C30 guard column

The carotenoid extracts were characterized and quantified by high-performance liquid chromatography-diode array detection (HPLC-DAD), which scans the entire UV-visible light spectrum during analysis, as described by Rodrigo et al. (2015) [22 (link)]. The liquid chromatography system was equipped with a 600E pump, a DAD model 2998 and Empower3 software (Waters^®, Barcelona, Spain). A C₃₀-carotenoid column (250 × 4.6 mm, 5 μm) coupled to a C₃₀ guard column (20 × 4.0 mm, 5 μm) (YMC, Teknokroma, Spain) was used. Samples were prepared for HPLC by dissolving the dried carotenoid extracts in chloroform:MeOH:acetone (3:2:1, v:v:v). A ternary gradient elution with MeOH, water and MTBE was used for carotenoid separation [8 (link),10 (link),23 (link)]. The carotenoids were identified by absorbance spectra and retention time. For each elution, a Maxplot chromatogram was obtained which integrated each carotenoid peak at its corresponding maximum absorbance wavelength and their contents were calculated using the appropriate calibration curves, as described elsewhere [8 (link),10 (link),23 (link)].

The carotenoid composition of each sample was analysed by HPLC with a Waters liquid chromatography system equipped with a 600E pump and a photodiode array detector (DAD) model 2998, and Empower3 software (Waters, Spain). A C30 carotenoid column (250 × 4.6 mm, 5 μm) coupled to a C30 guard column (20 × 4.0 mm, 5 μm) (YMC, Teknokroma, Spain) was used. The chromatographic conditions are described in Lado et al. (2015) (link) and Rodrigo et al. (2015) (link). Absorbance spectra and retention time identified each carotenoid, peaks were integrated at their individual maximal wavelength, and their contents were calculated using the appropriate calibration curves, as described elsewhere (Lado et al., 2015 (link); Rodrigo et al., 2015 (link)).