In the Classroom
Using the Melamine Contamination of Foods To Enhance the Chemistry Classroom Doris Renate Kimbrough* and Anna Chick Jensen Department of Chemistry, University of Colorado at Denver, Denver, Colorado 80217 *
[email protected] A common complaint from students learning chemistry is its lack of relevance in the real world. One of our goals as educators is to find ways to show the importance of chemistry to our students' daily lives (1-3). One example of the societal significance of chemistry is the study of food contamination, such as coumarin contamination in vanilla (4) or melamine contamination in milk products and pet food. News reports regarding the tainting of foodstuffs with the triazine compound, melamine, C3H6N6, began in 2007 with contaminated pet food sold in the United States and Canada and continued in 2008 with milk and infant formula contamination, predominantly in China. Melamine (I) has the structure shown in Figure 1. It has a number of important industrial uses: it is a component in adhesives and molding compounds; when mixed with resins, it has fire-retardant properties (5); and it can be copolymerized with formaldehyde to produce plastics and polymeric cleaning agents (6, 7). Melamine is also a metabolite of cyromazine, a pesticide used in plants and rat poison and a parasiticide in goats. During the aforementioned pet food scandal, the wheat gluten and rice protein, which are common dog and cat food ingredients, were found to be tainted by melamine. Thousands of pets died and more were sickened in the incident, many with severe kidney failure (8). Numerous pet food recalls took place to prevent further melamine-tainted pet food from reaching the market. The more recent infant formula contamination once again brought this chemical into the public eye with thousands of children in China hospitalized with kidney stones. It is believed that a few farmers in China diluted milk products to increase profit margins (9, 10). In both instances, melamine was believed to have been added at some point in the manufacturing process to boost the apparent protein content. The public attention that melamine has attracted makes its chemistry very topical. Herein, we discuss the chemistry of melamine within the context of various chemistry courses. Its rich chemistry can be presented at many levels, and we offer sample problems and questions for different courses from high school, both college-prep (HCP) and consumer-based (HCon), to college-level general chemistry (Gen), nonmajors chemistry (NM), organic (Or), biochemistry (Bio), and analytical (An). A complete set of examples is provided in the supporting information. Nitrogen in Food Nutritive organic molecules are classically divided into four categories: carbohydrates (including fiber), fats, proteins, and 496
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Figure 1. Melamine (I) is a cyclic trimer of cyanamide (triazine). Scheme 1. At a Neutral pH, the Amino Acid Exists in Its Zwitterionic Form as Both Carboxyl and Amino Groups Are Ionized (Upper Equation)a
a
The lower equation shows the reaction in the nonionic form, which may be easier for less experienced students to understand.
vitamins (11). Both carbohydrates and fats have molecular formulas that can be described as CxHyOz and contain no nitrogen. Some vitamins present in food, such as vitamin B1 and B12, contain nitrogen, but vitamins are typically present in relatively small quantities compared to the first three categories and are considered to have zero caloric value (although they have important nutritional roles). Proteins are polypeptide molecules. Water is formed during protein formation as peptide bonds are created between the amine group of one amino acid and the carboxylic acid group of another (Scheme 1). Thus, because most proteins have general formulas of CwHxNyOz, (with small amounts of S), the nitrogen content of foods can be directly linked to protein content, although the relationship is approximate. (Sample problems are provided in the supporting information.) Examination of the structure of melamine (Figure 1) reveals a high mole and mass percent of nitrogen relative to the carbon and hydrogen; thus, its addition to foodstuffs confounds analysis for protein.
