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Characterization and Measurement of UNIT F1.

2
Anthocyanins by UV-Visible Spectroscopy
Anthocyanin pigment content has a critical role in the color quality of many fresh and
processed fruits and vegetables. Thus, accurate measurement of anthocyanins, along with
their degradation indices, is very useful to food technologists and horticulturists in
assessing the quality of raw and processed foods. Since many natural food colorants are
anthocyanin derived (e.g., grape-skin extract, red-cabbage extract, purple-carrot extract),
the same measurements can be used to assess the color quality of these food ingredients.
In addition, there is intense interest in the anthocyanin content of foods and nutraceuticals
because of possible health benefits such as reduction of coronary heart disease (Bridle
and Timberlake, 1996), improved visual acuity (Timberlake and Henry, 1988), antioxidant
activities (Takamura and Yamagami, 1994; Wang et al., 1997), and anticancer activities
(Karaivanova et al., 1990; Kamei et al., 1995). Substantial quantitative and qualitative
information can be obtained from the spectral characteristics of anthocyanins. The
protocols described in this unit rely on the structural transformation of the anthocyanin
chromophore as a function of pH, which can be measured using optical spectroscopy. The
pH-differential method, a rapid and easy procedure for the quantitation of monomeric
anthocyanins, is first described (see Basic Protocol 1). In addition, other auxiliary
spectrophotometric techniques are used to measure the extent of anthocyanin polymeri-
zation and browning (see Basic Protocol 2).

TOTAL MONOMERIC ANTHOCYANIN BY THE pH-DIFFERENTIAL BASIC


METHOD PROTOCOL 1
Anthocyanin pigments undergo reversible structural transformations with a change in pH
manifested by strikingly different absorbance spectra (Fig. F1.2.1). The colored oxonium
form predominates at pH 1.0 and the colorless hemiketal form at pH 4.5 (Fig. F1.2.2).
The pH-differential method is based on this reaction, and permits accurate and rapid
measurement of the total anthocyanins, even in the presence of polymerized degraded
pigments and other interfering compounds.

2.0
1.8
1.6
1.4
Absorbance

pH 1.0
1.2
1.0
0.8
0.6
0.4
0.2 pH 4.5
0.0
260 360 460 560 660 760
Wavelength (nm)

Figure F1.2.1 Spectral characteristics of purified radish anthocyanins (acylated pelargonidin-3-


sophoroside-5-glucoside derivatives) in pH 1.0 and pH 4.5 buffers.
Anthocyanins
Contributed by M. Mnica Giusti and Ronald E. Wrolstad F1.2.1
Current Protocols in Food Analytical Chemistry (2001) F1.2.1-F1.2.13
Copyright 2001 by John Wiley & Sons, Inc.
R1 R1
OH OH

HO O HO O
R2 H+ +
R2

O-gly O-gly
O-gly O-gly
quinonoidal base: blue flavylium cation (oxonium form): orange to purple
pH = 7 pH = 1

+H2O H+

R1
H OH
R1 O
HO OH OH HO O
O-gly R2

R2 O-gly
O-gly O O-gly
chalcone: colorless carbinol pseudo-base (hemiketal form): colorless
pH = 4.5 pH = 4.5

Figure F1.2.2 Predominant structural forms of anthocyanins present at different pH levels.

Materials
0.025 M potassium chloride buffer, pH 1.0 (see recipe)
0.4 M sodium acetate buffer, pH 4.5 (see recipe)
1. Turn on the spectrophotometer. Allow the instrument to warm up at least 30 min
before taking measurements.
2. Determine the appropriate dilution factor for the sample by diluting with potassium
chloride buffer, pH 1.0, until the absorbance of the sample at the vis-max (Table F1.2.1)
is within the linear range of the spectrophotometer (i.e., for most spectrophotometers
the absorbance should be less than 1.2). Divide the final volume of the sample by the
initial volume to obtain the dilution factor (DF; for example see step 7).
IMPORTANT NOTE: In order to not exceed the buffers capacity, the sample should not
exceed 20% of the total volume.
3. Zero the spectrophotometer with distilled water at all wavelengths that will be used
(vis-max and 700 nm).
Many spectrophotometers will allow for a rapid baseline correction to zero by using
baseline adjust.

