Laboratory Experiment

pubs.acs.org/jchemeduc

Paper Chromatography and UV−Vis Spectroscopy To Characterize

Anthocyanins and Investigate Antioxidant Properties in the Organic
Teaching Laboratory
Kelli R. Galloway, Stacey Lowery Bretz, and Michael Novak*
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States
*
S Supporting Information

ABSTRACT: A variety of fruits and vegetables, including raspberries, blueberries,

Concord grapes, blackberries, strawberries, peaches, eggplant, red cabbage, and red
onions, contain flavonoid compounds known as anthocyanins that are responsible for the
blue-red color and the astringent taste associated with such foods. In addition,
anthocyanins exhibit a wide range of chemical properties, such as radical scavenging,
metal chelation, pH-dependent color changes, and intramolecular stabilization. Two
experiments have been developed for anthocyanin-containing extracts isolated from
freeze-dried berries to teach students the skills of solid−liquid extraction, paper
chromatography, UV−vis spectroscopic characterization, and detection and evaluation of
radical scavenging properties.

KEYWORDS: Second-Year Undergraduate, Organic Chemistry, Laboratory Instruction, Collaborative/Cooperative Learning,

Chromatography, Free Radicals, Natural Products, UV-Vis Spectroscopy, Hands-On Learning/Manipulatives, Food Science

■ INTRODUCTION
Rapidly evolving research and technology necessitates
granola, allowing students to make connections to foods they
eat regularly. In the second experiment, students perform an
continued re-evaluation and redesign of laboratory curriculum.1 antioxidant assay to investigate the potential of berry extracts to
In a year-long organic chemistry nonmajors laboratory course, scavenge the stable free radical produced by one-electron
oxidation of 2,2′-azinobis(3-ethyl-benzothiazoline-6-sulfonic
renovation of laboratory rooms prompted modernization of
acid (ABTS+̇ ). Pairs of students investigate one of three
experiments to emphasize real-world connections and the
berries, and students pool results to compare findings across all
relevance of organic chemistry. Potential benefits of con-
three berries to determine if a correlation exists between
sumption of fruits and berries were integrated into the design of
anthocyanin content and antioxidant potential. Because the
two experiments. Discoveries about antioxidant properties of
berry extracts contain other soluble antioxidants, not all of the
certain foods have led to investigations to understand how
antioxidant potential of the extracts is due to their anthocyanin
polyphenols, anthocyanins, and related compounds contribute
content. Students must consult the literature to reconcile
to antioxidant properties and to potential health benefits. Two
experimental antioxidant data of the second experiment with
recent studies documented a decreased risk of myocardial
anthocyanin concentrations determined in the first experiment.
infarction and arterial stiffness in women with a consistent
By reconciling the differences between expected results for a
intake of foods high in anthocyanins.2,3 Anthocyanins have
pure sample and the data obtained from their real world
been used to develop laboratory experiments and outreach
samples, students have the opportunity to develop inquiry and
activities4−11 and are included in an effort to use natural
analysis skills that the Association of American Colleges &
products to teach chemical analysis.12 The experiments
Universities (AAC&U) recognizes as vital to higher educa-
described herein teach students the techniques of solid−liquid
tion.13

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extraction, reflux, paper chromatography, UV−vis spectroscopy,
and radical scavenging assays within the context of natural BACKGROUND
products they frequently ingest.
Two experiments were developed for students to gain Anthocyanins are water-soluble, acid stable, pigments found in
experience with anthocyanins. The first experiment involves the vacuoles of many plants.14,15 They are responsible for the
extraction of anthocyanins from freeze-dried blueberries, red-blue color of many fruits and vegetables, ranging from
blackberries, and raspberries; hydrolysis of the anthocyanin strawberries to blackberries, and eggplant to red onion.16
extracts to their anthocyanidins; and characterization and Anthocyanins are in the flavonoid family, which has long been
quantification using UV−vis spectroscopy and paper chroma-
tography. These berries are often present in cereal, trail mix, or Published: October 8, 2014
© 2014 American Chemical Society and
Division of Chemical Education, Inc. 183 dx.doi.org/10.1021/ed400520n | J. Chem. Educ. 2015, 92, 183−188
Journal of Chemical Education Laboratory Experiment

studied for its potential health benefits.15 In naturally occurring increases and the pH-dependent equilibrium shifts. The pKa
anthocyanins, hydroxy and/or methoxy substituents are present typically ranges from 2.5 to 3.5 depending on substituents.
on all three rings and the hydroxy substituents are often While anthocyanins do not have the density of aromatic rings
modified by glycosidic linkages or acetyl groups (Figure 1). or hydroxyl group of true polyphenols, they do share many of
Over 600 anthocyanins have been identified in nature due to the chemical properties of polyphenols, including the ability to
the number of variations possible with mono- or diglycosides of scavenge free radicals (Scheme 2).21−24 Students explore the
mono-, di-, or trisaccharides.15,16 radical scavenging potential of the berry extracts by performing
an antioxidant assay.22,25

