This paper mainly introduces the method of using Agilent Prep reverse phase column to realize the gradient separation of corticosteroids. Different diameter Agilent Prep reverse phase columns show nearly linear scale-up for the gradient separation of corticosteroids. With as much as 8 mg of total corticosteroid injected onto a 4.6 mm column and 320 mg onto a 30 mm column, the loadability of the columns is quite good.
This document describes the scalability and loadability of Agilent Prep C18 reversed-phase HPLC columns for the separation of corticosteroids. It details a method developed on an analytical scale using a 4.6-mm column and demonstrates how to linearly scale up to larger preparative columns with internal diameters of 21.2 mm and 30.0 mm while maintaining resolution and purity.
The note provides specific chromatographic conditions, including mobile phase compositions (water and a mixture of acetonitrile, methanol, and isopropanol), gradient profiles, and flow rates for different column sizes. It also defines loadability as the maximum sample mass that can be injected without compromising purity and presents experimental data showing the capacity of the 30-mm column to handle up to 320 mg of total corticosteroid.
| Analytical Column ID | 4.6 mm |
|---|---|
| Preparative Column IDs | 21.2 mm and 30.0 mm |
| Column Length | 150 mm |
| Mobile Phase A | Water |
| Mobile Phase B | 50% Acetonitrile: 17% Methanol: 33% Isopropanol |
| Injection Solvent | DMSO |
| Analytical Flow Rate | 1 mL/min |
| 21.2-mm Column Flow Rate | 21.2 mL/min |
| 30.0-mm Column Flow Rate | 42.4 mL/min |
| Maximum Loadability (30-mm) | 320 mg |
The column can handle up to 320 mg of total corticosteroid before overloading begins.
Dimethylsulfoxide (DMSO) is recommended because it dissolves organic compounds well and elutes rapidly.