## 2 Chromatographic Techniques for Carotenoids Analysis
### High-Performance Liquid Chromatography  
HPLC is the most widely employed method for carotenoid analysis due to its precision and ability to separate individual carotenoids effectively. Table 1 compares the determination of carotenoids in different foods using HPLC coupled with different detection systems [12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25]. Reversed-phase liquid chromatography (RP-LC) is particularly popular, using C18 and C30 columns with non-polar stationary phases to achieve excellent resolution [12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25]. C18 phases, such as Spherisorb ODS-2 and BEH C18, are suitable for hydrophobic carotenoids [12; 13; 18; 19; 20; 22; 25], while C30 phases, like YMC C30 and Prontosil C30, offer superior separation of structurally similar compounds and geometric isomers [13; 14; 15; 16; 17; 21; 23; 24]. As seen in Kinetex C18, core-shell technology enhances separation efficiency, whereas sub-2 \(\upmu\)m particles in BEH C18 facilitate UHPLC applications, reducing analysis time while maintaining high sensitivity [13; 18; 19; 20; 25]. The choice of mobile phases in HPLC depends on the polarity and solubility of carotenoids. Commonly used solvents include methanol (MeOH), acetonitrile (ACN), methyl tert-butyl ether (MTBE), tetrahydrofuran (THF), acetone, and water. Some methods incorporate EtOAc for improved solubility, while ammonium acetate (NH\({}_{4}\)OAc) is used as a buffer modifier [13; 16]. Environmental concerns arise from solvents such as ACN, MeOH, and MTBE, which pose toxicity and waste disposal issues [26]. MTBE, in particular, is environmentally persistent and a potential groundwater contaminant [26]. Sustainable alternatives, such as ethyl acetate, are being explored to reduce environmental impact [25; 26; 27]. Modifiers like trifluoroacetic acid and trimethylamine improve peak shape but require careful handling due to their corrosiveness and volatility [14; 20; 28; 29]. Column dimensions in HPLC vary depending on the analytical needs. Standard 4.6 mm LD. columns with lengths between 150 and 250 mm provide high-resolution separations. In comparison, narrow-bore (2.1 mm LD.) columns, such as BEH C18, are used in UHPLC for increased sensitivity and lower solvent consumption [18; 20]. Flow rates range from 0.3 to 2.0 mL/min, with higher rates for larger-particle columns and lower for sub-2 \(\upmu\)m particle columns. The analysis time spans from 6 to 140 min, with shorter columns and smaller particle sizes leading to faster analyses, while longer columns ensure better separation for complex matrices. \begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline
**Sample** & **Analytes** & **Stationary Phase** & **Mobile Phase** & \begin{tabular}{c} **Flow Rate** \\ **(mL/min)** \\ \end{tabular}  & \begin{tabular}{c} **Detector** \\ **System** \\ \end{tabular}  & \begin{tabular}{c} **Analysis** \\ **Time** \\ **(min)** \\ \end{tabular}  & \begin{tabular}{c} **LOD** \\ **(ng/g)** \\ \end{tabular}  & \begin{tabular}{c} **LOQ** \\ **(ng/g)** \\ \end{tabular}  & \begin{tabular}{c} **Recovery** \\ **(\%)** \\ \end{tabular}  & **Ref. ** \\ \hline Red cell peppers (9 varieties) & Capsanthin, Zexexanthin, & \begin{tabular}{c} Spheresoft DC-2 \\ Cl8 (250 mm x 4.6 mm, 5 mm) \\ \end{tabular}  & \begin{tabular}{c} MoOH/THF/ \\ \(\mathrm{ACN}/\)Acetone \\ \end{tabular}  & 0.5/1.0/1.5 & DAD & 25 & 20-63 & 67–209 & 88–107 & [12] \\ \hline Legunes (7 species, including beams, peas, and lentils) & Latetin, Zexanthin, & \begin{tabular}{c} Cl30 (250 \(\times\) \\ 6-Carroene, e-Actene, \\ \end{tabular}  & \ \begin{tabular}{c} MPE, \\ 4.6 mm LD, \\ 5-Crytoplasmin \\ \end{tabular}