Characterization 4. Prepare two dilutions of the sample, one with potassium chloride buffer, pH 1.0, and
and Measurement the other with sodium acetate buffer, pH 4.5, diluting each by the previously
of Anthocyanins
by UV-Visible determined dilution factor (step 2). Let these dilutions equilibrate for 15 min.
Spectroscopy

F1.2.2
Current Protocols in Food Analytical Chemistry
Table F1.2.1 Reported Molar Absorptivity of Anthocyanins

Molar
Anthocyanina Solvent system vis-max (nm) Reference
absorptivity ()
Cyanidin (Cyd)
Cyd 0.1% HCl in ethanol 510.5 24600 Schou, 1927
0.1% HCl in ethanol 547 34700 Ribereau-Gayon, 1959
Cyd-3-ara 15:85 0.1 N HCl/ethanol 538 44400 Zapsalis and Francis,
1965
15:85 0.1 N HCl/ethanol 535 44460 Fuleki and Francis,
1968a
Cyd-3,5-diglu 0.1 N HCl 520 30175 Niketic-Aleksic and
Hrazdina, 1972
Methanolic HCl 508.5 35000 Brouillard and El Hache
Chahine, 1980
Cyd-3-gal 0.1% HCl in methanol 530 34300 Siegelman and
Hendricks, 1958
15:85 0.1 N HCl/ethanol 535 44900 Sakamura and Francis,
1961
15:85 0.1 N HCl/ethanol 535 46200 Zapsalis and Francis,
1965
15:85 0.1 N HCl/ethanol 535 46230 Fuleki and Francis,
1968a
HCl in methanol 530 30200 Swain, 1965
Cyd-3-glu Aqueous buffer, pH 1 510 26900 Jurd and Asen, 1966
0.1 N HCl 520 25740 McClure, 1967
1% HCl in methanol 530 34300 Siegelman and
Hendricks, 1958
10% ethanol, pH 1.5 512 18800 Heredia et al., 1998
Cyd-3-rut Aqueous buffer, pH 0.9 510 7000 Figueiredo et al., 1996
1% HCl 523 28840 Swain, 1965
Cyd-3-sam-5-glu Aqueous buffer, pH 0.9 522 3600 Figueiredo et al., 1996
Cyd-3-sam-5-glu + sinapic + Aqueous buffer, pH 0.9 538 21200 Figueiredo et al., 1996
caffeic + malonic
Cyd-3-sam-5-glu + sinapic + Aqueous buffer, pH 0.9 528 15100 Figueiredo et al., 1996
ferulic
Cyd-3-sam-5-glu + sinapic + Aqueous buffer, pH 0.9 538 20100 Figueiredo et al., 1996
ferulic + malonic
Cyd-3-sam-5-glu + sinapic + Aqueous buffer, pH 0.9 536 19000 Figueiredo et al., 1996
p-coum + malonic
Cyd-3-soph-5-glu Methanolic HCl 524 37150 Hrazdina et al., 1977
Cyd-3-soph-5-glu + malonic Methanolic HCl 528 32360 Hrazdina et al., 1977
Cyd-3-soph-5-glu + sinapic Methanolic HCl 528 37150 Hrazdina et al., 1977
Cyd-3-soph-5-glu + di-sinapic Methanolic HCl 530 38020 Hrazdina et al., 1977
Cyd-3-soph-5-glu + ferulic Methanolic HCl 528 32360 Hrazdina et al., 1977
Cyd-3-soph-5-glu + di-ferulic Methanolic HCl 530 34670 Hrazdina et al., 1977
Cyd-3-soph-5-glu + p-coumaric Methanolic HCl 526 38020 Hrazdina et al., 1977
Cyd-3-soph-5-glu + Methanolic HCl 528 32360 Hrazdina et al., 1977
di-p-coumaric
Delphinidin (Dpd)
Dpd 0.1% HCl in ethanol 522.5 34700 Schou, 1927

continued

F1.2.3
Current Protocols in Food Analytical Chemistry
Table F1.2.1 Reported Molar Absorptivity of Anthocyanins, continued

Molar
Anthocyanina Solvent system vis-max (nm) Reference
absorptivity ()
Dpd-3-glu 1% HCl in methanol 543 29000 Asen et al., 1959
10% ethanol, pH 1.5 520 23700 Heredia et al., 1998
Malvidin (Mvd)
Mvd 0.1% HCl in ethanol 520 37200 Schou, 1927
0.1% HCl in ethanol 557 36200 Ribereau-Gayon, 1959
Mvd-3,5-diglu 0.1% HCl in ethanol 519 10700 Schou, 1927
0.1% HCl in ethanol 545 10300 Ribereau-Gayon, 1959
0.1 N HCl 520 37700 Niketic-Aleksic and
Hrazdina, 1972
Mvd-3-glu 0.1% HCl in methanol 546 13900 Somers, 1966
0.1% HCl in methanol 538 29500 Koeppen and Basson,
1966
0.1 N HCl 520 28000 Niketic-Aleksic and
Hrazdina, 1972
Methanol, pH 1.0 535 36400 Metivier et al., 1980
10% ethanol, pH 1.5 520 20200 Heredia et al., 1998
Mvd-3-glu + p-coum 0.1% HCl in methanol 536 30200 Koeppen and Basson,
1966
Pelargonidin (Pg)
Pg 0.1% HCl in ethanol 504.5 17800 Schou, 1927
0.025 M potassium 505 18420 Giusti et al., 1999
chloride buffer, pH 1.0
0.1% HCl in methanol 524 19780 Giusti et al., 1999
Pg-3,5-diglu HCl in methanol 510 32360 Swain, 1965
Pg-3-(dicaffeoylglu)-soph-5-glu Aqueous buffer, pH 0.8 512 28000 Dangles et al., 1993
Pg-3-glu 1% HCl in H2O 496 27300 Jorgensen and
Geissman, 1955
36600 Wrolstad et al., 1970
1% HCl 513 22390 Swain, 1965
1% HCl in ethanol 516 31620 Swain, 1965
0.025 M potassium 496 15600 Giusti et al., 1999
chloride buffer, pH 1.0
0.1% HCl in methanol 508 17330 Giusti et al., 1999
Pg-3-rut-5-glu + p-coumaric 0.025 M potassium 504 32080 Giusti et al., 1999
chloride buffer, pH 1.0
0.1% HCl in methanol 511 39591 Giusti et al., 1999
Pg-3-soph-5-glu Aqueous buffer, pH 0.8 498 1800020000 Dangles et al., 1993
0.025 M potassium 497 25370 Giusti et al., 1999
chloride buffer, pH 1.0
0.1% HCl in methanol 506 30690 Giusti et al., 1999
Pg-3-soph-5-glu + ferulic 0.025 M potassium 506 24140 Giusti et al., 1999
chloride buffer, pH 1.0
0.1% HCl in methanol 507 29636 Giusti et al., 1999
Pg-3-soph-5-glu caffeoyl Aqueous buffer, pH 0.8 498 18000-20000 Dangles et al., 1993
derivatives
Pg-3-soph-5-glu + p-coumaric 0.025 M potassium 506 28720 Giusti et al., 1999
chloride buffer, pH 1.0
0.1% HCl in methanol 508 34889 Giusti et al., 1999

continued

F1.2.4
Current Protocols in Food Analytical Chemistry
Table F1.2.1 Reported Molar Absorptivity of Anthocyanins, continued

Molar
Anthocyanina Solvent system vis-max (nm) Reference
absorptivity ()

Pg-3-soph-5-glu + p-coumaric + 0.025 M potassium 508 33010 Giusti et al., 1999


malonic chloride buffer, pH 1.0
0.1% HCl in methanol 508 39785 Giusti et al., 1999
Pg-3-soph-5-glu + ferulic + 0.025 M potassium 508 31090 Giusti et al., 1999
malonic chloride buffer, pH 1.0
0.1% HCl in methanol 508 39384 Giusti et al., 1999
Peonidin (Pnd)
Pnd 0.1% HCl in ethanol 511 37200 Schou, 1927
15:85 0.1 N HCl/ethanol 532 40800 Sakamura and Francis,
1961
Pnd-3-ara 15:85 0.1 N HCl/ethanol 532 46100 Zapsalis and Francis,
1965
15:85 0.1 N HCl/ethanol 532 46070 Fuleki and Francis,
1968a
Pnd-3,5-diglu 0.1 N HCl 520 36654 Niketic-Aleksic and
Hrazdina, 1972
Pnd-3-gal 15:85 0.1 N HCl/ethanol 532 48400 Sakamura and Francis,
1961
15:85 0.1 N HCl/ethanol 532 48400 Zapsalis and Francis,
1965
15:85 0.1 N HCl/ethanol 531 48340 Fuleki and Francis,
1968a
Pnd-3-glu 0.1% HCl in methanol 536 11300 Somers, 1966
10% ethanol, pH 1.5 512 14100 Heredia et al., 1998
Petunidin (Ptd)
Ptd-3,5-diglu 0.1 N HCl 520 33040 Niketic-Aleksic and
Hrazdina, 1972
HCl in methanol 535 23440 Swain, 1965
Ptd-3-glu 0.1% HCl in methanol 546 12900 Somers, 1966
10% ethanol, pH 1.5 520 18900 Heredia et al., 1998
aAbbreviations: ara: arabinoside; gal: galactoside; glu: glucoside; rut: rutinoside; sam: sambubioside; soph: sophoroside.

