## 2 Chromatographic Techniques for Carotenoids Analysis
### Thin-Layer Chromatography  
Thin-layer chromatography (TLC) is a simple and cost-effective technique often used for the preliminary screening of carotenoids. It involves the separation of carotenoids on a stationary phase, such as silica gel, using solvent systems optimized for their polarity. While TLC lacks the precision and quantification capabilities of HPLC or GC, it remains valid for rapid qualitative analysis and initial profiling of carotenoid mixtures. Table 2 summarizes different TLC-based methods for carotenoid determination used since 2009.  
These studies employed various stationary phases, developing solvents, and detection techniques to identify carotenoids in different food matrices [35; 36; 37; 38; 39; 40; 41; 42; 43; 44]. Most studies used silica gel-based TLC plates, either standard (TLC Silica Gel 60) or high-performance variants (HPTLC Silica Gel 60 F254) [35; 36; 37; 40; 41; 43], with some employing aluminum-backed silica gel or nano-silica gel plates for better separation [39; 41]. Developing solvents varied significantly, with petroleum ether-based systems being common, while more polar solvents like THF/methylene chloride/n-hexane and chloroform/methanol/acetone/ammonium hydroxide were also used [36; 40]. Some solvents, such as methylene chloride and chloroform, pose environmental concerns due to their toxicity and disposal challenges, making greener alternatives a critical consideration [45]. Detection methods mainly relied on UV-Vis, with 425 and 461 nm detection wavelengths, sometimes supplemented by densitometry or Raman spectroscopy [35; 38]. More advanced approaches, such as TLC-HPLC-DAD-ESI-MS, were employed in certain studies to improve identification accuracy [38; 41].  
The number of carotenoids analyzed varied among studies. Simple food matrices, such as dietary supplements [36; 38], typically contained three to five carotenoids, whereas complex samples, like _Aesculus hippocastanum_ leaves, exhibited up to ten different carotenoids [37]. Advanced detection techniques were employed for more complex mixtures, while single carotenoids, such as \(\beta\)-carotene in fruit juices, were identified using more straightforward UV-Vis detection [39]. The foods analyzed included pollen, dietary supplements, fruit juices, leaves, spices, pastes, sauces, palm oils, shrimp, and orange peel waste. The main carotenoids identified were \(\beta\)-carotene, lutein, zeaxanthin, astrocytum, bacterioruberin, and several xanthophyll derivatives.  
A recent study by [PERSON] et al. presents an innovative approach using preparative TLC for purifying xanthophyll carotenoids from lettuce [46]. This method utilizes Hyflo-Super-Cel-MgO (Heavy)-calcium sulfate hemihydrate (9:9:2 \(w/w\)) as an adsorbent and acetone- hexane (1:1) as the mobile phase, achieving 95-96% purity in a single step without interference from chlorophylls or minor carotenoids. This approach simplifies the purification process while maintaining high purity levels, making it suitable for analytical and preparative applications. Additionally, the study explored the anticancer potential of the purified carotenoids, emphasizing their health benefits. This analysis highlights the versatility of TLC in carotenoid research, from preliminary screening to purification and bioactivity studies. While TLC cannot replace more advanced chromatographic techniques, it remains a valuable tool for qualitative analysis and initial compound separation.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline