BS ISO 7358:2021 pdf download – Essential oils of bergamot, lemon, bitter orange and lime, fully or partially reduced in bergapten — Determination of bergapten content by high- performance liquid chromatography (HPLC) 8.2.1.2 External standard method Follow the procedure for the external standard method as specified in ISO 8432. 8.2.2 HPLC in reversed phase 8.2.2.1 Preparation of the reference solutions for calibration 8.2.2.1.1 General For each calibration, use the stock solutions of bergapten and coumarin to prepare the standard solution. Store the standard solution in a cool place at 4 °C. 8.2.2.1.2 Stock solutions As accurately as possible weigh 50 mg of bergapten into a 100 ml flask and make up to volume with the dilution solvent. Homogenize the solution then leave it for 3 min in the ultrasonic bath to get stock solution. This stock solution contains almost exactly 500 mg/l of bergapten. As accurately as possible weigh 50 mg of coumarin into a 50 ml flask and make up to volume with the elution solvent. Homogenize the solution then leave it for 3 min in the ultrasonic bath to get stock solution. This solution contains almost exactly 1 000 mg/l of coumarin. 8.2.2.1.3 Working solutions Using the stock solutions, prepare a standard range of eight calibration points for bergapten concentration: 1 mg/l, 2,5 mg/l, 5 mg/l, 10 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200 mg/l. — 1 mg/l standard solution: into a 50 ml flask, transfer 100 µl of bergapten stock solution and 5 ml of coumarin stock solution then make up to volume with the dilution solvent. — 2,5 mg/l standard solution: into a 50 ml flask, transfer 250 µl of bergapten stock solution and 5 ml of coumarin stock solution then make up to volume with the dilution solvent. — 5 mg/l standard solution: into a 50 ml flask, transfer 500 µl of bergapten stock solution and 5 ml of coumarin stock solution then make up to volume with the dilution solvent. — 10 mg/l standard solution: into a 50 ml flask, transfer 1 ml of bergapten stock solution and 5 ml of coumarin stock solution then make up to volume with the dilution solvent. — 25 mg/l standard solution: into a 50 ml flask, transfer 2,5 ml of bergapten stock solution and 5 ml of coumarin stock solution then make up to volume with the dilution solvent. — 50 mg/l standard solution: into...

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