Infrared spectra of glycosaminoglycans in deuterium oxide and deuterium chloride solution: Quantitative evaluation of uronic acid and acetamidodeoxyhexose moieties

I.r. spectra in the “carbonyl region” (1800-1400 cm^?1) have been obtained for aqueous solutions of a number of glycosaminoglycans and model compounds. In D₂O solution, the uronic carboxylate and acetamido groups absorb at characteristic frequencies (ν_asCOO^?, 1605 ±5; ν₅COO^?, 1420 ±5; Amide I, 1623 ±3; and Amide II, 1480 ±2 cm^?1). In acidic (m DCl) solution, the amide bands remain substantially unmodified, whereas those of the carboxylate anion disappear and are replaced by a single band due to the undissociated carboxyl (νCOOH, 1723 ±3 cm^?1). These bands can be used for quantitative evaluation of the uronic acid and acetamidodeoxyhexose moieties of glycosaminoglycans, using either standard polysaccharides or the corresponding monosaccharides as reference compounds. For D₂O solutions, the absorbances of the carboxylate and acetamido groups have been measured by graphical extrapolation of the corresponding most-intense bands (ν_asCOO^? and Amide I). In DCl, the two analytical bands (νCOOH and Amide I) are well-resolved, and analyses have been performed directly from the absorbances recorded. I.r. data for the uronic acid and 2-acetamido-2-deoxyhexose content of various reference samples of glycosaminoglycans are in reasonable agreement with those expected from the “established” structures, as well as with those obtained by colorimetric and conductimetric methods. When sodium d-glucuronate was used as the reference standard, the i.r. data gave relatively low values for the uronic acid content of heparin. The apparent acid dissociation constants of chondroitin 4-sulfate and heparin were estimated from the absorbance of νCOO^? and/or the νCOOH bands at different pH (pD) values.