Journal of Chromatography A, 1149 (2007) 38–45 Study of molar response of dextrans in electrochemical detection Franco Abballe a , Mila Toppazzini b , Cristiana Campa b,∗ , Fulvio Uggeri c , Sergio Paoletti d a Dionex SpA, Via XXV Aprile 6, 20097 San Donato Milanese, Milano, Italy b Bracco Imaging SpA, CRB Trieste, AREA Science Park, Building Q, SS14, Km 163.5, 34012 Basovizza (Trieste), Italy c Bracco Imaging SpA, via E. Folli 50, 20134 Milano, Italy d Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, via L. Giorgieri 1, 34127 Trieste, Italy Available online 22 December 2006 Abstract In this work, a methodological approach is reported, aimed at assessing the electrochemical response of some model gluco-oligosaccharides (dextrans). Such strategy is based on the complementary use of both anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD) and capillary zone electrophoresis coupled with UV detection (CZE–UV). Unlike HPAEC–PAD, CZE–UV required derivati- zation with a chromophoric dye (i.e., 8-aminonaphtalene-1,3,6-trisulphonic acid, ANTS) to enhance UV response and separation selectivity. From the comparison between chromophore response and PAD signal, the reliability of HPAEC–PAD for quantitative evaluation of dextran mixtures containing mainly oligomers with polymerization degree (DP) up to 18 could be proved, due to the fairly constant molar response. For higher DPs (up to 41), a maximum in the trend of the molar responses was observed followed by a steep decrease for DPs higher than about 30–35; indeed, an underestimation of weight-average molecular weight of dextran mixtures containing such oligomers was noticed. © 2006 Elsevier B.V. All rights reserved. Keywords: Dextrans; Oligosaccharide; Anion-exchange chromatography; Pulsed amperometric detection; Capillary zone electrophoresis 1. Introduction Structure elucidation of carbohydrates has a high poten- tial in biomedicine and pharmaceutics, for designing new and specific diagnostic and therapeutic tools. Due to their struc- tural complexity, carbohydrates require multiple complementary analytical approaches. Among the liquid chromatographic techniques, high-performance anion-exchange chromatography coupled with pulsed electrochemical detection (HPAEC–PED) (more commonly known as pulsed amperometric detection, HPAEC–PAD) deserves a special mention, since it pro- vides selective separation and sensitive detection without derivatization [1–4]. The electrochemical behaviour of carbo- hydrates, however, can be affected by molecular weight and structure [4–7]; this has been demonstrated for various carbohy- drate oligomers, including, for instance, gluco-oligosaccharides [8–13], and oligo-mannuronic acids [14]. Indeed, as stated by LaCourse [4] “all quantitative applications of LC–PED must be ∗ Corresponding author. Tel.: +39 040 3757830; fax: +39 040 3757831. E-mail addresses: cristiana.campa@bracco.com, cri cam@yahoo.com (C. Campa). based on careful calibration plots using standard solutions for each sample component”. The achievement of oligosaccharide standards of high purity is quite challenging, especially for those compounds having a degree of polymerization (DP) higher than 15–20. Some meth- ods have been proposed to face this problem, in order to achieve PAD molar response factors. For instance, enzyme-based post- column reactors have been used for the quantitative analysis of malto-oligosaccharides [8] after their HPAEC separation; glucose produced by the post-column reactor was used for quan- tification purposes and the results were compared with those achieved with PAD detection of oligomers; starch was also suc- cessfully hydrolyzed in an analogue flow-injection system [15]. Another approach is based on partial fractionation of polysac- charide hydrolysates, HPAEC–PAD analysis, and evaluation of the total carbohydrate content by the anthrone method [11] or by enzymatic hydrolysis to the monomeric units [10]. These meth- ods of evaluation of the carbohydrate content, however, do not allow for the quantification of individual oligomers; therefore they are not reliable as an independent route for the evaluation of the molar composition of fractionated oligosaccharides. In the present work, an experimental strategy is pre- sented, aimed at assessing the PAD response of (1 → 6) 0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.11.086