Detection of Sudan Dyes in Red Chilli Powder by Thin Layer Chromatography

Abstract

Sudan I, II, III & IV are suspected genotoxic carcinogens and reports have shown the adulteration of chilli and chilli containing products with these azo dyes in recent past. In this study, a Simple Thin Layer Chromatographic method is described for the qualitative detection of Sudan-III & IV in Red chilli powder. The study includes extraction of sample, preparation of Thin Layer Chromatography (TLC) plates and Separation cum detection of dyes on TLC plates. The dyes were extracted with solvent Acetonitrile. Sample extracts were filtered and analyzed by simple thin layer chromatography in benzene, hexane and acetic acid solvent system. The chromatograms of sample, standard and spiked samples were evaluated in term of Rf and relative Rf values. The relative Rf values of four samples analysed, relative to the two standards of Sudan (IIIS2 and IV S1) were less than one (P1-0.13,0.25; P2-0.1 9,0.35; P3-0.30,0.35; P4-0.15,0.26 and P5-0.20,0.38), confirming the absence of Sudan III and IV in these samples. The efficiency of the method was checked by internal standard method, in which pure sample was spiked by known weight of standard dye. The relative Rf values of spiked sample was almost equal to one (b1-0.93, b2-0.80). Limit of detection (5 mg/kg) was calculated on the basis of visual evaluation of spots (i.e. spot visible or not at given concentration of standard dye) under normal light without any derivetization. The chilli samples analysed though varied in color intensity, but none of them was found to contain Sudan III or IV. with Sudan IV (less often with Sudan I) and originates from various African countries [2]. Sudan dyes are fat soluble azo dyes but solubility data of Sudan dyes is not abundant in literature. Sudan dyes are insoluble in water and soluble in various organic solvents (1.49 mol/dm3 in trichloromethane, 0.57 mol/dm3 in dichloromethane, 0.30 mol/dm3 in toluene, 0.17 mol/dm3 in benzene, 0.04 mol/dm3 in acetonitrile, 0.02 mol/dm3 in ethanol and 0.017 mol/dm3 in methanol. There is no solubility information for Sudan II. Sudan III is 0.015 and Sudan IV is 0.09 mol/dm3 soluble in ethanol. The log-P value of Sudan I is 5.86 meaning that it is highly lipophilicity compound. No data is available for Sudan II-IV but given their molecular structures, their log-P values are expected to be similar or somewhat higher. UV-spectra of Sudan dyes I-IV differ from each other (Figure 3). The absorption maxima are at the following wavelengths; Sudan I 475 nm, Sudan II-489 nm, Sudan III-503 nm and Sudan IV-513 nm and 515 nm. Sudan dyes are weak acids, however the azo group in the I position that acts as a hydrogen bond acceptor leads to the formation of an intra-molecular hydrogen bond with the phenol-OH. This additional stabilization of the neutral Sudan molecule is responsible for rather high pKa value 11.65 of Sudan I and II [3]. The pka values of Sudan III and IV are expected to be similar. Sudan dyes thus can be considered to behave as neutral in most of the analytical methods. Sudan dyes are indirect carcinogens (classified as category 3 carcinogens by IARC) and are therefore banned from the use in foods