SOP: Separation and Estimation of Individual Anthocyanins
GIFSQ — Global Institute for Food Safety & Quality
Principle
Individual anthocyanins must be separated before each one can be quantified. With fresh or frozen fruits the crude pigment may be separated without purification; with sweetened products, preliminary purification (basic lead acetate precipitation or Amberlite CG-50 ion-exchange chromatography) is essential to remove or reduce the sugar content and concentrate the anthocyanins. The pigments are separated by multiple ascending chromatography on Whatman No. 1 paper with n-butanol–benzene–formic acid–water (100 : 19 : 10 : 25), and the individual bands are measured densitometrically (preferred) or eluted and measured colorimetrically. The method as developed by Fuleki and Francis for cranberry anthocyanins is given; a similar procedure may be used for other materials.
Apparatus required
- Fritted glass funnel, medium porosity, with filter aid
- Ion-exchange chromatographic column: all-glass, 300 × 10 mm, fitted with a fritted glass filter and a polyvinylchloride (Polyvic) filter disc on top
- Chromatography tanks (30 × 30 × 60 cm)
- Micropipette
- Densitometer with filters
- Centrifuge tubes
- Abbe refractometer
- Magnetic stirrer; pH electrodes
Reagents
a. Basic lead acetate (basic lead subacetate, Pb(C₂H₃O₂)₂·2Pb(OH)₂ — reconstructed from heavily garbled OCR in the source; the standard formula for basic lead subacetate): prepare a saturated solution (pH 8.4).
b. Approximately 0.5 N NaOH; approximately 2 N HCl; dilute HCl solution (for electrode storage).
c. Amberlite CG-50, a weak acidic cation exchange resin of 100–200 mesh (other resins tried: IR-4B of Rohm and Hass Co., Rexyn 102 of Fisher Scientific Co., AG-50 of Biorad Lab., Zeo-Karb, Permutit Q, H-70 of Permutit Co.).
d. Distilled water.
e. Methanol containing 0.259% HCl; 5% HCl in methanol.
f. Solvent: upper phase of n-butanol–benzene–formic acid–water (100 : 19 : 10 : 25) mixture, aged 3 days; lower phase of the same mixture for equilibration.
g. 6 N HCl (for fuming the chromatograms).
Procedure
Purification with basic lead acetate
Prepare a saturated solution of basic lead acetate (pH 8.4)
↓
To the juice, add sufficient lead acetate solution drop-wise with vigorous stirring to precipitate the anthocyanins — the juice turns to a green slurry when the reaction is complete
↓
Adjust the pH to 7.0–7.1 with drop-wise addition of approximately 0.5 N NaOH and vigorous stirring of the slurry with a magnetic stirrer
↓
Filter the precipitate with suction on a medium porosity fritted glass funnel using a thin layer of filter aid
↓
Check the completeness of precipitation: add a few drops of approximately 2 N HCl to the bottom of the beaker in which the filtrate is collected before starting filtration — the appearance of a pink colour indicates anthocyanins in the filtrate
↓
Transfer the precipitate to a centrifuge tube; add 5% HCl in methanol, stir and centrifuge
↓
Collect the supernatant liquid and make up to a known volume (any degraded anthocyanins present would be further degraded when alkali is added)
Purification on ion-exchange resin column
Hydrate the Amberlite CG-50 resin by soaking in water, decanting the supernatant liquid occasionally to remove undersize particles
↓
Pour a sufficient amount of resin slurry into the all-glass chromatographic column (300 × 10 mm, fitted with a fritted glass filter and a Polyvic filter disc) to form a 15–18 cm column after the resin settles
↓
Pipette 5–10 ml of the juice onto the settled column and allow to adsorb
↓
Wash the sugar down from the column with distilled water (7 ml is sufficient) — check the completeness of sugar washing with an Abbe refractometer
↓
Elute the anthocyanins with methanol containing 0.259% HCl, adding the methanolic HCl to the column in small increments
↓
Collect the dense part of the anthocyanin band eluted with methanol and make up to a known volume for the determination of individual anthocyanins by chromatography
