Summary Advanced Food Chemistry – Phenolics
Structure & biosynthesis
• Phenolics are in fruits, vegetables and nuts.
• Phenolics are present in low concentrations but reactive so large effect on quality!
• Organic compounds containing an aromatic hydrocarbon ring with at least 1 OH group.
1 OH = monophenol, 2 OH = diphenol, >3 OH = polyphenol
• Synthesized from the activated building blocks (precursors) p-coumaryl-CoA and malonyl-
CoA → ratio depends subclass of phenol
• OH group can be methylated → strictly no phenolic anymore
• Phenols can form oligomeric structures by
o Covalent linkages: carbon-carbon, ether (C-O), ester bonds or amide bonds
o Con-covalently linkages: stacking, metal chelation by OH groups, hydrophobic
interactions, H-bonds
Hydroxycinnamic acid (1 ring)
• Building block of a lot of compounds
• Contains a COOH group so it can form an ester with alcohol. For instance ester of caffeic acid
with quinic acid → chlorogenic acid
• They can form phenol-amides (contain an NH2 group)
Stilbene
Stilbenes (2 rings)
• Ratio: 1 p-coumaryl-CoA : 3 malonyl-CoA
Flavonoids (3 rings)
• Ratio: 1 p-coumaryl-CoA : 3 malonyl-CoA
Flavonoid
Analysis & isolation
Step 1: extraction
• Defatting with hexane before extraction → higher recovery of phenolics & cleaner extract
• Select a suitable extraction solvent (organic solvent) → comparable polarity with phenols and
low boiling point (easy solvent evaporation after extraction). For instance methanol. Mixing of
extraction solvents is possible
• Re-extract the residual material of the extraction → continue until absorbance of last extract
is less than 5% of the sum of the absorbances of all previous extracts. Measure this with UV-
Vis at 280nm. Proteins are also show absorbance at this wavelength so you might overestimate
the amount of phenolics that is still in the residue. Too low wavelength: too much interference
because a lot of compounds show absorbance. Too high wavelength: not all compounds show
absorbance, too specific.
• Ratio between solvent and dry matter: important for extraction efficiency. With a little
solvent, you have to do more cycles
Step 2: sample clean up (removal of impurities)
• Solid-phase extraction (SPE):
o Apolar, carbon based (C18) stationary phase is used so the apolar phenolics bind to
this. Polar mobile phase elutes oligosaccharide contaminants. Mobile phase is polar so
reversed-phase SPE.
o After removal of oligosaccharides, the bound phenolics can be eluted by an apolar
mobile phase. First less apolar molecules elute. Most apolar molecules elute latest.
• Liquid-liquid extraction (LLE) / partitioning:
o Vigorous mixing → impurities and phenolics have the opportunity to distribute
themselves over two phases. Larger solubility difference = easier separation
o For instance carbohydrates in water phase and phenolics in ethyl acetate phase.
o You can also use this method (1) to defat the extract. Or (2) to remove e.g. sugars
, o Or (3) to transfer your compounds to organic solvent → easier to remove than by
freeze drying to remove water
• In both methods, pH of the extraction solvent is important because it depends the charge of
the phenolics! High pH = COO- instead of COOH → COO- makes the phenolic polar…
• Removal of the organic solvent after SPE or LLE:
o Longer stability of phenolics at lower water content & easier quantification
o Remove organic solvents by rotating evaporator. After that, dissolve them in water
and freeze dry them. Direct freeze-drying is not possible because their boiling point is
too low and this would lead to loosing phenolic material.
