This Sample Protocol presents the full text of a cutting-edge Unit from Current Protocols in Food Analytical Chemistry, including expert commentary sections with critical information designed to ensure the success of your experiments.
 

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From Unit F1.2

Characterization and Measurement of Anthocyanins by UV-Visible Spectroscopy

Ronald E. Wrolstad,
Oregon State University
Corvallis, Oregon

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).
 

BASIC PROTOCOL 1

Anthocyanin pigments undergo reversible structural transformations with a change in pH manifested by strikingly different absorbance spectra. The colored oxonium form predominates at pH 1.0 and the colorless hemiketal form at pH 4.5. 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.

Materials

0.025 M potassium chloride buffer, pH 1.0 (see recipe)

0.4 M sodium acetate buffer, pH 4.5 (see recipe)

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 l_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 7).

IMPORTANT NOTE: In order to not exceed the buffer's capacity, the sample should not exceed 20% of the total volume.

Many spectrophotometers will allow for a rapid baseline correction to zero by using baseline adjust.

5. Measure the absorbance of each dilution at the l_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.

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

A = (A_{l vis-max}– A₇₀₀)_{pH 1.0} –   (A_{l 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)/(e´1)

IMPORTANT NOTE: The MW ande used in this formula correspond to the predominant anthocyanin in the sample. Use the e reported in the literature for the anthocyanin pigment in acidic aqueous solvent. If the e 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 e = 26,900 (see Background Information, discussion of Molar Absorptivity).

BASIC PROTOCOL 2

INDICES FOR PIGMENT DEGRADATION, POLYMERIC COLOR, AND BROWNING

Materials

Bisulfite solution (see recipe)

0.025 M potassium chloride buffer, pH 1.0 (see recipe)

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 l_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, l_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.

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).

6. Calculate the color density of the control sample (treated with water) as follows:

Color density = [(A_{420 nm}– A_700nm) + (A_{l vis-max}– A_{700 nm})]  ´DF

7. Calculate the polymeric color of the bisulfite bleached sample as follows:

Polymeric color = [(A_{420 nm} – A_{700 nm}) + (A_{l vis-max}– A_{700 nm})]  ´DF

Percent polymeric color = (polymeric color/color density)  ´100
 

REAGENTS AND SOLUTIONS

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 CH₃CO₂Na·3 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

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 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. 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 l_vis-max  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, 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 author's 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 l_vis-max  and at 420 nm on the bisulfite bleached and control samples. Color density is the sum of the absorbances at the l_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 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 thel_vis-max, or as a molar absorption coefficient. Absorptivity coefficients of some known anthocyanins have been reported by different researchers. 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).

Spectral characteristics

Substantial information can be obtained from the spectral characteristics of anthocyanins. 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 l_vis-max , ranging from 520 nm for pelargonidin to 546 nm for delphinidin, and their monoglucosides exhibit their l_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 l_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, and the ratio of absorbance at 310 to 360 nm to the absorbance at the visible l_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 l_vis-max  that is typical of all samples (i.e., 520 nm). The visible absorbance peak is broad, and measuring a few nanometers off l_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 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 l_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%).

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 mono- or 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. Anthocyanin-based 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 e utilized.

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.

Somers and Evans, 1974. See above.

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

Wrolstad et al., 1982. See above.

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