Continuous Temperature-Dependent Raman Spectroscopy of Melamine and Structural Analog Detection in Milk Powder Walter F. Schmidt,a,* C. Leigh Broadhurst,a Jianwei Qin,a Hoyoung Lee,a Julie K. Nguyen,a Kuanglin Chao,a Cathleen J. Hapeman,b Daniel R. Shelton,a Moon S. Kima a Environmental Microbiology and Food Safety Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Building 303, BARC-East, 10300 Baltimore Blvd., Beltsville, MD 20705 USA b Hydrology and Remote Sensing Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Building 001, BARCWest, 10300 Baltimore Avenue, Beltsville, MD 20705 USA
Hyperspectral Raman imaging has the potential for rapid screening of solid-phase samples for potential adulterants. We can improve mixture analysis algorithms by defining a temperature range in which the contaminant spectrum changes dramatically and uniquely compared with unadulterated material. Raman spectra were acquired for urea, biuret, cyanuric acid, and melamine (pure and at 1% in dried milk powder) from 50 to 310 8C with a gradient of 1 8C min1. Adulterants were clearly indentified in the milk powder. Specific frequencies that were mainly associated with ring breathing, stretching, and in-plane deformation shifted with respect to temperature up to 12 cm1 in all four molecules. Specific frequencies significantly increased/decreased in intensity within narrow temperature ranges independent of whether the amine was mixed in milk. Correlation of Raman and differential scanning calorimetry data identified structural components and vibrational modes, which concur with or trigger phase transitions. Index Headings: Raman spectroscopy; Biuret; Cyanuric acid; Melamine; Hyperspectral imaging; Temperature-dependent Raman spectroscopy.
INTRODUCTION Extending Raman imaging spectroscopy from microscale or nanoscale point measurements and ambient temperatures to a technique capable of two-dimensional surface scans across wide temperature ranges provides a new avenue for the rapid acquisition of both practical and theoretical chemical data. A practical application our laboratory recently developed is a hyperspectral Raman imaging system with sufficient spectral and spatial resolution to simultaneously identify and map four adulterant particles (ammonium sulfate, dicyandiamide, melamine, urea) mixed into dry milk at concentration levels from 0.1 to 5.0%.1 The hyperspectral imaging system maps a 25 3 25 mm2 area in about 1 hr. Rapid and accurate authentication of food ingredients is important for safety and quality evaluation. Raman chemical imaging coupled with appropriate mixture analysis algorithms can be used for simultaneous detection of multiple adulterants in many types of powdered food and nonfood materials. This system can also be used for macroscale imaging of food and Received 15 May 2014; accepted 4 September 2014. * Author to whom correspondence should be sent. E-mail: walter. [email protected]. DOI: 10.1366/14-07600
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agricultural products, such as scanning cross-sections of cut tomatoes for maturity evaluation.2,3 Significant wavenumber shifts in infrared and Raman spectra of solids with respect to temperature have been observed previously, and such data have the potential to readily identify contaminants, phase dissolution, and phase transitions.4–8 For example, over the temperature range 30 to 22 8C, Unger et al.6 could identify characteristic absorptions for both solid-state and liquid–solid phase transitions in oleic acid with Fourier transform infrared spectroscopy. Extending this study to Raman could simplify the spectral interpretation for this major edible lipid, which is abundant in olive and peanut oils and most nuts and meats. We further developed the technique of continuous temperature-dependent Raman spectroscopy to investigate isomerization of the organochlorine pesticide endosulfan between the nonsymmetric a and symmetric b-diastereomers.9 Raman spectra were acquired at 1 8C intervals from 50 to 102 8C for a-endosulfan, b-endosulfan, and a 60/40 mixture. For most vibrational modes discrete temperature-dependent changes occurred above 97 8C. Dramatic changes occurred at 970, 1027, 1092, and 1350 cm1, where spectral density increased, peaks shifted, and peaks broadened substantially. The response of these modes indicated that the corresponding molecular sites are the most temperature sensitive and therefore the most flexible. Results also preclude a to b isomerization. A phase transition observed at 97– 102 8C for b-endosulfan corresponded to the largest changes in the temperature-dependent Raman spectra. Temperature-dependent Raman provides a novel and very straightforward technique to identify theoretically proposed molecular rearrangements that occur just prior to phase transitions. In 2007, a widespread recall of pet foods occurred after thousands of dogs and cats in the United States experienced kidney failure. The United States Food and Drug Administration later determined that a Chinesesourced wheat gluten ingredient was contaminated with melamine. In 2008, some 300 000 Chinese children experienced kidney problems, including six fatalities, from melamine economic adulteration of infant formula produced by a major Chinese dairy company.10 In addition to melamine, economic adulteration of milk powder with fertilizers is facile and potentially introduces urea, biuret, triuret, and cyanamide to dairy products.11
0003-7028/15/6903-0398/0 Q 2015 Society for Applied Spectroscopy
APPLIED SPECTROSCOPY
FIG. 1. Schematic of continuous temperature-dependent Raman spectroscopy analytical system.
Raman spectroscopy has been used previously to detect adulterants in dry milk including melamine,2,12 whey,13 ammonium sulfate, dicyandiamide, and urea.1,14 Surface-enhanced Raman with Au or Au-coated colloidal microsphere greatly improves the detection limits for melamine in solution but has not been developed for dry mixtures.15,16 Hyperspectral imaging has the potential for inexpensive rapid throughput screening of multiple samples with minimal preparation and without solvents. If it is determined that any of the suspect contaminants occur in a particular sample, more precise, but also more expensive and time-consuming analyses10,11,17–19 can be performed for individual components in those samples. Temperature-dependent Raman can potentially improve the mixture analysis algorithms by defining a narrow temperature range in which the spectrum of a contaminant changes dramatically and uniquely in comparison with the unadulterated or background material. In this contribution we present TDR spectra for urea, biuret, cyanuric acid, and melamine; pure and mixed at 1% in dried milk powder.
MATERIALS AND METHODS The temperature-dependent Raman system (Fig. 1) uses a Raman spectrometer (Raman Explorer 785, Headwall Photonics, Fitchburg, MA) fitted with a 16-bit charge-coupled device camera (1024 3 256 pixels; Newton DU920N-BR-DD, Andor Technology, South Windsor, CT). The spectrometer detects a Raman shift range
of 102.2 to 2538.1 cm1 with a spectral resolution of 3.7 cm1. A 785 nm laser module (I0785MM0350MF-NL, Innovative Photonic Solutions, Monmouth Junction, NJ) served as the excitation source. A fiber optic Raman probe (RPB, InPhotonics, Norwood, MA) was used to focus the laser and acquire the Raman signals. A bifurcated fiber bundle delivers the laser radiation to the probe and transmits the Raman signals to the spectrometer. Laboratory-grade reagents were as follows: melamine (C3H6N6, 99.0%, Aldrich), urea (CH4N2O, 98.0%, Sigma), cyanuric acid (C3H3N3O3, 98.0%, Aldrich), and biuret (C2H5N2O2, 98.0%, J.T. Baker). Organic skim dry milk (Organic Valley, La Farge, WI) was purchased from a local supermarket. The four reagents were mixed into the milk powder at 1.0% in 50 ml polypropylene centrifuge tubes. A vortex mixer was used to ensure uniform distribution of the adulterant particles. Powder samples were placed in copper sample holders and heated on a ceramic hotplate. Copper heat sinks were placed between the ceramic heat surface and the sample holder to slow the rate of temperature increase. Two K-type thermocouple probes were attached to two sides of the sample area and connected to a dual-input thermometer (EasyView EA15, Extech Instruments, Nashua, NH). The sample temperature was defined as the average value of the two probes. The Raman probe, hotplate, and sample materials were placed in a closed black box to avoid ambient light. The
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FIG. 2. Amine structural analogs used: (a) urea; (b) biuret; (c) cyanuric acid; (d) melamine.
