This work describes a novel sequential injection chromatography (SIC) method combined with linear gradient elution for the separation and determination of three main methylxanthines (theobromine, theophylline and caffeine) using a short C18 monolithic column. The method utilizes a hybrid manifold which exploits zone fluidics for solution manipulation and programmable fluidics for the implementation of a two-solvent linear gradient elution protocol. This approach offers a high degree of automation and enables faster separation of the three target methylxanthines with respect to isocratic elution as well as better chromatographic efficiency. The limits of detection of the three methylxanthines ranged from 0.18 to 0.45 μmol L, the instrumental repeatability (at the 10 μmol L level of the target compounds, n = 6) was ≤2% and the separation time was 3.3 min. The SIC method was validated and applied to the analysis of coffee, chocolate and tea samples.
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