5. Measure the absorbance of each dilution at the vis-max and at 700 nm (to correct for
haze), against a blank cell filled with distilled water.
All measurements should be made between 15 min and 1 hr after sample preparation, since
longer standing times tend to increase observed readings.
Absorbance readings are made against water blanks, even if the samples are in buffer or
bisulfite solutions, as buffer or bisulfite absorbance is nil at the measured wavelengths.
The authors have compared the values obtained by using water as a blank as compared
with buffer or bisulfite as blanks in different systems and have found no difference in the
final values obtained for monomeric and/or polymeric anthocyanin content; on the other
hand, reading the diluted samples against the corresponding buffer and/or bisulfite solution
is more time-consuming and extends the procedure unnecessarily.
The samples to be measured should be clear and contain no haze or sediments; however,
some colloidal materials may be suspended in the sample, causing scattering of light and
a cloudy appearance (haze). This scattering of light needs to be corrected for by reading
at a wavelength where no absorbance of the sample occurs, i.e., 700 nm.
Anthocyanins

F1.2.5
Current Protocols in Food Analytical Chemistry
Table F1.2.2 Molecular Weights of Anthocyanidins, Anthocyanins, and Acylating Groups Commonly Found
in Naturea

Anthocyanidins Pelargonidin Cyanidin Peonidin Delphinidin Petunidin Malvidin


271 287 301 303 317 331
Hex 180.2 180.2 180.2 180.2 180.2 180.2
Hex H2Ob 162.2 162.2 162.2 162.2 162.2 162.2
Acd + 1 hex 433.2 449.2 463.2 465.2 479.2 493.2
Acd + 2 hex 595.4 611.4 625.4 627.4 641.4 655.4
Acd + 3 hex 757.6 773.6 787.6 789.6 803.6 817.6
Pent 150.0 150.0 150.0 150.0 150.0 150.0
Pent H2Ob 132.0 132.0 132.0 132.0 132.0 132.0
Acd + 1 pent 403.0 419.0 433.0 435.0 449.0 463.0
Acd + 1 hex + 1 pent 565.2 581.2 595.2 597.2 611.2 625.2
Rhamnose 164.2 164.2 164.2 164.2 164.2 164.2
Rutinose 326.2 326.2 326.2 326.2 326.2 326.2
Rutinose H2Ob 308.2 308.2 308.2 308.2 308.2 308.2
Acd + rutinose 579.2 595.2 609.2 611.2 625.2 639.2
Acd + rutinose + 1 hex 741.4 757.4 771.4 773.4 787.4 801.4
Acd + rutinose + 1 pent 711.2 727.2 741.2 743.2 757.2 771.2
Common acylating groups
H2Ob
p-Coumaric acid 164.2 146.2
Caffeic acid 180.2 162.2
Ferulic acid 194.2 176.2
Sinapic acid 224 206
Acetic acid 82 64
Propionic acid 96.1 78.1
Malonic acid 104.1 86.1
Succinic acid 118.1 100.1
aAbbreviations: hex: hexose; pent: pentose; acd: anthocyanidin.
bH O indicates a dehydrated sugar (water is lost upon forming a glycosidic bond).
2

