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How to improve preparative HPLC by mass­­-directed fractionation

In preparative HPLC, it is common to monitor and control the purification via refractive index or UV signal. Although both detection principles can detect peaks, they are not specific enough to identify, for example, co-elution.
Exact analysis is usually carried out after separation by mass spectrometry.

Applying a mass spectrometer (MS) already during the preparative purification is therefore a promising option. It allows both a more accurate fractionation and the analysis directly at the separation.

The compact mass spectrometer 4000 MiD is perfectly suited for this purpose. It represents a perfect complement to preparative standard HPLC systems with its small footprint, integrated  vacuum pump and easy handling.

Intelligent fractionation for accurate and fast purification

The specialized purification software PurityChrom® now supports the mass spectrometer 4000 MiD. With PurityChrom the spectrometer can be used with manual control and also within methods. The available MS modes are SIM, Scan & XIC and MIX mode (see Basics below).
The extensive fractionation possibilities offered by control software PurityChrom allow an intelligent combination of different signals. Thus, a quick and resource-saving purification can be implemented.

In the PurityChrom software, all data channels that are recorded in the system can be monitored at the same time and be used for fractionation. For example, a peak can be isolated based on the UV and MS signals by triggering fractionation when a given UV signal appears in combination with a defined mass. However, it can also be arranged that the simultaneous measurement of an impurity in the mass spectrometer prevents fractionation.

For purifications performed in XIC or MIX mode, the post-run analysis can be used to examine the entire spectrum for each measurement point. Here at the latest, unwanted impurities or by-products can be detected. Thus, the subsequent analysis can be limited to the pure product fractions, which not only saves time and labor, but also solvents.

Mass spectrometer (MS) modes

Mass-triggered purification of epigallocatechin gallate from green tea extract

The green tea antioxidant, referred to as epigallocatechin gallate (EGCG), is said to have beneficial effects on human health. Therefore, it is of interest as a dietary supplement.

By using the mass spectrometer 4000 MiD and the software PurityChrom, the purification of epigallocatechin gallate from green tea extract could be carried out easily and quickly.

Grafics:
Preparative purification of a green tea extract.

Top: UV channel.

Bottom: MS channel for 457.4 m/z. With SIM for the mass of the desired EGCG.

 

Basic preparative system with mass spectrometer

The AZURA Prep Pilot 250 LPG with mass spectrometer 4000 MiD is a low pressure gradient (LPG) HPLC system for the mass-directed purification of samples in the laboratory scale. The AZURA Preparative HPLC system is designed and optimized for the purification of milligram to gram samples.

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