Preparation of halloysite nanotube-based monolithic column for small molecules and protein analysis

Abstract

s: This study aimed to prepare a new separation medium, silane coupling agent KH570- modified halloysite nanotube (MPS-HNT) monolithic column, with excellent separation performance for small molecular compounds and macromolecular proteins. This was prepared using the principle of redox polymerization with modified HNTs as monomers. The optimal monomer proportion was obtained by optimizing the ratio of monomer, cross-linker, and pore-forming agent, which was evaluated using scanning electron microscopy, nitrogen adsorption, and mercury intrusion. The monolithic column exhibited a relatively homogeneous pore structure and good separation performance and permeability. As a high-performance liquid chromatography stationary phase, six small aromatic molecules were successfully separated in 6 min with a theoretical plate count of 35, 640 plates m⁻¹ for benzene. Twenty-one peaks were separated from the fermentation mattress extract containing lipopeptide antibiotics on the MPS-HNT column. The separated chromatographic peaks further identified three lipopeptide antibiotics using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. Twelve chromatographic peaks were isolated from chicken egg whites, and the eighth peak was identified as the lysozyme. The methodological validation of the lysozyme in chicken egg white showed that the linear correlation coefficient was 0.9996. The intraday and inter-day relative standard deviations were 3.1–4.0 % and 1.9–3.4 %, respectively. The spike recovery rate of lysozyme was 94.20–103.31 %. The overall column displayed good stability: the relative standard deviations of the peak area of six aromatic compounds and retention time were < 3.28 %.