hot plate heater was set for a gradient of about 1 8C min1. Raman spectra were acquired from 50 to 250 8C for urea and biuret and 50 to 310 8C for cyanuric acid and melamine. If dramatic chemical–thermal rearrangement occurred at a temperature prior to 250 8C, that temperature was the maximum used. We did not use a set time schedule for spectral acquisition, but rather acquired spectra each time the sample temperature increased 1 8C. System software was developed using LabVIEW (National Instruments, Austin, TX) to fulfill functions such as camera control, data acquisition, temperature measurement, and synchronization.2 Differential Scanning Calorimetry (DSC). One milligram of each amine was placed in an aluminum sample pan with a crimped lid. An empty pan was used as control. Samples were placed in a DSC Q200 differential scanning calorimeter (TA Instruments, New Castle, DE) and scanned from 50 8C to approximately 20 8C above the melting point (mp). A thermocouple under each pan recorded the rate of heat absorbed versus the rate applied. Heating rate was 5 or 10 8C min1 depending on the melting temperature. Samples were protected from oxidation using a continuous supply of N2, which is heated at the same rate as the sample, then pumped into the heating chamber.
RESULTS AND DISCUSSION Urea, biuret, cyanuric acid, and melamine contain overlapping structural components (Fig. 2); however, each structural analog has markedly different physical properties (e.g., melting point urea, 133 8C; biuret, 193 8C; cyanuric acid, 330 8C; melamine, 345 8C). Figure 3 shows that 1% melamine in milk powder is clearly detectable, as are the other three amines. Figures 4 and 5 are contour plots showing the progressive Raman frequency shifts with increasing temperature. Shifts are significant with respect to line bandwith and are specific to individual molecules and individual molecular sites; spectra are truncated at melting. Figure 6 shows DSC data for three of the molecules over the temperature range of the temperature-dependent Raman experiments. Assignments of chemical structure to temperature-dependent Raman vibrational modes agree with
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previously reported Raman frequencies observed at a constant temperature (Table I).9,20–36 Urea. The major temperature-dependent Raman vibrational modes in urea (CH4N2O) at 50 8C are 548, 1006, 1543, and 1649 cm1. All shift in frequency with temperature; the three largest shifts are a decrease at 544 cm1 (in-plane deformation), and increases at 1543 and at 1645 cm1 (amide III N–H stretching and NH2 asymmetric bending). Urea is symmetrical about the O=C axis and at all four H sites; the flexibility of N–H groups is equal. Gradually, both 1543 cm1 (N–H asymmetrical stretching) and 1649 cm1 (NH2 asymmetrical bending) increase in frequency. A structural explanation for the temperature-dependent Raman data is that one of the four N–H sites become longer than (i.e., unequal to) the other three, and therefore asymmetrical vibrational modes increase in intensity. In Fig. 6 these shifts correspond to the shoulder of the sharp peak of increasing heat flow indicating the melting of urea. Biuret. The temperature-dependent Raman and DSC data for biuret are very different from urea, even though chemically biuret is the dimer of urea. The major vibrational modes at 50 8C are 281, 456, 703, 954, 1007, 1116, 1517, and 1605 cm1. Despite their similar structure, only one frequency is within 5 cm1 of those in urea (1005 cm1). A second peak in biuret shows up at 1550 cm1 only above about 130 8C, whereas the [O=C– N–H] NH asymmetric bending frequency in urea occurs only below 130 8C. DSC peaks (Fig. 6) indicate two solid-state phase transitions (at 70 and 130 8C) prior to the peak for melting at 193 8C. This correlates with Fig. 4 and Table I (see footnotes), where some observed frequencies decrease sharply when 70 8C and then 130 8C are reached, yet others increase after 130 8C. The center of the biuret molecule is an N–H group between two O=C sites. Because each site is an amide (O=C–N–H), the two sites can be coplanar, and the NH will always be either above or below this plane. The increase in frequency at 456 cm1 and change in intensity at 954 cm1 (C–N–C bending) is consistent with a change in twist between the two O=C sites. Biuret has a discrete temperature profile: at 90 8C the 1605 cm1 peak intensity decreases dramatically (O=C–N–H symmetrical NH bending) at 110 8C, 1005 cm1 peak intensity (N–C–N stretching) is at its maximum; concurrently at 130 8C, peaks at 462 cm1 and 990 cm1 appear (C–O inplane bending and symmetrical skeletal stretching, respectively). The simplest structural interpretation is that at 50 8C, the two amide groups are not coplanar, but at 110 8C, they are. The intensity of the 1698 cm1 peak increases above 80 8C and is maximized at 130 8C. Since this peak is assignable to a planar b-turn in an amide, this also indicates biuret is planar at that temperature. The changes near 990 cm1 and at 1120 cm1 would then correspond to changes in flexibility of NH2, NH, or both moieties above and below the plane. Cyanuric acid. The three most intense temperaturedependent Raman frequencies in cyanuric acid at 110 8C are 547, 700, and 1729 cm1. Four other significant frequencies (Fig. 5) are also observed (411, 987, 1416, and 1470 cm1). Structurally, cyanuric acid is a cyclic trimer of urea, and the peak at 547 cm1 assignable to in-
FIG. 3. Contour plot of intensity and frequency of selected major Raman spectral feature vs. temperature for 1% melamine in nonfat milk powder and control milk powder.
plane deformation is similar to 549 cm1 in urea. The peak at 411 cm1 is assignable to ring deformation. The narrowing of temperature-dependent Raman peaks with minimal or no frequency shift at 1727 cm1 (O=C stretching) and at 700 cm1 (ring N–H site flexibility) indicates these sites in the ring (O=C–NH–(O=C)–NH) become more rigid with increasing temperature, and since the peak at 987 cm1 decreases, the applied thermal stress also reduces symmetrical ring stretching. The temperature-dependent Raman frequency shift in cyanuric acid at 539 cm1 (in-plane deformation) is
nonuniform, narrowing to 240 8C and then broadening to 290 8C. Concurrent with the broadening is an increase in intensity at 403 cm1 (ring deformation) and an additional peak at 458 cm1 (C–O in-plane bending). Thus at about 270 8C, the N=C–N site in the ring is deformed, and C–O bending at this C site becomes planar. The N–C=N bond sequence becomes a less planar–asymmetrical component of the ring resonance structure above 250 8C. One explanation for the asymmetry and frequency shift in the temperature-dependent Raman peak at 547 cm1 from 110 8C is that at the lower temperature, one of two
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FIG. 4. Overview contour plots of intensity and frequency of selected major Raman spectral features vs. temperature for urea, biuret, cyanuric acid, and melamine.
O=C–NH is more flexible than the other. The increasing concurrence in the coplanar conformation of the two O=C–NH sites with temperature explains the very high thermal stability of cyanuric acid. Spectra are observed over 290 8C in Fig. 4. The DSC curve (Fig. 6) indicates the
onset of melting at 330 8C as reported; however, the thermal absorption curve is broad with a shallow slope and extends nearly 70 degrees, in concurrence with a 12 cm1 shift in the ring stretching frequency, which is the largest shift we observed overall.
FIG. 5. Detail of significant frequency shifts vs. temperature from Fig. 4
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.
FIG. 6. Differential scanning calorimetry heat absorption curves for urea, biuret, cyanuric acid, and melamine. Note multiple absorptions in biuret prior to and after melting (193 8C).
