SOP: Separation and Identification of Polyphenols by Paper Chromatography

GIFSQ — Global Institute for Food Safety & Quality

Principle

The phenolic fractions obtained by extraction are mixtures of many closely related compounds. Two-dimensional paper chromatography separates them on the basis of their partition between the stationary aqueous phase held by the paper and the moving solvent, and the separated spots are identified by their fluorescence in ultraviolet light, their colour reactions with specific spray reagents, and their R_f values (the ratio of the distance travelled by the compound to that travelled by the solvent front). Only spots that give a colour reaction with the ferric chloride–ferricyanide reagent are taken as polyphenols, since some fluorescent spots seen under ultraviolet light are not phenolic.

Apparatus required

• Chromatographic tank with ancillary equipment for the simultaneous development of a large number of chromatograms
• Dent and Dutta frame for multiple papers (for routine work)
• Filter paper, 9 × 10 inch square (or a full-sized Whatman paper or equivalent grade for special separations)
• Petri dish
• Large glass troughs
• Ultraviolet lamp

Reagents

a. Solvent systems for paper chromatography (selected systems; compositions as volume ratios):

• n-Butanol : acetic acid : water, 4:1:5 (v/v) — upper layer: polyphenols, sugars, acids
• n-Butanol : acetic acid : water, 4:1:2.2 (v/v) — miscible: polyphenols
• n-Butanol : 2 N HCl, printed as “131” * (v/v) — upper layer: anthocyanins
• n-Butanol : pyridine : water, 6:3:1 (v/v) — upper layer: sugars
• Benzene : acetic acid : water, 2:2:1 (v/v) — upper layer: polyphenols
• Ethyl acetate : acetic acid : water, printed as “9:22:32” * (v/v) — miscible: polyphenols, sugars
• Acetic acid : water : conc HCl, printed as “30:10:33” * (v/v) — miscible: anthocyanidins
• Formic acid : conc HCl : water, printed as “52223” * (v/v) — miscible: aglycones
• n-Butanol : benzene : formic acid : water, 100:19:10:25 (v/v) — upper layer: sugars, anthocyanins
• n-Pentanol : acetic acid : water, ratio illegible in the extracted source * — miscible: acylated anthocyanins
• Chloroform : acetic acid : water, ratio illegible in the extracted source * (v/v) — lower layer: same (acylated anthocyanins)
• Ethanol : acetic acid : water, 9:2:5 (v/v) — miscible: flavonols, flavonol glycosides
• Acetic acid : water, printed as “15385” * (v/v) — miscible: flavonol glycosides
• Acetic acid : water, printed as “2:88” * (v/v) — miscible: polyphenols
• n-Propanol : conc ammonia, printed as “723” * (v/v) — miscible: acyl groups

Starred entries are carried exactly as read from the extracted source, where the printed ratio is ambiguous (more parts than components) or illegible; they are not verified printed values. The 4:1:5 and 4:1:2.2 n-butanol–acetic acid–water systems are confirmed by the book’s own procedure text.

b. Ferric chloride–potassium ferricyanide reagent: prepare stock solutions of ferric chloride, FeCl₃·6H₂O, and potassium ferricyanide, K₃Fe(CN)₆, each containing a drop of potassium permanganate, separately. Mix the solutions freshly and dilute with water so that the concentration of each is 0.5%.

c. Ferric reagent: dissolve 3 g of AR ammonium ferric sulphate (iron alum), FeNH₄(SO₄)₂·12H₂O, in 100 ml of water and use immediately.

d. Ammoniacal silver nitrate reagent: dissolve 14 g of silver nitrate in 100 ml of water and add 6 N ammonium hydroxide until the silver oxide formed just dissolves.

e. Bis-diazotised benzidine reagent: (a) weigh 5 g of benzidine or 6 g of benzidine hydrochloride, add to 14 ml of conc HCl, stir and dissolve the suspension in 980 ml of water; (b) prepare a 10% solution of sodium nitrite. Mix 2 parts of (a) with 3 parts of (b) and use immediately.

f. Vanillin reagent: dissolve 2 g of vanillin and 1 g of toluene-p-sulphonic acid in 100 ml of absolute ethyl alcohol.

g. Toluene-p-sulphonic acid reagent: prepare a 3% solution of toluene-p-sulphonic acid in absolute alcohol.

h. 0.25 N HCl; 0.1% sodium thiosulphate solution; ammonia.

