Roberto Sebastiano 1 Martha Elena Mendieta 2 Paolo Antonioli 2 Alessandra Bossi 2 Pier Giorgio Righetti 1, 2 Attilio Citterio 1 1 Department of Chemistry, Materials and Engineering Chemistry, Politecnico di Milano, Milan, Italy 2 Department of Agricultural and Industrial Biotechnology, University of Verona, Verona, Italy Received September 15, 2005 Revised December 21, 2005 Accepted December 23, 2005 Research Article Organic and inorganic di-cations for capillary silica coating and EOF modulation in CE: Example of application in PEG analysis EOF measurements, by using 1,4-di-(4-aza-1-azonia-bicyclo[2.2.2]octane)butane diiodide, barium and strontium tetraborate as silica wall modifiers, are reported and, as an example of application, analysis of PEG (PEG 400–2000) polydisperse preparations in free solution CZE is shown. PEGs have been derivatized with phthalic anhydride so as to form singly or doubly charged derivatives with strong UV absorbance at 214 nm. Whereas separations in plain tetraborate buffer, pH 9.0, without any EOF control, did not lead to good resolution of all-size oligomers and suffered from long analysis times, excellent resolution of all oligomers up to 40 ethylene oxide (EO) units could be obtained under EOF control. Such EOF modulation was engendered by addition of 1 mM M7C4M7, a doubly charged organic cation able to stick tenaciously to the silica wall. Further modulation of EOF and silica surface modification could be achieved also by addition of inorganic cations, notably those of group II, whereas monovalent cations did not seem to affect much the EOF flux. Among the doubly charged cations investi- gated, Ca 11 , Mg 11 , Sr 11 and Ba 11 , the latter did seem to offer best EOF control and reproducible runs. A judicious blend of M7C4M7 (0.33–1 mM range) with barium (10– 20 mM range) allowed baseline resolution of all PEG oligomers investigated up to PEG 2000 and .40 EO units in length. In this last case, best results in terms of repro- ducibility and separation efficiency of the more heavy homologues were obtained using Li 1 salt in small amounts. Keywords: Capillary electrophoresis / Electroendoosmotic flow / Quaternarized piperazines / Silica / Surface coating DOI 10.1002/elps.200500691 1 Introduction Linear PEGs are hydrophilic compounds that are widely employed as intermediates in the manufacture of non- ionic surfactants and as additives in the pharmaceutical industry. Characterization of PEGs, in terms of their chemical composition and polydispersity (average mo- lecular mass, M r , and M r distribution), is an important issue in controlling their manufacturing process and in identifying additives in commercial products. Classical methods for PEG analysis include normal phase (NP) and RP-HPLC [1], supercritical fluid chromatography [2] and size-exclusion chromatography [3]. More modern and highly reliable methods adopt mass spectrometric (MS) tools, such as MALDI-TOF [4]. From this point of view, also CZE, due to its requirements for minute sample sizes, fast analysis times and very high resolution [5], could be a formidable tool for analysis of PEGs. In fact, the molecular mass distribution of synthetic PEGs by CZE has been already reported by Kok et al. [6] and Engelhardt and Grosche [7]. Both Bullock [8] and Vanhoenacker et al. [9] demonstrated CZE analysis of PEG derivatives, up to a molecular mass of 3000 Da, after derivatization with phthalic anhydride (PhAH). These analyses are carried out in free solution, and separation of oligomers having identical charge but increasing mass values, due to variable numbers of ethylene oxide (EO) units, is obtained due to their differing M r /z values. For larger size oligomers, where separation in free solution would tend to level off, acceptable results can be still obtained by filling the capillary lumen with sieving liquid polymers [10]. Correspondence: Dr. Roberto Sebastiano, Department of Chemis- try, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, Via Mancinelli 7, Milano 20131, Italy E-mail: roberto.sebastiano@polimi.it Fax: 139-0223993080 Abbreviations: EO, ethylene oxide; M7C4M7, 1,4-di-(4-aza-1-azo- nia-bicyclo[2.2.2]octane)butane diiodide; PhAH, phthalic anhydride; R.B., running buffer Electrophoresis 2006, 27, 1495–1501 1495  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com