The loaded amount of lysozyme was investigated using the indirect method by reverse-phase high-pressure liquid chromatography (HPLC) with a method previously reported and validated by our group [12 (link)] using an Ultimate 3000 HPLC System (Thermo Fisher Scientific, Waltham, MA, USA) with a quaternary pump, a column oven, a LichroSphere® 100 RP 18 column (Merck KGaA, Darmstadt, Germany), and a UV–vis detector. The mobile phase consisted of solvent A: 10% acetonitrile, 90% MiIli-Q® water and 0.1% trifluoro acetic acid and B: 90% acetonitrile, 10% Milli-Q® water and 0.1% trifluoro acetic acid. The flow rate was 0.8 mL/min, and a gradient was set from 100% A to 100% B in 15 min. The concentration was determined using a calibration row by different lysozyme concentrations and the resulting AUC/min extracted from the chromatogram covering the relevant concentrations.
Lichrosphere 100 rp 18 column
Manufactured by Merck Group
Sourced in Germany
The LichroSphere® 100 RP 18 column is a reversed-phase liquid chromatography column. It is designed for the separation and purification of a wide range of organic compounds.
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2 protocols using lichrosphere 100 rp 18 column
1
Quantifying Lysozyme Levels by HPLC
2
Carotenoid Content Quantification by HPLC
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The content of carotenoids was determined by high performance liquid chromatography (HPLC) using a LaChrom chromatograph (Merck-Hitachi) equipped with a L-7450 diode array detector, a L-7100 gradient pump, a L-7250 autosampler and a D7000 interface. Separations were carried out on a LiChrosphere 100 RP-18 column (5 μm; 250 × 4 mm; Merck Darmstadt Germany). The column temperature was maintained at 30°C. Mobile phases consisted of methanol/acetonitryle (9:1 v/v), acetone and 1M ammonium acetate. Extraction of samples and separations at a flow rate of 1 mL/min in gradient manner were performed according to Fiutak et al. (2019) . Detection of carotenoids was conducted at 350-750 nm. The content of carotenoids was evaluated on the basis of standard curves.
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