Analysis of Peptone Samples Using the Agilent InfinityLab 2D‑LC Solutions with Active Solvent Modulation

Significance of the Topic

Peptones are complex peptide and amino acid mixtures essential for cell culture media in biopharma. Batch-to-batch variability affects productivity and quality. Two-dimensional liquid chromatography (2D-LC) combining size exclusion (SEC) and reversed-phase (RPLC) offers enhanced resolution for characterizing such complex samples. The use of active solvent modulation (ASM) addresses solvent incompatibility between dimensions and preserves chromatographic performance.

Objectives and Study Overview

This study demonstrates the application of an Agilent 1290 Infinity II 2D-LC system with ASM for analyzing commercial and research casein hydrolysates. Multiple heart-cutting and high-resolution sampling modes were compared. The aim was to evaluate chromatography performance, injection precision, and to reveal batch differences in peptone samples via combined DAD and MS detection.

Methodology and Instrumentation

• SEC in the first dimension separated peptides by size under isocratic conditions (70/30 water/acetonitrile with 0.1% TFA) using a 4.6×300 mm column.
• RPLC in the second dimension separated by hydrophobicity with gradient elution on a 3.0×50 mm C18 column.
• Multiple heart-cutting collected 40 µL loops at defined retention times; high-resolution sampling used shorter sampling windows for detailed mapping.
• Active Solvent Modulation diluted injected fractions with weak solvent before RPLC to prevent breakthrough and distortion.
• Detection by diode array (214 nm) and ESI single quadrupole MS (100–1750 m/z) established identity and intensity improvements.

Applied Instrumentation

• Agilent 1290 Infinity II 2D-LC system with ASM and multiple valve drives
• Agilent AdvanceBio SEC 130 Å, 4.6×300 mm, 2.7 µm column
• Agilent ZORBAX Eclipse Plus C18 RRHD columns (3.0×50 mm and 3.0×100 mm, 1.8 µm)
• Agilent 6130 single quadrupole LC–MS with ESI source
• Diode array detectors (Max-Light and HDR cells)
• Agilent OpenLab CDS ChemStation and 2D-LC software

Results and Discussion

• One-dimensional SEC and RPLC highlighted batch differences: commercial samples had higher low-molecular-weight fractions and simpler RPLC profiles than research samples.
• High-resolution and heart-cutting 2D-LC preserved retention time repeatability (<0.1% RSD) and peak area precision (<5% RSD).
• Without ASM, early eluting polar compounds exhibited breakthrough and peak distortion in RPLC; ASM restored signal intensity and peak shape by up to fivefold.
• Comparative 2D maps revealed distinct peptide distributions among research batches; targeted MS confirmed differences in specific m/z traces (e.g., m/z 449.8).

Benefits and Practical Applications

• Improved peak capacity and quantitative precision for complex peptide mixtures.
• Efficient identification of batch-to-batch variability in peptone quality control.
• Compatibility of ASM with DAD and MS detection ensures reliable profiling without sample loss or distortion.
• Adaptable heart-cutting and high-resolution modes allow focused analysis or comprehensive mapping.

Future Trends and Potential Applications

• Integration of high-resolution MS and ion mobility for deeper peptide structure elucidation.
• Automation of 2D-LC workflows for real-time process monitoring in biomanufacturing.
• Expansion to other complex matrices such as protein digests, monoclonal antibody fragments, and metabolomics samples.
• Development of machine learning tools to interpret multidimensional chromatographic data.

Conclusion

The Agilent InfinityLab 2D-LC system with Active Solvent Modulation provides robust and precise analysis of complex peptone samples. By combining SEC and RPLC, and leveraging ASM, the method overcomes solvent incompatibility to deliver high peak capacity, reliable quantitation, and clear batch differentiation. This approach enhances quality control workflows in biopharmaceutical manufacturing.

References

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