6. Calculate the absorbance of the diluted sample (A) as follows:


A = (A vis-max A700)pH 1.0 (A vis-max A700)pH 4.5
7. Calculate the monomeric anthocyanin pigment concentration in the original sample
using the following formula:
Monomeric anthocyanin pigment (mg/liter) = (A MW DF 1000)/( 1)
where MW is the molecular weight (Table F1.2.2), DF is the dilution factor (for
example, if a 0.2 ml sample is diluted to 3 ml, DF = 15), and is the molar absorptivity
(Table F1.2.1).
IMPORTANT NOTE: The MW and used in this formula correspond to the predominant
anthocyanin in the sample. Use the reported in the literature for the anthocyanin pigment
Characterization
in acidic aqueous solvent. If the of the major pigment is not available, or if the sample
and Measurement composition is unknown, calculate pigment content as cyanidin-3-glucoside, where MW =
of Anthocyanins 449.2 and = 26,900 (see Background Information, discussion of Molar Absorptivity).
by UV-Visible
Spectroscopy The equation presented above assumes a pathlength of 1 cm.
F1.2.6
Current Protocols in Food Analytical Chemistry
INDICES FOR PIGMENT DEGRADATION, POLYMERIC COLOR, AND BASIC
BROWNING PROTOCOL 2
Indices for anthocyanin degradation of an aqueous extract, juice, or wine can be derived
from a few absorbance readings of a sample that has been treated with sodium bisulfite.
Anthocyanin pigments will combine with bisulfite to form a colorless sulfonic acid adduct
(Figure F1.2.3). Polymerized colored anthocyanin-tannin complexes are resistant to
bleaching by bisulfite, whereas the bleaching reaction of monomeric anthocyanins will
rapidly go to completion. The absorbance at 420 nm of the bisulfite-treated sample serves
as an index for browning. Color density is defined as the sum of absorbances at the vis-max
and at 420 nm. The ratio between polymerized color and color density is used to determine
the percentage of the color that is contributed by polymerized material. The ratio between
monomeric and total anthocyanin can be used to determine a degradation index.
Materials
Bisulfite solution (see recipe)
0.025 M potassium chloride buffer, pH 1.0 (see recipe)
1. Turn on the spectrophotometer and allow the instrument to warm up at least 30 min
before taking measurements.
2. Determine the appropriate dilution factor for the sample by diluting with 0.025 M
potassium chloride buffer, pH 1.0 until the absorbance of the sample at the vis-max is
within the linear range of the spectrophotometer (i.e., for most spectrophotometers
the absorbance should be less than 1.2). Divide the final volume of the sample by the
initial volume to obtain the dilution factor (DF; for example see step 6).
3. Zero the spectrophotometer with distilled water at all wavelengths that will be used
(420 nm, vis-max, 700 nm).
Many spectrophotometers will allow for a rapid baseline correction to zero by using
baseline adjust.
4. Dilute the sample with distilled water using the dilution factor already determined
(step 2). Transfer 2.8 ml of the diluted sample to each of two cuvettes. Add 0.2 ml of
bisulfite solution to one and 0.2 ml distilled water to the other. Equilibrate for 15 min.
It is critical that the pH not be adjusted to highly acidic conditions (e.g., pH 1) but rather
be in the typical pH range of fruit juices and wines, or higher (e.g., pH 3). Highly acidic
conditions will reverse the bisulfite addition reaction and render the measurement invalid.
5. Measure the absorbance of both samples at 420 nm, vis-max, and 700 nm (to correct
for haze), against a blank cell filled with distilled water.
All measurements should be made between 15 min (see step 4) and 1 hr after sample
preparation and bisulfite treatment. Longer standing times tend to increase observed
readings.
Absorbance readings are made against water blanks, even if the samples are in buffer or
bisulfite solutions, as buffer or bisulfite absorbance is nil at the measured wavelengths.
The authors have compared the values obtained by using water as a blank as compared
with the use buffer or bisulfite as a blank in different systems and have found no difference
in the final values obtained for monomeric and/or polymeric anthocyanin content; on the
other hand, reading the samples against the corresponding buffer and/or bisulfite solution
is more time-consuming and extends the procedure unnecessarily.
The samples to be measured should be clear and contain no haze or sediments; however,
some colloidal materials may be suspended in the sample, causing scattering of light and
a cloudy appearance (haze). This scattering of light needs to be accounted for by reading
at a wavelength where no absorbance of the sample occurs (i.e., 700 nm). Anthocyanins

F1.2.7
Current Protocols in Food Analytical Chemistry
6. Calculate the color density of the control sample (treated with water) as follows:
Color density = [(A420 nm A700nm) + (A vis-max A700 nm)] DF
where DF is the dilution factor (for example, if 0.2 ml sample diluted to 3 ml, DF =
15)
7. Calculate the polymeric color of the bisulfite bleached sample as follows:
Polymeric color = [(A420 nm A700 nm) + (A vis-max A700 nm)] DF
8. Calculate the percent polymeric color using the formula:
Percent polymeric color = (polymeric color/color density) 100

REAGENTS AND SOLUTIONS


Use deionized or distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Bisulfite solution
Dissolve 1 g of potassium metabisulfite (K2S2O5) in 5 ml of distilled water.
This reagent must be prepared the same day as the readings; otherwise, it develops a yellow
color that will contribute to the absorbance readings and interfere with the quantitation.
Potassium chloride buffer, 0.025 M, pH 1.0
Mix 1.86 g KCl and 980 ml of distilled water in a beaker. Measure the pH and adjust
to 1.0 with concentrated HCl. Transfer to a 1 liter volumetric flask and fill to 1 liter
with distilled water.
The solution should be stable at room temperature for a few months, but the pH should be
checked and adjusted prior to use (see Critical Parameters).
Sodium acetate buffer, 0.4 M, pH 4.5
Mix 54.43 g CH3CO2Na3 H2O and 960 ml distilled water in a beaker. Measure
the pH and adjust to 4.5 with concentrated HCl. Transfer to a 1 liter volumetric flask
and fill to 1 liter with distilled water.
The solution should be stable at room temperature for a few months, but the pH should be
checked and adjusted prior to use (see Critical Parameters).

