Study of chromatographic properties of the developed hyperbranched zwitterionic silica-based stationary phase for hydrophilic interaction liquid chromatography
Abstract
The aim of this work was to obtain a separation material with hyperbranched functional layer containing zwitterionic groups for hydrophilic interaction liquid chromatography and to characterize solute-adsorbent interactions using test compounds of various acid-base properties. A set of mobile phases with varying acidity containing 90 vol.% of acetonitrile were used for testing the interactions of analytes with the stationary phase. The concentration of the eluting ion was varied in order to evaluate the contribution of ion exchange to the retention of charged compounds at the extremes of considered pH values.
It was found that throughout the entire range of mobile phase from 2.85 to 5.76 ( 5.5-9.2), the adsorbent provided predominantly anion-exchange properties. It was caused by the quaternary amino groups formed in the first and second functional layers on the surface of the substrate. As a result, low retention factors under all the conditions for highly hydrophilic cation thiamine (log D = -4.20) were obtained. However, the retention factors and ion exchange contribution for organic anions decreased significantly with increasing pH. It was likely caused by an increase in dissociation of carboxyl, residual silanol and protonated amino groups of the stationary phase. At the same time, a decrease of the neutral analytes’ retention was noted. This was probably caused by a decrease of the adsorbed aqueous layer thickness of the stationary phase, which was directly involved in partitioning mechanism. Moreover, a possible change in the strength of adsorption solute-adsorbent interactions was involved.
Unexpected retention dependences were obtained for weak acids, when the composition of the mobile phase was varied. For benzoic acid, practically undissociated at 2.85 of the eluent, a high contribution of ion exchange to its retention was established up to 80% at 1.25 mM concentration of the eluting ion in the mobile phase. A significant increase in retention factors and the contribution of electrostatic interactions was shown for xanthine with increasing eluent pH, which was not consistent with its value of 7.60. The observations indicated in favor of using aqueous-organic pH scale when working in the hydrophilic interaction liquid chromatography mode and taking into account the influence of ion exchange on the retention of analytes even at a low degree of their dissociation.
Thus, the developed method for creating a hyperbranched zwitterionic functional layer on the surface of 3-aminopropylsilica lead to the production of a stationary phase with predominant anion-exchange properties in the working pH range. Varying the acidity and concentration of the buffer solution of the mobile phase allowed to select the conditions suitable for the separation of a multicomponent mixture containing substances of different hydrophilicity and charge.
The aim of this work was to obtain a separation material with hyperbranched functional layer containing zwitterionic groups for hydrophilic interaction liquid chromatography and to characterize solute-adsorbent interactions using test compounds of various acid-base properties. A set of mobile phases with varying acidity containing 90 vol.% of acetonitrile were used for testing the interactions of analytes with the stationary phase. The concentration of the eluting ion was varied in order to evaluate the contribution of ion exchange to the retention of charged compounds at the extremes of considered pH values.
It was found that throughout the entire range of mobile phase from 2.85 to 5.76 ( 5.5-9.2), the adsorbent provided predominantly anion-exchange properties. It was caused by the quaternary amino groups formed in the first and second functional layers on the surface of the substrate. As a result, low retention factors under all the conditions for highly hydrophilic cation thiamine (log D = -4.20) were obtained. However, the retention factors and ion exchange contribution for organic anions decreased significantly with increasing pH. It was likely caused by an increase in dissociation of carboxyl, residual silanol and protonated amino groups of the stationary phase. At the same time, a decrease of the neutral analytes’ retention was noted. This was probably caused by a decrease of the adsorbed aqueous layer thickness of the stationary phase, which was directly involved in partitioning mechanism. Moreover, a possible change in the strength of adsorption solute-adsorbent interactions was involved.
Unexpected retention dependences were obtained for weak acids, when the composition of the mobile phase was varied. For benzoic acid, practically undissociated at 2.85 of the eluent, a high contribution of ion exchange to its retention was established up to 80% at 1.25 mM concentration of the eluting ion in the mobile phase. A significant increase in retention factors and the contribution of electrostatic interactions was shown for xanthine with increasing eluent pH, which was not consistent with its value of 7.60. The observations indicated in favor of using aqueous-organic pH scale when working in the hydrophilic interaction liquid chromatography mode and taking into account the influence of ion exchange on the retention of analytes even at a low degree of their dissociation.
Thus, the developed method for creating a hyperbranched zwitterionic functional layer on the surface of 3-aminopropylsilica lead to the production of a stationary phase with predominant anion-exchange properties in the working pH range. Varying the acidity and concentration of the buffer solution of the mobile phase allowed to select the conditions suitable for the separation of a multicomponent mixture containing substances of different hydrophilicity and charge.
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References
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