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Characterization and Measurement of

Anthocyanins by UVVisible Spectroscopy
CHAPTER JULY 2001
DOI: 10.1002/0471142913.faf0102s00

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Characterization and Measurement of

Anthocyanins by UV-Visible Spectroscopy

UNIT F1.2

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 polymerization and browning (see Basic Protocol 2).
TOTAL MONOMERIC ANTHOCYANIN BY THE pH-DIFFERENTIAL
METHOD
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.

BASIC
PROTOCOL 1

2.0
1.8
Absorbance

1.6
1.4

pH 1.0

1.2
1.0
0.8
0.6
0.4
0.2
0.0
260

pH 4.5
360

460
Wavelength (nm)

560

660

760

Figure F1.2.1 Spectral characteristics of purified radish anthocyanins (acylated pelargonidin-3sophoroside-5-glucoside derivatives) in pH 1.0 and pH 4.5 buffers.
Contributed by M. Mnica Giusti and Ronald E. Wrolstad
Current Protocols in Food Analytical Chemistry (2001) F1.2.1-F1.2.13
Copyright 2001 by John Wiley & Sons, Inc.

Anthocyanins

F1.2.1

R1

R1

OH

OH
O

HO

R2

H+

HO

R2

O-gly

O-gly
O-gly

O-gly

flavylium cation (oxonium form): orange to purple

pH = 1

quinonoidal base: blue

pH = 7

H+

+H2O

R1
H
O

R1
HO

OH
O-gly

OH

HO

chalcone: colorless
pH = 4.5

R2
O-gly

R2
O-gly

OH

O-gly
carbinol pseudo-base (hemiketal form): colorless
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
and Measurement
of Anthocyanins
by UV-Visible
Spectroscopy

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

F1.2.2
Current Protocols in Food Analytical Chemistry

Table F1.2.1

Reported Molar Absorptivity of Anthocyanins

Anthocyanina
Cyanidin (Cyd)
Cyd
Cyd-3-ara

Cyd-3,5-diglu

Cyd-3-gal

Cyd-3-glu

Cyd-3-rut
Cyd-3-sam-5-glu
Cyd-3-sam-5-glu + sinapic +
caffeic + malonic
Cyd-3-sam-5-glu + sinapic +
ferulic
Cyd-3-sam-5-glu + sinapic +
ferulic + malonic
Cyd-3-sam-5-glu + sinapic +
p-coum + malonic
Cyd-3-soph-5-glu
Cyd-3-soph-5-glu + malonic
Cyd-3-soph-5-glu + sinapic
Cyd-3-soph-5-glu + di-sinapic
Cyd-3-soph-5-glu + ferulic
Cyd-3-soph-5-glu + di-ferulic
Cyd-3-soph-5-glu + p-coumaric
Cyd-3-soph-5-glu +
di-p-coumaric
Delphinidin (Dpd)
Dpd

Solvent system

vis-max (nm)

Molar
Reference
absorptivity ()

0.1% HCl in ethanol

0.1% HCl in ethanol
15:85 0.1 N HCl/ethanol

510.5
547
538

24600
34700
44400

15:85 0.1 N HCl/ethanol

535

44460

0.1 N HCl

520

30175

Methanolic HCl

508.5

35000

0.1% HCl in methanol

530

34300

15:85 0.1 N HCl/ethanol

535

44900

15:85 0.1 N HCl/ethanol

535

46200

15:85 0.1 N HCl/ethanol

535

46230

HCl in methanol
Aqueous buffer, pH 1
0.1 N HCl
1% HCl in methanol

530
510
520
530

30200
26900
25740
34300

10% ethanol, pH 1.5

Aqueous buffer, pH 0.9
1% HCl
Aqueous buffer, pH 0.9
Aqueous buffer, pH 0.9

512
510
523
522
538

18800
7000
28840
3600
21200

Schou, 1927
Ribereau-Gayon, 1959
Zapsalis and Francis,
1965
Fuleki and Francis,
1968a
Niketic-Aleksic and
Hrazdina, 1972
Brouillard and El Hache
Chahine, 1980
Siegelman and
Hendricks, 1958
Sakamura and Francis,
1961
Zapsalis and Francis,
1965
Fuleki and Francis,
1968a
Swain, 1965
Jurd and Asen, 1966
McClure, 1967
Siegelman and
Hendricks, 1958
Heredia et al., 1998
Figueiredo et al., 1996
Swain, 1965
Figueiredo et al., 1996
Figueiredo et al., 1996

