# Combining NMR Spectroscopy and Chemometrics to Monitor Structural Features of Crude Hep-arin
## 2 Results
### One-Dimensional (1D) Spectra Library and PCA  
The proton spectra of eighty-eight crude heparin samples were registered by the NMR analysis protocol indicated in the experimental section. The spectra show very high variability of composition, both in terms of non-GAGs components (broad signals at both aromatic and aliphatic regions due to DNA/RNA and other impurities, Figure S1) and galactosaminoglycans components, mainly dermatan and chondro sulfate (Figure 1). Due to this variability and the difficulties in establishing quantitative ratios among diagnostic signals, the spectral complexity was reduced to the basic elements which best define the crucial differences between samples by employing chemometric techniques. One of the most common tools used to explore a complex dataset is principal component analysis (PCA). Typically, this tool takes a large number of correlated variables and transforms the data into a smaller number of uncorrelated variables (principal components), while extracting the maximal amount of variation, thus making it easier to analyse the data and make predictions [13]. The score plot of the first two components, generated by the PCA of 88 \({}^{1}\)H-NMR spectra of the crude samples, is shown in Figure 2. The graph, displaying the variance accounted for by the principal components, shows that only two components are necessary to account for more than 80% of the total variance, indicating that the differences among the samples are mainly distributed along the components **1** and **2**. Figure 1: \({}^{1}\)H-NMR spectra. Acetyl region of proton spectra of samples of group A, group B, and group C (see Figure 2) registered at 600 MHz, showing methyl signals of Dermatan (2.08 ppm), Heparin (2.05 ppm), and Chondoritin (2.02 ppm) components. In the loading plot of the first principal component, the negative signal of the acetyl protons of dermatan sulfate was observed (Figure 3). By contrast, in the loading of the second principal component, signals belonging to the trisulfated disaccharide (corresponding to the most abundant dissacharide of heparin chains) are positive, while the other heparin and chondro acetyl signals are negative. The analysis of the loading plots suggested that samples having a positive score in component 1 (Figure 2, group A) contain a low amount of dermatan sulfate, while those having negative score in component 2 (Figure 2, group C) contain highly acetylated heparin with a larger amount of chondro. The samples contained within the group B have instead a higher degree of sulfation, together with a large amount of dermatan. The proton spectra of the samples belonging to these groups confirm what was observed by PCA, showing that the spectra of samples belonging to the group A, in the absence of dermatan and chondro acetyl signals, match with those of purified heparin (Figure 1). Since the major differences among the crude samples defined by PCA are due to the content of galactosaminoglycan and the acetylation degree of the heparin component, the PCA was performed also on part of the anomeric region of the proton spectra (5.75-4.94 ppm). In this region, with the exclusion of the weak H1 of I2S of dermatan sulfate (5.18 ppm), no signals belonging to dermatan and chondro are present. Before the PCA analysis, the selected region of the proton spectra was aligned with a custom algorithm (Table S14 and Figure S9), because without alignment the shift effect was dominant in the loading plot of the first principal component. After the alignment, PCA differentiates samples on the basis of the sulfation pattern of the heparin/heparan sulfate components. The score plot generated by the PCA of this spectral region is shown in Figure 4. No evident clusters are visible, and the loading plot of the first principal component (Figure S3) can be interpreted in terms of the degree of sulfation of the samples, where the samples positioned in the negative score of the first principal component are more highly sulfated.