672, 980, and 1557 cm1. The only temperature-dependent Raman vibrational mode similar between melamine and cyanuric acid is a relatively weak symmetrical skeletal stretching frequency (981 cm1 in melamine,
Melamine. Melamine is a structural analog of cyanuric acid in which –NH2 sites replace –OH sites or N=C–N2 sites replaces O=C–N2. Five major temperature-dependent Raman frequencies are observed at 50 8C: 385, 582,
TABLE I. Raman frequencies (cm1), temperature-dependent shift, and structural assignments. Melamine 50 8C 385
582 672
980
Cyanuric acid
250 8C
Shift
379
6
582 674
978
50 8C
1551
Shift
411
403
7
547
538
7
700
696
4
987
989
þ2
50 8C
170 8C
281
281a
456
462a
Urea Shift
50 8C
130 8C
Shift
þ6 548
544
4
1006
1003
3
1543
1549
þ6
1645
1649
þ4
þ2 2
1416 1470 1557
250 8C
Biuret
1408 1458
703 954
700b 948 990c
3 6
1007d 1116e
1125
þ9
1517f
1512
5
8 12
6 1557g 1605h 1698g 1729
1729
Assignments Ring C=O deformation35 C–N bending out of plane34 Ring deformation35 C–O in-plane bending29 In-plane deformation24 Ring breathing30 NH2 in-plane bending31 Ring breathing33 C–N–C out-of-plane bending35 Sym. skeletal stretching29 N–C–N stretching28 C–N stretching (in CNH2)31 Ring breathing (N–H bending)26 Ring stretching29 C=N–C bending23 N=C–NH NH sym. stretching,23 amide III32 O=C–NH NH asym. stretching,22 amide II27 O=C–NH site (N–H bending)21 NH2 asym. bending20,36 b turns (C=O stretching)9 O=C–NH site (C=O stretching)20
a
Loss in intensity at 115 8C. Maximum intensity about 110 8C. c Observed starting .130 8C. d Loss of intensity 130 8C. e Starts shifting 130 8C. f Shift begins 90 8C. g Low intensity at 50 8C and maximum 130 8C. h Clear loss in intensity 100 8C. b
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FIG. 7.
Normalized relative intensity of the cyanuric acid 537 cm1 in-plane deformation peak vs. temperature, showing increased intensity 130 8C.
987 cm1 in cyanuric acid). Melamine, urea, and biuret all have vibrational modes of NH and NH2 sites in common near 1557 cm1. Structurally, melamine has no N–H ring sites, but three N=C–N–H2 sites are present per molecule. The spectral lines at 582 cm1 and 980 cm1 are ring breathing and symmetrical skeletal stretching, respectively. Their signal intensities are very weak compared with 385 cm1 (C–N bending out of plane), 672 cm1 (NH2 in-plane bending) and 1557 cm1 (NH symmetrical stretching). Melamine has the highest reported melting temperature of the four amines but may decompose near melting, which agrees with Fig. 4. The sites that absorb thermal energy are C–N and C–N–H sites external to ring structure. The 385 cm1 frequency can be one of three sites; if the N in C–N–H is bending out of plane, NH sites also will be out of plane. With increasing temperature, the peak at 1557 cm1 shifts to 1551 cm1 and also the peak sharpens. In-plane NH2 bending (672 cm1) shifts upfield 2 cm1, and ring breathing at 582 cm1 steadily decreases. An explanation consistent with these results is that with increasing temperature, the ring structure overall becomes more rigid, and two of the NH2 sites become more planar, but one C–N site bends more out of plane. This molecular site becomes progressively more unequal, i.e., above or below the stable ring plane. The increase with temperature in out-of-plane bending may
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disrupt the uniformity of packing between melamine molecules, resulting in the broad melt/decomposition peak in the DSC data starting at about 250 8C and continuing to 345 8C (Fig. 6). Mechanism for Temperature-Dependent Raman Selectivity. Melting and other thermally induced phase transitions correspond at the molecular level to an increase in flexibility in response to thermal stress. Near their individual melting points, molecules become more flexible. Specific sites within a molecule may become flexible before the entire molecule responds. Under thermal stress, more rigid molecules or sites respond less quickly than more flexible sites. The pattern in which vibrational modes respond to thermal stress is unique to a specific molecular structure. Physical phase transitions in polymers,4 biopolymers,5 and oleic acid6 spectroscopy correlated with temperature have been reported using infrared or Raman spectroscopy. However in these studies measurements were made at discrete, discontinuous temperature steps under steady state conditions. Our spectra were collected with a continuous temperature gradient, which minimizes the time (and molecular pathways) in which thermal relaxation between individual temperature measurement steps can occur. A temperature gradient consistently perturbs steady state conditions, resulting in thermal stress. Molecular sites more sensitive to
thermal stress will absorb thermal energy at lower temperatures than less sensitive sites. For example, although the stereoisomers and bendosulfan contain identical structural components, each has discrete marker vibrational modes.9 In the bisomer, the seven-membered ring is symmetrical, whereas the ring is asymmetrical in the a-isomer. The flexibility of the asymmetrical seven-membered ring accounts for its physical properties: the a-isomer melts at 108 8C and environmentally concentrates in air. The rigidity of the symmetrical conformation in the b-isomer accounts for its melting point of 208 8C and the fact that it is found as an environmental contaminant in water, not air. In Fig. 7 the intensity of the cyanuric acid 537 cm1 inplane deformation peak is nearly doubled at temperatures over 130 8C. Similar magnitude changes in peak intensity were described for biuret above. Although temperature-dependent Raman spectra of melamine can be observed to 310 8C, 1% melamine in milk is not detected above 170 8C. At 120 8C milk proteins and sugars will begin to react irreversibly, forming Maillard or ‘‘browning’’ products. Upon heating, significant peaks in 1% melamine in milk occur in the 1200–1400 cm1 range, which are not observed for either of the component structures. Further research could confirm if these are sites where melamine and milk protein chemically react. Thus temperature-dependent Raman spectroscopy can enable detection of melamine even if it chemically reacts with a food product during processing. These are just a few of many examples of temperaturedependent Raman frequencies that increase, decrease, appear, or disappear within specific temperature ranges that often correlate with known phase transitions and may correlate with chemical reactions. With further development we undoubtedly can improve hyperspectral mixture analysis algorithms because we have a straightforward method to rule out structural analogs that can cause considerable uncertainty, time, and effort with other analytical methods. Recently, vibrational spectroscopy in the THz range has been used to identify melamine in several food products.37 A fingerprint spectrum can be assignable to a portion of a chemical structure or to some physical form of a chemical structure. Structural elucidation is a fundamental and critical component in spectroscopic techniques to avoid both false positive and false negative analytical results. Temperature-dependent Raman spectroscopy may also be useful for identifying environmental triazine contamination in soils and composts.38 Soils are more complex substrates than milk powder; however, response to increasing temperature for organic soil constituents will undoubtedly differ from that of clay minerals, potentially enhancing detection of individual organic species. Fractions bound tightly to soils may also be identified more easily than with other methods. Temperature-dependent Raman spectra are not identical to constant temperature Raman spectra because temperature-dependent spectra are transient. In constant temperature experiments any unequal absorption of thermal energy absorbed selectively site-to-site within a molecular structure will relax and redistribute evenly.
