# Ultra-Sensitive Bioanalytical Separations Using a New 4-Tritylphenyl Methacrylate-Based Monolithic Nano-Column with an Inner Diameter of 20 µm for Nano-LC

## TL;DR

A new ultra-sensitive nano-column for liquid chromatography was developed to improve the detection and separation of biomolecules.

## Contribution

A novel 4-tritylphenyl methacrylate-based monolithic nano-column with 20 µm inner diameter was developed for enhanced bioanalytical separations.

## Key findings

- The nano-column showed run-to-run and column-to-column reproducibility of <2.51% and 2.4–3.2%, respectively.
- The column successfully separated six standard proteins and three dipeptides.
- The nano-column enabled high-resolution peptide analysis in the MCF-7 cell line.

## Abstract

Low-flow liquid chromatography has become the primary tool for advanced chromatographic analysis and is an indispensable technique for the sensitive detection of biomolecules. In this study, we developed a new 4-tritylphenyl methacrylate-based monolithic nano-column with an internal diameter of 20 µm for bioanalytical separations in nano-liquid chromatography (nano-LC). The composition of the monolith was optimized with regard to the monomer and porogenic solvent. The column was characterized using Fourier Transformed Infrared Spectroscopy (FT-IR) spectroscopy, scanning electron microscopy (SEM) and chromatographic analyses. Chromatographic characterization was performed using homologous alkylbenzenes (ABs) and polyaromatic hydrocarbons (PAHs), which facilitate hydrophobic and π–π interactions. Run-to-run and column-to-column reproducibility values were found to be <2.51% and 2.4–3.2%, respectively. The final monolith was then used to separate six standard proteins, including β-lactoglobulin A, carbonic anhydrase, ribonuclease A (RNase A), α-chymotrypsinogen (α-chym), lysozyme (Lys), cytochrome C (Cyt C) and myoglobin (Myo), as well as three dipeptides: Alanine-tyrosine (Ala-Tyr), Glycine-phenylalanine (Gly-Phe) and L-carnosine. The nano-column was then applied to profiling peptides and proteins in the MCF-7 cell line, enabling high-resolution peptide analysis.