Subtracting and Combining Spectra
Difference spectroscopy is useful when two spectra are almost identical and you want to reveal small changes,
for example after a reaction, a titration, or the addition of a ligand.
You can subtract one spectrum from another to highlight their differences.
Addition and other linear combinations are equally straightforward.
Subtracting B from A to get: C = A - B
- Bring document A to the front. Expand the region(s) with the most important peaks; in this way you can monitor what's happening.
- Choose Format > Overlay.
- You will see the list of the other windows. Check the box on the left of the name of B. Now the spectrum B is drawn over A.
- Set the pre-multiplication factor for B (shown in the last column, “amplif.”). A positive value subtracts B from A. A negative value adds B to A. You can type the value or modify it with the little arrows at the bottom of the same column.
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Click Subtract to apply the operation.
The displayed spectrum is updated immediately and you can still adjust the pre-multiplication factor if necessary. The subtraction too is reversible. - When you are satisfied with the result, close the dialog. B disappears and only the difference spectrum remains.
- It is not enough to save A, because iNMR never overwrites the original spectrum. If you want to store the difference, choose File > Export and create a new iNMR document.
Not all overlays are suitable for subtraction. The two spectra must have the same number of dimensions and belong to the same domain (time or frequency). The digital resolution (ratio between the number of points and the spectral width in ppm) must be the same. In case of two-dimensional spectra, the number of points must correspond in each dimension. When the two spectra are mono-dimensional and their baselines have both been corrected with polynomial functions, the difference spectrum will contain the difference of the two baseline corrections. It is not, however, necessary that both baselines have been corrected.
To obtain the highest accuracy, bring the amplification factor near to its optimal value before beginning the subtraction.