方案摘要
方案下载应用领域 | 制药/生物制药 |
检测样本 | 其他 |
检测项目 | |
参考标准 | 暂无 |
Synthesizing novel compounds or isolating natural products can be a laborious and time-consuming process. After analyzing the precious sample on an analytical UHPLC system, the crucial step is to transfer the method to a preparative system with a minimal risk of losing valuable work or collecting impure compounds. This Technical Overview describes a practical way to optimize the scale-up process for reversed-phase chromatography from an analytical UHPLC system to preparative LC systems using a focused gradient to increase the sample load on the preparative column to achieve optimum purity.
Abstract
Synthesizing novel compounds or isolating natural products can be a laborious and time-consuming process. After analyzing the precious sample on an analytical UHPLC system, the crucial step is to transfer the method to a preparative system with a minimal risk of losing valuable work or collecting impure compounds.
This Technical Overview describes a practical way to optimize the scale-up process for reversed-phase chromatography from an analytical UHPLC system to preparative LC systems using a focused gradient to increase the sample load on the preparative column to achieve optimum purity.
Introduction
Generic gradients are suitable to cope with a large variety of sample types when there is no capacity or time to optimize the separation. Each gradient can be divided into four different steps. After the injection, an isocratic hold step can be applied to remove the injected solvent from the column and to improve resolution especially for polar compounds. The second step is a linear slope, which will be applied to separate effectively based on the chromatographic properties of the target compounds, followed by a purge phase. In the last step, the column is re-equilibrated at the initial solvent composition for the next sample analysis or purifi cation run. To improve resolution around the target compound, the linear slope has to be modifi ed.
In preparative chromatography, most often the goal is to isolate, effi ciently, a large amount of one or a few target compounds out of a crude mixture. Ideally, the chromatographic resolution around the target peak and the column load are increased without signifi cantly increasing the separation runtime. From the crude sample, an optimized method can be generated with the goal to extend the resolution between the target peak (green peak, Figures 1 and 2) and its neighbor compounds.
A basic approach to generate optimized preparative methods can be to divide the linear generic gradient method into time slices (described in the Technical Note 5991-3070EN1 ). This approach generates a set of preparative methods that can be used in any further sample purifi cation simply by identifying the time slice where the target peak elutes.
In this Technical Overview, an approach with focused gradients on target peak was used. This approach generates then a unique and dedicated method to a concerned target peak which has the advantage to increase the resolution better than the time slices method.
All optimization steps occur on the analytical system. After obtaining the fi rst chromatographic information of the crude mixture by using a generic gradient, the resolution is optimized by fl attening the slope followed by a loading study to determine the maximum column load before scaling up to the preparative column dimensions.
Conclusion
A scale-up from a 2.1-mm id column on a UHPLC system to a 1260 Infinity Preparative scale system equipped with a 21.2-mm id column was successfully developed.
For all scale-up situations, a correct method transfer was required to keep the resolution constant. This ensured maximum purity and recovery from the precious sample.
The steps in Table 3 summarize the process.
使用 Agilent 8890 气相色谱和 TCD/FID 系统分析氢气中的氦气、 氩气、氮气和烃类杂质
使用 Agilent InfinityLab 在线液相色谱 系统实现对流动化学反应器中反应过 程的实时监测
使用配备氦气节省模块以进行载气 切换的 Agilent 8850 气相色谱系统 分析香草提取物的方法转换
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