In our temperature-dependent Raman experiments, the temperature gradient is continuous; therefore assigned frequencies that are unaffected by temperature can be distinguished from vibrational modes that alter with temperature, especially within the temperature range in which phase transitions occur. Within this gradient, any thermal sequence in which individual molecular sites become more or less flexible can be determined. This information is an identifying component unique to that individual molecular structure. Since temperature-dependent Raman enables the temperature-dependent components of Raman spectra to be identified, these vibrational modes can be assigned to chemical structures. This in turn enables identification of individual vibrational modes and chemical structure components that concur with or trigger the start of a phase transition. 1. J. Qin, K. Chao, M.S. Kim. ‘‘Simultaneous Detection of Multiple Adulterants in Dry Milk Using Macro-Scale Raman Chemical Imaging’’. Food Chem. 2013. 138(2-3): 998-1007. 2. J. Qin, K. Chao, M.S. Kim. ‘‘Raman Chemical Imaging System for Food Safety and Quality Inspection’’. Trans. ASABE. 2010. 53(6): 1873-1882. 3. J. Qin, K. Chao, M.S. Kim. ‘‘Investigation of Raman Chemical Imaging for Detection of Lycopene Changes in Tomatoes During Postharvest Ripening’’. J. Food Eng. 2011. 107(3-4): 277-288. 4. C. Engert, A. Materny, W. Kiefer. ‘‘Temperature-Dependent Raman Spectra of Polydiacetylene Single Crystals Excited in Near Infrared’’. Chem. Phys. Lett. 1992. 198(3-4): 395-399. 5. M. Unger, H. Sato, Y. Ozaki, D. Fischer, H.W. Siesler. ‘‘Temperature-Dependent Fourier Transform Infrared Spectroscopy and Raman Mapping Spectroscopy of Phase-Separation in a Poly(3Hydroxybutyrate)-Poly(L-Lactic Acid) Blend’’. Appl. Spectrosc. 2013. 67(2): 141-148. 6. M. Unger, D. Chaturvedi, S. Mishra, P. Tandon, H.W. Siesler. ‘‘TwoDimensional Correlation Analysis of Temperature-Dependent FT-IR Spectra of Oleic Acid’’. Spectrosc. Lett. 2013. 46(1): 21-27. 7. R.X. Silva, C.W.A. Paschoal, R.M. Almeida, M. Carvalho-Castro Jr., A.P. Ayala, J.T. Auletta, M.W. Lufaso. ‘‘Temperature-Dependent Raman Spectra of Bi2Sn2O7 Ceramics’’. Vib. Spectrosc. 2013. 64(1): 172-177. 8. M. Carvalho-Castro Jr., E.F. Viana de Carvalho, W. Paraguassu, A.P. Ayala, F.C. Snyder, M.W. Lufaso, C.W.A. Paschoal. ‘‘Temperature-Dependent Raman Spectra of Ba2BiSbO6 Ceramics’’. J. Raman Spectrosc. 2009. 40(9): 1205-1210. 9. W.F. Schmidt, C.J. Hapeman, L.L. McConnell, S. Mookherji, C.P. Rice, J.K. Nguyen, J. Qin, H. Lee, K. Chao, M.S. Kim. ‘‘TemperatureDependent Raman Spectroscopic Evidence of and Molecular Mechanism for Irreversible Isomerization of b-Endosulfan to aEndosulfan’’. J. Agric. Food Chem. 2014. 62(9): 2023-2030. 10. N. Yan, L. Zhou, Z. Zhu, X. Chen. ‘‘Determination of Melamine in Dairy Products, Fish Feed, and Fish by Capillary Zone Electrophoresis with Diode Array Detection’’. J. Agric. Food Chem. 2009. 57(3): 807-811. 11. G. Abernathy, K. Higgs. ‘‘Rapid Detection of Economic Adulterants in Fresh Milk by Liquid Chromatography-Tandem Mass Spectrometry’’. J. Chromatogr. A. 2013. 1288: 10-20. 12. S. Okazaki, M. Hiramatsu, K. Gonmori, O. Suzuki, A. Tu. ‘‘Rapid Nondestructive Screening for Melamine in Dried Milk by Raman Spectroscopy’’. Forensic Toxicol. 2009. 27(2): 94-97. 13. M.R. Almeida, K.D. Oliveira, R. Stephani, L.F.C. de Oliveira. ‘‘Fourier Transform Raman Analysis of Milk Powder: A Potential Method for Rapid Quality Screening’’. J. Raman Spectrosc. 2011. 42(7): 1548-1552. 14. K. Chao, J. Qin, M.S. Kim, C.Y. Mo. ‘‘A Raman Chemical Imaging System for Detection of Contaminants in Food’’. Proc. SPIE. 2011. 8027: 802710. doi:10.1117/12.884498. 15. L. He, Y. Liu, M. Lin, J. Awika, D.R. Ledoux, H. Li, A. Mustapha. ‘‘A New Approach to Measure Melamine, Cyanuric Acid, and Melamine Cyanurate Using Surface Enhanced Raman Spectroscopy Coupled with Gold Nanosubstrates’’. Sens. Instrum. Food Qual. 2008. 2(1): 66-71.
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16. E. Ka¨mmer, T. Do¨rfer, A. Csa´ki, W. Schumacher, P.A. Da Costa Filho, N. Tarcea, W. Fritzsche. , P. Ro¨sch, M. Schmitt, J. Popp. ‘‘Evaluation of Colloids and Activation Agents for Determination of Melamine Using UV-SERS’’. J. Phys. Chem. C. 2012. 116(10): 60836091. 17. P. Lutter, M.-C. Savoy-Perroud, E. Campos-Gimenez, L. Meyer, T. Goldmann, M.-C. Bertholet, P. Mottier, A. Desmarchelier, F. Monard, C. Perrin, F. Robert, T. Delatour. ‘‘Screening and Confirmatory Methods for the Determination of Melamine in Cow’s Milk and Milk-Based Powdered Infant Formula: Validation and Proficiency-Tests of ELISA, HPLC-UV, GC-MS and LC-MS/MST’’. Food Control. 2011. 22(6): 903-913. 18. P.M. Santos, E.R. Pereira-Filho, L.E. Rodriguez-Saona. ‘‘Rapid Detection and Quantification of Milk Adulteration Using Infrared Microspectroscopy and Chemometrics Analysis’’. Food Chem. 2013. 138(1): 19-24. 19. S. Jawaid, F.N. Talpur, S.T.H. Sherazi, S.M. Nizamani, A.A. Khaskheli. ‘‘Rapid Detection of Melamine Adulteration in Dairy Milk by SB-ATR—Fourier Transform Infrared Spectroscopy’’. Food Chem. 2013. 141(3): 3066-3071. 20. S.Y. Venyaminov, N.N. Kalnin. ‘‘Quantitative IR Spectrophotometry of Peptide Compounds in Water (H2O) Solutions. I. Spectral Parameters of Amino Acid Residue Absorption Bands’’. Biopolymers. 1990. 30(13-14): 1243-1257. 21. H. Ishizaki, P. Balaram, R. Nagaraj, Y.V. Venkatachalapathi, A.T. Tu. ‘‘Determination of Beta-Turn Conformation by Laser Raman Spectroscopy’’. Biophys. J. 1981. 36(3): 509-517. 22. K.M. Vogel, T.G. Spiro, D. Shelver, M.V. Thorsteinsson, G.P. Roberts. ‘‘Resonance Raman Evidence for a Novel Charge Relay Activation Mechanism of the CO-Dependent Heme Protein Transcription Factor CooA’’. Biochemistry. 1999. 38(9): 2679-2687. 23. X. Sun, L. Rintoul, Y. Bian, D.P. Arnold, R. Wang, J. Jiang. ‘‘Raman Spectroscopic Characteristics of Phthalocyanine and Naphthalocyanine in Sandwich-Type Phthalocyaninato and Porphyrinato Rare Earth Complexes. Part 5—Raman Spectroscopic Characteristics of Naphthalocyanine in Mixed [Tetrakis(4-Tert-Butylphenyl)Porphyrinato] (Naphthalocyaninato) Rare Earth Double-Deckers’’. J. Raman Spectrosc. 2003. 34(4): 306-314. 24. Q. Yao, B. You, S. Zhou, M. Chen, Y. Wang, W. Li. ‘‘Inclusion Complexes of Cypermethrin and Permethrin with Monochlorotriazinyl-Beta-Cyclodextrin: A Combined Spectroscopy, TG/DSC and DFT Study’’. Spectrochim. Acta, Part A. 2014. 117: 576-586. 25. K.H. Michaelian, S.L. Zhang, R.H. Hall, J.T. Bulmer. ‘‘Fourier Transform Raman Spectroscopy of Syncrude Sweet Blend Distillation Fractions: The 200–1800 cm1 Region’’. Spectrochim. Acta, Part A. 2003. 59(5): 895-903.