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In the Classroom
Analytical Methods for Protein Content Quantification The Kjeldahl and Dumas methods are commonly employed to quantify the crude protein content in foods. However, neither method determines protein content directly; instead, each measures the nitrogen content. By assuming virtually all nitrogen comes from the peptide bonds of protein molecules, a correlation to protein content can be made, using a conversion factor. A typical conversion factor is 6.25 (equivalent to 0.16 g of nitrogen per gram of protein); however, this conversion factor is only approximate as different amino acid compositions can present different nitrogen content (12). Different conversion factors are used in different food analyses as information about the specific amino acid composition is considered. Several amino acid side chains contain one or more N atoms, (arginine, asparagine, glutamine, histidine, lysine, and tryptophan). Proteins that contain large amounts of these amino acid residues will have higher nitrogen percentages by mass. In addition, amino acids with small side chains such as glycine (R = -H) or alanine (R = -CH3) have a higher ratio of nitrogen mass to overall molecular mass than amino acids with larger side chains such as phenylalanine (R = -CH2-C6H5) or tyrosine (R = -CH2C6H4OH). Without knowing the exact amino acid composition of a given foodstuff (which is often extremely difficult, expensive, and unnecessary), the protein determination remains an approximation. The percent protein measured in these procedures, known as crude protein, also fails to account for other nitrogen-containing nonprotein molecules in the original sample, which is how addition of melamine can produce apparent protein percentages that are higher than the food actually contains. The Kjeldahl method was developed by Johan Kjeldahl in the late 1800s (13, 14). It utilizes a strong acid, typically sulfuric acid, to “digest” the food sample. The weighed sample is heated in sulfuric acid in the presence of sodium or potassium sulfate and a catalyst, such as copper(II) selenite (eq 1) (15, 16): H2 SO4
Cw Hx Ny Oz f ðNH4 Þ2 SO4 þ CO2 þ H2 O þ other byproducts catalyst
ð1Þ During this digestion process, organic nitrogen is converted into ammonium sulfate (NH4)2SO4. To separate ammonium ions from the unwanted byproduct, ammonia gas is then generated as the result of distilling ammonium sulfate with a base, such as sodium hydroxide (NaOH) (eq 2): ð2Þ NH4þ þ OH - f NH3 ðgÞ þ water There are two titration methods to determine ammonium concentration: indirect titration and back-titration. In the case of indirect titration, ammonia gas is removed and reacted with excess boric acid (H3BO3) solution to form a complex (eq 3): NH3 ðgÞ þ H3 BO3 f fNH4þ þ H2 BO3- gðcomplexÞ þ excess H3 BO3
ð3Þ The resulting solution is titrated with a standardized hydrochloric acid (HCl) solution (eq 4) to determine borate concentration, which can then be traced back to calculate the total nitrogen content in the original sample. fNH4þ H2 BO3- gðcomplexÞ þ Hþ f H3 BO3 þ NH4þ ð4Þ
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The amount of hydrochloric acid required to reach equivalence point is equal to the amount of nitrogen in the original food. The ammonium ion produced from the digestion process (eq 1) cannot be titrated directly as the other compounds resulting from the digestion process will interfere. (A sample problem is provided in the supporting information.) In the back-titration method, the NH3 is generated in the same manner as described above and then treated with excess hydrochloric or sulfuric acid (eq 5). The solution is then backtitrated with a standardized solution of NaOH (eq 6). (Sample problem is provided in the supporting information.) H3 ðgÞ þ HCl f NH4 ClðaqÞ þ excess HClðaqÞ ð5Þ Excess HClðaqÞ þ NaOHðaqÞ f H2 O þ NaClðaqÞ
ð6Þ
The Dumas method, also known as the nitrogen-combustion method, measures nitrogen content more efficiently than the Kjeldahl method and is analogous to other methods of elemental analysis. Developed by Jean-Baptiste Dumas in the early 19th century, the method is based on combusting organic nitrogen compounds at high temperature (in the presence of excess oxygen) to produce nitrogen oxides (NxOy) (eq 7), which are subsequently reduced to nitrogen gas (N2) using metal catalysis, such as Cu (eq 8). Λ
