Separation and Pulsed Amperometric Detection of Alditols and Carbohydrates by Anion-Exchange Chromatography Using Alkaline Mobile Phases Modified with Ba(II), Sr(II), and Ca(II) Ions Tommaso R. I. Cataldi,* Diego Centonze, and Giovanna Margiotta Dipartimento di Chimica, Universita ` degli Studi della Basilicata, Via N. Sauro, 85, 85100 Potenza, Italy The effect of some divalent nonelectroactive cations (DNCs) in the anion-exchange chromatographic separa- tions with alkaline mobile phases of carbohydrates and alditols was investigated; the ions examined at a typical concentration of 1 mM were Ca(II), Ba(II), and Sr(II). The use of these cations in the eluent as their corresponding acetates or nitrates has been found to yield at least a two- fold effect. First, the peak symmetry and concurrently the column efficiency is greatly improved. Second, the pres- ence of Ba(II) or Sr(II) significantly enhances the response of all analytes investigated in pulsed amperometry at a gold working electrode. The action of the DNCs on the separation seems to be related to a very effective removal of carbonate ion from the alkaline eluent and, especially, their ability to complex cyclic and acyclic polyhydroxy compounds. Efficiency estimated from data calculated at 1 0 % of peak height can increase by as much as 2 5 % with a comparably lower RSD (15%, n ) 9). From the viewpoint of the separation efficiency and reproducibility of chromatographic data, all divalent inorganic ions employed were well-behaved. However, only the use of Ba(II) or Sr(II) is recommended since alkaline mobile phases containing Ca(II) ion negatively affect the gold electrode response of some analytes. In the last ten years, several papers have appeared concerning the pulsed amperometric detection (PAD) of sugars and alditols (also known as sugar alcohols) at gold electrodes in strongly basic solutions. 1-12 The use of PAD is required because polyhydroxy- lated compounds are scarcely electroactive and their oxidation at a Au electrode in alkaline solutions is characterized by fouling of surface by accumulated detection products. 3-5 Moreover, the attractiveness of such a detection mode stems from the fact that carbohydrates and CHOH-bearing molecules, which are weakly ionizable compounds, can be successfully separated in high- performance anion-exchange chromatography (HPAEC) and capil- lary electrophoresis. 12-14 Both these separation methods in alkaline media combine very well with PAD. Actually, HPAEC is particularly appropriate for the separation of sugars and alditols provided that the alkaline mobile phase is prepared by carbonate-free sodium hydroxide solutions. 15 Of particular concern is the role that carbonate ion plays in limiting the column performances; its effects are sufficient to warrant removal from the alkaline eluent. Indeed, the presence of carbonate in the mobile phase poses some practical problems in terms of irreproducible effects on retention times and column efficiency when its concentration is unknown or frequently changed. A current strategy for minimizing the uptake of carbon dioxide, and thus of carbonate in the mobile phase, is to sparge the eluent and to pressurize the reservoir with an inert gas such as helium or nitrogen. Whereas such a strategy is apparently consistent to apply, it does not guarantee the complete absence of carbonate ion in the column. Even if commercially available carbonate-free 50%NaOH solutions are employed, the use of no well-degassed water in which carbon dioxide is present may significantly affect the separation of alditols and carbohydrates. Thus, the aim of this investigation was to explore the use of an innovative procedure for improving the chromatographic separation and detection of alditols and carbohydrates. The common knowledge that some divalent nonelectroactive cations (DNCs) such as Ca(II), Ba(II), or Sr(II) rapidly form insoluble carbonate salts has led us to consider a competitive, and more straightforward approach of minimizing the CO 3 2- level in the mobile phase. Instead of preventing the introduction of atmo- spheric carbon dioxide during preparation, use, and storage of the eluent, our strategy is to affect the solubility of carbonate by the formation of sparingly soluble salts. There is currently no report of DNCs used for improving the separation of carbohy- drates and alditols in HPAEC with PAD under any conditions. It is well-known, however, that cation-exchange supports loaded with Ag + , Pb 2+ , or Ca 2+ counterions have been widely applied to * Corresponding author: (Fax) 39-971-474223; (E-mail) cataldi@ unibas.it. (1) Hughes, S.; Johnson, D. C. Anal. Chim. Acta 1981 , 132, 11. (2) Olechno, J.; Carter, S. R.; Edwards, W. T.; Gillen, D. G. Am. Biotechnol. Lab. 1987 , 5, 38. (3) Johnson, D. C.; LaCourse, W. R. Anal. Chem. 1990 , 62, 589A. (4) Johnson, D. C.; LaCourse, W. R. Electroanalysis 1992 , 4, 367. (5) Johnson, D. C.; Dobberpuhl, D. A.; Roberts, R. A.; Vandeberg, P. J. J. Chromatogr. 1993 , 640, 79. (6) Rocklin, R. D.; and Pohl, C. A. J. Liq. Chromatogr. 1983 , 6, 1577. (7) Welch, L. E.; LaCourse, W. R.; Mead, D. A., Jr.; Johnson, D. C.; Hu, T. Anal. Chem. 1989 , 61, 555. (8) Jackson, W. A.; LaCourse, W. R.; Dobberpuhl, D. A.; Johnson, D. C. Electroanalysis 1991 , 3, 607. (9) LaCourse, W. R.; Jackson, W. A.; Johnson, D. C. Anal. Chem. 1991 , 63, 134. (10) Lee, Y. C. Anal. Biochem. 1990 , 189, 151. (11) LaCourse, W. R.; Johnson, D. C. Anal. Chem. 1993 , 65, 50. (12) Johnson, D. C.; LaCourse, W. R. In Carbohydrate Analysis. High Performance Liquid Chromatography and Capillary Electrophoresis; El Rassi, Z., Ed.; Elsevier: Amsterdam, 1995; Chapter 10. (13) Lu, W.; Cassidy, R. M. Anal. Chem. 1993 , 65, 2878. (14) O’Shea, T. J.; Lunte, S. M.; LaCourse, W. R. Anal. Chem. 1993 , 65, 948. (15) Dionex Corp. Installation, Instructions and Troubleshooting Guide, 1992 ; Document 034752. Anal. Chem. 1997, 69, 4842-4848 4842 Analytical Chemistry, Vol. 69, No. 23, December 1, 1997 S0003-2700(97)00374-0 CCC: $14.00 © 1997 American Chemical Society