COMMENTARY
Background Information 1994; Wang et al., 1997) and anticancer prop-
Anthocyanin pigments are responsible for erties (Karaivanova et al., 1990; Kamei et al.,
the attractive red to purple to blue colors of 1995). Anthocyanins have also found consid-
many fruits and vegetables. Anthocyanins are erable potential in the food industry as safe and
relatively unstable and often undergo degrada- effective food colorants (Strack and Wray,
tive reactions during processing and storage. 1994); interest in this application has increased
Measurement of total anthocyanin pigment in recent years. In 1980, the annual world pro-
content along with indices for the degradation duction had been estimated as reaching 10,000
of these pigments are very useful in assessing tons from grapes alone (Timberlake, 1980).
the color quality of these foods. Interest in the Quantitative and qualitative anthocyanin com-
anthocyanin content of foods and nutraceutical position are important factors in determining
preparations has intensified because of their the feasibility of the use of new plant materials
possible health benefits. They may play a role as anthocyanin-based colorant sources.
in reduction of coronary heart disease (Bridle Frequently, it is desirable to express antho-
Characterization
and Measurement and Timberlake, 1996) and increased visual cyanin determinations in terms that can be com-
of Anthocyanins acuity (Timberlake and Henry, 1988), and also pared with the results from different workers.
by UV-Visible have antioxidant (Takamura and Yamagami, The best way to express these results is in terms
Spectroscopy

F1.2.8
Current Protocols in Food Analytical Chemistry
R1 R1
OH OH

HO O HO O
R2 R2
+
O-gly O-gly
Strong acid
OH OH SO3H
flavylium cation: red bisulfite addition compound: colorless

Figure F1.2.3 Formation of colorless anthocyanin-sulfonic acid adducts.

of absolute quantities of anthocyanins present will decolor anthocyanins but not affect inter-
(Fuleki and Francis, 1968a). fering materials. A measurement of the absor-
The total anthocyanin content in crude ex- bance at the visible maximum is obtained, fol-
tracts containing other phenolic materials has lowed by bleaching and remeasuring to give a
been determined by measuring absorptivity of blank reading (Jackman et al., 1987). The two
the solution at a single wavelength. This is most used bleaching agents are sodium sulfite
possible because anthocyanins have a typical (Somers and Evans, 1974; Wrolstad et al.,
absorption band in the 490 to 550 nm region of 1982) and hydrogen peroxide (Swain and Hil-
the visible spectra (Figure F1.2.1). This band lis, 1959).
is far from the absorption bands of other phe- By using both of these spectral procedures,
nolics, which have spectral maxima in the UV accurate measurement of the total monomeric
range (Fuleki and Francis, 1968a). In many anthocyanin pigment content can be obtained,
instances, however, this simple method is inap- along with indices for polymeric color, color
propriate because of interference from antho- density, browning, and degradation. To deter-
cyanin degradation products or melanoidins mine total anthocyanin content, the absorbance
from browning reactions (Fuleki and Francis, at pH 1.0 and 4.5 is measured at the vis-max
1968b). In those cases, the approach has been (Table F1.2.1) and at 700 nm, which allows for
to use differential and/or subtractive methods haze correction. The bisulfite bleaching reac-
to quantify anthocyanins and their degradation tion is utilized to generate the various degrada-
products (Jackman and Smith, 1996). tion indices. While monomeric anthocyanins
The differential method (see Basic Protocol are readily bleached by bisulfite at product pH
1) measures the absorbance at two different pH (Fig. F1.2.3), the polymeric anthocyanin-tan-
values, and relies on the structural transforma- nin and melanoidin pigments are resistant and
tions of the anthocyanin chromophore as a will remain colored. Somers and Evans (1974)
function of pH (Fig. F1.2.1 and Fig. F1.2.2). used this reaction in developing spectral meth-
This concept was first introduced by Sondhe- ods for assessing the color quality of wines. The
imer and Kertesz in 1948, who used pH values authors laboratory has found them useful for
of 2.0 and 3.4 for analyses of strawberry jams tracking color quality in a wide range of antho-
(Francis, 1989). Since then, the use of other pH cyanin-containing foods (Wrolstad et al., 1982,
values has been proposed. Fuleki and Francis 1995). Absorbance measurements are taken at
(1968b) used pH 1.0 and 4.5 buffers to measure the vis-max and at 420 nm on the bisulfite
anthocyanin content in cranberries, and modi- bleached and control samples. Color density is
fications of this technique have been applied to the sum of the absorbances at the vis-max and
a wide range of commodities (Wrolstad et al., at 420 nm of the control sample, while poly-
1982, 1995). The pH differential method has meric color is the same measurement for the
been described as fast and easy for the quanti- bisulfite treated sample. A measure of percent
tation of monomeric anthocyanins (Wrolstad et polymeric color is obtained as the ratio between
al., 1995). these two indexes. The absorbance at 420 nm
Subtractive methods (see Basic Protocol 2) of the bisulfite-treated sample is an index for
are based on the use of bleaching agents that browning, as the accumulation of brownish Anthocyanins

