# Combining NMR Spectroscopy and Chemometrics to Monitor Structural Features of Crude Hep-arin
## 2 Results  
### Protocol for Sample Preparation  
Crude heparin samples are often not completely soluble in water. Therefore, to avoid the presence of insoluble material in the NMR tube, a protocol of sample separation by centrifugation was developed. Three parameters of the separation protocol were evaluated for their effect on the supernatant content, including: the amount of insoluble material present, crude concentration, and the method of solution equilibration (with or without stirring). Each of the eight combinations of the parameters was tested. Moreover, three separations were performed for a single parameter set to evaluate the precision of the data, giving a total of ten separations. For each separation, two parameters were measured after lyophilization of the supernatant or the pellet: the total weight loss and the amount of precipitate, both expressed as a percentage of the original weight (Table 1). With the exclusion of one result, the loss of water due to lyophilization was around 10%, regardless of sample concentration. While the stirring procedure during solution equilibration did not affect the yield on a percentage basis, the amount of precipitate was related to the sample concentration, showing that the precipitate still contains soluble material. To verify that the procedure does not result in the selective separation of glycosaminoglycan species, a precipitate obtained at a higher concentration (80 mg/mL) was re-suspended in 1 mL of D\({}_{2}\)O, centrifuged, and the spectrum of the supernatant compared with that obtained after the first solubilization. While no differences in the glycosamonoglycan pattern were found, higher non-GAG signals in the re-suspended precipitate were observed (Figure S1). Based on these results, to collect enough soluble material for the NMR characterization, an aliquot of 60 mg of crude heparin was determined to be sufficient for analysis in 1.5 mL of D\({}_{2}\)O. The average weights of the 88 crude samples are shown in Table S1. The proton chemical shifts of heparin are affected both by the pH and concentration of the solution [11; 12]. Crude heparin does not contain only heparin, but also other GAGs (heparan, dermatan, and chondro sulfate), as well as non-GAG material, mainly DNA and RNA, some proteinaceous material, and a variable amount of water and salts. For these reasons, the proton spectra of crude heparins in D\({}_{2}\)O were not fully reproducible, and a shift of signals, particularly those of anomeric glucosamine and iduronic acid residues, was often observed (Figure S2). The maximum shift of both peaks observed was about 10-12 Hz (0.015-0.020 ppm), presumably due to the different concentration of GAG components in the samples, which affect the pH of the solution. These spectra cannot be easily compared, particularly when they are analysed by multivariate analysis (e.g., PCA). To remove the effect of pH variability, the samples were solubilized in 0.15 M of phosphate buffer at pH 7.1, containing 3 mM of perdeuterated EDTA. The complexation of bivalent or paramagnetic ions induced by the addition of perdeuterated EDTA provides narrower lines and improved resolution. A comparison of crude proton spectra recorded in water and the buffer soluti