Spotting Fraction Impurities with More Confidence Using the Agilent 1290 Infinity II Preparative Open-Bed Sampler/Collector

Significance of the Topic

The ability to detect trace impurities in preparative chromatography fractions is critical for ensuring the purity of collected samples and minimizing downstream errors. Automated homogenization prior to fraction reanalysis reduces manual handling, improves representativeness of the sample, and increases confidence in purity assessment.

Objectives and Study Overview

This study evaluated the impact of an integrated automated homogenization feature in the Agilent 1290 Infinity II Preparative Open-Bed Sampler/Collector on detecting fraction impurities. Two fractions, each containing a target compound and a co-collected impurity, were reanalyzed with and without homogenization to compare apparent and actual purities.

Methodology

A preparative LC separation was performed using a binary gradient of 0.1% formic acid in water and acetonitrile at 20 mL/min. Two fractions were collected based on a UV trigger threshold of 10 mAU. Reanalysis employed an analytical gradient at 1 mL/min with default injection presets. One set of reanalysis runs omitted mixing; the other used 15 air-mixing cycles and a 1:20 dilution to prevent detector saturation.

Used Instrumentation

• Agilent 1290 Infinity II Preparative Open-Bed Sampler/Collector (G7158B)
• Agilent 1290 Infinity II Preparative Binary Pump (G7161B) with 200 mL pump heads
• Agilent 1290 Infinity II Preparative Column Compartment (G7163B)
• Agilent 1260 Infinity II DAD WR preparative and analytical detectors (G7115A)
• Agilent 1260 Infinity II Quaternary Pump (G7111B)
• Preparative column: ZORBAX SB-C18 PrepHT, 21.2×50 mm, 5 µm
• Analytical column: ZORBAX Eclipse XDB-C18, 4.6×50 mm, 5 µm
• OpenLab CDS ChemStation C.01.10 for LC/LC-MS data handling

Main Results and Discussion

Preparative fractionation grouped compounds A/B in fraction 1 and C/D in fraction 2 due to incomplete baseline separation. Reanalysis without homogenization indicated purity ≥99% for both fractions, as only the major peaks were sampled. After automated mixing, the co-eluted impurities became detectable, revealing actual purities of 92% and 93%, respectively. Chromatogram overlays clearly demonstrate the necessity of homogenization to achieve representative sampling.

Benefits and Practical Applications

• Combines purification and reanalysis in one platform with minimal manual intervention
• Automated homogenization ensures uniform mixing and reliable impurity detection
• Reduces sample handling errors and saves analyst time
• Facilitates scale-up workflows, scouting, and preparative workflows with integrated fraction verification

Future Trends and Opportunities

Advancements may include optimized mixing algorithms tailored to fraction volume and vessel geometry, integration of real-time analytics for dynamic fraction gating, and AI-driven workflow optimization. Expanding compatibility with diverse solvent systems and column formats will further broaden applicability in complex purification schemes.

Conclusion

The Agilent 1290 Infinity II Preparative Open-Bed Sampler/Collector’s automated homogenization feature is essential for accurate fraction purity assessment. Incorporating mixing and dilution within the same module eliminates manual steps, uncovers hidden impurities, and enhances confidence in preparative workflows.