F1.2.9
Current Protocols in Food Analytical Chemistry
degradation products increases the absorption much for anthocyanins with glycosidic substi-
in the 400 to 440 nm range. The absorption of tutions in position 3 as compared to those with
these compounds are in general not affected by substitutions in positions 3 and 5 or position 5
the addition of a bisulfite solution. only. The presence of glycosidic substitutions
at other positions (e.g., 3,7-diglycosides) can
Molar absorptivity be recognized because they exhibit a different
Regardless of the method used for anthocy- spectral curve from those of anthocyanins with
anin quantitation, the determination of the common substitution patterns. The presence of
amount present requires an absorptivity coeffi- cinnamic acid acylation is revealed by the pres-
cient. Absorptivity coefficients have been re- ence of a third absorption band in the 310 to
ported as the absorption of a 1% solution meas- 360 nm range (Figure F1.2.1), and the ratio of
ured through a 1-cm path at the vis-max, or as a absorbance at 310 to 360 nm to the absorbance
molar absorption coefficient. Absorptivity co- at the visible vis-max will give an estimation of
efficients of some known anthocyanins have the number of acylating groups (Harborne,
been reported by different researchers (Table 1967; Hong and Wrolstad, 1990). The solvent
F1.2.1). Through the years, there has been a used for spectral determination will affect the
lack of uniformity on the values of absorptivity position of the absorption bands, and therefore
reported, mainly due to the difficulties of pre- must be taken into consideration when compar-
paring crystalline anthocyanin, free from im- ing available data.
purities, in sufficient quantities to allow reliable
weighing under optimal conditions (Fuleki and Critical Parameters and
Francis, 1968a; Francis, 1982; Giusti et al., Troubleshooting
1999). Other problems are that the anthocyanin The pH of buffers should always be checked
mixtures may be very complicated, and not all and adjusted prior to use. The use of buffers
absorptivity coefficients may be known. Even with lower or higher pH levels will result in
when they are known, it is necessary to first under- or overestimations of the pigment con-
evaluate if the objective is the estimation of total tent.
anthocyanin content or the determination of The accuracy of the results will be greatly
individual pigments, and then to decide which affected by the accuracy of the volumetric
absorption coefficient(s) to use. The absorptiv- measurements. Make sure that any volumetric
ity is dependent not only on the chemical struc- flasks or pipets used for obtaining the appro-
ture of the pigment but also on the solvent used; priate dilutions are calibrated correctly.
preferably, the coefficient used should be one For the methodologies described in this unit,
obtained in the same solvent system as the one all spectral measurements should be made be-
used in the experiment. If the identity of the tween 15 min and 1 hr after the dilutions have
pigments is unknown, it has been suggested that been prepared. The observed readings tend to
it can be expressed as cyanidin-3-glucoside, increase with time.
since that is the most abundant anthocyanin in When working with several different sam-
nature (Francis, 1989). ples, it may be acceptable to use one common
approximate vis-max that is typical of all sam-
Spectral characteristics ples (i.e., 520 nm). The visible absorbance peak
Substantial information can be obtained is broad, and measuring a few nanometers off
from the spectral characteristics of anthocyan- vis-max will not significantly alter the estimated
ins (Fig. F1.2.1). Two distinctive bands of ab- final values.
sorption, one in the UV-region (260 to 280 nm) Serial dilutions are recommended to ensure
and another in the visible region (490 to 550 accurate measurements of highly concentrated,
nm) are shown by all anthocyanins. The differ- high density, or dried samples. Perform a
ent aglycons have different vis-max, ranging weight-by-volume dilution with distilled water
from 520 nm for pelargonidin to 546 nm for to obtain a single-strength solution (e.g., usu-
delphinidin, and their monoglucosides exhibit ally around 10 Brix for fruit juices; UNIT H1.4),
their vis-max at about 10 to 15 nm lower (Strack followed by a second dilution using 0.025 M
and Wray, 1989). The shape of the spectrum potassium chloride buffer, pH 1.0. Both dilu-
may give information regarding the number tion factors must be considered when calculat-
Characterization and position of glycosidic substitutions and ing monomeric anthocyanin content.
and Measurement number of cinnamic acid acylations. The ratio For example, 1 g of a 75 Brix juice concen-
of Anthocyanins
by UV-Visible between the absorbance at 440 nm and the trate was diluted to a final volume of 10 ml with
Spectroscopy absorbance at the vis-max is almost twice as distilled water (dilution factor = 10; assuming

