在制备型液相色谱中自动生成聚焦梯度曲线——使用Agilent 1260 Infinity 自动化液质联用纯化系统进行样

Abstract 

With the Agilent 1260 Infinity automated LC/MS purifi cation system, the purification process has reached a new level of automation. A tailored-gradient profile for each target compound was generated to increase the resolution, increase columnload, and reduce the cycle runtime. The Agilent automated purifi cation software automatically generated the different tailored-gradient profi les. 

This Technical Overview describes an automated purifi cation process of a compound mixture, and shows cross-platform compatibility between different analytical LC systems and the Agilent 1260 Infi nity automated LC/MS purifi cation system. A standard mix was analyzed using four different analytical (U)HPLC systems, using different gradient profi les and different column dimensions. The four analytical result fi les were used to calculate gradient profi les, and purify target compounds. Through the use of this intelligent software algorithm, we obtained automated tailored-gradient profi les for all four samples using the purifi cation process. We purified 100 mg of a sample mix containing 24 mg of the target compound. We obtained 90 % recovery and 99 % purity of the target compound after purifi cation.

Introduction 

The Agilent 1260 Infi nity automated LC/MS purifi cation system is an easy-to-use, intelligent solution to automate the purifi cation process of compounds after their synthesis. The process starts with an analytical scouting run followed by a nonlinear scale up process that generates a focused gradient profi le to isolate dedicated target compounds. The aim of using focused gradient profi les is to increase: 

• The chromatographic resolution 

• The injection volume 

• The effi ciency of the overall process by reducing the cycle runtime 

Streamlining workfl ows by defi ning user privileges 

The Agilent automated purifi cation software guides users, with varying levels of chromatographic knowledge, to achieve successful target compound isolation in a highly streamlined process. Sample submission and software features can be adjusted according to the user’s expertise level. Two different user modes are available: the Expert mode for the experienced chromatographer, and the Easy-Prep mode for the less experienced user. By selecting the Expert mode, a chromatographer can create template fi les that contain all parameters required for the purifi cation process. Required parameters are for example, the system dwell volumes, extra column volumes, column dimensions, and customized focused gradient profiles, which have been created from an experienced chromatographer to purify different compound libraries showing specifi c chromatographic behaviors.

When switching to the Easy-Prep mode, the user can choose from a list of predefi ned templates covering their needs, then start the purifi cation process. Table 1 list the different user privileges.

This Technical Overview describes the automated purifi cation process of a compound mixture, and demonstrates a cross-platform compatibility between different analytical HPLC and UHPLC instrumentation and the purifi cation system. A standard mix was analyzed using four different analytical LC systems with different gradient profiles and column dimensions. The analytical result files were used to calculate tailored-gradient profiles, and purify the target compounds.

Conclusion 

This Technical Overview shows the analysis of a crude sample using four different HPLC and UHPLC systems with different column dimensions, fl ow rates, and gradient lengths. All analytical data fi les were processed using Agilent automated purifi cation software to generate tailored focused gradient profi les. To demonstrate the methodology, a preparative injection was performed to purify the target compound. In this Technical Overview, 22 mg of the target compound, corresponding to 90 % of recovery, were recovered. After fraction re-analysis, a purity level of 99 % purity was obtained. A streamlined and automated purifi cation process is supported, even when the analytical result fi les have been acquired on different HPLC and UHPLC systems.