Aqueous buffer, pH 0.9

528

15100

Figueiredo et al., 1996

Aqueous buffer, pH 0.9

538

20100

Figueiredo et al., 1996

Aqueous buffer, pH 0.9

536

19000

Figueiredo et al., 1996

Methanolic HCl
Methanolic HCl
Methanolic HCl
Methanolic HCl
Methanolic HCl
Methanolic HCl
Methanolic HCl
Methanolic HCl

524
528
528
530
528
530
526
528

37150
32360
37150
38020
32360
34670
38020
32360

Hrazdina et al., 1977

Hrazdina et al., 1977
Hrazdina et al., 1977
Hrazdina et al., 1977
Hrazdina et al., 1977
Hrazdina et al., 1977
Hrazdina et al., 1977
Hrazdina et al., 1977

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

Anthocyanina

Solvent system

vis-max (nm)

Molar
Reference
absorptivity ()

Dpd-3-glu

1% HCl in methanol
10% ethanol, pH 1.5

543
520

29000
23700

Asen et al., 1959

Heredia et al., 1998

0.1% HCl in ethanol

0.1% HCl in ethanol
0.1% HCl in ethanol
0.1% HCl in ethanol
0.1 N HCl

520
557
519
545
520

37200
36200
10700
10300
37700

0.1% HCl in methanol

0.1% HCl in methanol

546
538

13900
29500

0.1 N HCl

520

28000

Methanol, pH 1.0
10% ethanol, pH 1.5
0.1% HCl in methanol

535
520
536

36400
20200
30200

Schou, 1927
Ribereau-Gayon, 1959
Schou, 1927
Ribereau-Gayon, 1959
Niketic-Aleksic and
Hrazdina, 1972
Somers, 1966
Koeppen and Basson,
1966
Niketic-Aleksic and
Hrazdina, 1972
Metivier et al., 1980
Heredia et al., 1998
Koeppen and Basson,
1966

0.1% HCl in ethanol

0.025 M potassium
chloride buffer, pH 1.0
0.1% HCl in methanol
HCl in methanol
Aqueous buffer, pH 0.8
1% HCl in H2O

504.5
505

17800
18420

Schou, 1927
Giusti et al., 1999

524
510
512
496

19780
32360
28000
27300

513
516
496

36600
22390
31620
15600

Giusti et al., 1999

Swain, 1965
Dangles et al., 1993
Jorgensen and
Geissman, 1955
Wrolstad et al., 1970
Swain, 1965
Swain, 1965
Giusti et al., 1999

508
504

17330
32080

Giusti et al., 1999

Giusti et al., 1999

511
498
497

39591
1800020000
25370

Giusti et al., 1999

Dangles et al., 1993
Giusti et al., 1999

506
506

30690
24140

Giusti et al., 1999

Giusti et al., 1999

507
498

29636
18000-20000

Giusti et al., 1999

Dangles et al., 1993

506

28720

Giusti et al., 1999

508

34889

Giusti et al., 1999

Malvidin (Mvd)
Mvd
Mvd-3,5-diglu

Mvd-3-glu

Mvd-3-glu + p-coum
Pelargonidin (Pg)
Pg

Pg-3,5-diglu
Pg-3-(dicaffeoylglu)-soph-5-glu
Pg-3-glu

Pg-3-rut-5-glu + p-coumaric

Pg-3-soph-5-glu

Pg-3-soph-5-glu + ferulic

Pg-3-soph-5-glu caffeoyl
derivatives
Pg-3-soph-5-glu + p-coumaric

1% HCl
1% HCl in ethanol
0.025 M potassium
chloride buffer, pH 1.0
0.1% HCl in methanol
0.025 M potassium
chloride buffer, pH 1.0
0.1% HCl in methanol
Aqueous buffer, pH 0.8
0.025 M potassium
chloride buffer, pH 1.0
0.1% HCl in methanol
0.025 M potassium
chloride buffer, pH 1.0
0.1% HCl in methanol
Aqueous buffer, pH 0.8
0.025 M potassium
chloride buffer, pH 1.0
0.1% HCl in methanol

continued

F1.2.4
Current Protocols in Food Analytical Chemistry

Table F1.2.1

Reported Molar Absorptivity of Anthocyanins, continued

Anthocyanina

Solvent system

vis-max (nm)

Molar
Reference
absorptivity ()

