Determination of Beet Root Betanin in Dairy Products by High

Laboratory Experiment pubs.acs.org/jchemeduc

Determination of Beet Root Betanin in Dairy Products by HighPerformance Liquid Chromatography (HPLC) Fernando Gandía-Herrero,* Ana Simón-Carrillo, Josefa Escribano, and Francisco García-Carmona Departamento de Bioquímica y Biología Molecular A, Unidad Docente de Biología, Facultad de Veterinaria, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain S Supporting Information *

ABSTRACT: The food industry uses different additives to give foods and beverages the appearance expected by the consumer. Among them, pigments of natural origin are receiving increasing attention due to safety concerns about traditional colorants and the relevance of a healthy diet. This experiment describes the quantitative determination of the characteristic pigment of red beet roots in dairy foods such as conventional yogurt, drinking yogurt, and fromage frais of strawberry, blackberry, and apricot varieties. The laboratory experiment is suitable for an undergraduate instrument analysis course and introduces the principles involved in chromatographic techniques and the use of highperformance liquid chromatography (HPLC) equipment. It also opens the discussion to other concepts such as carbon chirality, absorbance, and the requirements of food additives. KEYWORDS: Second-Year Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Chirality/Optical Activity, Chromatography, Dyes/Pigments, Food Science, Quantitative Analysis, UV−Vis Spectroscopy

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he food industry uses different additives to give foods and beverages the appearance expected by the consumer. Color is an important feature responsible for consumer choice and a variety of colorant molecules can be used to give the desired hues to food products. Concerns about the safety of traditional food colorants and the relevance of a healthy diet are giving natural pigments new application perspectives.1 The use of red beet root extracts to give pink and violet colors to food products is based on the high absorbance of the main pigment betanin, which possesses a molar extinction coefficient of ε = 65,000 L mol−1 cm−1 (λ = 536 nm).2 Betanin is the best known member of the betalains, a family of watersoluble vegetable pigments whose consumption has been related with the inhibition of skin and liver tumor formation and γ radiation effects protection in animals.3,4 In addition, betanin is stable over a wide range of pH and its use is also supported by a strong antioxidant and antiradical activity.5,6 The present experiment has been designed to show the applications of reversed-phase chromatography and to train students in the use of high-performance liquid chromatography (HPLC) by analyzing the presence of beet root betanin in conventional yogurt, drinking yogurt, and fromage frais of strawberry, blackberry, and apricot varieties (Figure 1). In our teaching experience, the attention of students is quickly attracted by the idea of finding the beet root “fingerprint” in these daily life consumption products. This increases the student motivation7 and makes teaching the principles involved in chromatographic techniques easier.8,9 The experiment also opens the discussion to other concepts such as carbon chirality, absorbance, and the requirements of food additives. © 2012 American Chemical Society and Division of Chemical Education, Inc.

Figure 1. Commercial dairy products used in this study.

The established method for betalain separation10 has been shortened to reduce the analysis time necessary to achieve the separation of the two isomers of betanin (2S/S and 2S/R). Figure 2 shows the structure of the pigment. The presence of Published: March 23, 2012 660

dx.doi.org/10.1021/ed200397q | J. Chem. Educ. 2012, 89, 660−664

Journal of Chemical Education

Laboratory Experiment

information is expected from the visible region. A molar extinction coefficient at 536 nm of ε = 65,000 L mol−1 cm−1 was used for the quantification of standard betanin.2 For sample clarification, the centrifuge Sigma 2-16K was used. Reagents

Conventional yogurt (two strawberry and one blackberry varieties), drinking yogurt (strawberry variety), and fromage frais (strawberry and apricot varieties) were purchased from a superstore. Red beet roots were purchased from a local supermarket. E-162 colorant was obtained from Chr. Hansen. Trifluoroacetic acid (TFA) and sodium phosphate were obtained from Sigma. HPLC-grade acetonitrile was purchased from Labscan Ltd. Deionized water was purified using a Milli-Q system for HPLC use (alternatively HPLC-grade water can be purchased).

Figure 2. Structure of the pigment betanin with the chiral carbon responsible for isomerization circled.

the two isomers in betanin is derived from the chiral carbon present in the betalamic acid moiety of the pigment (circled in Figure 2). In fresh beets, the configuration of this chiral carbon is in a ratio of 95:5 (S:R).11 Heat and pressure treatment of beet extract promotes racemization and 50:50 mixtures are found in pasteurized colorants as used in the food industry. The other chiral carbon, found in the cyclo-dihydroxyphenylalanine moiety, possesses a unique S configuration and does not contribute to the existence of the isomers. However, its presence means the whole molecule is enantiomeric and isomer separation can be achieved by reversed-phase chromatography, instead of making it necessary to use chiral chromatography. The present experiment can be performed in one laboratory session of 4 h of an undergraduate instrument analysis course. Students prepare the samples from the dairy products and run the equipment to measure the concentration of the food pigment in some of them. Results for additional samples or replicas are presented in a following classroom session. The experiment has been successfully introduced in an instrumental techniques course with positive feedback from students.

