Spectroscopic Techniques for Structure Elucidation
Once a phytoconstituent has been isolated in pure form, its complete chemical structure must be elucidated using a complementary suite of spectroscopic techniques, each contributing distinct structural information that, combined, allows unambiguous structure determination even for entirely novel natural products.
UV-Vis spectroscopy reveals the wavelength of maximum absorption (λmax), which is diagnostic of the compound's chromophoric system; for flavonoids, the characteristic bathochromic (red) shift in λmax observed upon addition of shift reagents such as sodium hydroxide or aluminium chloride provides direct information about the pattern and position of hydroxyl substitution on the flavonoid skeleton.
FTIR spectroscopy identifies the functional groups present in a molecule by their characteristic vibrational absorption frequencies across the 4000–400 cm⁻¹ range, including broad O–H stretching around 3200–3550 cm⁻¹, carbonyl (C=O) stretching around 1700–1750 cm⁻¹, aromatic C=C stretching around 1450–1600 cm⁻¹, and C–O–C ether stretching around 1000–1300 cm⁻¹, providing a rapid functional-group fingerprint that complements the more detailed structural information obtained from NMR.
Proton (¹H) NMR, typically recorded at 400 or 600 MHz, reveals the chemical environment of each hydrogen atom through its characteristic chemical shift, coupling pattern, and coupling constant, providing detailed information about the connectivity and stereochemistry of the molecule; carbon (¹³C) NMR and the DEPT (Distortionless Enhancement by Polarisation Transfer) experiment together establish the complete carbon framework, distinguishing methyl, methylene, methine, and quaternary carbons. Two-dimensional NMR experiments provide the definitive structural evidence required for novel compound elucidation: COSY reveals proton-proton coupling networks, HSQC reveals direct one-bond carbon-hydrogen correlations, and HMBC reveals longer-range two- to three-bond carbon-hydrogen correlations that allow entire ring systems and substituent attachment points to be established unambiguously.
High-resolution mass spectrometry, commonly using electrospray ionisation coupled to a quadrupole time-of-flight (ESI-QTOF) analyser, establishes the exact molecular mass and, from it, the molecular formula of a compound with high confidence, while the fragmentation pattern observed provides further structural clues; characteristic neutral losses, such as 162 Da for a glucose unit or 146 Da for a rhamnose unit, are particularly diagnostic for glycosidic natural products and allow the sugar substitution pattern to be inferred directly from the mass spectrum.