Spectral parameter ranges

Ranges of the spectral parameters (chemical shifts and J-Couplings) limit how much the iterator can change the calculated values of the parameters. Ranges are defined for couplings and chemical shifts. The limits can be approximated by the following equation:

calculated limits = predicted value ± 0.5 * range

At the beginning of the analysis the ranges should be large enough (0.1 ppm for shifts, 4-10 Hz for couplings).

The most convenient way to edit the ranges is to select the chemical shifts with the rectangle tool:

Right click chemical shift label and select Edit Ranges… from the popup menu:

The following dialog will appear:

Ranges can also be viewed and edited from the J-Couplings and Shifts tables:

The Force Shift and Force Couplings profile parameters define how much the couplings and shifts are forced to their ranges. Ranges can be completely ignored by unchecking the Force to Ranges parameter. This might be reasonable at the beginning of the analysis. The parameters can be edited from the profile & control parameter editor.

Fixing and unfixing spectral parameters

Fixing and unfixing spectral parameters is essential part of the QMSA. When parameters are fixed, they are not involved into iterating procedure. The following parameters can be fixed/unfixed: 1) chemical shifts, 2) coupling constants, 3) populations (i.e. molar fractions of spin systems), 4) response factors (of chemical shifts) and 5) line widths (of chemical shifts). Response factors should be kept fixed most of the cases (see Optimizing response factors of chemical shifts). The parameters can be fixed 1) individually or 2) all at once.

The parameters can be fixed/unfixed individually by selecting the chemical shifts with left mouse or rectangle tool, right clicking the mouse and selecting Fix or Unfix from the opening popup menu.

All the parameters can be fixed/unfixed at once by editing profile parameters which can be found from the bottom left corner of the ChemAdder.

Most of the statuses of the parameters can be recognized from the spectrum window.

Parameter statuses are represented with different checkboxes in the parameter tables of the Spin Systems page.

Optimizing response factors of chemical shifts

Response factors of chemical shifts (RF) should be given the value of 1 and they should be kept fixed in most of the cases. However, there are situations where they can be optimized. For example, water suppression may reduce the signals close to the water signal. For example, the glycoside proton signal of glucose can be only 50% of its expected intensity, when compared to the intensity of the other protons – its RESPONSE FACTOR is then 0.5. The RFs may vary due to different relaxation times, etc. Thy may also vary between molecules but usually one can rather safely assume that they are similar for similar molecules and set the largest RF of each molecule = 1.0.

There are two ways to handle the RF problem: 1) The RFs can be optimized, or 2) the observed spectrum can be ‘normalized’ so that the area/protons are the same for all the multiplets. In spectral analysis of a pure compound the normalization can be used at the very end of the analysis or to remove large solvent suppression or relaxation effects. In analyzing a mixture containing some very concentrated component (glucose, creatinine, ...) it may be necessary to optimize the RFs of those protons.

RFs offer a way to describe isomeric mixtures: for example, the ratio of α- and β-glucose is normally close to 0.35/0.65. In metabolomics it is practical to describe the two glucoses as one molecule, having two kinds of protons - with response factors of 0.35 and 0.65