# A Review: Sample Preparation and Chromatographic Technologies for Detection of Aflatoxins in Foods
## 3 Chromatography  
### Thin Layer Chromatography  
As one of the early chromatographic techniques used for aflatoxin analysis, TLC was credited for its important role in the first purification and identification of aflat back in the 1960s [94; 167] and since then various TLC applications have been developed, in a large part due to its simple and economical nature [168]. As a simple but inefficient chromatographic technique coupled with non-specific detection [169], early TLC methods were vulnerable to fluorescence interferences and non-fluorescing materials of similar migration on the plate (R\({}_{\mathrm{f}}\) value) that could mask aflat [170; 171]. Numerous studies were conducted to improve separation capacity of TLC. Noteworthy developments in TLC are high-performance TLC (HPTLC), 2-dimentional TLC (2D-TLC) and over-pressured layer chromatography (OPTLC) [172; 173]. As an extension of conventional TLC, HPTLC increases the resolution of aflat by using high quality plates coated with stationary phase with uniform and fine particles. With automated sample application devices and densitometers for quantitative analysis, HPTLC provides more efficient separation and better sensitivity [174]. Using HPTLC and multiple developments with two solvent systems of different polarity, [PERSON] et al. [175] achieved base-line separation of aflatoxin B\({}_{1}\), B\({}_{2}\), G\({}_{1}\), G\({}_{2}\), M\({}_{1}\) and M\({}_{2}\) and the other seven mycotoxins within 1 hr. Detection limits of aflat were in the low picogram ranges by fluorescence. Quantitation in the nanogram range demonstrated good precision with RSDs ranging from 0.7 to 2.2%. In 2D-TLC analyses, the plate is turned 180\({}^{\circ}\) after the first development and aflat and matrix components are further separated in the 2 nd development. The comparison of 1D-TLC and 2D-TLC for aflatoxin M\({}_{1}\) and B\({}_{1}\) in fatty foods (milk and eggs) shows that the two techniques provide comparable recoveries, but RSDs associated with 2D-TLC are much lower (7%) than that of 1D-TLC (16%), suggesting better repeatability [176]. The author emphasized that with limited separation capacity, 2D-TLC-based determinations of aflatoxin M\({}_{1}\) and B\({}_{1}\) requires in-depth sample clean-up procedures for fatty samples but the TLC analysis itself is an easy operation for inexperienced chemists and a more economical choice for labs without a budget for capital instruments such as LC. Many AOAC official methods were developed based on 2D-TLC for aflatoxin analysis in complex matrices [169]. Invented in the 1970s [177], OPTLC has been commonly described as a planar layer version of HPLLC, in which the mobile phase is pumped through a thin column with a much wider cross-section than conventional LC columns [173]. The applications of OPTLC has been limited to research applications, most likely due to the complexity of the system. In order to perform OPTLC, one would need a pressurized chamber, a liquid-delivery system, a detector in addition to other ancillary components. Although, advanced features such as online sample cleanup and delivery could be incorporated in to OPTLC, over-engineering would eliminate the simplicity of TLC with underperforming separation efficiency and sensitivity compared to LC. To date, only a handful of OPTLC applications are reported for aflatoxin analysis [172; 178; 179]. Generally, in terms of identification, a comparison of migration distance ratio, R\({}_{\mathrm{f}}\) values (samples vs. standards) on the TLC plate can only provide presumptive evidence of the existence of aflat,and further confirmation tests are required, especially for compliance samples. [PERSON] and [PERSON] (2010) developed a confirmation procedure based on the fluorescent reaction products of aflatoxin B\({}_{1}\) with formic acid-thionyl chloride, acetic acid-thionyl chloride and trifluoroacetic acid. The procedure required a large on plate mass of aflatoxin B\({}_{1}\), yet it did not work for other aflat.