chemical shifts, coupling constants and populations are unfixed. Response factors should be fixed.

  5. Set broadening to 20 – 100 Hz.

    Set broadening between 20 and 100.
  6. Run Total Line Shape Fitting (With Broadening)

    Run Total Line Shape Fitting (With Broadening).
  7. If some signals do not overlap, check the parameters and run Total Line Shape Fitting (With Broadening) again.

  8. Run Total Line Shape Fitting.

    Run Total Line Shape Fitting.
  9. Release line widths and run Total Line Shape Fitting.

    Having released line widths run Total Line Shape Fitting again.
  10. The quantitative results of the analysis can be reported to excel or text file by selecting from the menu File → Report….

    Report results via File menu.

Quantitative 1D NMR integration

Quantitative 1D NMR integration

The following contains a basic protocol for quantitative 1D NMR integration. For a more detailed walkthrough about integration see our kinetics example.

  1. Select the spectrum files you want to integrate from the Data Browser, right click mouse and select Open from the popup menu.

    Select spectrum files you want to integrate in the Data Browser.
  2. Integrate the spectra by selecting the integration limits with the cursors and by clicking the integral button from the main toolbar. All visible spectra are integrated. Superimposed plotting mode may be convenient for integration.

    Integrate the spectra after you have set the limits.
  3. Edit the integrals from the Integrals page. Toggle the reference integral from the Is Reference column. Give titles, molecular weights and number of protons for the integrals (except for the reference). Baseline correction can be included from the B/L column.

    Integrals can be edited in the Integrals page.
  4. Edit the reference integral by double clicking the title of the integral.

    The reference integral is editable by double clicking the title.
  5. All integral values can be viewed from the stacked integral table. Select the displayed value from the drop-down menu. The values can be copied to excel by selecting the cells of the table and by pressing Ctrl + C.

    Stacked integral table shows all integral values.
  6. The integrals can be also viewed from the population charts page:

    Another way to view the integrals is the population charts page.
  7. Save the integrals into parameter files by selecting Save Parameter Files As… from File menu.

    Save the integrals via File menu.
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