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26. R.L. Frost, J. Kristof, L. Rintoul, J.T. Kloprogge. ‘‘Raman Spectroscopy of Urea and Urea-Intercalated Kaolinites at 77 K’’. Spectrochim. Acta, Part A. 2000. 56(9): 1681-1691. 27. T.C. Cheam, S. Krimm. ‘‘Vibrational Analysis of Crystalline Diketopiperazine—I. Raman and I.R. Spectra’’. Spectrochim. Acta, Part A. 1984. 40(6): 481-501. 28. M. Bao, R. Wang, L. Rintoul, Q. Liu, D.P. Arnold, C. Ma, J. Jiang. ‘‘Vibrational Spectroscopy of Phthalocyanine and Naphthalocyanine in Sandwich-Type (Na)Phthalocyaninato and Porphyrinato Rare Earth Complexes: Part 13. The Raman Characteristics of Phthalocyanine in Unsubstituted and Peripherally Octa(Octyloxy)Substituted Homoleptic Bis(Phthalocyaninato) Rare Earth Complexes’’. Polyhedron. 2006. 25(5): 1195-1203. 29. B.S. Yadav, I. Ali, P. Kumar, P. Yadav. ‘‘FTIR and Laser Raman Spectra of 2-Hydroxy-5-Methyl-3-Nitro Pyridine’’. Indian J. Pure Appl. Phys. 2007. 45(12): 979-983. 30. J. Bandekar, S. Krimm. ‘‘Vibrational Analysis of Peptides, Polypeptides, and Proteins: Characteristic Amide Bands of Beta-Turns’’. Proc. Natl. Acad. Sci. USA. 1979. 76(2): 774-777. 31. S. Xie, X. Zhang, S. Yang, M.C. Paau, D. Xiao, M.M.F. Choi. ‘‘Liesegang Rings of Dendritic Silver Crystals Emerging from Galvanic Displacement Reaction in a Liquid-Phase Solution’’. RSC Adv. 2012. 2(11): 4627-4631. 32. Y. Shyma Mary, P.J. Jojo, C. Van Alsenoy, M. Kaur, M.S. Siddegowda, H.S. Yathirajan, H.I.S. Nogueira, S.M.A. Cruz. ‘‘Vibrational Spectroscopic (FT-IR, FT-Raman, SERS) and Quantum Chemical Calculations of 3-(10,10-Dimethyl-Anthracen-9-Ylidene)N,N,N-Trimethyl Propanaminiium Chloride (Melitracenium Chloride)’’. Spectrochim. Acta, Part A. 2014. 120: 370-380. 33. P. Lagant, G. Vergoten, G. Fleury, M.-H. Loucheux-Lefebvre. ‘‘Raman Spectroscopy and Normal Vibrations of Peptides: Characteristic Normal Modes of A Type-II B Turn’’. Eur. J. Biochem. 1984. 139(1): 137-148. 34. P. Larkin. Infrared and Raman Spectroscopy: Principles and Spectral Interpretation. San Diego, CA: Elsevier, 2011. Pp. 212. 35. J. Kong, S. Yu. ‘‘Fourier Transform Infrared Spectroscopic Analysis of Protein Secondary Structures’’. Acta Biochim. Biophys. Sin. 2007. 39(8): 549-559. 36. S. Kumar, A.K. Rai, S.B. Rai, D.K. Rai, A.N. Singh, V.B. Singh. ‘‘Infrared, Raman and Electronic Spectra of Alanine: A Comparison with Ab Initio Calculation’’. J. Mol. Struct. 2006. 791(1-3): 23-29. 37. S.H. Baek, H.B. Lim, H.S. Chun. ‘‘Detection of Melamine in Foods Using Terahertz Time-Domain Spectroscopy’’. J. Agric. Food Chem. 2014. 62(24): 5403-5407. 38. Y. Tian, L. Chen, L. Gao, M. Wu, W.A. Dick. ‘‘Comparison of Three Methods for Detection of Melamine in Compost and Soil’’. Sci. Total Environ. 2012. 417-418: 255-262.
Hyperspectral Raman imaging has the potential for rapid screening of solid-phase samples for potential adulterants. We can improve mixture analysis algorithms by defining a temperature range in which the contaminant spectrum changes dramatically and uniquely compared with unadulterated material. Raman spectra were acquired for urea, biuret, cyanuric acid, and melamine (pure and at 1% in dried milk powder) from 50 to 310 8C with a gradient of 1 8C min1. Adulterants were clearly indentified in the milk powder. Specific frequencies that were mainly associated with ring breathing, stretching, and in-plane deformation shifted with respect to temperature up to 12 cm1 in all four molecules. Specific frequencies significantly increased/decreased in intensity within narrow temperature ranges independent of whether the amine was mixed in milk. Correlation of Raman and differential scanning calorimetry data identified structural components and vibrational modes, which concur with or trigger phase transitions. Index Headings: Raman spectroscopy; Biuret; Cyanuric acid; Melamine; Hyperspectral imaging; Temperature-dependent Raman spectroscopy.
INTRODUCTION Extending Raman imaging spectroscopy from microscale or nanoscale point measurements and ambient temperatures to a technique capable of two-dimensional surface scans across wide temperature ranges provides a new avenue for the rapid acquisition of both practical and theoretical chemical data. A practical application our laboratory recently developed is a hyperspectral Raman imaging system with sufficient spectral and spatial resolution to simultaneously identify and map four adulterant particles (ammonium sulfate, dicyandiamide, melamine, urea) mixed into dry milk at concentration levels from 0.1 to 5.0%.1 The hyperspectral imaging system maps a 25 3 25 mm2 area in about 1 hr. Rapid and accurate authentication of food ingredients is important for safety and quality evaluation. Raman chemical imaging coupled with appropriate mixture analysis algorithms can be used for simultaneous detection of multiple adulterants in many types of powdered food and nonfood materials. This system can also be used for macroscale imaging of food and Received 15 May 2014; accepted 4 September 2014. * Author to whom correspondence should be sent. E-mail: walter. [email protected]. DOI: 10.1366/14-07600
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agricultural products, such as scanning cross-sections of cut tomatoes for maturity evaluation.2,3 Significant wavenumber shifts in infrared and Raman spectra of solids with respect to temperature have been observed previously, and such data have the potential to readily identify contaminants, phase dissolution, and phase transitions.4–8 For example, over the temperature range 30 to 22 8C, Unger et al.6 could identify characteristic absorptions for both solid-state and liquid–solid phase transitions in oleic acid with Fourier transform infrared spectroscopy. Extending this study to Raman could simplify the spectral interpretation for this major edible lipid, which is abundant in olive and peanut oils and most nuts and meats. We further developed the technique of continuous temperature-dependent Raman spectroscopy to investigate isomerization of the organochlorine pesticide endosulfan between the nonsymmetric a and symmetric b-diastereomers.9 Raman spectra were acquired at 1 8C intervals from 50 to 102 8C for a-endosulfan, b-endosulfan, and a 60/40 mixture. For most vibrational modes discrete temperature-dependent changes occurred above 97 8C. Dramatic changes occurred at 970, 1027, 1092, and 1350 cm1, where spectral density increased, peaks shifted, and peaks broadened substantially. The response of these modes indicated that the corresponding molecular sites are the most temperature sensitive and therefore the most flexible. Results also preclude a to b isomerization. A phase transition observed at 97– 102 8C for b-endosulfan corresponded to the largest changes in the temperature-dependent Raman spectra. Temperature-dependent Raman provides a novel and very straightforward technique to identify theoretically proposed molecular rearrangements that occur just prior to phase transitions. In 2007, a widespread recall of pet foods occurred after thousands of dogs and cats in the United States experienced kidney failure. The United States Food and Drug Administration later determined that a Chinesesourced wheat gluten ingredient was contaminated with melamine. In 2008, some 300 000 Chinese children experienced kidney problems, including six fatalities, from melamine economic adulteration of infant formula produced by a major Chinese dairy company.10 In addition to melamine, economic adulteration of milk powder with fertilizers is facile and potentially introduces urea, biuret, triuret, and cyanamide to dairy products.11
0003-7028/15/6903-0398/0 Q 2015 Society for Applied Spectroscopy
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FIG. 1. Schematic of continuous temperature-dependent Raman spectroscopy analytical system.
