2.5. Quantification and Structural Confirmation of Carotenoid and Pectin

The wet carotenoid–pectin complex was recovered and weighed gravimetrically. The complex was then suspended in 1 mL of THF. The pectin was removed by a round of centrifugation at 4500 rpm for 10 min, and the THF liquid fraction containing carotenoid suspended in THF was collected for further analysis. The absorbance of the sample was measured using a spectrophotometer (Genesys 20, Thermo Scientific, Waltham, MA, USA) at 480 nm, and the concentration of carotenoid was calculated using the calibration curves prepared by β-carotene standard and lycopene standard in the concentration range of 7.81–1000 ug/mL. The results were expressed as total carotenoid content (mg/100 g wet sample). The purity of carotenoid was analyzed using an HPLC system (Agilent Technologies 1200) equipped with a C30 reversed-phase column (250 mm × 46 ID, 5 µm, Waters, Zellik, Belgium) and a diode array detector. An isocratic elution was conducted using a mobile phase prepared by mixing methanol, isopropyl alcohol, and THF at a ratio of 30:30:35 [17]. The column temperature was kept at 35 °C and the injection volume was 10 µl. The flow rate through the column was set at 0.5 mL/min. The extracted pectin was structurally confirmed using Fourier-transform infrared spectroscopy (FTIR). An FTIR spectrometer (Nicolet iS10, Thermo Scientific) equipped with an attenuated total reflectance sampling accessory was used. The FTIR spectra of the fractionated pectin were obtained via a wavelength scan (550 cm⁻¹ to 4000 cm⁻¹) with a spectral resolution of 4 cm⁻¹ in 64 scans.