Journal of Chromatography A, 1370 (2014) 255–262 Contents lists available at ScienceDirect Journal of Chromatography A j o ur na l ho me page: www.elsevier.com/locate/chroma Generalized polymer effective charge measurement by capillary isotachophoresis Joseph Chamieh a,1 , Duˇ san Koval b,1 , Adeline Besson a , Václav Kaˇ siˇ cka b , Hervé Cottet a,∗ a Institut des Biomolécules Max Mousseron (UMR 5247 CNRS—Université de Montpellier 1—Université de Montpellier 2), place Eugène Bataillon CC 1706, 34095 Montpellier Cedex 5, France b Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo n. 2, 166 10 Prague 6, Czech Republic a r t i c l e i n f o Article history: Received 14 June 2014 Received in revised form 3 October 2014 Accepted 9 October 2014 Available online 18 October 2014 Keywords: Polymer effective charge Polyelectrolyte Isotachophoresis Counter-ion condensation Capillary electrophoresis a b s t r a c t In this work, we have generalized the use of capillary isotachophoresis as a universal method for deter- mination of effective charge of anionic and cationic (co)polymers on ordinary capillary electrophoresis instruments. This method is applicable to a broad range of strong or weak polyelectrolytes with good repeatability. Experimental parameters (components and concentrations of leading and terminating elec- trolytes, capillary diameters, constant electric current intensity) were optimized for implementation in 100 m i.d. capillaries for both polyanions and polycations. Determined values of polymer effective charge were in a very good agreement with those obtained by capillary electrophoresis with indirect UV detection. Uncertainty of the effective charge measurement using isotachophoresis was addressed and estimated to be ∼5–10% for solutes with mobilities in the 20–50 × 10 -9 m 2 V -1 s -1 range. © 2014 Elsevier B.V. All rights reserved. 1. Introduction The effective charge of a macromolecule can be defined as the real charge of the macromolecule entity taking into account all the ionized groups and any ionic species tightly associated with it [1]. This parameter has a primary role in the control of interactions between charged species in solution [2–6]. It sets the strength of the relatively long-range electrostatic force between charged com- pounds. It also plays a great role in entropic effects such as those observed during the formation of polyelectrolyte complexes [7] due to counter-ion release. The determination of polymer effective charge remains a challenging issue because it depends on multiple parameters such as pH (dissociation or protonation of ionogenic moieties), counter-ion condensation [8,9] and possibly on specific interactions such as hydrophobic effects [10–12] and weak inter- actions [13]. Different methods have been investigated for polymer effec- tive charge determination including conductivity measurements [14], osmotic pressure [15], scattering techniques [16] and elec- trophoretic techniques [17–25]. Conductivity and osmotic pressure measurements are generally performed in the absence of addi- tional salts (or at very low ionic strength), while neutron scattering ∗ Corresponding author. Tel.: +33 4 6714 3427; fax: +33 4 6763 1046. E-mail address: hcottet@univ-montp2.fr (H. Cottet). 1 These authors contributed equally. techniques requires specific and restricted-access source of radi- ations. Electrophoretic techniques can be applied at a given ionic strength in the typical 5–100 mM range. Regarding electrophoretic methods, one can distinguish two kinds of approaches. A first group is based on electrophoretic mobility measurement combined to electrophoretic modeling, as described, e.g. in ref [25]. These approaches are generally based on the experimental deter- mination of the electrophoretic mobility and size (hydrodynamic radius) in combination with a theoretical model. This approach is well suited for small ions and nanoparticles (hardcore charged spheres) but is not yet applicable to polyelectrolytes due to the lack of suitable theoretical model. Numerical simulations can also be used [26–31] but these approaches are computationally time consuming and, as a consequence, limited so far to the study of oligomeric chains. A second group is basically relying on the Kohlrausch reg- ulating function (KRF) [19] or on electroneutrality and electric current conservation [12,17,20], allowing a direct determination of the effective charge by capillary electrophoresis using indirect UV detection mode (IUV) [19] or by capillary isotachophoresis (ITP) using conductometric or UV-absorption detection [12,17,20]. The IUV method consists in determining the effective charge from the transfer ratio (i.e. the quantity of chromophore displaced per mole of analyte), which is measured from the sensitivity of detection (i.e. from the peak area of solute knowing its injected concentration). Applicability of the IUV method relies on the availability of a chro- mophore that absorbs UV radiation at the wavelength, at which the http://dx.doi.org/10.1016/j.chroma.2014.10.025 0021-9673/© 2014 Elsevier B.V. All rights reserved.