Article pubs.acs.org/jchemeduc
Visualizing Capsaicinoids: Colorimetric Analysis of Chili Peppers Robert Q. Thompson,* Christopher Chu, Robin Gent, Alexandra P. Gould, Laura Rios, and Theresa M. Vertigan Department of Chemistry and Biochemistry, Oberlin College, Oberlin, Ohio 44074, United States S Supporting Information *
ABSTRACT: A colorimetric method for total capsaicinoids in chili pepper (Capsicum) fruit is described. The placental material of the pepper, containing 90% of the capsaicinoids, was physically separated from the colored materials in the pericarp and extracted twice with methanol, capturing 85% of the remaining capsaicinoids. The extract, evaporated and reconstituted in acetonitrile, was treated with Gibbs reagent to produce a beautiful blue product, easily detected by eye. Semiquantitative measurement was made by comparison to a printed color chart. The simple analysis required no electronic equipment or chromatographic separations, so is amenable to classroom demonstration, introductory laboratory, and the field. Accuracy was demonstrated by favorable comparison of results with those from a standard liquid chromatographic method for eight varieties of chili pepper. KEYWORDS: First-Year Undergraduate/General, High School/Introductory Chemistry, Analytical Chemistry, Demonstrations, Laboratory Instruction, Hands-On Learning/Manipulatives, Agricultural Chemistry, Consumer Chemistry, Food Science, UV−Vis Spectroscopy
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Table 1. Colorimetric Methods for Detecting Capsaicinoids
hili peppers (Capsicum) have attracted attention for centuries because of their spicy flavor, and today most secondary school and college students know of peppers and their nociceptive and exhilarating effects first-hand. The “hotness” of chili peppers may be assessed by the Scoville method, an organoleptic method that is the basis of the Scoville scale used to rank peppers.1 But taste tests are rather subjective and imprecise, so direct concentration-measuring methods have been developed. Liquid chromatography (LC) with mass spectrometric (MS) detection has become the standard research method for determining capsaicinoids in chili peppers and other matrixes.2,3 Because LC−MS or LC−MS−MS requires expensive instrumentation, it is not available to all chemistry students. A step down in analytical strength is liquid chromatography with ultraviolet detection (UV) of capsaicinoids at 280 nm, and this Journal has published two undergraduate laboratory experiments employing LC−UV for the analysis of chili peppers and hot sauces.4,5 Less instrument-intensive experiments require a colorimetric reaction. Capsaicinoids can be visualized by reactions of the vanillyl moiety of the capsaicinoid, either by oxidation of the phenolic group or substitution or addition on the aromatic ring. Neither mechanism is specific for capsaicinoids. A number of reactions have been developed over the past century (Table 1), and spot tests, semiquantitative methods, and complete analytical procedures based on the visible absorbance of the colored product have been reported. The first three methods listed in Table 1 give acceptable qualitative and quantitative results, but are cumbersome for routine use. The nitrosation of the vanillyl ring proceeds under mild conditionsacidified aqueous sodium nitrite solution mixed with a methanolic solution of capsaicinoidsand is attractive because the reagents are relatively safe and © 2012 American Chemical Society and Division of Chemical Education, Inc.
Method
Color of Product
Reference
Vanadium Folin−Ciocalteu Griess Nitrosation Gibbs
Various Blue Red Yellow Blue
6 7 8 9−11 see text
inexpensive. Unfortunately, difficulties exist with this method: the final solution color is similar to the color of the original chili extract; the lower molar absorptivity of the product yields lower sensitivity; side products can form.12 In the Gibbs method, 2,6dichloroquinone-4-chloroimide, Gibbs reagent, participates in an oxidation−reduction reaction with a semiaqueous solution of capsaicinoids to yield a beautiful blue-colored indophenol. The reaction has been used for over 20 years to detect capsaicinoids on thin-layer chromatography plates13−16 and for quantitative studies based on the absorbance of the reaction mixture at ∼600 nm.17−20 The difficulties of the nitrosation method are absent, albeit with use of more expensive and less environmentally benign reagents. Prior to the determination of capsaicinoids by LC−MS, LC− UV, or spectrophotometry, capsaicinoids are removed from the chili fruit or foodstuff and put into solution by liquid−solid extraction. UV-absorbing and colored substances from the sample are also extracted, making direct spectrophotometric analysis difficult, if not impossible. As a consequence, a recent experiment described in this Journal21 and all of the procedures cited in Table 1 include a separation step, such as column chromatography, prior to the absorbance measurement. Published: January 26, 2012 610
dx.doi.org/10.1021/ed200258r | J. Chem. Educ. 2012, 89, 610−612
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Article
or concentrated solutions and with hot chili peppers and their extracts. Adequate ventilation is required. Gibbs reagent and its products are chlorinated compounds and should be handled with care and disposed of properly in accordance with local regulations. Methanol is extremely flammable and toxic by inhalation. Methanol may be fatal or cause blindness if swallowed. Acetonitrile may be fatal if swallowed, inhaled, or absorbed through skin, may cause irritation to skin, eyes, and respiratory tract, and is flammable.
