Two step purification with the sample pump set up and PurityChrom 6

Importance of the Topic

Two-step purification strategies play a critical role in modern protein chromatography by combining two distinct chromatographic steps into a single automated workflow. This approach improves product quality and purity, reduces manual handling and cross-contamination risks, and increases throughput in bio-analytical and process development laboratories.

Objectives and Study Overview

This application note describes the implementation of an automated two-step protein purification using a sample pump setup on the PurityChrom 6 system. The aim is to demonstrate how independent chromatographic methods, each with a dedicated column, can be seamlessly coupled to achieve high-purity protein separations without manual intervention.

Methodology and Instrumentation

The workflow uses an AZURA Bio Lab System Advanced or equivalent equipped with:

• A sample pump with air sensor for automatic sample loading.
• A multi-injection valve for sample transfer and intermediate peak storage.
• A column selection valve to switch between ion exchange and desalting columns.
• An outlet valve to direct eluent to fraction collector or loop.
• UV detector and conductivity monitor for real-time peak detection and threshold-driven valve switching.
• PurityChrom 6 software to define two sequential methods and a combined sequence table.

First, a 1 ml ion exchange column is equilibrated and the protein sample is pumped in. The eluting peak triggers storage in an injection loop based on UV thresholds. Second, the looped fraction is redirected onto a 5 ml desalting column to polish the sample and remove salts. Automated valve switching ensures precise collection and minimal dead volume effects.

Main Results and Discussion

The sample pump configuration enabled loading of large volumes with high reproducibility. Threshold-based triggering of valve switching captured the target peak efficiently, with negligible cross‐contamination. The two methods executed in sequence delivered a highly pure protein fraction collected directly in small volumes or into a fraction collector.

Benefits and Practical Applications

• Fully automated, hands-free operation reduces operator workload.
• Improved reproducibility and consistency across runs.
• Minimized risk of sample carry-over by isolating intermediate peaks.
• Flexible configuration for different column chemistries and scales.

Future Trends and Possibilities for Use

Advances in valve technology, sensor integration, and software intelligence will further streamline multi-step purifications. Integration of mass-based detectors and inline dilution units could enable real-time process monitoring and adaptive control for biopharmaceutical manufacture.

Conclusion

The sample pump setup on the PurityChrom 6 platform offers a robust solution for automated two-step protein purifications. By combining ion exchange and desalting in one sequence, the method delivers high purity, flexibility, and throughput suitable for research and industrial QC applications.