Zorbax rx sil column

The High-Performance Liquid Chromatography (HPLC) method, as delineated by Reference (33 ), was executed employing a normal phase with direct saponification and n-hexane extraction as outlined by Prates (32 (link)).
The HPLC apparatus was outfitted with an Agilent Zorbax Rx-Sil column (5 μm particle diameter, 4.6 mm ID × 25 cm) maintained at a temperature of 20°C. The mobile phase comprised a hexane-isopropanol mixture (99.1 ratio) with a flow rate set at 1.0 mL/min. The injection volumes were configured at 10 and 100 μL for α-tocopherol and α-tocotrienol (vitamin E) analysis, respectively, and 100 μL for cholesterol, β-carotene, and other com-pound assessments.
The quantifications of cholesterol, β-carotene, and vitamin E were conducted using the external standard method. The samples were injected separately and identified by comparing the retention times. Specifically, for the tocopherol profile, four distinct calibration curves were utilized (corresponding to α-tocopherol, β-tocopherol, γ-tocopherol, and ∆-tocopherol), and similarly for tocotrienols, four calibration curves were applied (corresponding to α-tocotrienol, β-tocotrienol, γ-tocotrienol, and ∆-tocotrienol). For the remaining compounds, individual calibration curves were employed—one for cholesterol and another for β-carotene, aligning with the previous method of area versus concentration comparison.

Lipidomics experiments were performed at the Lipidomics Facility of Columbia University and analyzed by LC-MS. Lipids were separated via chloroform-methanol extraction and analyzed with a 6490 Triple Quadrupole LC-MS system (Agilent Technologies) according to the manufacturer’s instructions. Glycerophospholipids were separated with normal-phase HPLC using an Agilent Zorbax Rx-Sil column (inner diameter, 2.1 × 100 mm).

The separation of the lipid classes was accomplished in an HPLC system (model 1260; Agilent Technologies Inc. Palo Alto, CA, USA) coupled with an ELSD (SEDEX 85 model; Sedere SAS, Alfortville Cedex, France) while using prefiltered compressed air as the nebulizing gas at a pressure of 350 kPa at 60 °C; in addition, the gain was set at 3. Two columns in series (250 × 4.5 mm Zorbax Rx-SIL column with 5-μm particle diameter; Agilent Technologies Inc.) and a pre-column with the same packing were used. Before analysis, samples were dissolved in dichloromethane (5 mg/mL) and 50 μL was injected after column equilibration at 40 °C. The solvent gradient was conducted as detailed in Castro-Gómez et al., (2017) [37 (link)]. Both samples and standards were analyzed under the same conditions, using solvents that were freshly prepared.

Lipids were extracted from equal amounts of material (30 μg protein/sample). Lipid extracts were prepared via chloroform–methanol extraction, spiked with appropriate internal standards, and analyzed using a 6490 Triple Quadrupole LC/MS system (Agilent Technologies, Santa Clara, CA) as described previously (Chan et al, 2012). Cholesterol and cholesteryl esters were separated with normal‐phase HPLC using an Agilent Zorbax Rx‐Sil column (inner diameter 2.1 Å ~100 mm) under the following conditions: mobile phase A (chloroform:methanol:1 M ammonium hydroxide, 89.9:10:0.1, v/v/v) and mobile phase B (chloroform:methanol:water:ammonium hydroxide, 55:39.9:5:0.1, v/v/v/v); 95% A for 2 min, linear gradient to 30% A over 18 min and held for 3 min, and linear gradient to 95% A over 2 min and held for 6 min. Quantification of lipid species was accomplished using multiple reaction monitoring (MRM) transitions that were developed in earlier studies (Chan et al, 2012) in conjunction with referencing of appropriate internal standards. Values are represented as mole fraction with respect to total lipid (% molarity). For this, lipid mass of any specific lipid is normalized by the total mass of all lipids measured (Chan et al, 2012).

We used HPLC to examine the retinoid content of eyes of juvenile and adult lamprey. The eyes were homogenized under dim light conditions in cold saline with a glass dounce. Retinaldehydes were then derivatized by treatment with hydroxylamine (Sigma, 255580) and extracted with hexane. The extract was dried under a stream of nitrogen, resuspended in 120 µl of hexane, and 100 µl was injected into an Agilent 1100 series HPLC equipped with a Zorbax RX-SIL column (4.6 × 250 mm, 5 µm, Agilent). The samples were eluted with a gradient mobile phase consisting of 0.5% ethyl acetate in hexane for 5 min then a ramp up to 10% ethyl acetate in hexane from 5 to 20 min, followed by isocratic conditions through 35 min. The column was held at 25°C, and the flow rate was 1.4 ml min⁻¹ throughout the run. The samples were monitored with a photodiode array detector at 325, 350 and 380 nm, and retinoids were putatively identified by comparison to authentic standards or published accounts (electronic supplementary material, table S1) [26 (link)–29 (link)].

The samples were accurately weighed and dissolved in dichloromethane to a concentration of 5 mg/mL. Afterwards, samples were analysed on an HPLC (model 1260 Infinity II; Agilent Technologies, Santa Clara, CA, USA) attached to an evaporative light scattering detector (ELSD; 1290 Infinity II, Agilent Technologies, Santa Clara, CA, USA) using nitrogen as a nebulising gas coupled to a Zorbax RX-SIL column (Agilent; 2.1 × 150 mm, 5 µm). Analysis conditions were assayed as described by Abreu et al. [33 (link)], with some changes. Four mobile phases were used with the following compositions: A, isooctane:ethyl acetate (99.8:0.2, v/v); B, acetone:ethyl acetate (2:1, v/v) containing 0.1% acetic acid (v/v); C, 2-propanol:water (85:15, v/v) containing 0.013% acetic acid (v/v) and 0.031% of TEA v/v; and D, EtAc.
The flow rate was set at 0.275 mL/min with the gradient composition described in Table S1. and an injection volume of 20 µL. The detector was set as follows: the evaporator and nebuliser temperature was set to 60 °C with nitrogen as the nebulising gas at a 1.20 SLM flow rate.