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With the development of various physico-chemical analysis techniques so far, the basic knowledge about the structure of agar and agarose molecules has been inproved. Specific enzymatic activities like; hydrolysis of agar and agarose, the physico-chemical fractionation of these molecules, and specially the molecular structure defining techniques such as nuclear magnetic resonance spectroscopy have enhanced the basic chemical understanding about these substances.

In 1956, Araki and Arai used agarases, also known as agarose 4-glycanohydrolase, to determine the structure of agarobiose. Since then several agarases have been purified and used to study the agar structure of different origins. Therefore agarases helped a lot in predicting the accurate chemical structure of agar.

In 1983, Christiaen and Bodard used infrared spectroscopy to determine the sulfate content in G. verrucosa agar. He also used infrared spectroscopy to track the sulfate and 3,6-anhydrogalactose content variations in different agar fractions. Conversely infrared spectroscopy wasn’t precise enough to determine the definitive polysaccharide structures. Later in 1985 Whyte and his team studied a combination of infrared spectroscopy and high performance liquid chromatography (HPLC) of methanolysates of algal galactans and used it to classify them into agar and/or carrageenan.

The most important discoveries in the field of structural and chemical analysis of agar were made using nuclear magnetic resonance during the period of 1980 to 1991. Izumi in 1973 [30] and Welti in 1977 initially proposed the ¹H NMR spectroscopy of agar to study its quantitative data and to detect the minor concentration of particular repeating units of agarobiose. Although it is not generally used due to the complex spectra it provides, still its data quantification is possible. Nevertheless, identification of minor traces of desired repeating units is also possible. ¹³C NMR spectroscopy provides less complicated and easy to understand spectra by using one nicely resolved signal per carbon molecule. Even though ¹H NMR is more sensitive than the ¹³C NMR spectroscopy, but it delivers complicated spectra of molecules than the ¹³C NMR spectroscopy.

Therefore a combination of techniques can be used to precisely study the chemical, physico-chemical as well as the molecular structure of agar gel. Specifically extracting, fractioning, enzymatic hydrolyzing the agar, as well as ¹H and ¹³C Nuclear Magnetic Resonance spectroscopy techniques are club together to accurately study the critical chemical structures and distribution of the consecutive units of agarobiose and other similar molecules in different agars of various algae.

Since, 3,6-anhydrogalactose deposit of agar gets hydrolyzed due to acidic pH, thus lost during the procedure. Therefore gas chromatography was not suitable for studying agar components, before 1991 [12]. In 1991, a modified procedure was developed, using hydrolysis and reduction in a two-step process thereby not damaging 3,6-anhydrogalactose. This would retain the anhydro-sugar, which can be quantified conventional capillary gas chromatography.

Thus factors associated with the extensive and monotonous extraction and purification process are avoided during the direct quantification of polysaccharides, extracted from the cell wall of the algae. Helleur and his team in 1985, and Bird and his team in 1987, used the pyrolysis–gas chromatography, with or without mass spectrometry, to verify the critical chemical structure of algal cell-wall polysaccharides, either after extraction or in situ. Solid state ¹³C NMR spectroscopy chemical study of agarose, verified the spectrum of longer line-width although with the identical peak chemical change detected as from high resolution ¹³C NMR spectroscopy. These spectra also help in the analysis of polysaccharides of algal cell wall, which also helps in characterization of substituent like methoxyl and pyruvate groups.