High-performance liquid chromatography (HPLC) is one of the most widely used analytical techniques in food science, offering robust separation and quantification of non-volatile and thermally labile compounds. The choice between normal-phase and reversed-phase chromatography depends on analyte polarity: reversed-phase (C18, C8) dominates for mid-to-low polarity compounds such as vitamins, polyphenols, and preservatives, while normal-phase and HILIC columns are preferred for highly polar analytes like sugars and organic acids. Column selection also involves particle size (sub-2 µm for UHPLC), pore size, and bonded phase chemistry.
Detector selection determines sensitivity and selectivity. UV-Vis and diode-array detectors (DAD) are standard for chromophoric compounds, while refractive index (RID) is used for sugars and non-UV-absorbing analytes. Fluorescence detectors offer orders-of-magnitude higher sensitivity for aflatoxins and vitamins, and MS detection provides structural confirmation. Application areas include quantitative analysis of sugars (HILIC-RID or ELSD), water- and fat-soluble vitamins, artificial sweeteners (aspartame, acesulfame-K), preservatives (benzoates, sorbates), polyphenols in wine and tea, and mycotoxins (aflatoxins, ochratoxin A) with fluorescence or MS detection.
Sample preparation is critical for reliable HPLC results. Solid-phase extraction (SPE) using C18, ion-exchange, or mixed-mode sorbents concentrates analytes and removes matrix interferences. Derivatization may be required for analytes lacking chromophores or fluorescence, such as amino acids (OPA/FMOC) or fatty acids. Method validation parameters include linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ), following ICH or FDA guidelines. System suitability tests (retention time RSD, tailing factor, theoretical plates) ensure consistent performance.
Modern trends include ultra-high-performance LC (UHPLC) with sub-2 µm particles for faster separations, two-dimensional LC (LC×LC) for complex food matrices, and hyphenation with high-resolution mass spectrometry for untargeted profiling. Green HPLC approaches using ethanol-water mobile phases and shorter columns are gaining traction in quality control laboratories. HPLC complements gas chromatography for non-volatile analytes and is often coupled with mass spectrometry for confirmation. Applications include mycotoxin analysis and detection of biogenic amines.
