Polymeric Microbore Monolithic Column for the Fast Determination of Milk Whey Proteins in Powder Supplements by Reversed‐phase Liquid Chromatography

ABSTRACT A rapid reversed‐phase liquid chromatographic method was developed for the separation and quantification of major whey proteins in dietary supplements using a poly(butyl methacrylate–co–ethylene dimethacrylate) monolithic column. The column structure was characterized by scanning electron microscopy, revealing a homogeneous porous network composed of ∼1 µm polymer globules and large flow‐through pores, which together provide high permeability and low backpressure, suitable for fast separations. Under a steep acetonitrile gradient, α‐lactalbumin (α‐LA), bovine serum albumin (BSA), and β‐lactoglobulin (β‐LG) were separated within 4 min, showing good peak symmetry and repeatable retention factors. The method exhibited excellent linearity ( R 2 > 0.99) over the concentration range of 0.025–0.91 mg/mL, with limits of detection ranging from 2.7 µg/mL for BSA to 6.2 µg/mL for β‐LG. Furthermore, analysis of three commercial whey protein supplements showed high intermediate precision (evaluated over three different days), with relative standard deviations below 6.0% for the major proteins (α‐LA and β‐LG) and under 12.5% for the minor component (BSA). Spike‐and‐recovery experiments confirmed the accuracy and reliability of the proposed method, yielding recovery rates ranging from 97% to 113%. β‐LG was the predominant protein, followed by α‐LA and minor amounts of BSA. The proposed approach provided fast analysis and low consumption of solvent and sample, with acceptable chromatographic efficiency, demonstrating that polymeric monolithic microbore columns constitute a potential alternative to packed columns for routine quality control of whey‐based dietary supplements.