Temperature-responsive mixed-mode column for the modulation of multiple interactions

Abstract

In this study, mixed-mode chromatography columns have been investigated using multiple analyte interactions. A mixed-mode chromatography column was developed using poly(N-isopropylacrylamide) (PNIPAAm) brush-modified silica beads and poly(3-acrylamidopropyl trimethylammonium chloride) (PAPTAC) brush-modified silica beads. PNIPAAm brush-modified silica beads and PAPTAC brush-modified silica beads were prepared by atom transfer radical polymerization. The beads were then packed into a stainless-steel column in arbitrary compositions. The elution studies evaluated the column performance on hydrophobic, electrostatic, and therapeutic drug samples using steroids, adenosine nucleotide, and antiepileptic drugs as analytes, respectively. Steroids exhibited an increased retention time when the column temperature was increased. The retention of adenosine nucleotides increased with the increasing composition of the PAPTAC-modified beads in the column. The antiepileptic drugs were separated using the prepared mixed-mode columns. An effective separation of antiepileptic drugs was observed on a 10:1 PNIPAAm:PAPTAC column because the balance between the hydrophobic and electrostatic interactions with antiepileptic drugs was optimized for the bead composition. Oligonucleotides were also separated using mixed-mode columns through multiple hydrophobic and electrostatic interactions. These results demonstrate that the developed mixed-mode column can modulate multiple hydrophobic and electrostatic interactions by changing the column temperature and composition of the packed PNIPAAm and PAPTAC beads.

Figure 1

Temperature-responsive mixed-mode column using thermoresponsive-polymer-modified beads and cationic-polymer-modified beads. Schematic illustration of the (A) preparation of PNIPAAm (left) and PAPTAC (right) brush-modified beads, (B) preparation of the mixed-mode column by packing two types of beads, and (C) multiple types of interactions with the analyte. (Figures were drawn Microsoft PowerPoint 2019 Version 2112).

Figure 2

Characterizations of the prepared beads. (A) FT–IR spectra of the prepared beads. The dashed lines, (i) and (ii), indicate the peaks attributed to the C=O stretching and N–H bending vibrations, respectively. (B) Zeta potential of the prepared silica beads (n = 3). (C) SEM images of the prepared beads where the scale bars = 2 μm. (Figures were drawn Microsoft PowerPoint 2019 Version 2112).

Figure 3

Chromatograms of hydrophobic steroids using prepared mixed-mode columns. The mobile phase was pure water. The flow rate of the mobile phase was 1.0 mL/min, and detection was measured at a wavelength of 260 nm. Peak 1 = hydrocortisone, peak 2 = prednisolone, peak 3 = dexamethasone, peak 4 = hydrocortisone acetate, and peak 5 = testosterone. (Figures were drawn Microsoft PowerPoint 2019 Version 2112).

Figure 4

Chromatograms of adenosine nucleotides using prepared mixed-mode columns. The mobile phase is 33.3 mmol/L phosphate buffer solution (pH = 7.0). The flow rate of the mobile phase is 1.0 mL/min, and detection was measured at a wavelength of 260 nm. The column temperature is 40 °C. Peak 1 = AMP, peak 2 = ADP, and peak 3 = ATP. (Figures were drawn Microsoft PowerPoint 2019 Version 2112).

Figure 5

Chromatograms of the antiepileptic drugs on the prepared mixed-mode columns. The mobile phase is 10 mM CH₃COONH₄ buffer solution (pH 6.8) with a flow rate of 1.0 mL/min, and detection was measured at a wavelength of 260 nm. Peak 1 = zonisamide, peak 2 = carbamazepine, peak 3 = nitrazepam, and peak 4 = clonazepam. (Figures were drawn Microsoft PowerPoint 2019 Version 2112).

Figure 6

Retention times of the antiepileptic drugs on the prepared mixed-mode columns. (Figures were drawn Microsoft PowerPoint 2019 Version 2112).

Figure 7

van’t Hoff plots of the antiepileptic drugs on the prepared mixed-mode columns. The dashed line indicates the phase transition temperature of PNIPAAm. (Figures were drawn Microsoft PowerPoint 2019 Version 2112).

Figure 8

Chromatograms of oligonucleotides on prepared mixed-mode columns. (A) Mixture of d(T)₅ and d(T)₆. The mobile phase is 66.7 mM phosphate buffer solution (pH = 7.0) with a flow rate of 1.0 mL/min. Detection was measured at a wavelength of 260 nm. Peak 1 = d(T)₅ and peak 2 = d(T)₆. (B) Mixture of d(T)₅ and d(T)₁₀. The mobile phase is 66.7 mM phosphate buffer solution (pH = 7.0) + 100 mM NaCl with a flow rate of 1.0 mL/min. Detection was measured at a wavelength of 260 nm. Peak 1 = d(T)₅ and peak 2 = d(T)₁₀. (Figures were drawn Microsoft PowerPoint 2019 Version 2112).