Determination of possible Saffron adulteration via TLC? Thin-layer chromatography has been used to determine the grade, and possible adulteration of saffron [1,2]. Whilst mostly known for it's use for food, there is growing medicinal use of saffron for an array of disorders and if adulterated, may not offer the desired therapeutic effects, or display toxicity. This paper aims to see if rapid citizen TLC of saffron serves to distinguish some commercial saffron samples available in Australia, and if it highlights any potential adulteration. Some common adulterants include safflower, marigold, curcuminoids, tartrazine yellow and Allura red, Red 40 and Yellow 5 dyes [1]. In pure saffron, crocin and crocetin are the main carotenoids giving the colour and some of the medicinal effects, crocin - contrary to many carotenoids - is hydrophilic due to it's glycosidic structure Crocin Crocetin The two saffron samples Some examples of solvent systems for saffron TLC include mixtures of 1-butanol, acetic acid and water. As citizen TLC, a readily available solvent system was desired. 95% Ethanol, acetone, water and ethanol/water were initially selected. Very small samples of saffron from two brands (AU $7.50 per gram [still claimed to be "Saffron Filament - G1"] vs "Pure Saffron" @ $70 per gram) were selected and macerated in a small volume of 95% ethanol. The resultant solution, bright yellow for both was spotted on a TLC plate (Silica, 0.2mm, glass backed) and developed with 95% ethanol, acetone, water or water:ethanol 1:1. Whilst significant streaking of constituents was noted, 95% ethanol gave a UV fluorescent (orange-red) broad band prominent for only one saffron sample, the other was pigmented visually, of similar Rf but lacking the characteristic fluoresence. For acetone, a high Rf constituent of same fluorescence nearing the solvent front was seen only on the same (cheaper) sample. Elution with water gave differing results and some visible separation (with streaking), where the cheaper saffron had a lower Rf 1 constituent, that of the more expensive brand was somewhat higher Rf with still poor separation. 50% aq. ethanol gave fairly poor separation of the constituents Saffron samples with water as solvent Various solvents using aq. acetone were tried on an aqueous extract of saffron - 1:1, 3:1 etc. 3:1 acetone:water showed the predominate coloured hydrophilic pigment was, in part, potentially different in the cheaper sample with it mainly running extremely close along the solvent front, vs a large constituent in the more expensive saffron, distinctly, slightly below it. 3:1 water:acetone again showed differences in the major hydrophilic constituent, where it was of significantly lower Rf in the cheaper sample TLC (3:1 H 2 O:acetone) of the saffron aqueous extract showing differences in the hydrophilic constituents Conclusion Finding a suitable 'citizen' solvent system was challenging and requires further work. Whilst distinguishing the samples differences with aqueous solvents was somewhat challenging, there was a striking presence of the UV fluorescent constituent on elution with acetone, or 95% ethanol in the cheaper sample. That said, given the limitations of citizen analysis, results should be interpreted cautiously. The cheaper (AU $7.50/gram) sample clearly demonstrated the presence of an atypical constituent that was fluorescent under UV that was absent in the more expensive brand. It is possible this may be an azo dye etc. It also seems the predominate hydrophilic pigment is somewhat slightly different in both References [1] Dai, H., Gao, Q., & He, L. (2020). Rapid Determination of Saffron Grade and Adulteration by Thin-Layer Chromatography Coupled with Raman Spectroscopy. Food Analytical Methods. doi:10.1007/s12161-020-01828-x [2] Djozan, D., Karimian, G., Jouyban, A., Iranmanesh, F., Gorbanpour, H., & Alizadeh-Nabil, A. (2014). Discrimination of saffron based on thin-layer chromatography and image analysis. Journal of Planar Chromatography – Modern TLC, 27(4), 274–280.. 2 doi:10.1556/jpc.27.2014.4.7 3