Understanding system peaks in GPC/SEC for accurate and reliable analysis

Significance of the Topic

In gel permeation chromatography (GPC) or size exclusion chromatography (SEC), unexpected signal artifacts known as system peaks can compromise data quality and lead to misinterpretation of polymer or biomolecule distributions. Recognizing and characterizing these peaks is critical for labs engaged in quality control, polymer research, and biopharmaceutical analysis.

Objectives and Overview of the Study

This work demonstrates two strategies to identify and assign system peaks in GPC/SEC using refractive index detection. The first approach overlays chromatograms from blank and sample injections to distinguish sample-specific analyte peaks from system artifacts. The second approach involves spiking the mobile phase with individual buffer components to pinpoint the origin of each system peak.

Methodology and Instrumentation

A phosphate buffered saline (PBS) mobile phase (pH 7.4) was used with a dual-column SEC configuration maintained at 30 °C and a flow rate of 1 mL/min. A 20 µL partial loop injection mode introduced citric acid and ethylene glycol standards. Chromatographic data were collected on a system comprising an isocratic pump, a refractive index detector, a diode array detector, an autosampler, and a column thermostat. Spiking experiments injected individual PBS salts (NaCl, KCl, KH2PO4, Na2HPO4) to assign specific system peaks.

Main Results and Discussion

Blank injections revealed two consistent system peaks at the elution range where analyte peaks appear. Overlaying with citric acid and ethylene glycol chromatograms highlighted peak coelution and signal distortion by system artifacts. Individual salt spikes confirmed the identity of each peak, enabling unambiguous separation of true analyte signals from system-induced distortions.

Benefits and Practical Applications

• Improves accuracy of molecular weight distributions in polymers and proteins.
• Reduces risk of misassigning peaks during QA/QC processes.
• Guides method optimization by adjusting buffer composition and injection parameters.

Future Trends and Potential Applications

Advances may include automated peak recognition algorithms, integration of gradient elution SEC, novel detector technologies with enhanced sensitivity, and coupling with mass spectrometry or light scattering for comprehensive polymer characterisation.

Conclusion

System peaks are inherent in GPC/SEC due to transient disturbances in phase equilibrium. Utilizing blank overlays and targeted mobile phase spiking provides a robust framework for identifying and mitigating these artifacts, ensuring more reliable chromatographic analysis.

References

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2. Levin S., Grushka E. System peaks in liquid chromatography: origin, formation, importance. Anal. Chem. 1986, 58(8), 1602–1607.
3. Held D., Gores F. Tips & Tricks GPC/SEC: System peaks or ghost peaks. LCGC Int. 2019.