Two step purification with a basic set up

Importance of the topic

The purification of biomolecules, particularly proteins, requires high selectivity and reproducibility. Two-step automated chromatography workflows reduce manual handling, improve yield and purity, and ensure consistency in production and research settings. This approach is particularly valuable in biopharmaceutical development, quality control laboratories, and academic research where sample integrity and throughput are critical.

Study objectives and overview

This application note describes a basic configuration for automated two-step purification using a KNAUER AZURA Bio Lab system. The first chromatographic step employs ion exchange to capture the target protein, and the second step uses a desalting column to remove salts and low-molecular-weight impurities. The workflow is executed without manual intervention by transferring the intermediate elution peak through a sample loop and switching valves.

Methodology and instrumentation

The basic setup integrates:

• AZURA Bio Lab system with a system pump and sample pump
• Multi-injection valve for sample injection and intermediate peak collection
• Column selection valve to switch between first and second columns
• UV detector and conductivity monitor to detect protein peaks and salt gradients
• Outlet valve directing flow to waste, fraction collector, or back to the loop
• 1 mL ion exchange column and 5 mL desalting column
• PEEK capillaries for fluid connections and inline filters to protect valves

The workflow is controlled in PurityChrom software by defining two methods: an ion exchange method with a UV threshold trigger and a desalting method for final polishing.

Main results and discussion

The basic two-step protocol demonstrated reliable capture of the protein peak on the ion exchange column and quantitative transfer to the desalting column. UV threshold detection at 60 mAU enabled automated collection of the target peak into the sample loop. The second step efficiently removed buffer salts, yielding fractions with high purity. Limitations include small sample volume dictated by the loop size and diffusion losses, which can be mitigated by injecting larger volumes with the major pump and using high-capacity filters.

Benefits and practical applications

• Automated operation reduces hands-on time and operator variability.
• High reproducibility of retention and elution profiles.
• Flexible configuration allows scaling from small research samples to larger preparative runs.
• Suitable for protein purification, desalting, and buffer exchange in bioprocessing and QA/QC environments.

Future trends and opportunities

Advances may include integration with inline mass spectrometry, real-time analytics, and expanded valve configurations for multistep workflows. Further automation of sample loading by peristaltic or pressure-driven pumps and miniaturization of hardware will increase throughput and decrease solvent consumption.

Conclusion

The described basic two-step purification setup on the AZURA Bio Lab system offers an efficient, reproducible, and hands-free solution for protein isolation and desalting. By combining ion exchange and desalting columns with automated valve switching and UV-based peak detection, laboratories can achieve high-quality protein preparations with minimal manual intervention.

Reference

Krop U., Monks K. Two step purification with a basic set up. KNAUER Wissenschaftliche Geräte GmbH; 12/2020. VTN0014