Mechanism of action of quaternary diamino quenchers in capillary zone electrophoresis

Abstract

The synthesis of two novel amino compounds, able to quench and reverse the electroosmotic low (EOF) in capillary electrophoresis is here reported. These chemicals are derivatives of two previously described quaternarized piperazines, 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide (M1C4I) and 1-(4-iodobutyl)4-aza-1-azoniabicyclo[2.2.2] octane iodide (M7C4I), believed to bind covalently to the silica surface via alkylation of ionized silanols through their terminal, reactive iodine. The novel compounds, although unable to form a covalent bond, due to lack of the o-butyl iodine, are found to be very efficient in suppressing protein interaction with the wall and reversing the EOF. On the basis of their behavior in solution, the minimal structural motifs for strong binding of amino compounds to the silica wall have been derived and are thought to be: (i) the presence of two quaternary nitrogens in the molecule; (ii) a correct distance between said charged nitrogens, represented by a butyl chain (C(4)); (iii) an hydrophobic "decoration" of the molecules, consisting on a high CH(2)/N ratio (in the present case 8:1).

Phospholipid‐lysozyme coating for chiral separation in capillary electrophoresis

“…M1C4, which has just recently been introduced in CE, has a similar chemical structure to HEPES (see Fig. 1), but unlike HEPES it can effectively suppress and even reverse the EOF [44][45][46][47], which would be a beneficial property for enhancing the chiral resolution. In this study, M1C4 coating was employed to stabilize the phospholipid coating and to improve the chiral separation based on lysozyme.…”

“…1) [44][45][46][47]. As a kind of novel trifunctional diamine, the M1C4 coating can effectively quench the EOF in three ways: it forms ionic bonds with dissociated silanols via its quaternary nitrogen, hydrogen bonds via its tertiary nitrogen and, most importantly, covalent bonds via alkylation of ionized silanols through the terminal iodine atom in the butyl chain [44,45,47].…”

“…Use of 20 mM Tris at pH 9.0, instead of 20 mM Tris at pH 7.4, as the buffer solution for the M1C4 coating, resulted in a lower and more stable EOF. This was due to strong covalent binding of M1C4 to the capillary surface at higher pH values [44][45][46][47]. The M1C4 coating layer was stable after extended washing with 20 mM Tris at pH 7.4, which was used as the buffer solution for the subsequent phospholipid coating.…”

Recent applications of affinity interactions in capillary electrophoresis

“…A composite method for chiral separations in CE involving phospholipid lysozyme stably immobilized on the capillary wall through a derivative of quaternarized piperazine called M1C4 [48] successfully separated D-and L-tryptophan, and may be useful as a general approach for chiral separations based on phospholipid-protein wall coatings [49].…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…Purified mAb stocks (51.1 mg/mL) were used as samples. The (M7C4I) reagent was synthesized according to previous reports [277]. IdeS was purchased from Genovis (Lund, Sweden).…”