Raman spectroscopy has been used previously to detect adulterants in dry milk including melamine,2,12 whey,13 ammonium sulfate, dicyandiamide, and urea.1,14 Surface-enhanced Raman with Au or Au-coated colloidal microsphere greatly improves the detection limits for melamine in solution but has not been developed for dry mixtures.15,16 Hyperspectral imaging has the potential for inexpensive rapid throughput screening of multiple samples with minimal preparation and without solvents. If it is determined that any of the suspect contaminants occur in a particular sample, more precise, but also more expensive and time-consuming analyses10,11,17–19 can be performed for individual components in those samples. Temperature-dependent Raman can potentially improve the mixture analysis algorithms by defining a narrow temperature range in which the spectrum of a contaminant changes dramatically and uniquely in comparison with the unadulterated or background material. In this contribution we present TDR spectra for urea, biuret, cyanuric acid, and melamine; pure and mixed at 1% in dried milk powder.
MATERIALS AND METHODS The temperature-dependent Raman system (Fig. 1) uses a Raman spectrometer (Raman Explorer 785, Headwall Photonics, Fitchburg, MA) fitted with a 16-bit charge-coupled device camera (1024 3 256 pixels; Newton DU920N-BR-DD, Andor Technology, South Windsor, CT). The spectrometer detects a Raman shift range
of 102.2 to 2538.1 cm1 with a spectral resolution of 3.7 cm1. A 785 nm laser module (I0785MM0350MF-NL, Innovative Photonic Solutions, Monmouth Junction, NJ) served as the excitation source. A fiber optic Raman probe (RPB, InPhotonics, Norwood, MA) was used to focus the laser and acquire the Raman signals. A bifurcated fiber bundle delivers the laser radiation to the probe and transmits the Raman signals to the spectrometer. Laboratory-grade reagents were as follows: melamine (C3H6N6, 99.0%, Aldrich), urea (CH4N2O, 98.0%, Sigma), cyanuric acid (C3H3N3O3, 98.0%, Aldrich), and biuret (C2H5N2O2, 98.0%, J.T. Baker). Organic skim dry milk (Organic Valley, La Farge, WI) was purchased from a local supermarket. The four reagents were mixed into the milk powder at 1.0% in 50 ml polypropylene centrifuge tubes. A vortex mixer was used to ensure uniform distribution of the adulterant particles. Powder samples were placed in copper sample holders and heated on a ceramic hotplate. Copper heat sinks were placed between the ceramic heat surface and the sample holder to slow the rate of temperature increase. Two K-type thermocouple probes were attached to two sides of the sample area and connected to a dual-input thermometer (EasyView EA15, Extech Instruments, Nashua, NH). The sample temperature was defined as the average value of the two probes. The Raman probe, hotplate, and sample materials were placed in a closed black box to avoid ambient light. The
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FIG. 2. Amine structural analogs used: (a) urea; (b) biuret; (c) cyanuric acid; (d) melamine.
hot plate heater was set for a gradient of about 1 8C min1. Raman spectra were acquired from 50 to 250 8C for urea and biuret and 50 to 310 8C for cyanuric acid and melamine. If dramatic chemical–thermal rearrangement occurred at a temperature prior to 250 8C, that temperature was the maximum used. We did not use a set time schedule for spectral acquisition, but rather acquired spectra each time the sample temperature increased 1 8C. System software was developed using LabVIEW (National Instruments, Austin, TX) to fulfill functions such as camera control, data acquisition, temperature measurement, and synchronization.2 Differential Scanning Calorimetry (DSC). One milligram of each amine was placed in an aluminum sample pan with a crimped lid. An empty pan was used as control. Samples were placed in a DSC Q200 differential scanning calorimeter (TA Instruments, New Castle, DE) and scanned from 50 8C to approximately 20 8C above the melting point (mp). A thermocouple under each pan recorded the rate of heat absorbed versus the rate applied. Heating rate was 5 or 10 8C min1 depending on the melting temperature. Samples were protected from oxidation using a continuous supply of N2, which is heated at the same rate as the sample, then pumped into the heating chamber.
RESULTS AND DISCUSSION Urea, biuret, cyanuric acid, and melamine contain overlapping structural components (Fig. 2); however, each structural analog has markedly different physical properties (e.g., melting point urea, 133 8C; biuret, 193 8C; cyanuric acid, 330 8C; melamine, 345 8C). Figure 3 shows that 1% melamine in milk powder is clearly detectable, as are the other three amines. Figures 4 and 5 are contour plots showing the progressive Raman frequency shifts with increasing temperature. Shifts are significant with respect to line bandwith and are specific to individual molecules and individual molecular sites; spectra are truncated at melting. Figure 6 shows DSC data for three of the molecules over the temperature range of the temperature-dependent Raman experiments. Assignments of chemical structure to temperature-dependent Raman vibrational modes agree with
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previously reported Raman frequencies observed at a constant temperature (Table I).9,20–36 Urea. The major temperature-dependent Raman vibrational modes in urea (CH4N2O) at 50 8C are 548, 1006, 1543, and 1649 cm1. All shift in frequency with temperature; the three largest shifts are a decrease at 544 cm1 (in-plane deformation), and increases at 1543 and at 1645 cm1 (amide III N–H stretching and NH2 asymmetric bending). Urea is symmetrical about the O=C axis and at all four H sites; the flexibility of N–H groups is equal. Gradually, both 1543 cm1 (N–H asymmetrical stretching) and 1649 cm1 (NH2 asymmetrical bending) increase in frequency. A structural explanation for the temperature-dependent Raman data is that one of the four N–H sites become longer than (i.e., unequal to) the other three, and therefore asymmetrical vibrational modes increase in intensity. In Fig. 6 these shifts correspond to the shoulder of the sharp peak of increasing heat flow indicating the melting of urea. Biuret. The temperature-dependent Raman and DSC data for biuret are very different from urea, even though chemically biuret is the dimer of urea. The major vibrational modes at 50 8C are 281, 456, 703, 954, 1007, 1116, 1517, and 1605 cm1. Despite their similar structure, only one frequency is within 5 cm1 of those in urea (1005 cm1). A second peak in biuret shows up at 1550 cm1 only above about 130 8C, whereas the [O=C– N–H] NH asymmetric bending frequency in urea occurs only below 130 8C. DSC peaks (Fig. 6) indicate two solid-state phase transitions (at 70 and 130 8C) prior to the peak for melting at 193 8C. This correlates with Fig. 4 and Table I (see footnotes), where some observed frequencies decrease sharply when 70 8C and then 130 8C are reached, yet others increase after 130 8C. The center of the biuret molecule is an N–H group between two O=C sites. Because each site is an amide (O=C–N–H), the two sites can be coplanar, and the NH will always be either above or below this plane. The increase in frequency at 456 cm1 and change in intensity at 954 cm1 (C–N–C bending) is consistent with a change in twist between the two O=C sites. Biuret has a discrete temperature profile: at 90 8C the 1605 cm1 peak intensity decreases dramatically (O=C–N–H symmetrical NH bending) at 110 8C, 1005 cm1 peak intensity (N–C–N stretching) is at its maximum; concurrently at 130 8C, peaks at 462 cm1 and 990 cm1 appear (C–O inplane bending and symmetrical skeletal stretching, respectively). The simplest structural interpretation is that at 50 8C, the two amide groups are not coplanar, but at 110 8C, they are. The intensity of the 1698 cm1 peak increases above 80 8C and is maximized at 130 8C. Since this peak is assignable to a planar b-turn in an amide, this also indicates biuret is planar at that temperature. The changes near 990 cm1 and at 1120 cm1 would then correspond to changes in flexibility of NH2, NH, or both moieties above and below the plane. Cyanuric acid. The three most intense temperaturedependent Raman frequencies in cyanuric acid at 110 8C are 547, 700, and 1729 cm1. Four other significant frequencies (Fig. 5) are also observed (411, 987, 1416, and 1470 cm1). Structurally, cyanuric acid is a cyclic trimer of urea, and the peak at 547 cm1 assignable to in-
FIG. 3. Contour plot of intensity and frequency of selected major Raman spectral feature vs. temperature for 1% melamine in nonfat milk powder and control milk powder.
