Quantitative analysis of binary amino acid mixtures via drop-coating deposition Raman spectroscopy: feasibility and...

Drop-coating deposition Raman (DCDR) spectroscopy is a promising approach for enhancing Raman signal sensitivity for dilute biological samples. However, its reliability for the quantitative analysis of multicomponent mixtures remains insufficiently explored due to potential solute fractionation effects during droplet drying. In this work, we investigate the limits of applicability of the DCDR method for quantifying binary mixtures of proteinogenic amino acids. Raman spectra of dried droplets containing individual amino acids and their mixtures were measured and analyzed using a linear combination approach combined with least-squares fitting. As a model system, the L-alanine/l-glutamine mixture was first examined to determine optimal spectral processing parameters and sampling region within the "coffee-ring" structure. The approach was subsequently extended to binary mixtures of L-alanine with the remaining 19 proteinogenic amino acids. The results reveal that estimated amino acid relative concentrations strongly depend on the physicochemical properties of the mixture components, including solubility and molecular mobility, which affect their spatial fractionation during droplet drying. These results provide important insights into the applicability of DCDR spectroscopy for complex biological samples paving the way for future analyses of multicomponent systems under physiologically relevant conditions.