■ EXPERIMENTAL PROCEDURES
Students work in pairs. Experiment 1 is done in one 3-h lab
period and Experiment 2 is done in two 3-h lab periods.
Detailed procedures are in the Supporting Information.
Experiment 1: Extraction and Characterization18,26,27
Students grind freeze-dried berries to a powder in a mortar and
pestle. A solid−liquid extraction is performed on the berry
Figure 1. Variability in the structure of the common anthocyanins and powder with acidic methanol solution. An aliquot of the extract
anthocyanidins. is set aside for analysis and the remaining solution is hydrolyzed
with an equal volume of HCl (2 M) at reflux for 45 min.
The aglycosidic forms of anthocyanins are called anthocya- During reflux, students perform paper chromatography on the
nidins. They are formed by acid catalyzed hydrolysis of the anthocyanin extract by spotting blueberry, blackberry, and
anthocyanins (Scheme S-1 in Supporting Information).16 Only raspberry extracts. Students characterize their sample with
17 anthocyanidins are found in nature, 6 of which are common paper chromatography before and after hydrolysis.16,26,28 The
in plants.15,16 These common anthocyanidins vary by place- colors of anthocyanins and anthocyanidins are used for
ment of hydrogen, hydroxy or methoxy substituents at the 3′- visualization of spots. Students acquire a UV−vis spectrum of
and 5′-positions of the B ring (Table 1), while all have the the hydrolyzed extract.
hydroxy substituent at the 4′-position. Experiment 2: Investigation of Antioxidant Potential29,30
Students perform an antioxidant assay with ABTS+• on
Table 1. Structural Variability of the Common unhydrolyzed acidic berry extract from Experiment 1. A
Anthocyanidins constant volume (12 mL) of ABTS+• solution (see the
Anthocyanidin R1 R2 Instructor Notes in Supporting Information) is pipetted into
Pelargonidin H H
each assay tube, followed by variable volumes of diluted (1/
Cyanidin OH H
100) berry extract (0 (control), 0.5, 1.0, or 1.5 mL), and
Delphinidin OH OH
sufficient acidified methanol to keep the total assay volume
Peonidin OMe H constant in each assay tube (15 mL). Details of solution
Petunidin OMe OH preparation for the assay are provided in Supporting
Malvidin OMe OMe Information Table S-1 and accompanying text in the Instructor
Notes. The concentration of ABTS+•̇ is monitored in each assay
sample by UV−vis spectroscopy 10 min after sample
Although anthocyanins are found in many fruits and preparation. Plots of ABTS+• concentration as a function of
vegetables, their identities and amounts vary. Cyanidin is berry extract volume are prepared and analyzed.

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present in the majority of anthocyanin-containing foods, while
petunidin and malvidin are uncommon.17,18 Students inves-
tigate blueberries, blackberries, and raspberries due to their HAZARDS
complementary anthocyanidin profiles (Table 2). Blackberry Standard laboratory safety procedures should be followed
and raspberry primarily contain different amounts of cyanidin, including wearing gloves and goggles. All operations should be
with smaller amounts of pelargonidin. Blueberry contains all of performed in fume hoods. Methanol and butanol are
the common anthocyanidins except pelargonidin. flammable. Hydrochloric and formic acids are caustic and
The colors of anthocyanins and anthocyanidins are pH- should not be inhaled. ABTS is toxic if inhaled or ingested.
dependent.19,20 In acidic solution, the extended conjugation of Anthocyanins and their aglycones are not dangerous as they are
the flavylium cation (Scheme 1) is responsible for the present in many common food items. They are highly colored
substituent-dependent visible absorbance at 465−550 nm, dyes, but the colors fade with time, so they will not generally
giving rise to variable colors. The color disappears as pH leave permanent stains on clothing.

Table 2. Anthocyanidin Content of Berries17

Anthocyanidin (mg/100 g of fresh or consumed form)

Food Cyanidin Delphinidin Pelargonidin Peonidin Petunidin Malvidin
Wild Blueberry 66.3 141 36.9 87.6 154.6
Blackberry 244 0.7
Raspberry 90.2 1.9

184 dx.doi.org/10.1021/ed400520n | J. Chem. Educ. 2015, 92, 183−188

Journal of Chemical Education Laboratory Experiment

Scheme 1. pH-Dependent Equilibrium of the Flavylium Cation and the Pseudo-Base/Anhydro-Base

Scheme 2. Radical Scavenging by Polyphenols and spectrum is shown in Figure 2. Students recorded λmax in the
Anthocyanins visible region and calculate an approximate concentration of

Figure 2. Typical student results for the UV−vis spectrum for

blackberry anthocyanidins mixture, λ max = 530 nm. (Note:
anthocyanidins have UV absorbance, but the intensity of the UV
peak is also attributable to other compounds in the extract.)