Including such a step makes the procedure less amenable to applications such as field work and classroom demonstrations. Here a simple analytical method is described to determine the total quantity of capsaicinoids in chili pepper fruit that is based on the Gibbs reaction, that does not require a cleanup step, and that uses the human eye to assess the color of the reaction mixture (colorimetry). The additional chromatography is avoided by discarding the colored pericarp (outer shell) of the pepper and extracting only the placenta, the seedcontaining tissues at the center of the fruit.22 Because the placenta is the site of capsaicinoids synthesis23 and has been found to contain a large fraction (about 90%) of the total mass of capsaicinoids in a chili fruit,24,25 analysis of only the placental material should not be a barrier to accurate semiquantitative results. With nearly all of the interfering colored materials removed and the deep blue color of the Gibbs product generated, the eye is selective and sensitive enough to measure the quantity of capsaicinoids. This method was developed in part as a response to a several entreaties from middle school and high school teachers, received by the lead author over the past few years, for a way to analyze chili peppers simply and easily.
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DISCUSSION Eight varieties of ripe chili peppers, covering the range of peppers typically available at grocery stores, were analyzed by colorimetry for their total capsaicinoids content. For comparison, the same extracts were also measured by a standard LC− UV method26 for which the values were taken as true. The results are given in Table 2. The colorimetric results showed Table 2. The Total Quantity of Capsaicinoids in Eight Chili Peppers As Determined by LC−UV and Colorimetry Total Quantity of Capsaicinoids
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EXPERIMENTAL PROCEDURE The experiment involved removal of the placental material from a chili pepper fruit, followed by liquid−solid extraction with methanol, filtering of the methanol extract, evaporation of 0.5 mL of extract and reconstitution in 0.5 mL of acetonitrile, reaction with Gibbs reagent, and finally measurement of the color produced. Full details are given in the Supporting Information. Because the placenta contained about 90% of the total capsaicinoids and the extraction removed 85% of the capsaicinoids from the placenta, the sample preparation as a whole captured about 75% of the “heat” of chili pepper fruit. Semiquantitative measurement of total capsaicinoids was accomplished by comparison of sample reaction mixtures to a color chart (Figure 1). The chart was prepared by reacting
Peppers C. annuum Anaheim Long Hot Poblano Yellow Banana Big Bomb Jalapeno C. chinense Habanero A Habanero B
LC−UV Semiquantitativeb
Colorimetry Semiquantitativeb
70 250 640 850
Low Low Low Low
Low Low Low Mid
2900 4100
Mid High
Mid High
4900 10,100
High Very High
High Very High
Microgram quantities are reported to ±5%. bThe semiquantitative labels were assigned as follows: low for 0−1000 μg; mid for 1000− 3000 μg; high for 3000−5000 μg; and very high for >5000 μg. a
high accuracy with only one pair of results (for the yellow banana pepper) failing to match. Errors likely arose from four sources: detecting and comparing colors in solution with those in the color chart; reproduction of solution color through photography and printing in preparation of the color chart; reaction of Gibbs reagent with other phenols, such as capsinoids,27 in the extracts; and use of simple volumetric delivery devices, for example, disposable graduated pipets. Although each of these error sources can be eliminated, none can be eliminated without making the method more complex, which mitigates the goal of simplicity.
Figure 1. Color chart for comparison to reaction solutions and the determination of the quantity of total capsaicinoids in the chili pepper fruit.
Gibbs reagent with known concentrations of a capsaicinoid (Nvanillylnonanamide) in orange sweet pepper extract. Sweet pepper extract, which did not contain capsaicinoids, was used to account for the slight color of the placental extracts of chili peppers. Reaction solutions were added to a 96-well plate and digitally photographed. Colors were adjusted using computer software to best represent the actual solutions colors. It must be noted that sauces and other foods containing ground chili pepper may not be amenable to the procedure described here, because the food likely contains both placenta and pericarp.
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LC−UV/μga
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CONCLUSION
The method described here for determining the total capsaicinoids was demonstrated to be a simple, fast, and effective means for the semiquantitative analysis of chili peppers that is appropriate for use in the classroom, laboratory, or field. The results were semiquantitative due to the use of a color chart to assess the capsaicinoid concentration and the inherent imprecision in human color perception. By this method, chili pepper “heat” can be easily and accurately estimated without resorting to taste tests or, at the other extreme, expensive and instrumentally intensive procedures.
HAZARDS
Capsaicinoids are nociceptive compounds and should be used with all reasonable care. Gloves, safety glasses, and masks should be worn when working with capsaicinoids as pure solids 611
dx.doi.org/10.1021/ed200258r | J. Chem. Educ. 2012, 89, 610−612
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(27) Singh, S.; Jarret, R.; Russo, V.; Majetich, G.; Shimkus, J.; Bushway, R.; Perkins, B. J. Agric. Food Chem. 2009, 57, 3452−3457.
ASSOCIATED CONTENT
S Supporting Information *
A step-by step procedure to determine the total quantity of capsaicinoids in chili pepper fruit by colorimetry; additional explanatory information and results from method development, including analysis by visible spectrophotometry. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected].
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ACKNOWLEDGMENTS This research was funded in part by Oberlin College through its Research & Development Committee, Grants-in-Aid Program, and Scholars & Artists Fund.
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REFERENCES
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