Procedure

Examine the total alcoholic extract (after removal of chlorophyll and waxy matter), the ether extract, the ethyl acetate extract and the residual aqueous concentrate by two-dimensional paper chromatography

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Use n-butanol–acetic acid–water (4:1:5 or 4:1:2.2) in the first direction and 2% acetic acid in the second direction for general separations; ascending as well as descending chromatography may be used

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After development, allow the chromatograms to air dry and observe visually and under ultraviolet light

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Expose the chromatogram to ammonia vapour — place ammonia in a petri dish and hold the paper slightly above it in a chamber saturated with ammonia vapour — then observe visually and again under ultraviolet light

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Identify the spots by colour reactions with the spray reagents below, by their fluorescence and by their R_f values

Spray reactions

• Ferric chloride–potassium ferricyanide: dip the chromatogram in reagent (b). Spots develop almost immediately. Rinse with 0.25 N HCl and wash with distilled water to remove excess reagent. o- and p-Dihydroxy, o-trihydroxy and some other easily oxidised phenols give a Prussian blue (or pale blue) spot on a white background. Only spots giving this reaction are considered polyphenols.
• Ferric reagent: use reagent (c). It gives sharp blues with pyrogallol and greens with catechol groups. Mono- and meta-hydroxyphenols present in excessively high concentrations give weak purple colourations.
• Ammoniacal silver nitrate: spray reagent (d) on the chromatograms and wash three times with chloride-free water (or distilled water) in a large glass trough. Dip in 0.1% sodium thiosulphate solution for 3–4 min and finally wash for 20 min in running tap water. Carry out all washings in the dish and air dry. The reagent gives brown or black spots with compounds containing pyrogallol or catechol nuclei: metallic grey with catechol, protocatechuic acid and some other catechol units at sufficiently high concentrations; black with pyrogallol units at high concentrations; brown with pyrogallol units at low concentrations.
• Bis-diazotised benzidine: use reagent (e). Allow 1–3 min for full colour development and wash immediately for 20 min in running tap water to avoid a yellow background from excess reagent. Air dry. The colours are the sum of the A and B phenolic moieties: resorcinol + catechol gives pale to very pale yellow (e.g. fisetin, fustin, leucofisetinidin, fisetinidol); resorcinol + pyrogallol gives deeper yellow to canary yellow; phloroglucinol + catechol or pyrogallol gives intense claret maroon (e.g. catechins, flavan-3,4-diols). Exceptions: 2,3-dihydroflavonols of the phloroglucinol series tend towards yellow; melacacidin (7,8,3′,4′-tetrahydroxy flavan-3,4-diol) consistently gives pale yellow from the free catechol groups of both nuclei.
• Vanillin: spray reagent (f), air dry and heat in a hot-air oven at 80–100°C for 5–10 min. Flavonoids with a phloroglucinol nucleus (catechins, gallocatechins, leucocyanidin, leucodelphinidin, dihydromyricetin) give a strong violet-red; those with a resorcinol nucleus (robinetinidol, leucofisetinidin, fustin) give a weak pink after more prolonged heating.
• Toluene-p-sulphonic acid: spray reagent (g), air dry and heat at 80–100°C for 5–10 min. This reagent indicates the 3,4-diol of the heterocyclic ring: flavan-3,4-diols hydroxylated in the 5 position (leucodelphinidin, leucocyanidin) give yellow-red colours after prolonged heating; those not hydroxylated in the 5 position (melacacidin, leucorobinetinidin, leucofisetinidin) give scarlet. It also converts 2,3-dihydroflavonols into flavonols on heating, whose fluorescence can be seen under ultraviolet light.

Note: the source’s Table 3-2 (colour reactions of polyphenolic compounds) is illegible in the extracted source.

R_f values of authentic polyphenolic compounds (first solvent n-butanol–acetic acid–water; second solvent 2% acetic acid)

Starred values: the printed figures for epicatechin (BAW) and vanillic acid are ambiguous in the extracted source (“Ona” and “©.86″/”OnS7”) and are carried as interpreted; the protocatechuic acid UV+NH₃ column is blank as printed.

References

Harborne, J.B., J. Chromatog., 1, 473 (1959) — cited in the book.

Pridham, J.B., Anal. Chem., 29, 1167 (1957) — cited in the book.

Bate-Smith, E.C., Biochem. J., 58, 122 (1954) — cited in the book.

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.