F1.2.10
Current Protocols in Food Analytical Chemistry
Table F1.2.3 Anthocyanin Content of Some Common Fruits and Vegetables

Pigment content
Source Reference
(mg/100 g fresh weight)
Apples (Scugog) 10 Mazza and Miniati, 1993
Bilberries 300320 Mazza and Miniati, 1993
Blackberries 83326 Mazza and Miniati, 1993
Black currants 130400 Timberlake, 1988
Blueberries 25495 Mazza and Miniati, 1993
Red cabbage 25 Timberlake, 1988
Black chokeberries 560 Kraemer-Schafhalter et al., 1996
Cherries 4450 Kraemer-Schafhalter et al., 1996
Cranberries 60200 Timberlake, 1988
Elderberry 450 Kraemer-Schafhalter et al., 1996
Grapes 6600 Mazza and Miniati, 1993
Kiwi 100 Kraemer-Schafhalter et al., 1996
Red onions 721 Mazza and Miniati, 1993
Plum 225 Timberlake, 1988
Red radishes 1160 Giusti et al., 1988
Black raspberries 300400 Timberlake, 1988
Red Raspberries 2060 Mazza and Miniati, 1993
Strawberries 1535 Timberlake, 1988
Tradescantia pallida 120 Shi et al., 1992
(leaves)

a density of 1 g/ml for juice). Then, the appro- Highly acylated anthocyanins may not re-
priate dilution factor for the sample was deter- spond to pH changes the same way as anthocy-
mined by diluting 0.2 ml of the solution with anins with no or few acylating groups, and may
2.8 ml of 0.025 M potassium chloride buffer, not decolor as much as nonacylated or mono-
pH 1.0 (dilution factor = 15). To calculate or diacylated anthocyanins do at pH 4.5.
monomeric anthocyanin content, color density,
or polymeric color, the dilution factor to use Anticipated Results
would be: DF = (10 15) = 150. The anthocyanin content of different com-
The methodologies used to measure color mon fruits and vegetables is presented in Table
density and polymeric color were developed for F1.2.3. Anthocyanin-containing fruit or vege-
fruit juices, which naturally have an acidic pH. table juices typically have pigment content
If the material to be measured has a pH in the ranging from 50 to 500 mg/liter. Anthocyanin-
neutral or alkaline range, the pH of the solution based natural colorants and nutraceuticals may
should be lowered with a weak acid. In these have a much higher pigment concentration, on
cases, the authors recommend the use of a 0.1 the order of a few grams/liter.
M citric acid buffer, pH 3.5, instead of distilled Fresh fruit or vegetable juices should have
water to prepare the different dilutions. a low percentage of polymeric color (usually
Some potential interfering materials are less than 10%), while processed samples and
other red pigments: FD&C Red No. 40, FD&C materials subjected to storage abuse will be
Red No. 3, cochineal, and beet powder (betalain much higher (30% or more). This is highly
pigments). The presence of alternative color- variable, dependent on the commodity, proc-
ants may be suspected if the vis-max at pH 1.0 essing conditions, and storage history.
is high (550 nm, more typical of betalain pig- Always express anthocyanin pigment con-
ments), or if a bright red coloration is found at tent in terms of the specific anthocyanin used
pH 4.5 (potential presence of artificial dyes). for calculation, and specify molecular weight
The presence of ethanol does not interfere and utilized.
with the assay at the levels typically encoun-
tered in wines (10% to 14%). Anthocyanins

F1.2.11
Current Protocols in Food Analytical Chemistry
Time Considerations Hong, V. and Wrolstad, R.E. 1990. Use of HPLC
Quantitation of anthocyanins can be separation/photodiode array detection for char-
acterization of anthocyanins. J. Agric. Food
achieved in <1 hr. It is necessary to wait for the
Chem. 38:708-715.
spectrophotometer to warm up, and for the
Hrazdina, G., Iredale, H., and Mattick, L.R. 1977.
diluted samples to equilibrate at least 15 min.
Anthocyanin composition of Brassica oleracea
The absorbance readings take a few minutes. cv. Red Danish. Phytochemistry 16:297-301.
Jackman, R.L. and Smith, J.L. 1996. Anthocyanins
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F1.2.12
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Anthocyanins

F1.2.13
Current Protocols in Food Analytical Chemistry