Therefore, we can assign proton 10 as 5.209 ppm and proton 11 as 3.754 ppm. To differentiate protons 10 and 11, take a look at our COSY table 3.754 ppm shows two COSY correlations, while 5.209 ppm only shows one. From this list, we can easily assign proton 8 as the peak at 2.068 ppm based on its integration of 2 protons. Based on the COSY, proton 9 couples protons at 2.068 ppm (2H), 3.754 ppm (1H), and 5.209 ppm (1H). Thymidine’s structure suggests that proton 9 should couple protons 8, 10, and 11. Now that proton 9 has been assigned, the fun really begins. The remaining protons are doublets, triplets, and multiplets that can be assigned by 2-dimensional COSY. The peak also integrates to 1 proton, supporting the assignment. The chemical shift of 11.256 ppm supports this assignment, as imide protons often show up far downfield. The only proton that should show up as a singlet is proton 6, as it has no neighboring protons that would split the peak (the nearest proton is 5 bonds away!). There is only one singlet in the ¹H-NMR spectrum. Therefore, the peak at 7.690 ppm must represent proton 4! The integration and chemical shift support the assignment, as proton 4 is the only aromatic proton in the structure. The long-range coupling constant observed for proton 3 (J=1.2 Hz, split into a doublet by proton 4) is reflected in the coupling constant for proton 4 (J=1.2 Hz, split into a quartet by proton 3). Protons that are coupled to each other should exhibit the same coupling constant. The peak is split into a doublet with a coupling constant of 1.2 Hz, reflecting the long-range coupling between protons 3 and 4, which also supports this assignment. The high field chemical shift supports this assignment. The only peak with an integration of 3 is the doublet at 1.770 ppm. Proton 3 is the only methyl group in the structure, and therefore must integrate to 3 protons. In this tutorial the widespread equidistant binning and the non-equidistant binning, which becomes increasingly popular, are shown.To begin, let’s start with proton 3. Two approaches have been proposed to handle variations in chemical shifts, whereby the first approach bases on a reduction of the spectral resolution by equidistant or non-equidistant binning methods, and the second approach bases on an alignment of peaks with the help of genetic algorithms, beam searches or sophisticated correlations. It is visible that the location of the singlet of para-hydroxyphenylacetate at 3.455 ppm remains very stable for all samples whereas the location of the triplet of taurine varies between 3.407 ppm 3.435 ppm from sample to sample.Īlthough it has been demonstrated in a recent publication that peak shifts can be beneficial for a separation of different groups of samples under certain circumstances, this effect is unwanted for most applications. As the pH of the samples was constant due to buffer added, the shifts of the taurine triplet are caused by different concentrations of salts, of metal ions, of metabolites, or by varying dilutions of the samples.Ĭlipping between 3.35 ppm and 3.49 ppm of 1H-NMR spectra of 30 samples from a human metabonomic study. The position of the singlet is very stable at 3.455 ppm whereas the position of the taurine triplet varies between 3.407 ppm 3.435 ppm. The clipping between 3.35 ppm and 3.49 ppm shows one singlet belonging to para-hydroxyphenylacetate and one triplet belonging to taurine. In the figure an example with 1HNMR spectra of 30 samples from a human metabonomic study is shown. Relative concentrations of specific ions, relative concentration of specific metabolites and many more.Īll these parameters can influence the shifts of peaks, whereby not all peaks are affected and different peaks are affected to a different extentĮven when belonging to the same metabolite. Thereby many parameters play a role such as changes of the pH, changes of the salt concentration, overall dilution of the sample, As the matrix of biofluids and in particular urine is highly varying, the local environment of protons also varies. One of the major challenges for automated data analyses and peak assignments for NMR spectra of biofluids from metabonomic studies is the effect Matrix Effects and Peak Shifts of NMR Spectra
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