plane deformation is similar to 549 cm1 in urea. The peak at 411 cm1 is assignable to ring deformation. The narrowing of temperature-dependent Raman peaks with minimal or no frequency shift at 1727 cm1 (O=C stretching) and at 700 cm1 (ring N–H site flexibility) indicates these sites in the ring (O=C–NH–(O=C)–NH) become more rigid with increasing temperature, and since the peak at 987 cm1 decreases, the applied thermal stress also reduces symmetrical ring stretching. The temperature-dependent Raman frequency shift in cyanuric acid at 539 cm1 (in-plane deformation) is
nonuniform, narrowing to 240 8C and then broadening to 290 8C. Concurrent with the broadening is an increase in intensity at 403 cm1 (ring deformation) and an additional peak at 458 cm1 (C–O in-plane bending). Thus at about 270 8C, the N=C–N site in the ring is deformed, and C–O bending at this C site becomes planar. The N–C=N bond sequence becomes a less planar–asymmetrical component of the ring resonance structure above 250 8C. One explanation for the asymmetry and frequency shift in the temperature-dependent Raman peak at 547 cm1 from 110 8C is that at the lower temperature, one of two
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FIG. 4. Overview contour plots of intensity and frequency of selected major Raman spectral features vs. temperature for urea, biuret, cyanuric acid, and melamine.
O=C–NH is more flexible than the other. The increasing concurrence in the coplanar conformation of the two O=C–NH sites with temperature explains the very high thermal stability of cyanuric acid. Spectra are observed over 290 8C in Fig. 4. The DSC curve (Fig. 6) indicates the
onset of melting at 330 8C as reported; however, the thermal absorption curve is broad with a shallow slope and extends nearly 70 degrees, in concurrence with a 12 cm1 shift in the ring stretching frequency, which is the largest shift we observed overall.
FIG. 5. Detail of significant frequency shifts vs. temperature from Fig. 4
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.
FIG. 6. Differential scanning calorimetry heat absorption curves for urea, biuret, cyanuric acid, and melamine. Note multiple absorptions in biuret prior to and after melting (193 8C).
672, 980, and 1557 cm1. The only temperature-dependent Raman vibrational mode similar between melamine and cyanuric acid is a relatively weak symmetrical skeletal stretching frequency (981 cm1 in melamine,
Melamine. Melamine is a structural analog of cyanuric acid in which –NH2 sites replace –OH sites or N=C–N2 sites replaces O=C–N2. Five major temperature-dependent Raman frequencies are observed at 50 8C: 385, 582,
TABLE I. Raman frequencies (cm1), temperature-dependent shift, and structural assignments. Melamine 50 8C 385
582 672
980
Cyanuric acid
250 8C
Shift
379
6
582 674
978
50 8C
1551
Shift
411
403
7
547
538
7
700
696
4
987
989
þ2
50 8C
170 8C
281
281a
456
462a
Urea Shift
50 8C
130 8C
Shift
þ6 548
544
4
1006
1003
3
1543
1549
þ6
1645
1649
þ4
þ2 2
1416 1470 1557
250 8C
Biuret
1408 1458
703 954
700b 948 990c
3 6
1007d 1116e
1125
þ9
1517f
1512
5
8 12
6 1557g 1605h 1698g 1729
1729
Assignments Ring C=O deformation35 C–N bending out of plane34 Ring deformation35 C–O in-plane bending29 In-plane deformation24 Ring breathing30 NH2 in-plane bending31 Ring breathing33 C–N–C out-of-plane bending35 Sym. skeletal stretching29 N–C–N stretching28 C–N stretching (in CNH2)31 Ring breathing (N–H bending)26 Ring stretching29 C=N–C bending23 N=C–NH NH sym. stretching,23 amide III32 O=C–NH NH asym. stretching,22 amide II27 O=C–NH site (N–H bending)21 NH2 asym. bending20,36 b turns (C=O stretching)9 O=C–NH site (C=O stretching)20
a
Loss in intensity at 115 8C. Maximum intensity about 110 8C. c Observed starting .130 8C. d Loss of intensity 130 8C. e Starts shifting 130 8C. f Shift begins 90 8C. g Low intensity at 50 8C and maximum 130 8C. h Clear loss in intensity 100 8C. b
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FIG. 7.
Normalized relative intensity of the cyanuric acid 537 cm1 in-plane deformation peak vs. temperature, showing increased intensity 130 8C.