Cw Hx Ny Oz þ O2 ðgÞ f Nu Ov ðgÞ þ CO2 ðgÞ þ H2 OðgÞ ð7Þ Cu
Nu Ov ðgÞ f N2 ðgÞ
ð8Þ
Traditionally, the total nitrogen content was measured volumetrically. More modern procedures, however, determine the total nitrogen content by a thermal conductivity detector (the enhanced Dumas method) (17, 18). Although the enhanced Dumas method is more efficient, it requires more specialized instrumentation and equipment (19). Furthermore, the thermal conductivity is less accessible (and more arcane) for the high school or college general chemistry population; however, the more traditional method would utilize a gas-law calculation that lends itself well to introductory chemistry courses. A similar conversion factor is utilized to correlate the nitrogen content to the crude protein percentage. (A sample problem is provided in the supporting information.) The brief descriptions of the Kjeldahl and Dumas methods display their shortcomings as analytical methods in the measurement of actual protein content, as melamine or any other nitrogen-containing organic compound will be falsely identified as protein. More advanced methods are necessary to determine the true protein content of foods as well as to determine the identity and concentration of potential contaminants to prevent similar tragedies in the food industry in the future. Instrumentation, such as high performance liquid chromatography (HPLC), hydrophilic interaction chromatography (HILIC), electrospray ionization mass spectrometry (ESI-MS), and Fourier transform infrared spectroscopy (FTIR), have all been employed in detection and analysis in the two contamination events discussed here (20). Use of this instrumentation to solve the mystery ailments in both the pet food and baby formula cases would be an interesting case-study topic for analytical chemistry students. Addition of Melamine Increases Apparent Crude Protein Values Melamine has the chemical formula C3H6N6 and a molar mass of 126.12 g/mol. Its percent mass of nitrogen is 66.6%, far
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higher than that of most foods containing protein, which are typically 2.8-5.5% of nitrogen by mass. Thus, its addition can boost the nitrogen content of foods making them appear to contain more protein. The following question, therefore, can be posed to the students (HCP, Gen, An): If corrupt milk producers diluted their milk to 90% by volume ( i.e., adding an additional 10% of water), how much melamine needs to be added to 1.00 L of milk to produce the “correct” values for protein analysis? Parameters for the calculations:
• Milk density = 1.030 g/mL at 20 °C • Average mass percentage of crude protein content in milk = 3.10% • Conversion factor for milk (which takes into account the particular proteins and ratios in cow's milk) = 6.38 (i.e., nitrogen content of protein = 1/6.38 = 15.7%) (21) • Molar mass of nitrogen = 14.01 g/mol
The answer can be calculated stepwise: The mass of nitrogen in 1.00 L of nondiluted milk is g milk 3:10 g crude protein 1000 mL milk 1:030 mL milk 100 g milk
¼ 5:00 g N If the milk were diluted by 10% (presumably with water), the mass of nitrogen would be 5:00 g N ð0:90Þ ¼ 4:50 g N Therefore, 5.00 g - 4.50 g = 0.50 g of N would have to be added to maintain the same quantity of nitrogen content in the diluted milk. The mass of melamine (C3H6N6) that will make up this 0.50 g nitrogen difference is 1 mol N 1 mol C3 H6 N6 0:50 g N 14:01 g N 6 mol N 126:12 g C3 H6 N6 1 mol C3 H6 N6
¼ 0:75 g C3 H6 N6 Thus, 0.75 g of C3H6N6 would need to be added per liter of diluted milk. An alternative calculation that uses the mass percent is 100 g C3 H6 N6 ¼ 0:75 g C3 H6 N6 0:50 g N 67 g N Variations on these calculations, depending upon student level or interest, could include changing the dilution factors, or using different food types with different conversion factors (e.g., infant formula, using milk as an partial ingredient in a different food, etc.). Synthesis, Structure, and Health Effects of Melamine Melamine was first synthesized by Justus von Liebig in the early 18th century. This early synthetic method involved 498
