Quantitative 1D NMR integration example
In this example we have kinetics related spectra for which we do quantitative 1D NMR integration.
Contents
- Selecting spectrum files
- Switching plotting mode
- Integrating the selected spectra
- Editing integrals
- Analysing integrals
Selecting spectrum files
In this section we will select all .jdx files in Kinetics folder and load them unchecked.
If you came here by selecting the 'integration/qQMSA Example' in 'Welcome' page, you can almost skip this first section as the files were already selected.
From the Data Browser we select the spectrum files we want to integrate (remembering that we can filter file types at the top of the data browser). If we want to select multiple files, we can either press ctrl when left clicking and selecting a file, or select the first file and then press shift when selecting the last file: all the files in between are selected as well.
Having selected the files, we right click one of them and a popup menu opens. In the menu we left click 'Open'.
Available Parameter Files dialog opens. Load Checked loads the parameter files checked, meaning that parameter values for e.g. integrals and shifts etc. are used and thus may have already been defined. Choosing Cancel loads the files unchecked. For this example we choose Cancel.
We have now loaded spectrum files.
Switching plotting mode
In this section we will change the plotting mode to 'Active'.
The files are now opened and we see that spectra are laid vertically on top of each other in the spectrum screen.
That is useful to get the general picture of the spectra but details are not easily seen. Let's focus on one spectra at the time.
Plotting modes for spectra can be changed in Spectra menu. 'Active' plotting mode is our choice. The active plotting mode can also be set with ctrl + D.
If you have several spectra, switching to the superimposed or active plotting mode found in Spectra menu in the menu bar on the top may be convenient when integrating.
We can switch to the following and previous spectrum with ctrl + mouse wheel.
When using the stacked plotting mode, notice how the vertical blue bar on the right shows which spectrum is the active. You can switch between the spectra by ctrl + mouse wheel. That also works in the active plotting mode.
We have now switched to the active plotting mode and thus we have only one spectrum shown.
Integrating the selected spectra
In this section we set the limits for an integral and then integrate.
To integrate the spectra we first need to set the integration limits. That is done by right clicking a spot on the spectra that we want to be the an end point. A vertical line and ppm value appears when we define an end point. To define the other end point, we will do the same. If we would want to refine the limits, we just repeat the process.
To get into the details zooming may be needed. Zooming works in a similar as setting the integration limits: right click to set one limit, then right click to set the other limit. Now you only need to double click the spectrum area and it is zoomed. To get back to the full spectrum view, click 'Fit Horizontally/Vertically' in the tool bar.
Now that we have the limits set, we integrate the spectra by clicking the integral button from the main toolbar, or pressing ctrl + I. All spectras are integrated despite the plotting mode. The first integral is normalized to have value of 1.00. The values are shown below the active spectrum. The title is given according to the limits.
Regardless of the plotting mode, all spectra are integrated.
We have now integrated one (or more) peaks in the spectra.
Editing integrals
In this section we edit an integral so that we can start analysing the spectra.
Selecting an integral is simple, we just left click it. Note how Format Spectrum panel on the left cornel changes to Format Integral panel.
To remove an integral, select it and press 'Delete' on your keyboard.
In addition to Format Integral panel, integrals can be edited in the Integrals page that can be found in the bottom tool bar (3 Integrals). Integrals page lists all the integrals, their types, molecular weights, etc.
Integrals can also be selected in Integrals page by clicking the corresponding row.
Let's calculate the mass concentration now. 'Mass Concentration' is initially selected so we only need to click 'Calculate' next to it.
Oops! It did not work. Issues page tells us that we forgot to set the reference. Let's do it next.
To set the reference, we first need its integral. Did you notice the peak around 0 ppm? We integrate it first using the skills we learned previously and then select the integral.
When we have the integral selected, we can set it as reference by double clicking Type cell on the row causing a downward arrow head appear in the cell. Left clicking the cell, selecting Reference and pressing 'Enter' on the keyboard (or left clicking something) sets it as the reference. Notice how the font color changes to red for references.
In addition to the reference we can set another properties as well. Titles and molecular weights (MW) can be set by clicking or double clicking a corresponding cell. To set the number of protons (H's) and the normalized integration value (normalized), we can simply type values in the window that is to the right of Integrals window. Baseline correction can be set on and off by by clicking the Baseline checkbox.
For now we rename the title as 'TSP', set the molecular weight as '146.26' and the number of protons as '9'.
In this part we have selected the reference integral, renamed it and set values for the molecular weight and the number of protons.
Analysing integrals
In this section we will analyze the spectra by calculating molar concentrations.
Analyses can be done after the reference has been set. ChemAdder can calculate mass concentrations, molar concentrations and purity. Users may also add and solve their own formulas.
Instead of mass concentration, let's calculate calculate molar concentration instead. On the top of Integrals page, we find the third tool bar button that has a downward arrow head and currently reads 'Mass Concentration'. Tog get the molar concentration, we left click it and select 'Molar Concentration'.
To get results we hit 'Calculate' button on the left. (We have set at least the number of protons for another integrals, have we not?) The page switches to Results Stack that shows results for all the spectra. Results for the currently active spectrum are shown bolded.
The integrals can also be viewed as a bar chart. To do so locate 'View' option in the top left corner of the spectra area, left click 'Spectrum' and select 'Population Chart'.
If we want to go back to Integrals page, we can just left click the leftmost tool bar button currently showing Stacked Results and select Integrals.
If needed information was missing, like reference or nuclides were not defined, as they were not before, Issues page will appear on the right. Just switch back to Integrals and fill the missing information.
To copy results (e.g. for Excel) we can select a column and press ctrl + c. If we are only interested to copy single results, we can switch to Results page in the same way as we would switch to Integrals but selecting 'Results' and then selecting the cells we want to copy and pressing ctrl + c. It is good to check that the right spectrum is active.
After we have edited integrals, we can save them by going to Files menu and selecting Save Parameter Files As….
A dialog opens which allows to choose the save format. Existing formats are shown in red (bst and 1st). We select 2nd and then press OK.
We have now calculated molar concentrations for the integrals and saved them as parameter files, which concludes our example.