Enhancing effectiveness of capillary electrophoresis as an analytical tool in the supramolecular acidity modification

A strategic modification of acidity (pK a values) by the non-covalent host-guest interactions is one of the most promising concepts in current supramolecular chemistry. This work is aimed at enhancing the effectiveness of capillary electrophoresis (CE) in determination of pK a shifts caused by such interactions and their thermal dependencies crucial in a deep thermodynamic description. We show how to (i) minimize the systematic errors related to Joule heating, (ii) minimize the influence of a voltage ramp time, (iii) speed up pK a shift identification and estimation, (iv) interpret thermal effects related to two overlapped dynamic equilibria, and (v) determine pK a shifts by an alternative spectrophotometric method (CE-DAD). The proposed solutions were implemented to examine the supramolecular pK a shifts of several coumarin derivatives, caused by a variety of structurally different cyclodextrins. It was revealed that a specific host substitution pattern determines the magnitude of apparent pK a shifts. Accordingly, heptakis(2,6-di-O-methyl)-β-cyclodextrin induces the much stronger shifts than both non-methylated-β-cyclodextrin and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin applied at the same concentration. We also show that insofar as the complexation of 4-hydroxycoumarin and its derivative (coumatetralyl) are similarly exothermic, the thermal effects accompanying the deprotonation process are remarkably different for both molecules. The pK a shift induced by complexation with calixarene was also for the first time determined by a CE method. These observations throw a new light on the background of acidity modification and confirm the applicability of CE as an analytical tool. Electronic supplementary material The online version of this article (doi:10.1007/s00216-017-0305-y) contains supplementary material, which is available to authorized users.

The amounts of the given ingredients were calculated to ensure the constant ionic strength of 50 mM. The pH values presented in the table refer to the host-free solutions (without cyclodextrins and calixarene). For the host-containing buffers the exact pH values were measured prior to CE analysis.

Calculation of local temperatures by SUMET
According to the Krylov's method [1]: where ΔTcore is the temperature rise in the thermostated capillary section, ΔTi nlet is the temperature rise in the non-thermostated section, Pav is the average electric power (W), Ltot is the total capillary length (0.60 m), while c, n, g, k and a are the parameters valid for the chosen capillary, in this case (50 µm i.d.): c = 6203, n = 0.147, g = 7608, k = 7.16 and a = 0.953 [1].
Pav was calculated as: where Unom is the nominal separation voltage (30 kV); tramp is the ramp time, 0.17 min; ttot is the migration time of a given analyte (for which electrophoretic mobility is calculated), Iexp is the current measured after the ramp time, at the 30 th second of run.
The actual (effective) temperature values, obtained using Eq.8 (main text), are presented below:

Calculation of ΔH° and ΔS°
The values of standard deprotonation enthalpy (ΔH°) and entropy (ΔS°) were calculated from the Van't Hoff model describing the relation between pKa and temperature: where R is the gas constant (8.3145 J·mol -1 ·K -1 ).
Accordingly, the pKa values determined at various temperature were plotted against the inverse absolute temperature (1/T) and fitted by the linear function. Subsequently the enthalpic and entropic terms were calculated from the slope and intercept, respectively, see Fig.S2. In our recent work we postulated that the eCAP TM amine capillary is an optimal capillary type for determination of pKa by CE owing to a great repeatability of electrophoretic mobility values, crucial in the minimization of the random errors [2]. In another work we showed that it also ensures a repeatable analysis in the micellar media [3]. In this work the amine capillary was for the first time applied to determine the supramolecular pKa shifts. A simple test was performed. We estimated repeatability of electrophoretic mobilities of four analytes, expressed by the RSD (%) values, without and after the addition of 9 structurally diverse CDs. We also compared two pH values, where the analytes occur in the partially ionized (pH 4.6) and totally ionized (pH 8.0) forms. The results are shown in Table S3. As it is seen the RSD values are very low, similarly for each analyte. Some differences between two pH values are noted, mostly in favor of the higher pH value. The repeatability is in general similarly good for the reference (host-free) and host-containing systems, the RSD values rarely surpass 1% for some CD-containing buffers. This confirms that the amine capillary exhibits a stable EOF and it is a suitable choice for the repeatable determination of electrophoretic mobility in various systems. It is especially important in regard to the media containing the macrocyclic molecules where electrophoretic mobility derives from various types of physicochemical interactions.
Another issue is that in the amine capillary the average migration times noted for the negatively ionized molecules are typically much shorter than in the uncoated bare silica capillary. It results from the stronger electroosmosis induced by the polyaminecoated inner capillary surface and its opposite direction, entailing migration of the negatively ionized analytes consistently with EOF direction. This enables their detection prior to the EOF marker, see Fig.3 in the main text. Therefore, the amine capillary may ensure both a very high repeatability and fast analysis of the acidic molecules.