o Disadvantage: phenolics are not well water-soluble after freeze drying → compact,
inhomogeneous material is obtained after freeze drying
o Solution: use tert-butanol (organic solvent that is solid at room temperature) → does
not boil during freeze drying and phenolics are well soluble in tert-butanol
Step 3: analyses
Simple analyses
• UV-Vis: phenolics absorb light between 260 and 280nm because of their aromatic ring + OH
o Disadvantage: also proteins show absorbance so not accurate in mixtures
• Folin-Ciocalteu (total phenolics assay)
o Reaction between reducing substance and reagent → blue complexes
o Disadvantage 1: overestimation because also other reducing compounds
o Disadvantage 2: gallic acid is used as standard but there may be other phenolics that
have a higher response factor (more blue colour) but are not more present
• DPPH assay (antioxidant activity)
Advanced analytical methods
• Reversed phase LC coupled to PDA (UV-Vis detection) and MS (separation based on mass)
o Separation and detection of single compounds by UV-Vis and MS
o Advantages:
▪ You can use it for complex mixtures because also separation on mass by MS
▪ More sensitive than NMR (= can measure lower concentrations)
o Disadvantages:
▪ More laborious than upper methods (but more accurate)
▪ For best result you need calibration curve (standard compounds) for each
phenolic compound. When not available: (1) use molar extinction coefficient (=
how much light absorbs it compared to the compound you DO have a standard
from) or (2) express the quantity as equivalent of another structurally related
compound
• NMR spectroscopy
o Advantages: standard way, can be used for a lot of structures
o Disadvantages: difficult to apply in mixtures, difficult to interpretate
o 1 dimensional: 1H and 13C NMR → analyse the protons and carbon atoms in your
structure
o 2 dimensional: correlations between different atoms in the structure. COSY:
correlations between protons and other protons in the structure.
o Peak area can be used to see how much of the atom is present.
Why to isolate pure phenolic compounds?
• To elucidate structures of new compounds by NMR
• To determine its chemical properties (e.g. molar extinction coefficient)
• To asses the bioactivity and perform QSAR studies
• To use the pure compound as standard for future quantification
How to isolate pure phenolic compounds from a plant extract?
• Extract of the plant extract (defatting and extraction with methanol)
• Sample clean up (SPE or LLE)
• Pre-purification (for instance by flash chromatography) → fractions high in a certain compound
• Purification by preparative LC
➔ It takes weeks to do this and you have low yields!
Structure & biosynthesis
• Phenolics are in fruits, vegetables and nuts.
• Phenolics are present in low concentrations but reactive so large effect on quality!
• Organic compounds containing an aromatic hydrocarbon ring with at least 1 OH group.
1 OH = monophenol, 2 OH = diphenol, >3 OH = polyphenol
• Synthesized from the activated building blocks (precursors) p-coumaryl-CoA and malonyl-
CoA → ratio depends subclass of phenol
• OH group can be methylated → strictly no phenolic anymore
• Phenols can form oligomeric structures by
o Covalent linkages: carbon-carbon, ether (C-O), ester bonds or amide bonds
o Con-covalently linkages: stacking, metal chelation by OH groups, hydrophobic
interactions, H-bonds
Hydroxycinnamic acid (1 ring)
• Building block of a lot of compounds
• Contains a COOH group so it can form an ester with alcohol. For instance ester of caffeic acid
with quinic acid → chlorogenic acid
• They can form phenol-amides (contain an NH2 group)
Stilbene
Stilbenes (2 rings)
• Ratio: 1 p-coumaryl-CoA : 3 malonyl-CoA
Flavonoids (3 rings)
• Ratio: 1 p-coumaryl-CoA : 3 malonyl-CoA
Flavonoid
Analysis & isolation
Step 1: extraction
• Defatting with hexane before extraction → higher recovery of phenolics & cleaner extract
• Select a suitable extraction solvent (organic solvent) → comparable polarity with phenols and
low boiling point (easy solvent evaporation after extraction). For instance methanol. Mixing of
extraction solvents is possible
• Re-extract the residual material of the extraction → continue until absorbance of last extract
is less than 5% of the sum of the absorbances of all previous extracts. Measure this with UV-
Vis at 280nm. Proteins are also show absorbance at this wavelength so you might overestimate
the amount of phenolics that is still in the residue. Too low wavelength: too much interference
because a lot of compounds show absorbance. Too high wavelength: not all compounds show
absorbance, too specific.