508

33010

Giusti et al., 1999

508
508

39785
31090

Giusti et al., 1999

Giusti et al., 1999

508

39384

Giusti et al., 1999

511
532

37200
40800

15:85 0.1 N HCl/ethanol

532

46100

15:85 0.1 N HCl/ethanol

532

46070

Pnd-3,5-diglu

0.1 N HCl

520

36654

Pnd-3-gal

15:85 0.1 N HCl/ethanol

532

48400

15:85 0.1 N HCl/ethanol

532

48400

15:85 0.1 N HCl/ethanol

531

48340

0.1% HCl in methanol

10% ethanol, pH 1.5

536
512

11300
14100

Schou, 1927
Sakamura and Francis,
1961
Zapsalis and Francis,
1965
Fuleki and Francis,
1968a
Niketic-Aleksic and
Hrazdina, 1972
Sakamura and Francis,
1961
Zapsalis and Francis,
1965
Fuleki and Francis,
1968a
Somers, 1966
Heredia et al., 1998

0.1 N HCl

520

33040

HCl in methanol
0.1% HCl in methanol
10% ethanol, pH 1.5

535
546
520

23440
12900
18900

Pg-3-soph-5-glu + p-coumaric + 0.025 M potassium

malonic
chloride buffer, pH 1.0
0.1% HCl in methanol
Pg-3-soph-5-glu + ferulic +
0.025 M potassium
malonic
chloride buffer, pH 1.0
0.1% HCl in methanol
Peonidin (Pnd)
Pnd
0.1% HCl in ethanol
15:85 0.1 N HCl/ethanol
Pnd-3-ara

Pnd-3-glu
Petunidin (Ptd)
Ptd-3,5-diglu

Ptd-3-glu

Niketic-Aleksic and
Hrazdina, 1972
Swain, 1965
Somers, 1966
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
in Naturea

Molecular Weights of Anthocyanidins, Anthocyanins, and Acylating Groups Commonly Found

Anthocyanidins

Pelargonidin

Cyanidin Peonidin Delphinidin

Petunidin

Malvidin

Hex
Hex H2Ob
Acd + 1 hex
Acd + 2 hex
Acd + 3 hex

271
180.2
162.2
433.2
595.4
757.6

287
180.2
162.2
449.2
611.4
773.6

301
180.2
162.2
463.2
625.4
787.6

303
180.2
162.2
465.2
627.4
789.6

317
180.2
162.2
479.2
641.4
803.6

331
180.2
162.2
493.2
655.4
817.6

Pent
Pent H2Ob
Acd + 1 pent
Acd + 1 hex + 1 pent

150.0
132.0
403.0
565.2

150.0
132.0
419.0
581.2

150.0
132.0
433.0
595.2

150.0
132.0
435.0
597.2

150.0
132.0
449.0
611.2

150.0
132.0
463.0
625.2

Rhamnose
Rutinose
Rutinose H2Ob
Acd + rutinose
Acd + rutinose + 1 hex
Acd + rutinose + 1 pent

164.2
326.2
308.2
579.2
741.4
711.2

164.2
326.2
308.2
595.2
757.4
727.2

164.2
326.2
308.2
609.2
771.4
741.2

164.2
326.2
308.2
611.2
773.4
743.2

164.2
326.2
308.2
625.2
787.4
757.2

164.2
326.2
308.2
639.2
801.4
771.2

Common acylating groups

H2Ob
p-Coumaric acid
Caffeic acid
Ferulic acid
Sinapic acid
Acetic acid
Propionic acid
Malonic acid
Succinic acid

164.2
180.2
194.2
224
82
96.1
104.1
118.1

146.2
162.2
176.2
206
64
78.1
86.1
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).

Characterization
and Measurement
of Anthocyanins
by UV-Visible
Spectroscopy

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
in acidic aqueous solvent. If the of the major pigment is not available, or if the sample
composition is unknown, calculate pigment content as cyanidin-3-glucoside, where MW =
449.2 and = 26,900 (see Background Information, discussion of Molar Absorptivity).
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

BROWNING

BASIC
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

Characterization
and Measurement
of Anthocyanins
by UV-Visible
Spectroscopy

Anthocyanin pigments are responsible for

the attractive red to purple to blue colors of
many fruits and vegetables. Anthocyanins are
relatively unstable and often undergo degradative reactions during processing and storage.
Measurement of total anthocyanin pigment
content along with indices for the degradation
of these pigments are very useful in assessing
the color quality of these foods. Interest in the
anthocyanin content of foods and nutraceutical
preparations has intensified because of their
possible health benefits. They may play a role
in reduction of coronary heart disease (Bridle
and Timberlake, 1996) and increased visual
acuity (Timberlake and Henry, 1988), and also
have antioxidant (Takamura and Yamagami,

