Triphenyl-Modified Mixed-Mode Stationary Phases With and Without Embedded Ion-Exchange Sites for High-Performance Liquid Chromatography

Abstract

The present work reports on the preparation, characterization, and evaluation of a set of novel triphenyl-modified silica-based stationary phases without and with embedded ion-exchange sites for mixed-mode liquid chromatography. The three synthesized triphenyl phases differed in additionally incorporated ion-exchange sites. In one embodiment, allyltriphenylsilane was bonded to thiol-modified silica by thiol-ene click reaction, leading to particles with no ion-exchange sites. A second stationary phase was obtained by thiol-yne click reaction of thiol silica with 2-propinyl-triphenylphosphonium bromide, yielding a strong anion-exchanger (SAX). A third stationary phase was obtained from this SAX phase by the oxidation of residual thiols to sulfonic acid moieties, leading to a zwitterionic surface. All synthesized materials were subjected to elemental analysis, ¹³C and ²⁹Si solid-state cross-polarization/magic angle spinning nuclear magnetic resonance (CP/MAS NMR) spectroscopy analysis, and pH-dependent ζ-potential determinations via electrophoretic light scattering. The prepared stationary phases were chromatographically evaluated under classical reversed-phase, ion-exchange, and hydrophilic interaction chromatography conditions and classified within a set of commercially available columns by principal component analysis of retention factors. Finally, the obtained stationary phases were applied for biomolecule separations (e.g., teicoplanin and siRNA patisiran). These LC tests proved the orthogonality of the three prepared stationary phases and indicated possible fields of application.

Keywords: mixed‐mode chromatography; oligonucleotide; silatrane; stationary phase; thiol‐yne/ene click reaction.

FIGURE 1

Surface chemistries of the prepared stationary phases. Synthesis procedures for C4‐SP and C18‐SP are described in the supplementary material (see Figure S3). All stationary phases were manufactured starting from the same spherical silica (3 µm, 300 Å, 100 m²/g).

FIGURE 2

Synthesis scheme for the preparation of the triphenyl‐SAX‐SP by thiol‐yne/ene click reaction. After thermally initiated radical formation, the radical is transferred from the decomposition product of the initiator to the thiol groups. The thiyl radical generated causes a hydrothiolation reaction on the double or triple bonds of the reaction partners. As a result, the formation of a thioether takes place subsequently. In this process, a carbon‐centered radical is formed intermediately, which provokes a prolonged reaction by transferring the radical to the next thiol group. Therefore, double bonds can react once with one thiol group, whereas triple bonds can react with up to two thiol groups.

FIGURE 3

(A) ²⁹Si CP/MAS NMR analysis of SH‐SP and (B) ¹³C CP/MAS NMR spectra for SH‐SP, triphenyl‐SAX‐SP and triphenyl‐ZWIX‐SP. (A) The ²⁹Si NMR spectrum of SH‐SP exhibits the three characteristic signals for modified silica (T1, T2, and T3) beside the typical signals for the silica support (Q3: free single and vicinal silanol groups, Q4: siloxane groups). T1, T2, and T3 can be assigned to the silicon atoms of the diverse 3‐mercaptopropyl siloxane bonds immobilized on the silica surface, giving information about the proportion of mono‐, di‐, and trifunctional siloxane‐linked moieties. (B) ¹³C NMR spectra illustrate the successful synthesis of SH‐SP, triphenyl‐SAX‐SP, and triphenyl‐ZWIX‐SP. Thus, the successful immobilization of the phenyl ligands is proven as well as the accomplished oxidation of the residual sulfhydryl groups. The signal at approximately 50 ppm (bright green) can be attributed to methoxy groups generated on the silica surface due to the methanolic solvent used in the synthesis.

FIGURE 4

ζ Potentials of the modified silica particles determined by electrophoretic light scattering at different pH values (10 mM KCl in 1 mM buffer).

FIGURE 5

Normalized radar plot based on chromatographic investigation under Tanaka test conditions for RP‐type phases. Further information concerning chromatographic conditions and parameters can be found in Figures S8 and S9 and Tables S1 and S2.

FIGURE 6

Evaluation of the ion exchange characteristics of the triphenyl‐SP, triphenyl‐SAX‐SP and triphenyl‐ZWIX‐SP at pH 3 and pH 7.5. Chromatographic conditions: The mobile phase consisted of MeOH/aqueous ammonium phosphate buffer (20 mM, adjusted to pH 3 or pH 7.5) (30/70, v/v), flow rate: 1 mL/min, temperature: 25°C, injection volume: 5 µL.

FIGURE 7

Score plot of principal component analysis (PCA) for column classification. The corresponding loading plot can be found in Figure S13.

FIGURE 8

(A) Analysis of the biomolecules teicoplanin and (B) patisiran. The chemical structure of the biomolecules can be found in Figures S14 and S15. (A) Analysis of teicoplanin subspecies utilizing the set of triphenyl‐modified SPs and comparative RP‐SPs. Chromatographic conditions: Mobile Phase A consisted of water containing 10 mM ammonium acetate (pH adjusted to pH 5.4), mobile Phase B consisted of mobile Phase A and acetonitrile (5/95, v/v), flow rate: 1.0 mL/min, gradient time: 20 min (10% B to 100% B) temperature: 30°C, injection volume: 3 µL, detection: 210 nm. (B) Comparison of patisiran sense and antisense strand analysis on the three triphenyl‐modified SPs. Chromatographic conditions: Mobile Phase A consisted of water containing 20 mM ammonium acetate, mobile Phase B consisted of mobile Phase A and methanol (90/10, v/v), flow rate: 0.6 mL/min, gradient time: 10 min (5% B to 20% B), temperature: 40°C, injection volume: 5 µL, detection: 254 nm.