Determination of free and total sulfate and phosphate in glycosaminoglycans by column-switching high-performance size-exclusion and ion chromatography and single-column ion chromatography.
Analytical procedures for the determination of free and total sulfate and phosphate in glycosaminoglycans by high-performance liquid chromatography were studied. A column-switching method coupling high-performance size-exclusion chromatography (HPSEC) and ion chromatography (IC) is proposed for the determination of free anions. Good run-to-run and day-to-day precision values (RSD) of < 4.7% were obtained for both anions. Total anion contents were determined after wet acid hydrolysis with nitric acid-hydrogen peroxide (5 + 1) by single-column IC and ICP-AES elemental analysis in order to validate the results. Recoveries ranging from 94.6 to 99.0% for sulfate and from 80.8 to 94.0% for phosphate were obtained. Both HPSEC-IC and single-column IC methods were applied to the analysis of a low molecular mass heparin, a non-fractionated heparin and a chondroitin 4-sulfate. From the free and total sulfate determinations, the content of linked sulfur was calculated and ranged from 5.1 to 12.2% m/m.
Departament de Química Analítica, Facultat de Química, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain.
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V
Ruiz-Calero
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L
Puignou
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M
Diez
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MT
Galceran