4.1 The composition and sequential structure of alginate determines the functionality of alginate in an application. For instance, the gelling properties of an alginate are highly dependent upon the monomer composition and sequential structure of the polymer. Gel strength will depend upon the guluronic acid content (F_G) and also the average number of consecutive guluronate moieties in G-block structures (N_G>1).

4.2 Chemical composition and sequential structure of alginate can be determined by ¹H- and ¹³C-nuclear magnetic resonance spectroscopy (NMR). A general description of NMR can be found in <761> of the USP 35-NF30. The NMR methodology and assignments are based on data published by Grasdalen et al. (1979, 1981, 1983).^{4, 5, 6} The NMR technique has made it possible to determine the monad frequencies F_M (fraction of mannuronate units) and F_G (fraction of guluronate units), the four nearest neighboring (diad) frequencies F_GG, F_MG, F_GM, F_MM, and the eight next nearest neighboring (triad) frequencies F_GGG, F_GGM, F_MGG, F_MGM, F_MMM, F_MMG, F_GMM, F_GMG. Knowledge of these frequencies enables number averages of block lengths to be calculated. N_G is the number average length of G-blocks, and N_G>1 is the number average length of G-blocks from which singlets (-MGM-) have been excluded. Similarly, N_M is the number average length of M-blocks, and N_M>1 is the number average length of M-blocks from which singlets (-GMG-) have been excluded. ¹³C NMR must be used to determine the M-centered triads and N_M>1. This test method describes only the ¹H NMR analysis of alginate. Alginate can be well characterized by determining F_G and N_G>1.

4.3 In order to obtain well-resolved NMR spectra, it is necessary to reduce the viscosity and increase the mobility of the molecules by depolymerization of alginate to a degree of polymerization of about 20 to 50. Acid hydrolysis is used to depolymerize the alginate samples. Freeze-drying, followed by dissolution in 99 % D₂O, and another freeze-drying before dissolution in 99.9 % D₂O yields samples with low ¹H₂O content. TTHA is used as a chelator to prevent traces of divalent cations to interact with alginate. While TTHA is a more effective chelator, other agents such as EDTA and citrate may be used. Such interactions may lead to line broadening and selective loss of signal intensity.

4.4 Samples are analyzed at a temperature of 80 ± 1°C. Elevated sample temperature contributes to reducing sample viscosity and repositions the proton signal of residual water to an area outside that of interest.