• Ratio between solvent and dry matter: important for extraction efficiency. With a little
solvent, you have to do more cycles
Step 2: sample clean up (removal of impurities)
• Solid-phase extraction (SPE):
o Apolar, carbon based (C18) stationary phase is used so the apolar phenolics bind to
this. Polar mobile phase elutes oligosaccharide contaminants. Mobile phase is polar so
reversed-phase SPE.
o After removal of oligosaccharides, the bound phenolics can be eluted by an apolar
mobile phase. First less apolar molecules elute. Most apolar molecules elute latest.
• Liquid-liquid extraction (LLE) / partitioning:
o Vigorous mixing → impurities and phenolics have the opportunity to distribute
themselves over two phases. Larger solubility difference = easier separation
o For instance carbohydrates in water phase and phenolics in ethyl acetate phase.
o You can also use this method (1) to defat the extract. Or (2) to remove e.g. sugars
, o Or (3) to transfer your compounds to organic solvent → easier to remove than by
freeze drying to remove water
• In both methods, pH of the extraction solvent is important because it depends the charge of
the phenolics! High pH = COO- instead of COOH → COO- makes the phenolic polar…
• Removal of the organic solvent after SPE or LLE:
o Longer stability of phenolics at lower water content & easier quantification
o Remove organic solvents by rotating evaporator. After that, dissolve them in water
and freeze dry them. Direct freeze-drying is not possible because their boiling point is
too low and this would lead to loosing phenolic material.
o Disadvantage: phenolics are not well water-soluble after freeze drying → compact,
inhomogeneous material is obtained after freeze drying
o Solution: use tert-butanol (organic solvent that is solid at room temperature) → does
not boil during freeze drying and phenolics are well soluble in tert-butanol
Step 3: analyses
Simple analyses
• UV-Vis: phenolics absorb light between 260 and 280nm because of their aromatic ring + OH
o Disadvantage: also proteins show absorbance so not accurate in mixtures
• Folin-Ciocalteu (total phenolics assay)
o Reaction between reducing substance and reagent → blue complexes
o Disadvantage 1: overestimation because also other reducing compounds
o Disadvantage 2: gallic acid is used as standard but there may be other phenolics that
have a higher response factor (more blue colour) but are not more present
• DPPH assay (antioxidant activity)
Advanced analytical methods
• Reversed phase LC coupled to PDA (UV-Vis detection) and MS (separation based on mass)
o Separation and detection of single compounds by UV-Vis and MS
o Advantages:
▪ You can use it for complex mixtures because also separation on mass by MS
▪ More sensitive than NMR (= can measure lower concentrations)
o Disadvantages:
▪ More laborious than upper methods (but more accurate)
▪ For best result you need calibration curve (standard compounds) for each
phenolic compound. When not available: (1) use molar extinction coefficient (=
how much light absorbs it compared to the compound you DO have a standard
from) or (2) express the quantity as equivalent of another structurally related
compound
• NMR spectroscopy
o Advantages: standard way, can be used for a lot of structures
o Disadvantages: difficult to apply in mixtures, difficult to interpretate
o 1 dimensional: 1H and 13C NMR → analyse the protons and carbon atoms in your
structure
o 2 dimensional: correlations between different atoms in the structure. COSY:
correlations between protons and other protons in the structure.
o Peak area can be used to see how much of the atom is present.
Why to isolate pure phenolic compounds?
• To elucidate structures of new compounds by NMR
• To determine its chemical properties (e.g. molar extinction coefficient)
• To asses the bioactivity and perform QSAR studies
• To use the pure compound as standard for future quantification
How to isolate pure phenolic compounds from a plant extract?
• Extract of the plant extract (defatting and extraction with methanol)
• Sample clean up (SPE or LLE)
• Pre-purification (for instance by flash chromatography) → fractions high in a certain compound
• Purification by preparative LC
➔ It takes weeks to do this and you have low yields!