1994; Wang et al., 1997) and anticancer properties (Karaivanova et al., 1990; Kamei et al.,
1995). Anthocyanins have also found considerable potential in the food industry as safe and
effective food colorants (Strack and Wray,
1994); interest in this application has increased
in recent years. In 1980, the annual world production had been estimated as reaching 10,000
tons from grapes alone (Timberlake, 1980).
Quantitative and qualitative anthocyanin composition are important factors in determining
the feasibility of the use of new plant materials
as anthocyanin-based colorant sources.
Frequently, it is desirable to express anthocyanin determinations in terms that can be compared with the results from different workers.
The best way to express these results is in terms

F1.2.8
Current Protocols in Food Analytical Chemistry

R1

R1

OH

OH
O

HO

HO

R2

O-gly
OH

Strong acid

flavylium cation: red

R2

O-gly
OH SO3H
bisulfite addition compound: colorless

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

of absolute quantities of anthocyanins present

(Fuleki and Francis, 1968a).
The total anthocyanin content in crude extracts containing other phenolic materials has
been determined by measuring absorptivity of
the solution at a single wavelength. This is
possible because anthocyanins have a typical
absorption band in the 490 to 550 nm region of
the visible spectra (Figure F1.2.1). This band
is far from the absorption bands of other phenolics, which have spectral maxima in the UV
range (Fuleki and Francis, 1968a). In many
instances, however, this simple method is inappropriate because of interference from anthocyanin degradation products or melanoidins
from browning reactions (Fuleki and Francis,
1968b). In those cases, the approach has been
to use differential and/or subtractive methods
to quantify anthocyanins and their degradation
products (Jackman and Smith, 1996).
The differential method (see Basic Protocol
1) measures the absorbance at two different pH
values, and relies on the structural transformations of the anthocyanin chromophore as a
function of pH (Fig. F1.2.1 and Fig. F1.2.2).
This concept was first introduced by Sondheimer and Kertesz in 1948, who used pH values
of 2.0 and 3.4 for analyses of strawberry jams
(Francis, 1989). Since then, the use of other pH
values has been proposed. Fuleki and Francis
(1968b) used pH 1.0 and 4.5 buffers to measure
anthocyanin content in cranberries, and modifications of this technique have been applied to
a wide range of commodities (Wrolstad et al.,
1982, 1995). The pH differential method has
been described as fast and easy for the quantitation of monomeric anthocyanins (Wrolstad et
al., 1995).
Subtractive methods (see Basic Protocol 2)
are based on the use of bleaching agents that

will decolor anthocyanins but not affect interfering materials. A measurement of the absorbance at the visible maximum is obtained, followed by bleaching and remeasuring to give a
blank reading (Jackman et al., 1987). The two
most used bleaching agents are sodium sulfite
(Somers and Evans, 1974; Wrolstad et al.,
1982) and hydrogen peroxide (Swain and Hillis, 1959).
By using both of these spectral procedures,
accurate measurement of the total monomeric
anthocyanin pigment content can be obtained,
along with indices for polymeric color, color
density, browning, and degradation. To determine total anthocyanin content, the absorbance
at pH 1.0 and 4.5 is measured at the vis-max
(Table F1.2.1) and at 700 nm, which allows for
haze correction. The bisulfite bleaching reaction is utilized to generate the various degradation indices. While monomeric anthocyanins
are readily bleached by bisulfite at product pH
(Fig. F1.2.3), the polymeric anthocyanin-tannin and melanoidin pigments are resistant and
will remain colored. Somers and Evans (1974)
used this reaction in developing spectral methods for assessing the color quality of wines. The
authors laboratory has found them useful for
tracking color quality in a wide range of anthocyanin-containing foods (Wrolstad et al., 1982,
1995). Absorbance measurements are taken at
the vis-max and at 420 nm on the bisulfite
bleached and control samples. Color density is
the sum of the absorbances at the vis-max and
at 420 nm of the control sample, while polymeric color is the same measurement for the
bisulfite treated sample. A measure of percent
polymeric color is obtained as the ratio between
these two indexes. The absorbance at 420 nm
of the bisulfite-treated sample is an index for
browning, as the accumulation of brownish

