# A 1064 nm Dispersive Raman Spectral Imaging System for Food Safety and Quality Evaluation
## 3 Application to Authenticate Turmeric Powder
### Detection of Metanil Yellow Contamination in Turmeric Powder  
Metanil yellow (70% dye, Aldrich, Carson City, NV, USA) and organic turmeric powder (Frontier Natural Products CO-OP, Norway, IA, USA) were mixed together in a vortex mixer (Scientific Industries Inc., Bohemia, NY, USA) for ten minutes to prepare sample mixture at 1%, 3%, 5%, 7%, and 10% metanil yellow concentration (\(w/w\)). The weight of each mixture sample was 0.27 g. Each mixture sample was packed into a shallow nickel-plated sample container (internal volume 25 mm \(\times\) 25 mm \(\times\) 1 mm), and its surface was leveled flush with the edge of the container. A Raman spectral image of each sample was obtained at 1 s exposure time and 80 mW laser power, covering a spatial area of 25 mm \(\times\) 25 mm with a step size of 0.25 mm, collecting a total of 10,000 spectral pixels. Figure 4 shows the Raman spectra of turmeric powder and metanil yellow. The yellow color appearance of turmeric and metanil yellow is due to the similarly extended conjugation in both the chemicals [32]. However, a different chemical composition results in different vibrational modes specific to their chemical structure. The sharp peaks of metanil yellow at 1147 cm\({}^{-1}\) and 1433 cm\({}^{-1}\), due to the N = N site are most definitive to its identification and quantification [67; 68; 69]. The 1631 cm\({}^{-1}\) peak in turmeric spectrum is due to the carbonyl group. The carbonyl group is not present in metanil yellow [32]. Therefore, the 1631 cm\({}^{-1}\) peak is most definitive for turmeric identification. Figure 5 shows the Raman spectral images of turmeric, metanil yellow, and turmeric-metanil yellow mixtures across all of the concentration at 1147 cm\({}^{-1}\) and 1433 cm\({}^{-1}\). In Figure 5a, the dark pixels in the spectral images of turmeric show low spectral intensity of turmeric at 1147 cm\({}^{-1}\) and 1433 cm\({}^{-1}\). However, spectral images of metanil yellow have white pixels revealing high spectral intensity at 1147 cm\({}^{-1}\) and 1433 cm\({}^{-1}\) (Figure 5a). The 1147 cm\({}^{-1}\) and 1433 cm\({}^{-1}\) are the highest intensity metanil yellow peaks. Figure 5b is the raw spectral images of turmeric-metanil yellow samples at different concentrations. The white pixels in the spectral images are due to the high spectral peak intensities at 1147 cm\({}^{-1}\) and 1433 cm\({}^{-1}\). Figure 4: Raman spectra of turmeric and metanil yellow. For identification of metanil yellow pixels, binary detection images were created. The 1147 cm\({}^{-1}\) and 1433 cm\({}^{-1}\) peaks were used to convert the Raman spectral images into two separate single-band binary images. An intensity threshold was used to convert all the pixels below the threshold value into background pixels of turmeric. It was observed that the intensities of some of the metanil yellow pixels were lower than the set threshold value (false negative), while the intensities of some turmeric pixels were higher than the set threshold value (false positive). To avoid the false-positive and false-negative cases, a pixel-to-pixel analysis was done to select the most appropriate threshold value. Each pixel in the binary image was evaluated with its corresponding spectrum. A final intensity threshold value of 550 was set. All the pixels below the threshold value were converted to background pixels, and the remaining pixels represented metanil yellow. Figure 6 shows the 1147 cm\({}^{-1}\) and 1433 cm\({}^{-1}\) binary detection images of the samples, in which the white pixels represent the detected metanil yellow particles. A gradual increase in the number of white pixels at higher concentration shows more metanil yellow particles were detected at increasing concentration. A total of 8, 19, 35, 55, and 80 metanil yellow pixels were detected in the 1147 cm\({}^{-1}\) binary images of 1%, 3%, 5%, 7%, and 10% concentration samples (Figure 6). A similar number of metanil yellow pixels were detected in the 1433 cm\({}^{-1}\) binary images.