Agilent 1260 prep als

The HPLC analysis of n-hexane extract and its fractions (F1 and F2) was performed using an Agilent 1260 infinity series HPLC-DAD system (Agilent Technologies, Waldbronn, Germany) equipped with a binary gradient Agilent 1260 prep pump (G1361A) and an autosampler Agilent 1260 prep ALS (G2260A). Agilent diode array detector 1260 DAD VL (G1315D) was employed for the detection of carotenoids. The separation was performed using an Agilent normal phase (NP) silica column (ZORBAX RX-Sil, 5μm, 4.6 X 150 mm). The following solvents (A) n-hexane and (B) acetone were used at a flow rate of 1 mL/min using a gradient between solvents A and B following the method of Prum et al. [24 (link)] with some modifications as follows: B was run at 0 to 30% for 5 min, 30 to 50% for 15 min, 50 to 100% for 3 min, and maintaining 100% of B until the end of the separation at 30 min. The peaks were integrated at 450 nm. β-carotene (Sigma-Aldrich) was used as a standard to identify the isolated β-carotene.

The HPLC analysis of SOCE and its fractions were performed using an Agilent 1260 infinity series HPLC–DAD system (Agilent Technologies, Waldbronn, Germany) equipped with a binary gradient Agilent 1260 prep pump (G1361A) and an autosampler Agilent 1260 prep ALS (G2260A). Agilent diode array detector 1260 DAD VL (G1315D) was employed for the detection of carotenoids. The separation was performed using an Agilent normal phase (NP) silica column (ZORBAX RX-Sil, 5 µm, 4.6 × 150 mm). The following solvents (A) n-hexane and (B) acetone were used at a flow rate of 1 mL/min using a gradient between solvents A and B following the method of Prum et al.^{28 (link)} with some modifications as follows: B was run at 0 to 30% for 5 min, 30 to 50% for 15 min, 50 to 100% for 3 min, and maintaining 100% of B until the end of the separation at 30 min. The peaks were integrated at 450 nm.

H. pluvialis crude extract and beta carotene and astaxanthin enrich fractions were subjected to an Agilent 1260 infinity series HPLC-DAD system (Agilent Technologies, Waldbronn, Germany) equipped with binary gradient Agilent 1260 prep pump (G1361A), an autosampler Agilent 1260 prep ALS (G2260A), and Agilent diode array detector 1260 DAD VL (G1315D) was employed for the detection of the separated β-carotene and zeaxanthin. Agilent 5 Prep-C18 Scalar column (5 μm, 150 mm × 4.6 mm) was utilized for separation. The following solvents were used at a flow rate of 1.25 ml/min: (A) acetone and (B) methanol: H2O (9 : 1 v/v) containing 0.05% BHT. The separation of β-carotene and astaxanthin was achieved by a gradient between solvents A and B for 40 min as follows: B was run at 80 to 20% for 25 min, 20% for 10 min, and 20 to 80%for 5 min [28 (link)]. The peaks were integrated at 450 nm to quantify β-carotene and astaxanthin. β-Carotene (C4582-5MG) and all-trans-astaxanthin (SML0982-50MG) were purchased from Sigma-Aldrich Co., USA, and used as standard. β-Carotene and all-trans-astaxanthin were identified and quantified by comparing retention time and the peak area of the unknown peak with the β-carotene and all-trans-astaxanthin standards. All the used solvents were HPLC grade from Sigma-Aldrich Co., USA.