Discrimination and Structural Analysis of Gluten-Containing and Gluten-Free Flours: A Combined 2D-COS Approach and FTIR-PCA

This study investigates molecular differences between gluten-containing and gluten-free flours using Fourier Transform Infrared (FTIR) spectroscopy combined with chemometric techniques. FTIR measurements were performed to obtain spectral profiles, followed by Two-Dimensional Correlation Spectroscopy (2D-COS) to identify key spectral regions, revealing significant differences between the protein-rich amide regions of wheat flours and the carbohydrate-dominated profiles of gluten-free samples. Strong correlations between amide I and amide II bands indicated sequential protein structural changes associated with gluten presence. Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were applied to classify the samples and evaluate gluten-related differences. PCA effectively discriminated gluten-containing and gluten-free flours, with blend samples showing a gradual transition depending on gluten content. The first two principal components explained 93% of the total variance, and the PCA score plot revealed a clear separation into three distinct groups, while HCA clustered samples based on structural similarities. The combined FTIR–2D-COS–chemometric approach provides a rapid, sensitive, and non-destructive method for distinguishing gluten-containing and gluten-free flours. Blend experiments demonstrated that spectral changes could be detected even at low gluten levels, which is particularly important for gluten-related disorders where the presence of gluten is more critical than its quantity. Therefore, this methodology shows strong potential for industrial applications, especially for routine quality control and rapid screening of gluten contamination in gluten-free products.