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anthocyanidins in their berry using a molar absorptivity
coefficient of 3.0 × 104 M−1 cm−1.16 Students estimated the
RESULTS AND DISCUSSION moles of anthocyanidin/anthocyanin per gram of freeze-dried
Experiment 1 fruit based on their dilution factor and mass of berry powder.
Typical student results are in the range of 3.0 × 10−6 to 2.0 ×
Students were assigned to one of two eluting mixtures,
10−5 mol/g of berry with raspberry extracts containing less
butanol/acetic acid/water (4:1:5, upper phase) or a formic
anthocyanin than the other two extracts. Calculations are
acid (formic acid/conc HCl/water, 5:2:3) mixture. These
summarized in the Instructor Notes in Supporting Information.
solvents were chosen because of differences in polarities. The
Students pool spectroscopy results and discuss and compare
more polar formic acid mixture achieves greater separation.
their results with published anthocyanidin content for each
Information on similarities and differences between TLC
berry. The paper chromatography data is pooled both by berry
(performed 2 weeks earlier) and paper chromatography were
and by eluting solvent to facilitate observations before and after
provided in the laboratory manual (Supporting Information).
hydrolysis.
Pure anthocyanins and anthocyanidins have a single
wavelength of maximum absorbance (λmax) in the visible Experiment 2
region, with a narrow absorbance band range of 80−100 nm. Neutral ABTS (1, Scheme 3) is colorless. One electron
Mixtures may show multiple λmax and/or wider absorbance oxidation of 1 by potassium persulfate yields the radical cation
bands.16,26 Because one anthocyanidin represents many ABTS+• (2). Solutions of 2 are a blue/teal color and absorb in
anthocyanins, students analyze only the anthocyanidins with the UV and also in the visible range at 415, 645, 734, and 815
UV−vis spectroscopy using 1:10 to 1:50 dilutions of their nm (Figure 3).22,29,30 Students compare a UV−vis spectrum of
hydrolyzed sample with acidic methanol. A typical student 2 with the anthocyanidin spectra collected during experiment 1
185 dx.doi.org/10.1021/ed400520n | J. Chem. Educ. 2015, 92, 183−188
Journal of Chemical Education Laboratory Experiment

Scheme 3. Reaction Scheme for the Antioxidant Assay

Figure 3. Typical student results for the antioxidant assay with

blackberry extract.

to choose a wavelength to monitor the assay. To measure Figure 4. Typical student plot of μM ABTS+̇ in solution versus volume
changes in concentration of 2 accurately without interference of berry extract; Blueberry (⧫); Blackberry (■); Raspberry (▲).
from anthocyanin, students should choose a longer wavelength
to monitor. For the experiment, 2 is prepared by the instructor
to have an absorbance around 0.7 at 734 nm to be concentrated student plot for each berry. Additional plots are in the
enough for students to measure changes upon addition of the Supporting Information. Students discuss the meaning of the
anthocyanin (see Instructor Notes in Supporting Information). trend line and its slope, including comparisons across berries,
The molar absorptivity coefficient is 12867 M−1 cm−1 at 734 and the relevance to the results of the previous experiment
nm.30 where the approximate anthocyanin content of each berry
extract was determined.

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After acidic extraction of the berries as in experiment 1, an
antioxidant assay is performed with 2 on the unhydrolyzed
extract. Upon addition of an antioxidant such as anthocyanin 3 TYPICAL STUDENT OUTCOMES
to the solution of 2, the radical abstracts hydrogen from the During fall 2012 and 2013, these experiments were performed
antioxidant and the blue/teal color fades as the radical is by 270−290 students in a first-semester organic chemistry
reduced to 4, the conjugate acid of 1. The antioxidant strength nonmajors laboratory course. The first experiment was
of the berry extracts are determined by the extent of completed during one 3-h laboratory class around midterm.
decolorization of 2 as measured by absorbance changes. Students wrote a formal report describing the purpose of the
Figure 3 shows typical student spectra for blackberry. Beer’s experiment and relating their data to the published literature.
Law is used to calculate the concentration of 2 remaining in Many students had difficulty interpreting the paper chromatog-
solution. Calculations are summarized in the Instructor Notes. raphy for the anthocyanins because multiple anthocyanins in
A plot of concentration of 2 remaining in solution vs the each berry caused the spots to smear and run together.
volume of berry extract is prepared with a least-squares trend Students were challenged by quantitative interpretation of the
line, and data is pooled for all berries. Figure 4 shows a typical UV−vis spectra because of various dilutions used for the
186 dx.doi.org/10.1021/ed400520n | J. Chem. Educ. 2015, 92, 183−188
Journal of Chemical Education Laboratory Experiment

extracts. Overall, students were able to interpret their results expressed in this material are those of the authors and do not
accurately and reach reasonable conclusions. necessarily reflect the views of the National Science
The second experiment was the last experiment of the Foundation. We also thank Professor Ann Hagerman for her
semester. Students were given two 3-h lab periods to complete assistance in optimizing the experiment 2 procedure.

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it. The second period was available for students to re-do the
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