Chromatographic separation of individual anthocyanins
Use the upper phase of the n-butanol–benzene–formic acid–water (100 : 19 : 10 : 25) mixture, aged 3 days, as solvent
↓
With a micropipette, apply a known volume of the anthocyanin extract (purified by either method above, or the direct extract from fresh or frozen fruit) on Whatman No. 1 paper as 4 cm long streaks, so that the direction of development is perpendicular to the machine direction of the paper
↓
Equilibrate the chromatograms with the lower phase in a tank for 12 hr
↓
Transfer to another tank (30 × 30 × 60 cm) and develop ascendingly with the upper phase — saturate the atmosphere in the jar with the upper phase of the solvent before development by lining the side of the jar with filter paper soaked in the developing solvent
↓
Develop the chromatograms until the solvent front reaches within 1 to 2 cm from the top of the paper
↓
Air-dry the chromatograms for 20 min and repeat the development a second time
Densitometric measurement (preferred)
Cut the paper into suitable strips
↓
Intensify the attenuation of the anthocyanins by fuming the chromatograms with 6 N HCl for 5 min — carry out the fuming by suspending the strips from a glass frame in a chromatography tank over the acid
↓
By scanning the chromatogram with different filters in the densitometer, find the filter which gives the greatest response (525 nm in the case of cranberry anthocyanins); find the slit width of the instrument which gives the best response
↓
Scan the chromatogram in the densitometer and measure the area under each peak (a planimeter may be used)
Colorimetric measurement (alternative)
Extract the anthocyanins from the individual spots with a suitable solvent (see the grape procedure in SOP: Extraction, Purification and Separation of Anthocyanin Pigments) and measure colorimetrically — the densitometric method is preferred
Calculation
- Find the total anthocyanin content in mg/100 g (or ml) by SOP: Estimation of Total Anthocyanins.
- Find the ratio of individual anthocyanins from the densitometric readings:
No. 1 anthocyanin, % = [Densitometric peak area for No. 1 anthocyanin ÷ Densitometric peak area for all the pigments] × 100
No. 1 anthocyanin content, mg/100 g (ml) = [Total anthocyanin in mg ÷ 100] × No. 1 anthocyanin, %
The content of each of the anthocyanins may be calculated similarly.
Where,
- Densitometric peak area for No. 1 anthocyanin = measured area under the peak of the individual anthocyanin
- Densitometric peak area for all the pigments = sum of the areas under all the peaks
- Total anthocyanin in mg = total anthocyanin content determined by SOP: Estimation of Total Anthocyanins
Precautions
- The precipitate may stick to the pH electrodes and poison them — wash electrodes after every measurement with 2 N HCl followed by generous rinsing with distilled water, and store the electrodes overnight in dilute HCl solution to dissolve any adsorbed lead salt.
- Use lead compounds with care: degraded anthocyanins are further degraded when alkali is added, and the pink colour test indicates anthocyanins remaining in the filtrate.
- Equilibrate the chromatograms for the full 12 hr before development, and develop twice for complete separation.
References
Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.
- Fuleki, T. & F.J. Francis, J. Food Sci., 33, 72 (1968).
- Zapsalis, C. & F.J. Francis, J. Food Sci., 30, 396 (1965).
- Chandler, B.V. & K.A. Harper, Aust. J. Chem., 14, 856 (1961); 15, 114 (1962).
- Spaeth, E.C. & D.H. Rosenblatt, Anal. Chem., 22, 1321 (1950).
- Lynn, D.Y.C. & B.S. Luh, J. Food Sci., 29, 735 (1964).
- Luh, B.S., K. Stachowicz & C.L. Hsia, J. Food Sci., 30, 300 (1965).
- Smith, R.M. & B.S. Luh, J. Food Sci., 30, 995 (1965).
- Dickinson, D. & J.H. Gawler, J. Sci. Food Agr., 7, 699 (1956).
- Swain, T. & W.E. Hillis, J. Sci. Food Agr., 10, 63 (1959).
- Fuleki, T. & F.J. Francis, J. Food Sci., 33, 78 (1968).
- Fuleki, T. & F.J. Francis, J. Food Sci., 33, 266 (1968).
- Fuleki, T. & F.J. Francis, J. Food Sci., 33, 471 (1968).