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Extraction and Quantitation of FD&C Red Dye #40 from Beverages Containing Cranberry Juice: A College-Level Analytical Chemistry Experiment
Henry F. Rossi, III, Jacqueline Rizzo, Devon C. Zimmerman, and Karyn M. Usher*
Department of Chemistry, West Chester University of Pennsylvania, West Chester, Pennsylvania 19383, United States
S Supporting Information *

ABSTRACT: A chemical separation experiment can be an interesting addition to an introductory analytical chemistry laboratory course. We have developed an experiment to extract FD&C Red Dye #40 from beverages containing cranberry juice. After extraction, the dye is quantied using colorimetry. The experiment gives students hands-on experience in using solid phase extraction to extract the red dye from other colorants in the red beverages. Students are also given experience with colorimetry, the BeerLambert law, and some wet chemical techniques. The recovery of the red dye from spiked cranberry juice was found to be 99% with a standard deviation of 2%.

KEYWORDS: Second-Year Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Chromatography, Dyes/Pigments, Food Science

any common fruity beverages that people drink on a regular basis contain anthocyanins and FD&C Red Dye #40 (Red 40).1 Anthocyanins are plant pigments commonly used in beverages to provide both natural pigment and health benets. They are strong antioxidants that can improve and protect bodily functions. They can protect damaged blood vessels, protect against neurological oxidation damage, and improve eyesight.24 A generalized structure of anthocyanins and the structure of Red 40 are shown in Figure 1.

cyanin content of dierent foods and beverages.610 Many papers have also described student labs that involved food dyes.1117 In the experiment described here, students in a college-level introductory analytical chemistry class used solid phase extraction (SPE) to determine which beverages contained Red 40. The students then quantitated the Red 40 isolated from beverages using colorimetric analysis. The following topics are emphasized: (1) chromatography, (2) sample preparation (specically solid phase extraction), (3) colorimetry (BeerLambert law), (4) proper dilution technique, and (5) preparation of calibration curves and unknown concentration calculations. The experiment provides students with hands-on experience of the analysis of common beverages that they will most likely be familiar with.

Figure 1. Structures of (A) Red 40 and (B) generalized anthocyanins.

Red 40, also known as Allura Red AC or E129, is a red azo dye that may cause serious health side eects. Studies have linked it to hyperactivity and ADHD in children.5 Children between the ages of three and nine are most sensitive to the eects of Red 40 because of their stage of development. The eect may be far-reaching because many beverages targeted for children contain large quantities of the dye. Many research papers have described the use of highperformance liquid chromatography to determine the antho 2012 American Chemical Society and Division of Chemical Education, Inc.

EXPERIMENTAL DEVELOPMENT The determination of the percent of cranberry juice in apple cranberry juice cocktail was described in a previously published experiment.18 For that experiment, it was not necessary to separate the components of the juices. The apple juice did not absorb strongly at the same wavelength that the cranberry juice did, so the authors took advantage of this to analyze apple cranberry juice cocktails for their cranberry juice content. Our experiment is novel because it involves the extraction of Red 40 from beverages that contain both Red 40 and anthocyanins. As both of these colorants absorb strongly at the same wavelength, it is necessary to isolate them from one another to determine
Published: October 12, 2012
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Journal of Chemical Education the concentration of the Red 40 in the beverages. This experiment was developed using a variety of beverages with dierent concentrations of Red 40 and/or cranberry juice (a common source of anthocyanins). A solid phase extraction method was developed to separate the Red 40 from the anthocyanins. The Red 40 was collected and the concentration was determined using colorimetry. Further analysis of the anthocyanins will not be discussed in this paper.

Laboratory Experiment

experiment were Bond Elute JR-C18, 500 mg cartridge (PN: 12162028B Agilent Technologies, Santa Clara, CA). The Supporting Information includes a detailed list of equipment, reagents, and supplies.

EXPERIMENTAL OVERVIEW

Procedure

The solid phase extraction procedure was performed using Agilent Bond Elute JR C-18 cartridges. The procedure was developed using information about methods previously published for isolation of FD&C dyes from foods19 and isolation of anthocyanins from fruits.9,10 A beverage containing both Red 40 and cranberry juice (3-C Cranberry Juice Cocktail) was chosen for the students to analyze. Red 40 was isolated from the beverage using the solid phase extraction procedure shown in Figure 2. Once the Red 40 was isolated, the collected fraction was analyzed using colorimetry.

