## 2 Chromatographic Techniques for Carotenoids Analysis
### Supercritical Fluid Chromatography  
Supercritical fluid chromatography (SFC) is gaining attention as an environmentally friendly alternative to traditional methods. It employs supercritical carbon dioxide as the mobile phase, reducing the use of organic solvents and offering efficient separation, particularly for non-polar carotenoids. Coupled with detectors like DAD and MS, SFC provides reliable quantification and identification while supporting sustainable analytical practices. Table 3 outlines different SFC-based carotenoid and vitamin determination methods over the past decade [44; 45; 46; 47; 48; 49; 50; 51].  
The stationary phases include HSS C18 SB, 1-AA, BEH-2-EP, Ascents Express C30, and Venusil XBP C30. These phases offer varied selectivity, with C30-based columns often preferred for carotenoid separation due to their affinity for non-polar compounds. The back pressure regulators used in these studies ranged from 124.1 to 160 bar, ensuring stable CO\({}_{2}\) conditions for efficient separation. The temperature settings varied from 35 \({}^{\circ}\)C to 60 \({}^{\circ}\)C, with higher temperatures generally improving analyte solubility and reducing retention times. The mobile phase typically consisted of MeOH combined with CO\({}_{2}\), though one study utilized isopropanol (IPA) as a modifier to enhance the solubility of specific carotenoids [51].  
The number of analytes detected varied significantly across studies. For example, tocopherools and tocotrienols in dietary supplements and oils [47; 49] included up to nine compounds. In contrast, carotenoid profiling in microalgae and rosehip [48] involved up to ten analytes, such as \(\alpha\)-carotene, \(\beta\)-carotene, lycopene, canthaxanthin, lutein, zeaxanthin, \(\beta\)-cryptoxanthin, astaxanthin, and violaxanthin. Detection systems ranged from DAD to advanced QTOF-MS and APCI-QqQ/MS, which allowed for precise mass-based identification. The study on egg yolk from hens supplemented with \(\beta\)-carotene utilized DAD-MS/MS, enabling spectral and structural confirmation of \(\beta\)-carotene, lutein, and zeaxanthin [51]. The food matrices analyzed included hemp seed oil, waste fish oil, dietary supplements, microalgae, rosehip, habanero peppers, and egg yolk. These diverse sources highlight the broad applicability of SFC for lipid-soluble vitamins and carotenoids. _Capsicum chinense_[50] was particularly interesting as it contained ten apocarotenoids, demonstrating SFC's ability to separate structurally similar compounds effectively. A recent study by [PERSON] et al. developed a two-dimensional separation method (SFC \(\times\) RP-UHPLC) to improve the analysis of carotenoids in red chili pepper (_Capsicum annuum_ L.) [52]. This approach combined SFC using an Ascents ES Cyano column (250 \(\times\) 1.0 mm, 5 \(\upmu\)m) from Supelco (Darmstadt, Germany) in the first dimension, followed by reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) on an Acquity UPLC BEH C18 column (50 \(\times\) 2.1 mm, 1.7 \(\upmu\)m) from Waters (MA, USA) in the second dimension. This method achieved a more efficient separation of 50 carotenoids, including \(\beta\)-carotene, capsanthin, lutein, zeaxanthin, phytofluene, neurosporene, \(\beta\)-cryptoxanthin, and capsorubin. This approach improved analytical accuracy by classifying carotenoids into 15 structural groups and resolving co-elution challenges. Additionally, combining DAD, Q-ToF MS, and ion mobility spectrometry (IMS) allowed for detailed cis/trans isomer differentiation [52; 53]. Compared to conventional NP-LC \(\times\) RP-LC, this method reduced analysis time from 120 to 60 min. It decreased solvent consumption by 90%, demonstrating the potential of SFC \(\times\) RP-UHPLC for advanced carotenoid profiling [52; 53]. This highlights the evolving role of SFC in food analysis, offering both sustainability and enhanced separation efficiency.