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converting calcium cyanamide into Dicyandiamide, then maintaining the solution at high temperature to produce melamine (22). Because of the cost of calcium cyanamide, this early method has since been replaced by thermal decomposition of urea, followed by polymerization (eqs 9 and 10). ð9Þ 6ðNH2 Þ2 CO f 6HCNO þ 6NH3 6HCNO f C3 H6 N6 þ 3CO2
ð10Þ
One can therefore pose the following question to students (H-Con, H-CP, Gen, NM): What is the overall reaction equation for melamine synthesis? The answer is (23): ð11Þ 6ðNH2 Þ2 CO f C3 H6 N6 þ 6NH3 þ 3CO2
1g N 6:38 g crude protein
Scheme 2. The Interaction of Melamine (I) with Cyanuric Acid (II) To Form a Hydrogen-Bonded Adduct (III)
Melamine has a history as an unintentional food additive and is deemed safe in low doses. The Food and Drug Administration (FDA) lists 2.5 ppm as a safe concentration in foods; however, melamine concentration in some tainted Chinese powered milk was found to be as high as 6196 ppm (24)! Furthermore, industrial melamine is commonly contaminated with cyanuric acid (C3H3N3O3) (II), a structural analogue of melamine. Melamine and cyanuric acid combine together to form a hydrogen-bonded adduct (III) (Scheme 2). The hydrogen bonding is reminiscent of the type of bonding one sees among base pairs in DNA and RNA. (Sample problems are provided in the supporting information.) Diluting milk products to improve profits robs the consumer (in this case pets or babies) of the nutrients contained in the food that are needed for proper growth and development. This alone could produce malnutrition. However, the addition of melamine to mask the dilution has a more insidious effect. The adduct, melamine-cyanurate (III), is highly insoluble in aqueous solution and is reportedly more toxic than either the melamine or cyanuric acid alone (5). It is believed that the adduct (III) is the cause of the kidney problems that affected thousands of pets and children. Melamine-cyanurate concentrates and precipitates (forming kidney stones) in the microtubules, causing kidney damage and malfunction that can be irreversible and even fatal. Cyanuric acid is thought to be a common contaminant in the industrial melamine that was illegally added to pet foods and milk and infant formula in China. Both compounds are interesting from an organic structural standpoint in that both are heteratomic cyclic compounds. Melamine (I) is aromatic, and cyanuric acid has several tautomeric structures, one of which is aromatic (IIB) (Scheme 3). The keto form II is not technically aromatic, although one can draw
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In the Classroom
Scheme 3. The Tautomeric Structures of Cyanuric Acid (II) That Are Aromatic (IIB and IIC)
an aromatic resonance form as shown in Scheme 3. It is the ketoform (II) that interacts with melamine on the basis of X-ray crystallography data (25, 26). Conclusion The application of chemistry to current events highlights the importance of the underlying chemical principles and brings the chemistry to life. Incorporating the chemistry of melamine into introductory (high school or college nonmajors), general, organic, or analytical chemistry enables students to be better informed “chemistry citizens” and provides a real-world context to the solving of organic structural and stoichiometry problems. Literature Cited 1. Crowley, J. P.; DeBoise, K. L.; Marshall, M. R.; Shaffer, H. M.; Zafar, S.; Jones, K. A.; Palko, N. R.; Mitsch, S. M.; Sutton, L. A.; Chang, M.; Fromer, I.; Kraft, J.; Meister, J.; Shah, A.; Tan, P.; Whitchuch, J. J. Chem. Educ. 2002, 79, 824–827. 2. Giffin, G. A.; Boone, S. R.; Cole, R. S.; Mckay, S. E. J. Chem. Educ. 2002, 79, 813–819. 3. Olsen, K. G.; Ulicny, L. J. J. Chem. Educ. 2001, 78, 941. 4. Sparks, L.; Bleasdell, B. D. J. Chem. Educ. 1986, 63, 638–639. 5. Melamine and Cyanuric Acid: Toxicity, Preliminary Risk Assessment and Guidance on Levels in Food; Guidance from WHO; 2008. http://www.who.int/foodsafety/fs_management/Melamine.pdf (accessed Feb 2010). 6. Salaun, F.; Vroman, I. Eur. Polym. J. 2008, 44, 849–860. 7. Zhao, X.; Ye, L.; Hu Polym. Adv. Technol. 2008, 19, 399–408. 8. ABC News Web site. http://abcnews.go.com/WNT/story?id=2974319 (accessed Feb 2010).
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9. AP Web site. http://ap.google.com/article/ALeqM5iCL58EMBN 1tqq6xujZlsaITAFpCQD93BHE880 10. CBC Web site. http://www.cbc.ca/consumer/story/2008/09/22/ f-melamine-faq.html (accessed Feb 2010). 11. Hamilton, E. M. N.; Whitney, E. N.; Sizer, F. S. Nutrition: Concepts and Controversies; West Publishing Co.: St. Paul, 1985; p 15. 12. Carrat, B.; Boniglia, C.; Salise, F.; Ambruzzi, A. A.; Sanzini, E. Food Chem. 2003, 81, 357–362. 13. Tan, K. T. Soil Sampling, Preparation, and Analysis; Marcel Dekker: New York, 1996; p 139. 14. Oesper, R. E. J. Chem. Educ. 1934, 11, 457–462. 15. Jung, S.; Rickert, D. A.; Deak, N. A.; Aldin, E. D.; Recknor, J.; Johnson, L. A.; Murphy, P. A. J. Am. Oil Chem. Soc. 2003, 80, 1169–1173. 16. Collins, E. M.; Shelton, R. W. A J. Chem. Educ. 1940, 17, 475. 17. Advanced Dairy Chemistry, Vol. 1, 3rd ed.; Fox, P. F., McSweeney, P. L. H., Ed. Springer: New York, 2003; pp 60-61. 18. Winkler, R.; Von Ramin, J.; Frister, H. GIT Lab. J. 2001, 5, 107– 109. 19. Protein determination by combustion, USDA website. http:// www.fsis.usda.gov/OPHS/clg/clg-pro4.02.pdf 20. Dobson, R. L. M.; Motlagh, S.; Quijano, M.; Cambron, R. T.; Baker, T. R.; Pullen, A. M.; Regg, B. T.; Bigalow-Kern, A. S.; Vennard, T.; Fix, A.; Reimschussel, R.; Overmann, G.; Shan, Y.; Daston, G. P. Toxicol. Sci. 2008, 106, 251–262. 21. Food Energy-Methods of Analysis and Conversion Factors; Report of a technical workshop from Food and Agriculture Organization of the United Nations; Rome, 2003. 22. Hetherington, H. C.; Braham, J. M. Ind. Eng. Chem. 1923, 15, 1060–1063. 23. Kinoshita, H. Rev. Phys. Chem. Jpn. 1954, 24, 29–27. 24. Epoch Times: http://en.epochtimes.com/n2/china/china-powderedmilk-tests-2500-times-higher-than-fda-interim-safe-level-report-5336. html (accessed Feb 2010). 25. Whitesides, G. M.; Simanek, E. E.; Mathias, J. P.; Seto, C. T.; Chin, D. N.; Mammen, M.; Gordon, D. M. Acc. Chem. Res. 1995, 28, 37– 44. 26. Ranganathan, A.; Pedireddi, V. R.; Rao, C. N. R J. Am. Chem. Soc. 1999, 121, 1752–1753.
Supporting Information Available A full set of sample problems is available for different courses from high school, both college-prep (HCP) and consumer-based (HCon) to college level general chemistry (Gen), nonmajors chemistry (NM), organic (Or), biochemistry (Bio) and analytical (An). This material is available via the Internet at http://pubs.acs.org.
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