Determination of Sulfate Anions in Heparin by Capillary Electrophoresis with Improved Indirect UV Detection

A simple, sensitive and environmentally friendly capillary electrophoresis method for the determination of free sulfate anions in heparin and low-molecular-weight heparin with indirect UV detection is reported. The background electrolyte consists of 20 mM nitrate (pH 5.5) containing 0.2 mM CTAB. The use of nitrate as the probe anion is based on the consideration that nitrate has a very close electrophoretic mobility to that of sulfate, leading to a very narrow peak shape. Moreover, the apparent molar absorptivity of nitrate at 202 nm (8413 L mol −1 cm −1 ) is much higher than that of the commonly used probe anion chromate at 254 nm (2400 L mol −1 cm −1 ). The combination of the narrow peak shape with the high apparent molar absorptivity of the probe ion improved the limit of detection by 2.4 times and the limit of quantitation by 3.2 times. The effect of various CE parameters on the separation of sulfate from chloride was investigated and optimized. The method displays linearity in the range from 0.05 to 20 mM. The RSD % for intraday ( n  = 6), and interday ( n  = 3) repeatability in terms of migration times and peak areas were all less than 3 %. The limit of detection ( S / N  = 3) and limit of quantitation ( S / N  = 10) for sulfate were determined as 18 and 53 µM, respectively. The method was successfully applied for the quantification of sulfate anions in heparin and low-molecular-weight heparins.