HAZARDS Hazards involved with this experiment are associated with the use of hydrochloric acid, methanol, 2-propanol, and Red 40. Hydrochloric acid, 2 M (CAS: 7647-01-0, UN: 1789, PN: A452-4, Fisher Scientic, Fairlawn, NJ) may be used outside of the hood. Concentrated hydrochloric acid must be used in the hood. Methanol (CAS 67-56-1, PN: A 452-4, Fisher Scientic, Fairlawn, NJ) is a ammable liquid. The 2-propanol (CAS: 6763-0, PN: 3032-08, Mallinkrodt Paris, KY) used to prepare the 4% and 5% 2-propanol washes is also a ammable liquid. If acid, methanol, or 2-propanol gets in contact with eyes, ush eyes thoroughly with water. Any solutions including methanol or 2-propanol must be placed in a waste container for proper disposal. Students should avoid inhalation of the methanol and 2-propanol fumes and should wear gloves to avoid skin contact. Avoid inhalation of the 92% pure Red 40 (CAS: 25956-17-6, Rainbow Colors, LLC Windsor, CT) and wash eyes thoroughly with water if exposed. Safety glasses should be worn at all times during this experiment. RESULTS The experiment was performed by 13 students registered in the introductory analytical laboratory in a four hour lab period. The class average for the concentration of Red 40 in 3-C Cranberry Juice Cocktail was 113 g/mL with a standard deviation of 5 g/mL. In this experiment, students were required to prepare standards, create a calibration curve, use SPE to isolate the Red 40 in the sample, and use colorimetry to analyze the samples of Red 40. Author data for samples of 13 dierent beverages is included in Table 1. Several beverages that only contained Red 40 and several beverages that only contained cranberry juice (no Red 40) were chosen to show that the SPE method eciently isolated the Red 40. This was necessary because the quantity of Red 40 in the beverages is proprietary, so it would be dicult to prove a separation of the Red 40 and anthocyanins if only beverages with both were used. The method was also validated by using a sample of cranberry juice that was spiked with Red 40 to give a concentration of 101.6 g Red 40/mL of cranberry juice. The recovery of Red 40 using the method presented was 99% with a standard deviation of 2%. DISCUSSION Many beverages that contained both anthocyanins and Red 40 were studied in development experiments (data not shown). Grape-avored beverages that contain appreciable quantities of real grape juice cannot be analyzed by this method because both Red 40 and the colorants from grape juice were eluted at the same time. Grape beverages that do not contain real grape juice, but do contain Red 40 and FD&C blue dye #1 may be used. Experiments were used to verify that Red 40 could be isolated from the anthocyanins in many fruit avored beverages and extracted with high precision. This was accomplished by performing the experiment using 13 beverages with dierent combinations and concentrations of Red 40 and cranberry juice
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Figure 2. Solid phase extraction procedure for extraction of Red 40 from cranberry-avored beverages.

Red 40 (92%) was used to prepare the stock solution. Serial dilutions of the stock (ranging from 1 to 15 g/mL) were prepared and used to create the calibration curve. The wavelength of maximum absorbance (max) for the dilutions was determined using a MicroLAB FS-522 spectrophotometer to be 502 nm. The ve stock dilutions were within the linear range of absorbance for Red 40 and the concentration of Red 40 in the solutions isolated from the Red beverages fell within this range. The molar absorptivity () and intercept were determined using a BeerLambert plot and this was used to determine the concentration in the unknowns.
Equipment

Equipment capable of doing colorimetry is needed for this experiment. The data presented were collected using a MicroLab FS-522 data acquisition interface with FAST scan 16-wavelength spectrophotometer (MicroLab, Inc., P.O. Box 7358, Bozeman, MT 59771) in conjunction with MicroLab Software v.5.4.9-2009-12-23. Other required supplies include vials or cuvettes to t the instrument, an analytical balance, 1 mL volumetric pipet, 10 mL syringes, and solid phase extraction cartridges. The cartridges used to perform this