Anthocyanins

F1.2.9
Current Protocols in Food Analytical Chemistry

degradation products increases the absorption

in the 400 to 440 nm range. The absorption of
these compounds are in general not affected by
the addition of a bisulfite solution.
Molar absorptivity
Regardless of the method used for anthocyanin quantitation, the determination of the
amount present requires an absorptivity coefficient. Absorptivity coefficients have been reported as the absorption of a 1% solution measured through a 1-cm path at the vis-max, or as a
molar absorption coefficient. Absorptivity coefficients of some known anthocyanins have
been reported by different researchers (Table
F1.2.1). Through the years, there has been a
lack of uniformity on the values of absorptivity
reported, mainly due to the difficulties of preparing crystalline anthocyanin, free from impurities, in sufficient quantities to allow reliable
weighing under optimal conditions (Fuleki and
Francis, 1968a; Francis, 1982; Giusti et al.,
1999). Other problems are that the anthocyanin
mixtures may be very complicated, and not all
absorptivity coefficients may be known. Even
when they are known, it is necessary to first
evaluate if the objective is the estimation of total
anthocyanin content or the determination of
individual pigments, and then to decide which
absorption coefficient(s) to use. The absorptivity is dependent not only on the chemical structure of the pigment but also on the solvent used;
preferably, the coefficient used should be one
obtained in the same solvent system as the one
used in the experiment. If the identity of the
pigments is unknown, it has been suggested that
it can be expressed as cyanidin-3-glucoside,
since that is the most abundant anthocyanin in
nature (Francis, 1989).

Characterization
and Measurement
of Anthocyanins
by UV-Visible
Spectroscopy

Spectral characteristics
Substantial information can be obtained
from the spectral characteristics of anthocyanins (Fig. F1.2.1). Two distinctive bands of absorption, one in the UV-region (260 to 280 nm)
and another in the visible region (490 to 550
nm) are shown by all anthocyanins. The different aglycons have different vis-max, ranging
from 520 nm for pelargonidin to 546 nm for
delphinidin, and their monoglucosides exhibit
their vis-max at about 10 to 15 nm lower (Strack
and Wray, 1989). The shape of the spectrum
may give information regarding the number
and position of glycosidic substitutions and
number of cinnamic acid acylations. The ratio
between the absorbance at 440 nm and the
absorbance at the vis-max is almost twice as

much for anthocyanins with glycosidic substitutions in position 3 as compared to those with
substitutions in positions 3 and 5 or position 5
only. The presence of glycosidic substitutions
at other positions (e.g., 3,7-diglycosides) can
be recognized because they exhibit a different
spectral curve from those of anthocyanins with
common substitution patterns. The presence of
cinnamic acid acylation is revealed by the presence of a third absorption band in the 310 to
360 nm range (Figure F1.2.1), and the ratio of
absorbance at 310 to 360 nm to the absorbance
at the visible vis-max will give an estimation of
the number of acylating groups (Harborne,
1967; Hong and Wrolstad, 1990). The solvent
used for spectral determination will affect the
position of the absorption bands, and therefore
must be taken into consideration when comparing available data.

Critical Parameters and

Troubleshooting
The pH of buffers should always be checked
and adjusted prior to use. The use of buffers
with lower or higher pH levels will result in
under- or overestimations of the pigment content.
The accuracy of the results will be greatly
affected by the accuracy of the volumetric
measurements. Make sure that any volumetric
flasks or pipets used for obtaining the appropriate dilutions are calibrated correctly.
For the methodologies described in this unit,
all spectral measurements should be made between 15 min and 1 hr after the dilutions have
been prepared. The observed readings tend to
increase with time.
When working with several different samples, it may be acceptable to use one common
approximate vis-max that is typical of all samples (i.e., 520 nm). The visible absorbance peak
is broad, and measuring a few nanometers off
vis-max will not significantly alter the estimated
final values.
Serial dilutions are recommended to ensure
accurate measurements of highly concentrated,
high density, or dried samples. Perform a
weight-by-volume dilution with distilled water
to obtain a single-strength solution (e.g., usually around 10 Brix for fruit juices; UNIT H1.4),
followed by a second dilution using 0.025 M
potassium chloride buffer, pH 1.0. Both dilution factors must be considered when calculating monomeric anthocyanin content.
For example, 1 g of a 75 Brix juice concentrate was diluted to a final volume of 10 ml with
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

Source
Apples (Scugog)
Bilberries
Blackberries
Black currants
Blueberries
Red cabbage
Black chokeberries
Cherries
Cranberries
Elderberry
Grapes
Kiwi
Red onions
Plum
Red radishes
Black raspberries
Red Raspberries
Strawberries
Tradescantia pallida
(leaves)

