Rapid Determination of Saffron Grade and Adulteration by Thin-Layer Chromatography Coupled with Raman Spectroscopy

Saffron, one of the most expensive spices around the world, is highly vulnerable to economic adulteration in its powder form. Plant materials and artificial colorants are commonly used as adulterants to reduce saffron concentration as well as maintaining color strength in powdered saffron products. However, current analytical methods lack the ability to test samples accurately onsite. Therefore, the objective of this study is to develop a fast screening and quantification method combining thin-layer chromatography (TLC) and Raman spectroscopy for saffron powder analysis, including quantifying the main colorant crocin content and identifying possible adulterants out of a lab setting. A droplet of aqueous extract of saffron powder was deposited on a TLC plate, exhibiting a uniform yellow round pattern on the TLC plate. The content of crocin was analyzed using the ISO 3632 method and Raman spectroscopy separately. A quantitative model based on Raman spectra was established and validated using partial least square (PLS) with R of 0.9955 and 0.9929 for calibration and validation of the PLS plot, respectively. Possible adulterants such as powders of safflower, turmeric powder, red 40 (allura red), and yellow 5 (tartrazine) were mixed with saffron powder at various degrees. Their water extracts exhibited unique patterns under bright ambient light and UV light that can be discriminated from that of pure saffron samples through visual observation and principal component analysis (PCA) based on the L*a*b* values. We also estimated the lowest adulteration degrees that can be detected were 2.59% for Allura red, 4.15% for tartrazine, 31.01% for safflower, and 41.98% turmeric, respectively, using the PLS analysis. Further identification was evaluated using Raman spectroscopy, which provided fingerprint spectra for all adulterants except safflower. Combining TCL pattern and Raman spectroscopy, samples with identical optical properties to pure saffron but adulterated with multiple adulterants were analyzed and successfully detected. This study demonstrates the potential practical application of this method in rapid analysis of saffron quality and adulteration.