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Mobile-Phase Solutions

Solvent A was placed in one reservoir and was composed of Milli-Q grade water with 0.05% (v/v) TFA. Solvent B was placed in a different reservoir and was composed of acetonitrile with 0.05% (v/v) TFA. A linear gradient was performed for 15 min from 0% B to 35% (v/v) B. Solvent B percentage was then maintained at 35% for 5 additional minutes, and then returned to the starting conditions (0% B) for 5 min more (25 min total run time).

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EXPERIMENTAL PROCEDURES

Standard Solution Preparation

Betanin can be purchased as the commercial E-162 colorant used by the food industry and diluted in sodium phosphate buffer 20 mM, pH 6.0, to a concentration 3.0 μM (0.195 absorbance units at the wavelength λ = 536 nm). The betanin isomer ratio will be 50:50, as commented above and shown in Figure 3A. Alternatively, betanin can be extracted from red beet root and further purified through Sephadex G-25 columns.12,13 A 3.0 μM dilution in sodium phosphate buffer 20 mM, pH 6.0, of the extract will yield the standard solution. In this case, the betanin isomer ratio will be 95:5, as shown in Figure 3B. In both cases, a suitable five-data point calibration curve can be performed with concentrations below 3.0 μM (Figure 3C). In our HPLC system, the following equation was obtained: total peak area = 78 556 μmol/L + 7012 (r = 0.9999).

EXPERIMENTAL SECTION

Apparatus and Materials

Chromatograms were collected using a Shimadzu LC-10A apparatus equipped with a SPD-M10A photodiode array detector (PDA). Reversed-phase chromatography was performed with a 250 × 4.6 mm Luna C18(2) column packed with 5 μm particles. The flow rate was 1 mL/min, operated at 25 °C. Injection volume was 25 μL. Although the system described is equipped with a diode array detector, this experiment can be performed on a single wavelength detector (λ = 536 nm). A Jasco V-630 spectrophotometer was used for absorbance spectroscopy. Measurements were made in water at 25 °C. Quartz 1 mL cuvettes of 1 cm path length were used, but optic glass or even plastic cuvettes can be used because the relevant

Sample Preparation

The samples were made by placing 4 g of each of the following dairy products in 10 mL beakers: blackberry yogurt (A), strawberry yogurt sample 1 (B), strawberry yogurt sample 2 (C), strawberry liquid yogurt (D), strawberry fromage frais (E),

Figure 3. HPLC analysis for betanin pigment standards obtained from (A) a commercial colorant and (B) natural beet root, showing the isomers 2S/S (11.0 min) and 2S/R (11.4 min). The calibration curve for concentration determination of betanin (C). 661




Figure 4. Absorbance spectra for the dairy products samples: (A) blackberry yogurt, (B) strawberry yogurt 1, (C) strawberry yogurt 2, (D) strawberry liquid yogurt, (E) strawberry fromage frais, and (F) apricot fromage frais.

Figure 5. Chromatograms recorded at λ = 536 nm, obtained for the analyses of dairy products. Twenty-five microliters of each sample was injected: (A) blackberry yogurt, (B) strawberry yogurt 1, (C) strawberry yogurt 2, (D) strawberry liquid yogurt, (E) strawberry fromage frais, and (F) apricot fromage frais.

HPLC Procedure

and apricot fromage frais (F). Strawberry yogurt samples 1 and 2 differ in the product brand used. Sodium phosphate buffer, 2 mL of 20 mM, pH 6.0, was added to the conventional yogurt samples (A, B, and C). No additional buffer was added to the liquid yogurt sample (D), and 4 mL of the previous buffer was added to the fromage frais samples (E and F). Each sample was mixed thoroughly and further centrifuged (5000g, for 15 min) and filtrated through 0.45 μm syringe filters for clarification before HPLC injection. Volumes and samples weights can be varied to manipulate bigger volumes.

