## 2 Evolution of NMR Methodologies
### Solution-State NMR for Polysaccharides
#### 2.1.1 Structural Elucidation  
The structure of a polysaccharide can be elucidated using NMR spectroscopy, revealing its chemical composition and molecular arrangement. For example, [PERSON] et al. used COSY and HSQC to analyze the structure of _Sterculia apetala_ gum polysaccharides and reveal the complex branching and functional groups of these polysaccharides [16]. Recognizing these branching patterns is essential, as they can have an impact on the rheological properties of polysaccharides, which are crucial for the applications in both the food and medical industries. These studies highlighted the power of 2D NMR in uncovering structural details that are hard to figure out with traditional methods. Similarly, [PERSON] et al. used \({}^{1}\)H and \({}^{13}\)C NMR to characterize the polysaccharides of _Streptococcus pneumoniae_ serotype 6C. They identified the immunogenic epitopes of these polysaccharides and found their potential uses in vaccine development [17]. The novel aspect of their work is that they combined structural research with biomedical applications, demonstrating how NMR can connect basic research and translational science. By combining structural analysis with translational research, they pointed out that NMR has the potential to be an innovative discovery tool in glycopology. Advanced technologies like HMBCs and NOESY have further improved structural elucidation. In a study about bacterial polysaccharides, [PERSON] et al. combined 1D and 2D NMR with methylation analysis to decode the O-antigen structure of _Escherichia albertii_, highlighting the versatility of NMR in microbial polysaccharide studies [18]. Integrating different technologies reveals a novel way to deal with the challenges of complex and overlapping signals. The methodology developed in this study represents a contemporary approach for resolving signal overlap challenges in structurally diverse microbial polysaccharides, like _Escherichia albertii_ O-antigens. These results provide a framework that can be copied to solve similar problems in glycobiology. The structural elucidation of polysaccharides, especially those derived from medicinal plants like _Radix Astragali_ (RAPS-3), as illustrated in Figure 1, presents significant chal \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Sample State** & **Techniques** & **Key Features** & **Optimal Applications** \\ \hline \multirow{3}{*}{Solid-state} & CP/MAS & Resolves rigid molecular structures; crystallinity analysis & Plant cell wall polysaccharides (cellulose–ligin interactions) \\  & DNP-NMR & 10–100\(\times\) sensitivity enhancement; low-abundance components & Biofilm matrix, fungal cell walls \\ \cline{1-1}  & HETCOR & Heteronuclear correlations; hydration studies & Food gel hydration mechanisms \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont._lenges due to their complex and heterogeneous structures [21]. For example, in RAPS-3, \({}^{1}\)H NMR spectroscopy alone revealed the presence of a backbone with \(\alpha\)-(1\(\rightarrow\)4)-linked D-glucan. However, the exact nature of the glycosidic linkages and the configuration of side chains could not be fully confirmed without further 2D NMR analysis. The application of 2D NMR experiments, specifically COSY, HSQC, and HMBC, was crucial to elucidating the detailed structure of these polysaccharides. In the HSQC spectrum of RAPS-3, the anomeric proton at \(\delta_{\rm H}\) 5.42 (H-1) showed a direct correlation with the carbon signal at \(\delta_{\rm C}\) 102.5, confirming the \(\alpha\)-(1\(\rightarrow\)4) linkage between glucose units in the backbone of the polysaccharide. HSQC also helped confirm the identity of side chain components like arabinose, as cross-peaks between the anomeric protons of arabinose at \(\delta_{\rm H}\) 5.20, 5.17, and 5.11 and their corresponding carbons at \(\delta_{\rm C}\) 109.8, 109.7, and 110.3 were clearly observed.