Abstract: Saffron, a plant from the category of Iridaceae, is derived from dried stigmas of Crocus Sativus L. It is the world’s most expensive spice [1]. Due to its high price and limitation of its growing, falsification of saffron used artificial colorants, such as Methyl-Orange that is one of the most common way for saffron adulteration. In this study, it was discovered that the bilinear second order data of Methyl-Orange could be obtained from the micelle concentration gradual change–visible absorption spectra. In the method described here, the components of the micelle/surfactant were changed gradually by adding of Triton X-100 surfactant into Methyl-Orange and the absorption spectra of Methyl-Orange in a series of water–Triton X-100 mixed were recorded. The novel obtained second order data were the foundation of second order calibration algorithms (including Parallel factor analysis (PARAFAC) , rank annihilation factor analysis (RAFA), unfolded partial least-squares/residual bilinearisation (U-PLS/RBL) and Bilinear least square residual bilinearisation (BLLS/RBL))[2] for analysis of Methyl-Orange in various adulterated saffron without any pre-separation. As well as, due to, the rank deficiency in the concentration mode, three-way variation matrix was generated by subtracting the first spectrum from other spectra in the data set[1,3-4]. It eradicates the ambiguity, which results from the rank deficiency. Then variation matrices were analyzed using the original second order calibrations method. The results were compared with those obtained from the raw data. This developed technique is simple, rapid, convenient and dependable for generation of second order data. Reasonable and satisfactory results were achieved.

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