Pigment content
Reference
(mg/100 g fresh weight)
10
300320
83326
130400
25495
25
560
4450
60200
450
6600
100
721
225
1160
300400
2060
1535
120

a density of 1 g/ml for juice). Then, the appropriate dilution factor for the sample was determined by diluting 0.2 ml of the solution with
2.8 ml of 0.025 M potassium chloride buffer,
pH 1.0 (dilution factor = 15). To calculate
monomeric anthocyanin content, color density,
or polymeric color, the dilution factor to use
would be: DF = (10 15) = 150.
The methodologies used to measure color
density and polymeric color were developed for
fruit juices, which naturally have an acidic pH.
If the material to be measured has a pH in the
neutral or alkaline range, the pH of the solution
should be lowered with a weak acid. In these
cases, the authors recommend the use of a 0.1
M citric acid buffer, pH 3.5, instead of distilled
water to prepare the different dilutions.
Some potential interfering materials are
other red pigments: FD&C Red No. 40, FD&C
Red No. 3, cochineal, and beet powder (betalain
pigments). The presence of alternative colorants may be suspected if the vis-max at pH 1.0
is high (550 nm, more typical of betalain pigments), or if a bright red coloration is found at
pH 4.5 (potential presence of artificial dyes).
The presence of ethanol does not interfere
with the assay at the levels typically encountered in wines (10% to 14%).

Mazza and Miniati, 1993

Mazza and Miniati, 1993
Mazza and Miniati, 1993
Timberlake, 1988
Mazza and Miniati, 1993
Timberlake, 1988
Kraemer-Schafhalter et al., 1996
Kraemer-Schafhalter et al., 1996
Timberlake, 1988
Kraemer-Schafhalter et al., 1996
Mazza and Miniati, 1993
Kraemer-Schafhalter et al., 1996
Mazza and Miniati, 1993
Timberlake, 1988
Giusti et al., 1988
Timberlake, 1988
Mazza and Miniati, 1993
Timberlake, 1988
Shi et al., 1992

Highly acylated anthocyanins may not respond to pH changes the same way as anthocyanins with no or few acylating groups, and may
not decolor as much as nonacylated or monoor diacylated anthocyanins do at pH 4.5.

Anticipated Results
The anthocyanin content of different common fruits and vegetables is presented in Table
F1.2.3. Anthocyanin-containing fruit or vegetable juices typically have pigment content
ranging from 50 to 500 mg/liter. Anthocyaninbased natural colorants and nutraceuticals may
have a much higher pigment concentration, on
the order of a few grams/liter.
Fresh fruit or vegetable juices should have
a low percentage of polymeric color (usually
less than 10%), while processed samples and
materials subjected to storage abuse will be
much higher (30% or more). This is highly
variable, dependent on the commodity, processing conditions, and storage history.
Always express anthocyanin pigment content in terms of the specific anthocyanin used
for calculation, and specify molecular weight
and utilized.
Anthocyanins

F1.2.11
Current Protocols in Food Analytical Chemistry

Time Considerations
Quantitation of anthocyanins can be
achieved in <1 hr. It is necessary to wait for the
spectrophotometer to warm up, and for the
diluted samples to equilibrate at least 15 min.
The absorbance readings take a few minutes.

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Key References
Giusti et al.,1999. See above.
Compares the molar absorptivity of many anthocyanins in different solvent systems.

Swain, T. and Hillis, W.E. 1959. The phenolic constituents of Prunus domestica. I. The quantitative
analysis of phenolic constituents. J. Sci. Food
Agric. 10:63-68.

Somers and Evans, 1974. See above.

Takamura, H. and Yamagami, A. 1994. Antioxidative activity of mono-acylated anthocyanins isolated from Muscat Bailey A grape. J. Agric. Food
Chem. 42:1612-1615.

Wrolstad et al., 1982. See above.

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beverages]occurrence, extraction and chemistry [coloring material]. Food Chem. 5:69-80.
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Res. 280:107-121.

Spectral methods are described for generating several color quality indices for wines.

Description of the pH differential method for determination of total anthocyanins and indices for anthocyanin degradation as applied to fruit juices and
wines.

Contributed by M. Mnica Giusti,

University of Maryland
College Park, Maryland
Ronald E. Wrolstad,
Oregon State University
Corvallis, Oregon

Anthocyanins

F1.2.13
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