Journal of Chemical Education Table 1. Determination of Red 40 in Beverages

Sample Name 3-C Cranberry Juice Cocktailb Ocean Spray Diet Cranberryb Ocean Spray Cranberry Juice Cocktail Ocean Spray Light Cranberry Ocean Spray Sparkling Cranberry Gatorade Strawberry Mondo Sierra Mist Cranberry Splash Crush Grape Soda Wild Harvest Natural Cranberry Very Fine Cranberry Juice Cocktail Very Fine Fruit Punch Little Hug Fruit Barrel
a

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x/ (g/mL)a 120.41 12.79 NDc NDc NDc 40.47 38.20 51.55 55.99 NDc NDc 33.03 48.31

s/ (g/mL) 2.87 1.27 0.61 2.31 0.35 2.23 1.16 1.92

% RSD 2.38 9.89 1.51 6.04 0.68 3.98 3.51 3.96

Red 40 Listed on Label? Yes Yes No No No Yes Yes Yes Yes No No Yes Yes

previous experiment also did not yield very precise or accurate results when samples of known dye concentration were used. Students who performed the experiment described in this paper, following the procedure given in the Supporting Information, stated that the directions were clear and that they had no trouble performing the experiment after the instructor demonstrated how to use the SPE cartridge and syringe assembly. The experiment was preferred over the previous experiment and gave students a better understanding of how chromatography works.

ASSOCIATED CONTENT

S Supporting Information *

Notes for the instructor, including equipment and supplies, the types of drinks that should be used (and some suggestions for which types to avoid), and lecture ideas are provided. Also included are editable student procedures for the preparation of standards and for the solid phase extraction procedure used to isolate the Red 40. This material is available via the Internet at http://pubs.acs.org.

Data from 3 trials was obtained for each sample. bOnly Ocean Spray Diet Cranberry and 3-C Cranberry Juice Cocktail contained both Red 40 and anthocyanins. cND in the table means that Red 40 was below the limit of detection.

AUTHOR INFORMATION

Corresponding Author

*E-mail: kusher@wcupa.edu.
Notes

The authors declare no competing nancial interest. and also by recovery from a spiked solution. The results are shown in Table 1. Students may be given a few drinks that contain only Red 40, only cranberry juice, or a combination of Red 40 and cranberry juice and asked to determine the concentration of Red 40 in each. The solid phase extraction method can be used to determine which beverages contain only Red 40. Beverages containing only Red 40 can be analyzed by a simple dilution followed by colorimetric analysis as there are no anthocyanins present to interfere with the analysis. Beverages with only anthocyanins need no further analysis. The beverages with both Red 40 and anthocyanins can be analyzed using the SPE procedure and colorimetry. This experiment was appropriate for college-level analytical chemistry laboratory students; however, it would also be appropriate for use in a general chemistry lab. At the general chemistry level, instructors may choose to have students work in pairs or provide the standards because students at that level would likely need more time to perform the experiment than more advanced students.

ACKNOWLEDGMENTS The authors would like to thank those that have provided support for this experiment. Melissa Philips and Catherine Rimmer at NIST provided guidance in working with anthocyanins. Joni Stevens, William Long, and Ron Majors at Agilent Technologies, Inc. arranged for the donation of the Bond Elut JR cartridges used in this experiment and gave technical support. Sidney Cohen and Emilie Ryley at Sartomer USA, LLC administered the Sartomer Environmental Award which provided nancial support for one of the authors and funds for supplies. We acknowledge Al Plant for IT assistance and Carolyn Flechsig for her assistance.

SUMMARY This experiment was appropriate for college-level analytical chemistry laboratory courses. Both solid phase extraction and colorimetric analysis were used to quantitatively determine Red 40 in common fruit beverages. The solid phase extraction procedure was validated by measuring the recovery of Red 40 from a spiked cranberry juice sample. The recovery was 99% with a standard deviation of 2%. This experiment was developed to replace another experiment in which SPE cartridges were used to extract blue and yellow food dyes from a solution containing both. The SPE cartridges used were not compatible with the syringe assembly, so gravity ow was used in the separation. The experiment took a long time and required that each student use three SPE cartridges to perform three separations simultaneously. The
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