987 cm1 in cyanuric acid). Melamine, urea, and biuret all have vibrational modes of NH and NH2 sites in common near 1557 cm1. Structurally, melamine has no N–H ring sites, but three N=C–N–H2 sites are present per molecule. The spectral lines at 582 cm1 and 980 cm1 are ring breathing and symmetrical skeletal stretching, respectively. Their signal intensities are very weak compared with 385 cm1 (C–N bending out of plane), 672 cm1 (NH2 in-plane bending) and 1557 cm1 (NH symmetrical stretching). Melamine has the highest reported melting temperature of the four amines but may decompose near melting, which agrees with Fig. 4. The sites that absorb thermal energy are C–N and C–N–H sites external to ring structure. The 385 cm1 frequency can be one of three sites; if the N in C–N–H is bending out of plane, NH sites also will be out of plane. With increasing temperature, the peak at 1557 cm1 shifts to 1551 cm1 and also the peak sharpens. In-plane NH2 bending (672 cm1) shifts upfield 2 cm1, and ring breathing at 582 cm1 steadily decreases. An explanation consistent with these results is that with increasing temperature, the ring structure overall becomes more rigid, and two of the NH2 sites become more planar, but one C–N site bends more out of plane. This molecular site becomes progressively more unequal, i.e., above or below the stable ring plane. The increase with temperature in out-of-plane bending may
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disrupt the uniformity of packing between melamine molecules, resulting in the broad melt/decomposition peak in the DSC data starting at about 250 8C and continuing to 345 8C (Fig. 6). Mechanism for Temperature-Dependent Raman Selectivity. Melting and other thermally induced phase transitions correspond at the molecular level to an increase in flexibility in response to thermal stress. Near their individual melting points, molecules become more flexible. Specific sites within a molecule may become flexible before the entire molecule responds. Under thermal stress, more rigid molecules or sites respond less quickly than more flexible sites. The pattern in which vibrational modes respond to thermal stress is unique to a specific molecular structure. Physical phase transitions in polymers,4 biopolymers,5 and oleic acid6 spectroscopy correlated with temperature have been reported using infrared or Raman spectroscopy. However in these studies measurements were made at discrete, discontinuous temperature steps under steady state conditions. Our spectra were collected with a continuous temperature gradient, which minimizes the time (and molecular pathways) in which thermal relaxation between individual temperature measurement steps can occur. A temperature gradient consistently perturbs steady state conditions, resulting in thermal stress. Molecular sites more sensitive to
thermal stress will absorb thermal energy at lower temperatures than less sensitive sites. For example, although the stereoisomers and bendosulfan contain identical structural components, each has discrete marker vibrational modes.9 In the bisomer, the seven-membered ring is symmetrical, whereas the ring is asymmetrical in the a-isomer. The flexibility of the asymmetrical seven-membered ring accounts for its physical properties: the a-isomer melts at 108 8C and environmentally concentrates in air. The rigidity of the symmetrical conformation in the b-isomer accounts for its melting point of 208 8C and the fact that it is found as an environmental contaminant in water, not air. In Fig. 7 the intensity of the cyanuric acid 537 cm1 inplane deformation peak is nearly doubled at temperatures over 130 8C. Similar magnitude changes in peak intensity were described for biuret above. Although temperature-dependent Raman spectra of melamine can be observed to 310 8C, 1% melamine in milk is not detected above 170 8C. At 120 8C milk proteins and sugars will begin to react irreversibly, forming Maillard or ‘‘browning’’ products. Upon heating, significant peaks in 1% melamine in milk occur in the 1200–1400 cm1 range, which are not observed for either of the component structures. Further research could confirm if these are sites where melamine and milk protein chemically react. Thus temperature-dependent Raman spectroscopy can enable detection of melamine even if it chemically reacts with a food product during processing. These are just a few of many examples of temperaturedependent Raman frequencies that increase, decrease, appear, or disappear within specific temperature ranges that often correlate with known phase transitions and may correlate with chemical reactions. With further development we undoubtedly can improve hyperspectral mixture analysis algorithms because we have a straightforward method to rule out structural analogs that can cause considerable uncertainty, time, and effort with other analytical methods. Recently, vibrational spectroscopy in the THz range has been used to identify melamine in several food products.37 A fingerprint spectrum can be assignable to a portion of a chemical structure or to some physical form of a chemical structure. Structural elucidation is a fundamental and critical component in spectroscopic techniques to avoid both false positive and false negative analytical results. Temperature-dependent Raman spectroscopy may also be useful for identifying environmental triazine contamination in soils and composts.38 Soils are more complex substrates than milk powder; however, response to increasing temperature for organic soil constituents will undoubtedly differ from that of clay minerals, potentially enhancing detection of individual organic species. Fractions bound tightly to soils may also be identified more easily than with other methods. Temperature-dependent Raman spectra are not identical to constant temperature Raman spectra because temperature-dependent spectra are transient. In constant temperature experiments any unequal absorption of thermal energy absorbed selectively site-to-site within a molecular structure will relax and redistribute evenly.
In our temperature-dependent Raman experiments, the temperature gradient is continuous; therefore assigned frequencies that are unaffected by temperature can be distinguished from vibrational modes that alter with temperature, especially within the temperature range in which phase transitions occur. Within this gradient, any thermal sequence in which individual molecular sites become more or less flexible can be determined. This information is an identifying component unique to that individual molecular structure. Since temperature-dependent Raman enables the temperature-dependent components of Raman spectra to be identified, these vibrational modes can be assigned to chemical structures. This in turn enables identification of individual vibrational modes and chemical structure components that concur with or trigger the start of a phase transition. 1. J. Qin, K. Chao, M.S. Kim. ‘‘Simultaneous Detection of Multiple Adulterants in Dry Milk Using Macro-Scale Raman Chemical Imaging’’. Food Chem. 2013. 138(2-3): 998-1007. 2. J. Qin, K. Chao, M.S. Kim. ‘‘Raman Chemical Imaging System for Food Safety and Quality Inspection’’. Trans. ASABE. 2010. 53(6): 1873-1882. 3. J. Qin, K. Chao, M.S. Kim. ‘‘Investigation of Raman Chemical Imaging for Detection of Lycopene Changes in Tomatoes During Postharvest Ripening’’. J. Food Eng. 2011. 107(3-4): 277-288. 4. C. Engert, A. Materny, W. Kiefer. ‘‘Temperature-Dependent Raman Spectra of Polydiacetylene Single Crystals Excited in Near Infrared’’. Chem. Phys. Lett. 1992. 198(3-4): 395-399. 5. M. Unger, H. Sato, Y. Ozaki, D. Fischer, H.W. Siesler. ‘‘Temperature-Dependent Fourier Transform Infrared Spectroscopy and Raman Mapping Spectroscopy of Phase-Separation in a Poly(3Hydroxybutyrate)-Poly(L-Lactic Acid) Blend’’. Appl. Spectrosc. 2013. 67(2): 141-148. 6. M. Unger, D. Chaturvedi, S. Mishra, P. Tandon, H.W. Siesler. ‘‘TwoDimensional Correlation Analysis of Temperature-Dependent FT-IR Spectra of Oleic Acid’’. Spectrosc. Lett. 2013. 46(1): 21-27. 7. R.X. Silva, C.W.A. Paschoal, R.M. Almeida, M. Carvalho-Castro Jr., A.P. Ayala, J.T. Auletta, M.W. Lufaso. ‘‘Temperature-Dependent Raman Spectra of Bi2Sn2O7 Ceramics’’. Vib. Spectrosc. 2013. 64(1): 172-177. 8. M. Carvalho-Castro Jr., E.F. Viana de Carvalho, W. Paraguassu, A.P. Ayala, F.C. Snyder, M.W. Lufaso, C.W.A. Paschoal. ‘‘Temperature-Dependent Raman Spectra of Ba2BiSbO6 Ceramics’’. J. Raman Spectrosc. 2009. 40(9): 1205-1210. 9. W.F. Schmidt, C.J. Hapeman, L.L. McConnell, S. Mookherji, C.P. Rice, J.K. Nguyen, J. Qin, H. Lee, K. Chao, M.S. Kim. ‘‘TemperatureDependent Raman Spectroscopic Evidence of and Molecular Mechanism for Irreversible Isomerization of b-Endosulfan to aEndosulfan’’. J. Agric. Food Chem. 2014. 62(9): 2023-2030. 10. N. Yan, L. Zhou, Z. Zhu, X. Chen. ‘‘Determination of Melamine in Dairy Products, Fish Feed, and Fish by Capillary Zone Electrophoresis with Diode Array Detection’’. J. Agric. Food Chem. 2009. 57(3): 807-811. 11. G. Abernathy, K. Higgs. ‘‘Rapid Detection of Economic Adulterants in Fresh Milk by Liquid Chromatography-Tandem Mass Spectrometry’’. J. Chromatogr. A. 2013. 1288: 10-20. 12. S. Okazaki, M. Hiramatsu, K. Gonmori, O. Suzuki, A. Tu. ‘‘Rapid Nondestructive Screening for Melamine in Dried Milk by Raman Spectroscopy’’. Forensic Toxicol. 2009. 27(2): 94-97. 13. M.R. Almeida, K.D. Oliveira, R. Stephani, L.F.C. de Oliveira. ‘‘Fourier Transform Raman Analysis of Milk Powder: A Potential Method for Rapid Quality Screening’’. J. Raman Spectrosc. 2011. 42(7): 1548-1552. 14. K. Chao, J. Qin, M.S. Kim, C.Y. Mo. ‘‘A Raman Chemical Imaging System for Detection of Contaminants in Food’’. Proc. SPIE. 2011. 8027: 802710. doi:10.1117/12.884498. 15. L. He, Y. Liu, M. Lin, J. Awika, D.R. Ledoux, H. Li, A. Mustapha. ‘‘A New Approach to Measure Melamine, Cyanuric Acid, and Melamine Cyanurate Using Surface Enhanced Raman Spectroscopy Coupled with Gold Nanosubstrates’’. Sens. Instrum. Food Qual. 2008. 2(1): 66-71.
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