Filtrated samples were placed in vials and injected in the HPLC system. The number of samples or replicas used in a session can be adjusted depending on the available time and the possibility of presenting additional results in a further classroom session. A small volume, 25 μL, of each sample is injected. The mobile phase consisted of a gradient mixture of water and acetonitrile, both supplemented with 0.05% TFA at a rate of 1 mL/min. The protocol implies a fully aqueous starting condition, which is necessary for proper isomer separation. Each run included a 662




Table 1. Peak Areas and Concentrations Determined for Betanin Pigment in Dairy Products Analysis Peak Areaa Sample

11.4 min (2S/R)

Total

μMb

μg/(g sample)

38099 38741 n.d. n.d. 79375 77922 39874 39936 64559 62106 9463 9411

21253 21473 n.d. n.d. 52796 51110 32236 30683 51690 51937 6883 7399

59352 60214 n.d. n.d. 132171 129032 72110 70619 116249 114043 16346 16810

0.85 ± 0.01

0.68 ± 0.01

n.d.

n.d.

1.75 ± 0.03

1.40 ± 0.02

1.00 ± 0.01

0.52 ± 0.01

1.56 ± 0.02

1.65 ± 0.02

0.30 ± 0.01

0.32 ± 0.01

A Blackberry yogurt B Strawberry yogurt 1 C Strawberry yogurt 2 D Strawberry liquid yogurt E Strawberry fromage frais F Apricot fromage frais a

Betanin Concentration

11.0 min (2S/S)

Duplicated student data. bBetanin concentration as determined from peak areas, without considering sample preparation and dilution.

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SUMMARY This experiment was designed for an undergraduate instrumental techniques course to illustrate analysis concepts and train students in the use of HPLC. Additionally, the laboratory session allows for discussion on concepts such as carbon chirality, structural isomers separations, absorbance, and the requirements of food additives. The use of food material the students are familiar with arouses their interest and facilitates the introduction to the chemical concepts.

final step set up for column re-equilibration after analysis. After each session, the column was washed with 50:50 acetonitrile/ water to clean the column for the next use.

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HAZARDS

Acetonitrile is flammable and harmful if ingested, inhaled, or in contact with the skin. TFA is corrosive and harmful by ingestion and inhalation. Gloves and safety goggles should be used during the preparation of the HPLC solutions in an area with good ventilation. No additional precaution is necessary when preparing samples and standards.

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ASSOCIATED CONTENT

* Supporting Information S

Directions for the students and notes for the instructor. This material is available via the Internet at http://pubs.acs.org.

RESULTS AND DISCUSSION

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A visible spectrum for each clarified sample before injection can be optionally recorded to complement the visual information on the food color observed by the students. Figure 4 shows the spectra recorded, showing an absorbance peak centered at wavelengths around λ = 536 nm. Pink color is obtained with substances absorbing at this wavelength. Additionally, another peak centered at λ = 450 nm, at which yellow substances absorb, is observed for samples B, D, and F. The highest contribution of the yellow peak with respect to the pink peak was observed for sample F, corresponding to fromage frais of the apricot variety. Chromatograms for the six samples used are shown in Figure 5. Both isomers of betanin (2S/S) and (2S/R) appeared in the chromatogram at 11.0 and 11.4 min, respectively. Only sample B, one of the two conventional yogurts used in the study, lacked detectable amounts of betanin. Both yogurts report the use of beet root extracts as well as similar amounts of strawberry fruits (10.1% in B, and 9.5% in C). Sample B reports the use of additional strawberry concentrated juice, which might be behind the use of undetectable amounts of beet root to complement the color. All the other samples allow a proper determination of betanin, with the presence of both isomers being observed. Concentrations are appropriate for quantification under the calibration curve developed. The presence of betanin in the extracts can be expressed as concentration units (μM), and the total amount of the pigment in the dairy foods can be calculated as μg/g, considering that the mass for the molecule is 550 Da. Results are shown in Table 1.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

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ACKNOWLEDGMENTS The authors are grateful for the support of Ministerio de Ciencia e Innovación (MCINN-FEDER, Spain) (AGL201017938), and Fundación Séneca, Agencia de Ciencia y Tecnologiá de la Región de Murcia (Grupos de Excelencia de ́ la Región de Murcia, 2007/2010). F. Gandia-Herrero holds a contract with “Programa Ramón y Cajal” (MICINN-FEDER, Spain).

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REFERENCES

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(9) Leacock, R. E.; Stankus, J. J.; Davis, J. M. J. Chem. Educ. 2010, 88, 232−234. (10) Gandía-Herrero, F; García-Carmona, F.; Escribano, J. J. Chromatogr., A 2005, 1078, 83−89. (11) Gandía-Herrero, F.; Escribano, J.; García-Carmona, F. Plant Physiol. 2005, 138, 421−432. (12) Escribano, J.; Pedreño, M. A.; García-Carmona, F.; Muñoz, R. Phytochem. Anal. 1998, 9, 124−127. (13) Pedreño, M. A.; Escribano, J. J. Biol. Educ. 2000, 35, 49−51.

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Determination of Beet Root Betanin in Dairy Products by High

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